# Patent

## Title
Isopentenyl transferase sequences and methods of use

## Abstract

Methods and compositions for modulating plant development are provided. Polynucleotide sequences and amino acid sequences encoding isopentenyl transferase (IPT) polypeptides are provided. The sequences can be used in a variety of methods including modulating root development, modulating floral development, modulating leaf and/or shoot development, modulating senescence, modulating seed size and/or weight, and modulating tolerance of plants to abiotic stress. Polynucleotides comprising an IPT promoter are also provided. The promoter can be used to regulate expression of a sequence of interest. Transformed plants, plant cell, tissues, and seed are also provided.

## Background
CROSS-REFERENCE
This application claims priority to, and hereby incorporates by reference, U.S. patent application Ser. No. 12/388,113, filed Feb. 18, 2009, U.S. patent application Ser. No. 11/228,659 filed Sep. 16, 2005, U.S. Provisional Patent Application Ser. No. 60/610,656 filed Sep. 17, 2004; 60/637,230 filed Dec. 17, 2004; and 60/696,405 filed Jul. 1, 2005.


FIELD OF THE INVENTION
The invention relates to the field of genetic manipulation of plants, particularly the modulation of gene activity to affect plant development and growth.
BACKGROUND OF THE INVENTION
Cytokinins are a class of N6 substituted purine derivative plant hormones that regulate cell division and influence a large number of developmental events, such as shoot development, sink strength, root branching, control of apical dominance in the shoot, leaf development, chloroplast development, and leaf senescence (Mok, et al., (1994) Cytokinins. Chemistry, Action and Function. CRC Press, Boca Raton, Fla., pp. 155-166; Horgan, (1984) Advanced Plant Physiology ed. M B., Pitman, London, UK, pp. 53-75; and Letham, (1994) Annual Review of Plant Physiol 34:163-197). In maize, cytokinins (CK) play an important role in establishing seed size, decreasing tip kernel abortion, and increasing seed set during unfavorable environmental conditions (Cheikh, et al., (1994) Plant Physiol. 106:45-51; Dietrich, et al., (1995) Plant Physiol Biochem 33:327-36). Active cytokinin pools are regulated by rates of synthesis and degradation.
Until recently, roots were believed to be the major site of cytokinin biosynthesis but evidence indicates that others tissues, such as shoot meristems and developing seeds, also have high cytokinin biosynthetic activity. It has been suggested that cytokinins are synthesized in restricted sites where cell proliferation is active. The presence of several AtIPT genes in Arabidopsis and their differential pattern of expression might serve this purpose.
The catabolic enzyme isopentenyl transferase (IPT) directs the synthesis of cytokinins and plays a major role in controlling cytokinin levels in plant tissues. Multiple routes have been proposed for cytokinin biosynthesis. Transfer RNA degradation has been suggested to be a source of cytokinin, because some tRNA molecules contain an isopentenyladenosine (iPA) residue at the site adjacent to the anticodon (Swaminathan, et al., (1977) Biochemistry 16:1355-1360). The modification is catalyzed by tRNA isopentenyl transferase (tRNA IPT; EC 2.5.1.8), which has been identified in various organisms such as Escherichia coli, Saccharomyces cerevisiae, Lactobacillus acidophilus, Homo sapiens, and Zea mays (Bartz, et al., (1972) Biochemie 54:31-39; Kline, et al., (1969) Biochemistry 8:4361-4371; Holtz, et al., (1975) Hoppe-Seyler's Z Physiol. Chem 356:1459-1464; Golovko, et al., (2000) Gene 258:85-93; and, Holtz, et al., (1979) Hoppe-Seyler's Z Physiol. Chem 359:89-101). However, this pathway is not considered to be the main route for cytokinin synthesis (Chen, et al., (1997) Physiol. Plant 101:665-673 and McGraw, et al., (1995) Plant Hormones, Physiology, Biochemistry and Molecular Biology. Ed. Davies, 98-117, Kluwer Academic Publishers, Dordrecht).
Another possible route of cytokinin formation is de novo biosynthesis of iPMP by adenylate isopentenyl transferase (IPT; EC 2.5.1.27) with dimethylallyl-diphosphate (DMAPP), AMP, ATP, and ADP as substrates. Our current knowledge of cytokinin biosynthesis in plants is largely deduced from studies on a possible analogous system in Agrobacterium tumefaciens. Cells of A. tumefaciens are able to infect certain plant species by inducing tumor formation in host plant tissues (Van Montagu, et al., (1982) Curr Top Microbiol Immunol 96:237-254; Hansen, et al., (1999). Curr Top Microbiol Immunol 240:21-57). To do so, the A. tumefaciens cells synthesize and secrete cytokinins which mediate the transformation of normal host plant tissues into tumors or calli. This process is facilitated by the A. tumefaciens tumor-inducing plasmid which contains genes encoding the necessary enzyme and regulators for cytokinin biosynthesis. Biochemical and genetic studies revealed that Gene 4 of the tumor-inducing plasmid encodes an isopentenyl transferase (IPT), which converts AMP and DMAPP into isopentenyladenosine-5′-monophosphate (iPMP), the active form of cytokinins (Akiyoshi, et al., (1984) Proc. Natl. Acad. Sci USA 81:5994-5998). Overexpression of the Agrobacterium ipt gene in a variety of transgenic plants has been shown to cause an increased level of cytokinins and elicit typical cytokinin responses in the host plant (Hansen, et al., (1999) Curr Top Microbiol Immunol 240:21-57). Therefore, it has been postulated that plant cells use machinery similar to that of A. tumefaciens cells for cytokinin biosynthesis. Arabidopsis IPT homologs have recently been identified in Arabidopsis and Petunia (Takei, et al., (2001) J. Biol. Chem. 276:26405-26410 and Kakimoto, (2001) Plant Cell Physiol. 42:677-685). Overexpression of the Arabidopsis IPT homologs in plants elevated cytokinin levels and elicited typical cytokinin responses in planta and under tissue culture conditions (Kakimoto, (2001) Plant Cell Physiol. 42:677-685).
Arabidopsis ipt genes are members of a small multigene family of nine different genes, two of which code for tRNA isopentenyl transferases, and seven of which encode a gene product with a cytokinin biosynthetic function. Biochemical analysis of the recombinant AtIPT4 protein showed that, in contrast to the bacterial enzyme, the Arabidopsis enzyme uses ATP as a substrate instead of AMP. Another plant IPT gene (Sho) was identified in Petunia hybrida using an activation tagging strategy (Zubko, et al., (2002) The Plant Journal 29:797-808).
In view of the influence of cytokinins on a wide variety of plant developmental processes, including root architecture, shoot and leaf development, and seed set, the ability to manipulate cytokinin levels in higher plant cells, and thereby drastically effect plant growth and productivity, offers significant commercial value (Mok, et al., (1994) Cytokinins. Chemistry, Action and Function. CRC Press, Boca Raton, Fla., pp. 155-166).
BRIEF SUMMARY OF THE INVENTION
Compositions and methods of the invention comprise and employ isopentenyl transferase (IPT) polypeptides and polynucleotides that are involved in modulating plant development, morphology and physiology.
Compositions further include expression cassettes, plants, plant cells and seeds having the IPT sequences of the invention. The plants, plant cells and seeds of the invention may exhibit phenotypic changes, such as modulated (increased or decreased) cytokinin levels; modulated floral development; modulated root development; altered shoot to root ratio; increased seed size or an increased seed weight; increased plant yield or plant vigor; maintained or improved stress tolerance (e.g., increased or maintained size of the plant, minimized tip kernel abortion, increased or maintained seed set); decreased shoot growth; delayed senescence or an enhanced vegetative growth, all relative to a plant, plant cell, or seed not modified per the invention.
Compositions of the invention also include IPT promoters, DNA constructs comprising the IPT promoter operably linked to a nucleotide sequence of interest, expression vectors, plants, plant cells and seeds comprising these DNA constructs.
Methods are provided for reducing or eliminating the activity of an IPT polypeptide in a plant, comprising introducing into the plant a selected polynucleotide. In specific methods, providing the polynucleotide decreases the level of cytokinin in the plant and/or modulates root development of the plant.
Methods are also provided for increasing the level of an IPT polypeptide in a plant comprising introducing into the plant a selected polynucleotide. In specific methods, expression of the IPT polynucleotide increases the level of a cytokinin in the plant; maintains or improves the stress tolerance of the plant; maintains or increases the size of the plant; minimizes seed abortion; increases or maintains seed set; increases shoot growth; increases seed size or seed weight; increases plant yield or plant vigor; modulates floral development; delays senescence; or increases leaf growth.
Methods are also provided for regulating the expression of a nucleotide sequence of interest. The method comprises introducing into a plant a DNA construct comprising a heterologous nucleotide sequence of interest operably linked to an IPT promoter of the invention.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 (1A-1C) provides an alignment of cytokinin biosynthetic enzymes from maize, petunia, and Arabidopsis. The amino acid sequences present in the alignment include ZmIPT1 (SEQ ID NO: 23), ZmIPT2 (SEQ ID NO: 2), ZmIPT4 (SEQ ID NO: 6), ZmIPT5 (SEQ ID NO: 9), ZmIPT6 (SEQ ID NO: 12), ZmIPT7 (SEQ ID NO: 15), ZmIPT8 (SEQ ID NO: 18), AtIPT1 (SEQ ID NO: 29), AtIPT3 (SEQ ID NO: 34), AtIPT4 (SEQ ID NO: 30), AtIPT5 (SEQ ID NO: 35), AtIPT6 (SEQ ID NO: 36), AtIPT7 (SEQ ID NO: 37), AtIPT8 (SEQ ID NO: 38) and Sho (SEQ ID NO: 31). Asterisks indicate amino acids conserved in many IPT proteins and the underlined amino acids represent a putative ATP/GTP binding site.
FIG. 2 provides a schematic of the structure of the ZmIPT1 gene from Mo17 (SEQ ID NO: 21). Coding regions are indicated by the thick arrows and the CAAT and a putative TATA box are shown.
FIG. 3 provides an amino acid sequence alignment of ZmIPT1 (SEQ ID NO: 23, referred to as ZmIPT-Mo17) and a variant of ZmIPT1 (SEQ ID NO: 27, referred to as ZmIPT-B73). The sequences have 98% amino acid sequence identity. The consensus sequence for the ZmIPT1 polypeptide is found in SEQ ID NO: 39.
FIG. 4 provides ppm values for ZmIPT1 in Lynx embryo libraries at various days after pollination (DAP).
FIG. 5A shows the detection of ZmIPT1 in different maize organs using RT-PCR.
FIG. 5B shows the detection of ZmIPT1 in developing kernels using RT-PCR.
FIG. 6 shows a Southern blot with B73 or Mo17 genomic DNA digested by 3 different restriction enzymes. 40 μg of genomic DNA was digested and run on a 0.8% agarose gel and transferred to a nylon membrane. The ZmIPT2-B73 gene coding sequence was used as a probe.
FIG. 7 shows a Northern blot and relative expression of the ZmIPT2 gene in different vegetative organs and in whole kernels at different days after pollination (DAP). Transcript levels were measured in leaves (L), stalks (S), roots (R), and in whole kernels at 0, 5, 10, 15, 20 and 25 days after pollination, and quantified relative to abundance of cyclophilin transcripts.
FIG. 8 provides a Northern blots and relative expression of the ZmIPT2 gene in kernels at different days after pollination. Transcript levels were measured in 0- to 5-DAP whole kernels and in 6- to 34-DAP kernels without pedicels, and quantified relative to abundance of cyclophilin transcripts. Zeatin riboside levels (the most abundant CK in corn kernels) were previously measured in the same samples and are indicated by the solid line (Brugière, et al., (2003) Plant Physiol 132:1228-1240).
FIG. 9 provides ppm values in Lynx embryo libraries for ZmIPT2.
FIG. 10 (10A-10G) provides an alignment of the amino acid sequences corresponding to Arabidopsis IPT proteins (AtIPT), the petunia IPT protein (Sho) and rice putative IPT proteins (OsIPT). The sequences in the alignment are as follows: OsIPT6 (SEQ ID NO: 57); OsIPT8 (SEQ ID NO: 41); OsIPT10 (SEQ ID NO: 59); OsIPT11 (SEQ ID NO: 43); OsIPT9 (SEQ ID NO: 61); OsIPT3 (SEQ ID NO: 63); OsIPT2 (SEQ ID NO: 46); OsIPT1 (SEQ ID NO: 49); OsIPT5 (SEQ ID NO: 52); OsIPT4 (34394150) (SEQ ID NO: 66); OsIPT7 (SEQ ID NO: 54); AtIPT1 (AB062607) (SEQ ID NO: 29); AtIPT3 (AB062610) (SEQ ID NO: 34); AtIPT4 (AB062611) (SEQ ID NO: 30); AtIPT5 (AB062608) (SEQ ID NO: 35); AtIPT6 (AB062612) (SEQ ID NO: 36); AtIPT7 (AB062613) (SEQ ID NO: 37); AtIPT8 (AB062614) (SEQ ID NO: 38); Sho (Petunia) (SEQ ID NO: 31); and, consensus (SEQ ID NO: 67).
FIG. 11 is a Northern blot that shows the relative expression of the ZmIPT2 gene at different days after pollination in different parts of the kernels. Transcript levels were measured in 0- to 25-DAP dissected kernels and quantified relative to abundance of 18S RNA transcripts.
FIG. 12 shows chromatograms related to the DMAPP::AMP isopentenyl transferase activity of Agrobacterium and maize purified recombinant protein.
FIG. 13 shows chromatograms related to further treatment of the reaction products of FIG. 12.
FIG. 14 shows chromatograms related to the DMAPP::ATP isopentenyl transferase activity of the maize purified recombinant protein.
FIG. 15 is a Western blot of whole maize kernels at various days after pollinations.
FIG. 16 is a graphic representation of the TUSC results.
FIG. 17 is a phylogenetic tree of plant IPT sequences.



DETAILED DESCRIPTION OF THE INVENTION
The present inventions now will be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown. Indeed, these inventions may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like numbers refer to like elements throughout.
Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which this invention pertains having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the invention is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.
Compositions
Compositions of the invention include isopentenyl transferase (IPT) polypeptides and polynucleotides that are involved in modulating plant development, morphology, and physiology. Compositions of the invention further include IPT promoters that are capable of regulating transcription. In particular, the present invention provides for isolated polynucleotides comprising nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO: 2, 6, 9, 12, 15, 18, 23, 27, 41, 43, 46, 49, 52, 54, 57, 59, 61, 63, 66 and 77. Further provided are isolated polypeptides having an amino acid sequence encoded by a polynucleotide described herein, for example those set forth in SEQ ID NO: 1, 3, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 21, 22, 24, 26, 28, 40, 42, 44, 47, 50, 53, 55, 58, 60, 62, 64, 45, 48, 51, 56, 65, 69, 70, 71, 72, 73, 74 or 77. Additional compositions include the IPT promoter sequences set forth in SEQ ID NO: 25 or 75, and promoter sequences as further isolated and characterized from the 5′ regions provided herein for ZmIPT4 (SEQ ID NO: 5), ZmIPT5 (SEQ ID NO: 8), ZmIPT6 (SEQ ID NO: 11), ZmIPT7 (SEQ ID NO: 14), ZmIPT8 (SEQ ID NO: 17), ZmIPT9 (SEQ ID NO: 20), OsIPT1 (SEQ ID NO: 47), OsIPT2 (SEQ ID NO: 44), OsIPT3 (SEQ ID NO: 62), OsIPT4 (SEQ ID NO: 64), OsIPT5 (SEQ ID NO: 50), OsIPT6 (SEQ ID NO: 55), OsIPT7 (SEQ ID NO: 53), OsIPT8 (SEQ ID NO: 40), OsIPT9 (SEQ ID NO: 60), OsIPT10 (SEQ ID NO: 58), and OsIPT11 (SEQ ID NO: 42).
The isopentenyl transferase polypeptides of the invention share sequence identity with members of the isopentenyl transferase family of proteins. Polypeptides in the IPT family have been identified in various bacteria and in Arabidopsis and Petunia. See, for example, (Kakimoto, (2001) Plant Cell Physiol 42:677-658); Takei, et al., (2001) The Journal of Biological Chemistry 276:26405-26410 and Zubko, et al., (2002) The Plant Journal 29:797-808). Members of the IPT family are characterized by having the consensus sequence GxTxxGK[ST]xxxxx[VLI]xxxxxxx[VLI][VLI]xxDxxQx{57,60}[VLI][VLI]xGG[ST] (SEQ ID NO: 32) (where x denotes any amino acid residue, [ ] any one of the amino acids shown in [ ], and x{m,n} m to n amino acid residues in number). See, Kakimoto, et al., (2001) Plant Cell Physiol. 42.677-85 and Kakimoto, et al., (2003) J. Plant Res. 116:233-9, both of which are herein incorporated by reference. IPT family members may also have ATP/GTP binding sites. An amino acid alignment of the maize IPT proteins along with Arabidopsis and petunia cytokinin biosynthetic enzymes is provided in FIG. 1, and an amino acid alignment of the rice IPT proteins with Arabidopsis and petunia cytokinin biosynthetic enzymes is provided in FIG. 10. Asterisks indicate a consensus sequences found in many cytokinin biosynthetic enzymes. The underlined amino acids indicate a putative ATP/GTP binding domains.
Isopentenyl transferase enzymes are involved in cytokinin biosynthesis, therefore the IPT polypeptides of the invention have “cytokinin synthesis activity.” By “cytokinin synthesis activity” is intended enzymatic activity that generates cytokinins, derivatives thereof, or any intermediates in the cytokinin synthesis pathway. Cytokinin synthesis activity therefore includes, but is not limited to, DMAPP:AMP isopentenyltransferase activity (the conversion of AMP (adenosine-5′-monophosphate) and DMAPP into iPMP (isopentenyladenosine-5′-monophosphate)), DMAPP:ADP isopentenyltransferase activity (the conversion of ADP (adenosine-5′-diphosphate) and DMAPP into iPDP (isopentenyladenosine-5′-diphosphate)); DMAPP:ATP isopentenyltransferase activity (the conversion of ATP (adenosine-5′-triphosphate) and DMAPP into iPTP (isopentenyladenosine-5′-triphosphate)), and DMAPP:tRNA isopentenyltransferase activity (the modification of cytoplasmic and/or mitrochondrial tRNAs to give isopentenyl). Cytokinin synthesis activity can further include a substrate comprising a second side chain precursor, other than DMAPP. Examples of side chain donors include compounds of terpenoid origin. For example, the substrate could be hydroxymethylbutenyl diphosphate (HMBPP) which would allow trans-zeatin riboside monophosphate (ZMP) synthesis. See, for example, Åstot, et al., (2000) Proc Natl Acad Sci 97:14778-14783 and Takei, et al., (2003) J Plant Res. 116(3):265-9.
Cytokinin synthesis activity further includes the synthesis of intermediates involved in formation of ZMP. Methods to assay for the production of various cytokinins and their intermediates can be found, for example, in Takei, et al., (2001) The Journal of Biological Chemistry 276:26405-26410, Zubo, et al., (2002) The Plant Journal 29:797-808; Kakimoto, et al., (2001) Plant Cell Physio. 42:677-658, and Sun, et al., (2003) Plant Physiology 131:167-176, each of which is herein incorporated by reference. “Cytokinin synthesis activity” also includes any alteration in a plant or plant cell phenotype that is characteristic of an increase in cytokinin concentration. Such cytokinin specific effects are discussed elsewhere herein and include, but are not limited to, enhanced shoot formation, reduced apical dominance, delayed senescence, delayed flowering, increased leaf growth, increased cytokinin levels in the plant, increased tolerance under stress, minimization of tip kernel abortion, increased or maintained seed set under stress conditions and a decrease in root growth. Assays to measure or detect such phenotypes are known. See, for example, Miyawaki, et al., (2004) The Plant Journal 37:128-138, Takei, et al., (2001) The Journal of Biological Chemistry 276:26405-26410, Zubo, et al., (2002) The Plant Journal 29:797-808; Kakimoto, et al., (2001) Plant Cell Physio. 42:677-658 and Sun, et al., (2003) Plant Physiology 131:167-176, each of which is herein incorporated by reference. Additional phenotypes resulting from an increase in cytokinin synthesis activity in a plant are discussed herein.
Compositions of the invention include IPT sequences that are involved in cytokinin biosynthesis. In particular, the present invention provides for isolated polynucleotides comprising nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO: 2, 6, 9, 12, 15, 18, 23, 27, 41, 43, 46, 49, 52, 54, 57, 59, 61, 63, 66 and 77. Further provided are polypeptides having an amino acid sequence encoded by a polynucleotide described herein, for example those set forth in SEQ ID NOS: 1, 3, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 21, 22, 24, 26, 28, 40, 42, 44, 47, 50, 53, 55, 58, 60, 62, 64, 45, 48, 51, 56, 65, 69, 70, 71, 72, 73 or 74 and fragments and variants thereof. In addition, further provided are promoter sequences, for example, the sequence set forth in SEQ ID NO: 25 or 75, variants and fragments thereof.
The invention encompasses isolated or substantially purified polynucleotide or protein compositions. An “isolated” or “purified” polynucleotide or protein, or biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the polynucleotide or protein as found in its naturally occurring environment. Thus, an isolated or purified polynucleotide or protein is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Optimally, an “isolated” polynucleotide is free of sequences (optimally protein encoding sequences) that naturally flank the polynucleotide (i.e., sequences located at the 5′ and 3′ ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived. For example, in various embodiments, the isolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequence that naturally flank the polynucleotide in genomic DNA of the cell from which the polynucleotide is derived. A protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5% or 1% (by dry weight) of contaminating protein. When the protein of the invention or biologically active portion thereof is recombinantly produced, optimally culture medium represents less than about 30%, 20%, 10%, 5% or 1% (by dry weight) of chemical precursors or non-protein-of-interest chemicals.
Fragments and variants of the disclosed polynucleotides and proteins encoded thereby are also encompassed by the present invention. By “fragment” is intended a portion of the polynucleotide or a portion of the amino acid sequence and hence protein encoded thereby. Fragments of a polynucleotide may encode protein fragments that retain the biological activity of the native protein and hence have cytokinin synthesis activity. Alternatively, fragments of a polynucleotide that are useful as hybridization probes generally do not encode fragment proteins retaining biological activity. Thus, fragments of a nucleotide sequence may range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length polynucleotide encoding the proteins of the invention.
A fragment of an IPT polynucleotide that encodes a biologically active portion of an IPT protein of the invention will encode at least 15, 25, 30, 50, 100, 150, 200, 225, 250, 275, 300, 310, 315 or 320 contiguous amino acids, or up to the total number of amino acids present in a full-length IPT protein of the invention (for example, 322, 364, 337, 338, 352, 388, 353, 352, 450, 590, 328, 325, 251, 427, 417, 585, 455, 344 and 347 amino acids for SEQ ID NO: 2, 6, 9, 12, 15, 18, 23, 27, 41, 43, 46, 49, 52, 54, 57, 59, 61, 63 and 66, respectively). Fragments of an IPT polynucleotide that are useful as hybridization probes or PCR primers generally need not encode a biologically active portion of an IPT protein.
Thus, a fragment of an IPT polynucleotide may encode a biologically active portion of an IPT protein, or it may be a fragment that can be used as a hybridization probe or PCR primer using methods disclosed below. A biologically active portion of an IPT protein can be prepared by isolating a portion of one of the IPT polynucleotides of the invention, expressing the encoded portion of the IPT protein (e.g., by recombinant expression in vitro), and assessing the activity of the encoded portion of the IPT protein. Polynucleotides that are fragments of an IPT nucleotide sequence comprise at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 950 or 965 contiguous nucleotides, or up to the number of nucleotides present in a full-length IPT polynucleotide disclosed herein (for example, 1495, 969, 2901, 2654, 1095, 4595, 1014, 1955, 1017, 1652, 1059, 3419, 1167, 1535, 3000, 1209, 1062, 1299, 1056, 4682, 8463, 4470, 4114, 2599, 1284, 5030, 8306, 7608, 5075, 4777, 984, 975, 753, 1254, 1044, 1035, 1284, 1353, 1368, 1758 and 1773 nucleotides for SEQ ID NO: 1, 3, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 21, 22, 24, 26, 28, 40, 42, 44, 47, 50, 53, 55, 58, 60, 62, 64, 45, 48, 51, 56, 65, 69, 70, 71, 72, 73 and 74, respectively).
“Variants” is intended to mean substantially similar sequences. For polynucleotides, a variant comprises a deletion and/or addition of one or more nucleotides at one or more sites within the native polynucleotide and/or a substitution of one or more nucleotides at one or more sites in the native polynucleotide. As used herein, a “native” polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively. For polynucleotides, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the IPT polypeptides of the invention. Naturally occurring variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques as outlined below. Variant polynucleotides also include synthetically derived polynucleotide, such as those generated, for example, by using site-directed mutagenesis but which still encode an IPT protein of the invention. Generally, variants of a particular polynucleotide of the invention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular polynucleotide as determined by sequence alignment programs and parameters described elsewhere herein.
Variants of a particular polynucleotide of the invention (i.e., the reference polynucleotide) can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide. Thus, for example, isolated polynucleotides that encode a polypeptide with a given percent sequence identity to the polypeptide of SEQ ID NO: 2, 6, 9, 12, 15, 18, 23, 27, 41, 43, 46, 49, 52, 54, 57, 59, 61, 63, 66 or 77 are disclosed. Percent sequence identity between any two polypeptides can be calculated using sequence alignment programs and parameters described elsewhere herein. Where any given pair of polynucleotides of the invention is evaluated by comparison of the percent sequence identity shared by the two polypeptides they encode, the percent sequence identity between the two encoded polypeptides is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.
“Variant” protein is intended to mean a protein derived from the native protein by deletion or addition of one or more amino acids at one or more sites in the native protein and/or substitution of one or more amino acids at one or more sites in the native protein. Certain variant proteins encompassed by the present invention are biologically active, that is they continue to possess the desired biological activity of the native protein, that is, cytokinin synthesis activity, as described herein. Such variants may result from, for example, genetic polymorphism or from human manipulation. Biologically active variants of a native IPT protein of the invention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence for the native protein as determined by sequence alignment programs and parameters described elsewhere herein. A biologically active variant of a protein of the invention may differ from that protein by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2 or even 1 amino acid residue.
The proteins of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants and fragments of the IPT proteins can be prepared by mutations in the DNA. Methods for mutagenesis and polynucleotide alterations are well known in the art. See, for example, Kunkel, (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel, et al., (1987) Methods in Enzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff, et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), herein incorporated by reference. Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be optimal.
Thus, the genes and polynucleotides of the invention include both the naturally occurring sequences as well as mutant forms. Likewise, the proteins of the invention encompass naturally occurring proteins as well as variations and modified forms thereof. Such variants will continue to possess the desired IPT activity. Obviously, the mutations that will be made in the DNA encoding the variant must not place the sequence out of reading frame and optimally will not create complementary regions that could produce secondary mRNA structure.
The deletions, insertions, and substitutions of the protein sequences encompassed herein are not expected to produce radical changes in the characteristics of the protein. However, when it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays. That is, the activity can be evaluated by assaying for cytokinin synthesis activity. See, for example, Takei, et al., (2001) The Journal of Biological Chemistry 276:26405-26410; Zubo, et al., (2002) The Plant Journal 29:797-808; Kakimoto, et al., (2001) Plant Cell Physio. 42:677-658; Sun, et al., (2003) Plant Physiology 131:167-176; and Miyawaki, et al., (2004) The Plant Journal 37:128-138, all of which are herein incorporated by reference.
Variant polynucleotides and proteins also encompass sequences and proteins derived from a mutagenic and recombinogenic procedure such as DNA shuffling. With such a procedure, one or more different IPT coding sequences can be manipulated to create a new IPT polypeptide possessing the desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. For example, using this approach, sequence motifs encoding a domain of interest may be shuffled between the IPT gene of the invention and other known IPT genes to obtain a new gene coding for a protein with an improved property of interest, such as an increased Km in the case of an enzyme. Strategies for such DNA shuffling are known in the art. See, for example, Stemmer, (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751; Stemmer, (1994) Nature 370:389-391; Crameri, et al., (1997) Nature Biotech. 15:436-438; Moore, et al., (1997) J. Mol. Biol. 272:336-347; Zhang, et al., (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri, et al., (1998) Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.
The compositions of the invention also include isolated polynucleotides comprising an IPT promoter nucleotide sequence as set forth in SEQ ID NO: 25 or 75, and promoter sequences as further isolated and characterized from the regions 5′ to the coding sequence provided as a part of SEQ ID NO: 5 (ZmIPT4), SEQ ID NO: 8 (ZmIPT5), SEQ ID NO: 11 (ZmIPT6), SEQ ID NO: 14 (ZmIPT7), SEQ ID NO: 17 (ZmIPT8), SEQ ID NO: 20 (ZmIPT9), SEQ ID NO: 47 (OsIPT1), SEQ ID NO: 44 (OsIPT2), SEQ ID NO: 62 (OsIPT3), SEQ ID NO: 64 (OsIPT4), SEQ ID NO: 50 (OsIPT5), SEQ ID NO: 55 (OsIPT6), SEQ ID NO: 53 (OsIPT7), SEQ ID NO: 40 (OsIPT8), SEQ ID NO: 60 (OsIPT9), SEQ ID NO: 58 (OsIPT10) and SEQ ID NO: 42 (OsIPT11).
By “promoter” is intended a regulatory region of DNA usually comprising a TATA box capable of directing RNA polymerase II to initiate RNA synthesis at the appropriate transcription initiation site for a particular polynucleotide sequence. A promoter may additionally comprise other recognition sequences generally positioned upstream or 5′ to the TATA box, referred to as upstream promoter elements, which influence the transcription initiation rate. The promoter sequences of the present invention regulate (i.e., repress or activate) transcription.
It is recognized that additional domains can be added to the promoter sequences of the invention and thereby modulate the level of expression, the developmental timing of expression, or tissue type that expression occurs in. See particularly, Australian Patent Number AU-A-77751/94 and U.S. Pat. Nos. 5,466,785 and 5,635,618.
Fragments and variants of the disclosed IPT promoter polynucleotides are also encompassed by the present invention. Fragments of a promoter polynucleotide may retain biological activity and hence retain transcriptional regulatory activity. Alternatively, fragments of a polynucleotide that are useful as hybridization probes generally do not retain biological activity. Thus, fragments of a promoter nucleotide sequence may range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides and up to the full-length polynucleotide of the invention.
Thus, a fragment of an IPT promoter polynucleotide may encode a biologically active portion of an IPT promoter, or it may be a fragment that can be used as a hybridization probe or PCR primer using methods disclosed below. A biologically active portion of the IPT promoter polynucleotides can be prepared by isolating a portion of one of the IPT promoter polynucleotide of the invention, and assessing the activity of the portion of the IPT promoter. Polynucleotides that are fragments of an IPT promoter comprise at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1,000, 1,050 or 1,080 contiguous nucleotides, or up to the number of nucleotides present in a full-length IPT promoter polynucleotide disclosed herein (for example, 1082 and 1920 nucleotides for SEQ ID NOS: 25 and 75, respectfully).
For a promoter polynucleotide, a variant comprises a deletion and/or addition of one or more nucleotides at one or more internal sites within the native polynucleotide and/or a substitution of one or more nucleotides at one or more sites in the native polynucleotide. Generally, variants of a particular promoter polynucleotide of the invention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular polynucleotide as determined by sequence alignment programs and parameters described elsewhere herein.
Variant polynucleotides also encompass sequences derived from a mutagenic and recombinogenic procedure such as DNA shuffling. With such a procedure, one or more different promoter sequences can be manipulated to create a new IPT promoter possessing the desired properties. Strategies for such DNA shuffling are described elsewhere herein.
Methods are available in the art for determining if a promoter sequence retains the ability to regulate transcription. Such activity can be measured by Northern blot analysis. See, for example, Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.), herein incorporated by reference. Alternatively, biological activity of the promoter can be measured using assays specifically designed for measuring the activity and/or level of the polypeptide being expressed from the promoter. Such assays are known in the art. Also, known promoter elements can be identified within a putative promoter sequence. For example, the IPT1 promoter (SEQ ID NO: 25) of the invention has a TATA-box at by 688. A TATA-box like sequence can be found 48 bp upstream of the transcription start site (between by 1035 and 1042). A potential CAAT box can be found between by 929 and 932.
The polynucleotides of the invention (i.e., the IPT sequences and the IPT promoter sequences) can be used to isolate corresponding sequences from other organisms, particularly other plants, more particularly other monocots. In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences based on their sequence homology to the sequences set forth herein. Sequences isolated based on their sequence identity to the entire IPT sequences or the IPT promoter sequences set forth herein or to variants and fragments thereof are encompassed by the present invention. Such sequences include sequences that are orthologs of the disclosed sequences. “Orthologs” is intended to mean genes derived from a common ancestral gene and which are found in different species as a result of speciation. Genes found in different species are considered orthologs when their nucleotide sequences and/or their encoded protein sequences share at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity. Functions of orthologs are often highly conserved among species. Thus, isolated polynucleotides that encode for an IPT protein or comprise an IPT promoter sequence and which hybridize under stringent conditions to the IPT sequences or the IPT promoter sequences disclosed herein, or to variants or fragments or complements thereof, are encompassed by the present invention.
In a PCR approach, oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any plant of interest. Methods for designing PCR primers and PCR cloning are generally known in the art and are disclosed in Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.). See also, Innis, et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially-mismatched primers and the like.
In hybridization techniques, all or part of a known polynucleotide is used as a probe that selectively hybridizes to other corresponding polynucleotides present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen organism. The hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group such as 32P, or any other detectable marker. Thus, for example, probes for hybridization can be made by labeling synthetic oligonucleotides based on the IPT polynucleotides or the IPT promoter sequences of the invention. Methods for preparation of probes for hybridization and for construction of cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).
For example, the entire IPT polynucleotide or the IPT promoter sequence disclosed herein, or one or more portions thereof, may be used as a probe capable of specifically hybridizing to corresponding IPT polynucleotides, messenger RNAs, or promoter sequences. To achieve specific hybridization under a variety of conditions, such probes include sequences that are unique among IPT polynucleotide sequences or IPT promoter sequences and are optimally at least about 10 nucleotides in length, and most optimally at least about 20 nucleotides in length. Such probes may be used to amplify corresponding IPT polynucleotides or IPT promoters from a chosen plant by PCR. This technique may be used to isolate additional coding sequences from a desired plant or as a diagnostic assay to determine the presence of coding sequences in a plant. Hybridization techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, for example, Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).
Hybridization of such sequences may be carried out under stringent conditions. By “stringent conditions” or “stringent hybridization conditions” is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, optimally less than 500 nucleotides in length.
Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffers may comprise about 0.1% to about 1% SDS. Duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours. The duration of the wash time will be at least a length of time sufficient to reach equilibrium.
Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl, (1984) Anal. Biochem. 138:267-284: Tm=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Tm is reduced by about 1° C. for each 1% of mismatching; thus, Tm, hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with >90% identity are sought, the Tm can be decreased 10° C. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3 or 4° C. lower than the thermal melting point (Tm); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9 or 10° C. lower than the thermal melting point (Tm); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15 or 20° C. lower than the thermal melting point (Tm). Using the equation, hybridization and wash compositions, and desired Tm, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a Tm of less than 45° C. (aqueous solution) or 32° C. (formamide solution), it is optimal to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen, (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, New York); and Ausubel, et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, New York). See, Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).
The following terms are used to describe the sequence relationships between two or more polynucleotides or polypeptides: (a) “reference sequence”, (b) “comparison window”, (c) “sequence identity” and (d) “percentage of sequence identity.”
(a) As used herein, “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.
(b) As used herein, “comparison window” makes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two polynucleotides. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100 or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence a gap penalty is typically introduced and is subtracted from the number of matches.
Methods of alignment of sequences for comparison are well known in the art. Thus, the determination of percent sequence identity between any two sequences can be accomplished using a mathematical algorithm. Non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller, (1988) CABIOS 4:11-17; the local alignment algorithm of Smith, et al., (1981) Adv. Appl. Math. 2:482; the global alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443-453; the search-for-local alignment method of Pearson and Lipman, (1988) Proc. Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul, (1990) Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karlin and Altschul, (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.
Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif., USA). Alignments using these programs can be performed using the default parameters. The CLUSTAL program is well described by Higgins, et al., (1988) Gene 73:237-244 (1988); Higgins, et al., (1989) CABIOS 5:151-153; Corpet, et al., (1988) Nucleic Acids Res. 16:10881-90; Huang, et al., (1992) CABIOS 8:155-65; and Pearson, et al., (1994) Meth. Mol. Biol. 24:307-331. The ALIGN program is based on the algorithm of Myers and Miller, (1988) supra. A PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used with the ALIGN program when comparing amino acid sequences. The BLAST programs of Altschul, et al., (1990) J. Mol. Biol. 215:403 are based on the algorithm of Karlin and Altschul, (1990) supra. BLAST nucleotide searches can be performed with the BLASTN program, score=100, wordlength=12, to obtain nucleotide sequences homologous to a nucleotide sequence encoding a protein of the invention. BLAST protein searches can be performed with the BLASTX program, score=50, wordlength=3, to obtain amino acid sequences homologous to a protein or polypeptide of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul, et al., (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. See, Altschul, et al., (1997) supra. When utilizing BLAST, Gapped BLAST, PSI-BLAST, the default parameters of the respective programs (e.g., BLASTN for nucleotide sequences, BLASTX for proteins) can be used. See www.ncbi.nlm.nih.gov. Alignment may also be performed manually by inspection.
Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using GAP Version 10 using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix; or any equivalent program thereof. By “equivalent program” is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.
GAP uses the algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443-453, to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in units of matched bases. GAP must make a profit of gap creation penalty number of matches for each gap it inserts. If a gap extension penalty greater than zero is chosen, GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty. Default gap creation penalty values and gap extension penalty values in Version 10 of the GCG Wisconsin Genetics Software Package for protein sequences are 8 and 2, respectively. For nucleotide sequences the default gap creation penalty is 50 while the default gap extension penalty is 3. The gap creation and gap extension penalties can be expressed as an integer selected from the group of integers consisting of from 0 to 200. Thus, for example, the gap creation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.
GAP presents one member of the family of best alignments. There may be many members of this family, but no other member has a better quality. GAP displays four figures of merit for alignments: Quality, Ratio, Identity, and Similarity. The Quality is the metric maximized in order to align the sequences. Ratio is the quality divided by the number of bases in the shorter segment. Percent Identity is the percent of the symbols that actually match. Percent Similarity is the percent of the symbols that are similar. Symbols that are across from gaps are ignored. A similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold. The scoring matrix used in Version 10 of the GCG Wisconsin Genetics Software Package is BLOSUM62 (see, Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915).
(c) As used herein, “sequence identity” or “identity” in the context of two polynucleotides or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity”. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif.).
(d) As used herein, “percentage of sequence identity” means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
The invention further provides plants having altered levels and/or activities of the IPT polypeptides of the invention. In some embodiments, the plants of the invention have stably incorporated into their genome the IPT sequences of the invention. In other embodiments, plants that are genetically modified at a genomic locus encoding an IPT polypeptide of the invention are provided. By “native genomic locus” is intended a naturally occurring genomic sequence. For some embodiments, the genomic locus is set forth in SEQ ID NO: 21, 40, 42, 44, 47, 50, 53, 55, 58, 60, 62 or 64. The genomic locus may be modified to reduce or eliminate the activity of the IPT polypeptide. The term “genetically modified” as used herein refers to a plant or plant part that is modified in its genetic information by the introduction of one or more foreign polynucleotides, and the insertion of the foreign polynucleotide leads to a phenotypic change in the plant. By “phenotypic change” is intended a measurable change in one or more cell functions. For example, plants having a genetic modification at the genomic locus encoding the IPT polypeptide can show reduced or eliminated expression or activity of the IPT polypeptide. Various methods to generate such a genetically modified genomic locus are described elsewhere herein, as are the variety of phenotypes that can result from the modulation of the level/activity of the IPT sequences of the invention.
The invention further provides plants having at least one DNA construct comprising a heterologous nucleotide sequence of interest operably linked to the IPT promoter of the invention. In further embodiments, the DNA construct is stably integrated into the genome of the plant.
As used herein, the term plant includes reference to whole plants, plant parts or organs (e.g. leaves, stems, roots), plant cells, and seeds and progeny of same. Plant cell, as used herein, includes, without limitation, cells obtained from or found in seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen and microspores, as well as plant protoplasts and plant cell tissue cultures, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, grain and the like. As used herein, “grain” refers to the mature seed produced by commercial growers for purposes other than growing or reproducing the species. Progeny, variants, and mutants of the regenerated plants are also included within the scope of the invention, provided that these parts comprise the introduced nucleic acid sequences.
Methods
I. Providing Sequences
The sequences of the present invention can be introduced/expressed in a host cell such as bacteria, yeast, insect, mammalian or optimally plant cells. It is expected that those of skill in the art are knowledgeable in the numerous systems available for the introduction of a polypeptide or a nucleotide sequence of the present invention into a host cell. No attempt to describe in detail the various methods known for providing proteins in prokaryotes or eukaryotes will be made.
By “host cell” is meant a cell which comprises a heterologous nucleic acid sequence of the invention. Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian or mammalian cells. Host cells can also be monocotyledonous or dicotyledonous plant cells. In certain embodiments, the monocotyledonous host cell is a maize host cell.
The use of the term “polynucleotide” is not intended to limit the present invention to polynucleotides comprising DNA. Those of ordinary skill in the art will recognize that polynucleotides can comprise ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. The polynucleotides of the invention also encompass all forms of sequences including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures and the like.
The IPT polynucleotides or the IPT promoters of the invention can be provided in expression cassettes for expression in the plant of interest. The cassette will include 5′ and 3′ regulatory sequences operably linked to an IPT polynucleotide of the invention. “Operably linked” is intended to mean a functional linkage between two or more elements. For example, an operable linkage between a polynucleotide of interest and a regulatory sequence (i.e., a promoter) is a functional link that allows for expression of the polynucleotide of interest. Operably linked elements may be contiguous or non-contiguous. When used to refer to the joining of two protein coding regions, by operably linked is intended that the coding regions are in the same reading frame. The cassette may additionally contain at least one additional gene to be cotransformed into the organism. Alternatively, the additional gene(s) can be provided on multiple expression cassettes. An expression cassette may be provided with a plurality of restriction sites and/or recombination sites for insertion of the IPT polynucleotide to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes.
In certain embodiments, the expression cassette will include in the 5′-3′ direction of transcription, a transcriptional and translational initiation region (i.e., a promoter), an IPT polynucleotide of the invention, and a transcriptional and translational termination region (i.e., termination region) functional in plants. The regulatory regions (i.e., promoters, transcriptional regulatory regions and translational termination regions) and/or the IPT polynucleotide of the invention may be native/analogous to the host cell or to each other. Alternatively, the regulatory regions and/or the IPT polynucleotide of the invention may be heterologous to the host cell or to each other. As used herein, “heterologous” in reference to a sequence is a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous polynucleotide is from a species different from the species from which the polynucleotide was derived, or, if from the same/analogous species, one or both are substantially modified from their original form and/or genomic locus, or the promoter is not the native promoter for the operably-linked polynucleotide. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence.
While heterologous promoters can be used to express the IPT sequences, the native promoter sequences or other IPT promoters (e.g., SEQ ID NO: 25 or 75) may also be used. Such constructs can change expression levels of IPT sequences in the plant or plant cell. Thus, the phenotype of the plant or plant cell can be altered.
The termination region may be native with the transcriptional initiation region, may be native with the operably-linked IPT polynucleotide of interest, may be native with the plant host, or may be derived from another source (i.e., foreign or heterologous with reference to the promoter), the IPT polynucleotide of interest, the plant host, or any combination thereof. Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also, Guerineau, et al., (1991) Mol. Gen. Genet. 262:141-144; Proudfoot, (1991) Cell 64:671-674; Sanfacon, et al., (1991) Genes Dev. 5:141-149; Mogen, et al., (1990) Plant Cell 2:1261-1272; Munroe, et al., (1990) Gene 91:151-158; Ballas, et al., (1989) Nucleic Acids Res. 17:7891-7903 and Joshi, et al., (1987) Nucleic Acids Res. 15:9627-9639.
Where appropriate, the polynucleotides may be optimized for increased expression in the transformed plant. That is, the polynucleotides can be synthesized using plant-preferred codons for improved expression. See, for example, Campbell and Gowri, (1990) Plant Physiol. 92:1-11 for a discussion of host-preferred codon usage. Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Pat. Nos. 5,380,831 and 5,436,391, and Murray, et al., (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.
Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats and other such well-characterized sequences that may be deleterious to gene expression. The G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.
The expression cassettes may additionally contain 5′ leader sequences. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5′ noncoding region) (Elroy-Stein, et al., (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie, et al., (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf Mosaic Virus) (Virology 154:9-20), and human immunoglobulin heavy-chain binding protein (BiP) (Macejak, et al., (1991) Nature 353:90-94); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling, et al., (1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie, et al., (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel, et al., (1991) Virology 81:382-385). See also, Della-Cioppa, et al., (1987) Plant Physiol. 84:965-968. Other methods known to enhance translation can also be utilized.
In preparing the expression cassette, the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved.
The expression cassette can also comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markers include phenotypic markers such as β-galactosidase and fluorescent proteins such as green fluorescent protein (GFP) (Su, et al., (2004) Biotechnol Bioeng 85.610-9 and Fetter, et al., (2004) Plant Cell 16.215-28), cyan fluorescent protein (CYP) (Bolte, et al., (2004) J. Cell Science 117:943-54 and Kato, et al., (2002) Plant Physiol 129:913-42), and yellow fluorescent protein (PhiYFPTM from Evrogen, see, Bolte, et al., (2004) J. Cell Science 117:943-54). For additional selectable markers, see generally, Yarranton, (1992) Curr. Opin. Biotech. 3:506-511; Christopherson, et al., (1992) Proc. Natl. Acad. Sci. USA 89:6314-6318; Yao, et al., (1992) Cell 71:63-72; Reznikoff, (1992) Mol. Microbiol. 6:2419-2422; Barkley, et al., (1980) in The Operon, pp. 177-220; Hu, et al., (1987) Cell 48:555-566; Brown, et al., (1987) Cell 49:603-612; Figge, et al., (1988) Cell 52:713-722; Deuschle, et al., (1989) Proc. Natl. Acad. Aci. USA 86:5400-5404; Fuerst, et al., (1989) Proc. Natl. Acad. Sci. USA 86:2549-2553; Deuschle, et al., (1990) Science 248:480-483; Gossen, (1993) Ph.D. Thesis, University of Heidelberg; Reines, et al., (1993) Proc. Natl. Acad. Sci. USA 90:1917-1921; Labow, et al., (1990) Mol. Cell. Biol. 10:3343-3356; Zambretti, et al., (1992) Proc. Natl. Acad. Sci. USA 89:3952-3956; Baim, et al., (1991) Proc. Natl. Acad. Sci. USA 88:5072-5076; Wyborski, et al., (1991) Nucleic Acids Res. 19:4647-4653; Hillenand-Wissman, (1989) Topics Mol. Struc. Biol. 10:143-162; Degenkolb, et al., (1991) Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidt, et al., (1988) Biochemistry 27:1094-1104; Bonin, (1993) Ph.D. Thesis, University of Heidelberg; Gossen, et al., (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Oliva, et al., (1992) Antimicrob. Agents Chemother. 36:913-919; Hlavka, et al., (1985) Handbook of Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill, et al., (1988) Nature 334:721-724. Such disclosures are herein incorporated by reference. The above list of selectable marker genes is not meant to be limiting. Any selectable marker gene can be used in the present invention.
A number of promoters can be used in the practice of the invention, including the native promoter of the polynucleotide sequence of interest. The promoters can be selected based on the desired outcome. The nucleic acids can be combined with constitutive, inducible, tissue-preferred, or other promoters for expression in plants.
Such constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odell, et al., (1985) Nature 313:810-812); rice actin (McElroy, et al., (1990) Plant Cell 2:163-171); ubiquitin (Christensen, et al., (1989) Plant Mol. Biol. 12:619-632 and Christensen, et al., (1992) Plant Mol. Biol. 18:675-689); pEMU (Last, et al., (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten, et al., (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026) and the like. Other constitutive promoters include, for example, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142 and 6,177,611.
Tissue-preferred promoters can be utilized to target enhanced IPT expression within a particular plant tissue. Tissue-preferred promoters include Yamamoto, et al., (1997) Plant J. 12(2):255-265; Kawamata, et al., (1997) Plant Cell Physiol. 38(7):792-803; Hansen, et al., (1997) Mol. Gen Genet. 254(3):337-343; Russell, et al., (1997) Transgenic Res. 6(2):157-168; Rinehart, et al., (1996) Plant Physiol. 112(3):1331-1341; Van Camp, et al., (1996) Plant Physiol. 112(2):525-535; Canevascini, et al., (1996) Plant Physiol. 112(2):513-524; Yamamoto, et al., (1994) Plant Cell Physiol. 35(5):773-778; Lam, (1994) Results ProbL Cell Differ. 20:181-196; Orozco, et al., (1993) Plant Mol Biol. 23(6):1129-1138; Matsuoka, et al., (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590 and Guevara-Garcia, et al., (1993) Plant J. 4(3):495-505. Such promoters can be modified, if necessary, for weak expression. See, also, US Patent Application Publication Number 2003/0074698, herein incorporated by reference.
Leaf-preferred promoters are known in the art. See, for example, Yamamoto, et al., (1997) Plant J. 12(2):255-265; Kwon, et al., (1994) Plant Physiol. 105:357-67; Yamamoto, et al., (1994) Plant Cell Physiol. 35(5):773-778; Gotor, et al., (1993) Plant J. 3:509-18; Orozco, et al., (1993) Plant Mol. Biol. 23(6):1129-1138; Baszczynski, et al., (1988) Nucl. Acid Res. 16:4732; Mitra, et al., (1994) Plant Molecular Biology 26:35-93; Kayaya, et al., (1995) Molecular and General Genetics 248:668-674; and Matsuoka, et al., (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590. Senecence regulated promoters are also of use, such as, SAM22 (Crowell, et al., (1992) Plant Mol. Biol. 18:459-466). See, also, U.S. Pat. No. 5,589,052 herein incorporated by reference.
Root-preferred promoters are known and can be selected from the many available from the literature or isolated de novo from various compatible species. See, for example, Hire, et al., (1992) Plant Mol. Biol. 20(2):207-218 (soybean root-specific glutamine synthetase gene); Keller and Baumgartner, (1991) Plant Cell 3(10):1051-1061 (root-specific control element in the GRP 1.8 gene of French bean); Sanger, et al., (1990) Plant Mol. Biol. 14(3):433-443 (root-specific promoter of the mannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao, et al., (1991) Plant Cell 3(1):11-22 (full-length cDNA clone encoding cytosolic glutamine synthetase (GS), which is expressed in roots and root nodules of soybean). See also, Bogusz, et al., (1990) Plant Cell 2(7):633-641, where two root-specific promoters isolated from hemoglobin genes from the nitrogen-fixing nonlegume Parasponia andersonii and the related non-nitrogen-fixing nonlegume Trema tomentosa are described. The promoters of these genes were linked to a β-glucuronidase reporter gene and introduced into both the nonlegume Nicotiana tabacum and the legume Lotus corniculatus, and in both instances root-specific promoter activity was preserved. Leach and Aoyagi, (1991) describe their analysis of the promoters of the highly expressed roIC and rolD root-inducing genes of Agrobacterium rhizogenes (see, Plant Science (Limerick) 79(1):69-76). They concluded that enhancer and tissue-preferred DNA determinants are dissociated in those promoters. Teeri, et al., (1989) used gene fusion to lacZ to show that the Agrobacterium T-DNA gene encoding octopine synthase is especially active in the epidermis of the root tip and that the TR2′ gene is root specific in the intact plant and stimulated by wounding in leaf tissue, an especially desirable combination of characteristics for use with an insecticidal or larvicidal gene (see, EMBO J. 8(2):343-350). The TR1′ gene, fused to nptII (neomycin phosphotransferase II) showed similar characteristics. Additional root-preferred promoters include the VfENOD-GRP3 gene promoter (Kuster, et al., (1995) Plant Mol. Biol. 29(4):759-772); rolB promoter (Capana, et al., (1994) Plant Mol. Biol. 25(4):681-691; and the CRWAQ81 root-preferred promoter with the ADH first intron (US Patent Application Publication Number 2005/0097633). See also, U.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,110,732 and 5,023,179.
“Seed-preferred” promoters refers to those promoters active during seed development and may include expression in seed initials or related maternal tissue. Such seed-preferred promoters include, but are not limited to, Cim1 (cytokinin-induced message); cZ19B1 (maize 19 kDa zein); milps (myo-inositol-1-phosphate synthase) (see, WO 00/11177 and U.S. Pat. No. 6,225,529; herein incorporated by reference). Gamma-zein is an endosperm-specific promoter. Globulin-1 (Glob-1) is a representative embryo-specific promoter. For dicots, seed-specific promoters include, but are not limited to, bean β-phaseolin, napin, β-conglycinin, soybean lectin, cruciferin, and the like. For monocots, seed-specific promoters include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, gamma-zein, waxy, shrunken 1, shrunken 2, globulin 1, etc. See also, WO 00/12733, where seed-preferred promoters from end1 and end2 genes are disclosed; herein incorporated by reference. Additional embryo specific promoters are disclosed in Sato, et al., (1996) Proc. Natl. Acad. Sci. 93:8117-8122; Nakase, et al., (1997) Plant J 12:235-46; and Postma-Haarsma, et al., (1999) Plant Mol. Biol. 39:257-71. Additional endosperm specific promoters are disclosed in Albani, et al., (1984) EMBO 3:1405-15; Albani, et al., (1999) Theor. Appl. Gen. 98:1253-62; Albani, et al., (1993) Plant J. 4:343-55; Mena, et al., (1998) The Plant Journal 116:53-62 and Wu, et al., (1998) Plant Cell Physiology 39:885-889.
Also of interest are promoters active in meristem regions, such as developing inflorescence tissues, and promoters which drive expression at or about the time of anthesis or early kernel development. This may include, for example, the maize Zag promoters, including Zag1 and Zag2 (see, Schmidt. et al., (1993) The Plant Cell 5:729-37; GenBank X80206; Theissen, et al., (1995) Gene 156:155-166 and U.S. patent application Ser. No. 10/817,483); maize Zap promoter (also known as ZmMADS; U.S. patent application Ser. No. 10/387,937; WO 03/078590); maize ckx1-2 promoter (US Patent Application Publication Number 2002/0152500 A1; WO 02/0078438); maize eep1 promoter (U.S. patent application Ser. No. 10/817,483); maize end2 promoter (U.S. Pat. No. 6,528,704 and U.S. patent application Ser. No. 10/310,191); maize lec1 promoter (U.S. patent application Ser. No. 09/718,754); maize F3.7 promoter (Baszczynski, et al., (1997) Maydica 42:189-201); maize tb1 promoter (Hubbarda, et al., (2002) Genetics 162:1927-1935 and Wang, et al., (1999) Nature 398:236-239); maize eep2 promoter (U.S. patent application Ser. No. 10/817,483); maize thioredoxinH promoter (U.S. Provisional Patent Application Ser. No. 60/514,123); maize Zm40 promoter (U.S. Pat. No. 6,403,862 and WO 01/2178); maize mLIP15 promoter (U.S. Pat. No. 6,479,734); maize ESR promoter (U.S. patent application Ser. No. 10/786,679); maize PCNA2 promoter (U.S. patent application Ser. No. 10/388,359); maize cytokinin oxidase promoters (U.S. patent application Ser. No. 11/094,917); promoters disclosed in Weigal, et al., (1992) Cell 69:843-859; Accession No. AJ131822; Accession No. Z71981; Accession No. AF049870; and shoot-preferred promoters disclosed in McAvoy, et al., (2003) Acta Hort. (ISHS) 625:379-385. Other dividing cell or meristematic tissue-preferred promoters that may be of interest have been disclosed in Ito, et al., (1994) Plant Mol. Biol. 24:863-878; Regad, et al., (1995) Mo. Gen. Genet. 248:703-711; Shaul, et al., (1996) Proc. Natl. Acad. Sci. 93:4868-4872; Ito, et al., (1997) Plant J. 11:983-992; and Trehin, et al., (1997) Plant Mol. Biol. 35:667-672, all of which are hereby incorporated by reference herein.
Inflorescence-preferred promoters include the promoter of chalcone synthase (Van der Meer, et al., (1990) Plant Mol. Biol. 15:95-109), LAT52 (Twell, et al., (1989) Mol. Gen. Genet. 217:240-245), pollen specific genes (Albani, et al., (1990) Plant Mol Biol. 15:605, Zm13 (Buerrero, et al., (1993) Mol. Gen. Genet. 224:161-168), maize pollen-specific gene (Hamilton, et al., (1992) Plant Mol. Biol. 18:211-218), sunflower pollen expressed gene (Baltz, et al., (1992) The Plant Journal 2:713-721), and B. napus pollen specific genes (Arnoldo, et al., (1992) J. Cell. Biochem, Abstract No. Y101204).
Stress-inducible promoters include salt/water stress-inducible promoters such as P5CS (Zang, et al., (1997) Plant Sciences 129:81-89); cold-inducible promoters, such as, cor15a (Hajela, et al., (1990) Plant Physiol. 93:1246-1252), cor15b (Wlihelm, et al., (1993) Plant Mol Biol 23:1073-1077), wsc120 (Ouellet, et al., (1998) FEBS Lett. 423-324-328), ci7 (Kirch, et al., (1997) Plant Mol Biol. 33:897-909), ci21A (Schneider, et al., (1997) Plant Physiol. 113:335-45); drought-inducible promoters, such as, Trg-31 (Chaudhary, et al., (1996) Plant Mol. Biol. 30:1247-57); osmotic inducible promoters, such as, Rab17 (Vilardell, et al., (1991) Plant Mol. Biol. 17:985-93) and osmotin (Raghothama, et al., (1993) Plant Mol Biol 23:1117-28); and, heat inducible promoters, such as, heat shock proteins (Barros, et al., (1992) Plant Mol. 19:665-75; Marrs, et al., (1993) Dev. Genet. 14:27-41), and smHSP (Waters, et al., (1996) J. Experimental Botany 47:325-338). Other stress-inducible promoters include rip2 (U.S. Pat. No. 5,332,808 and US Patent Application Publication Number 2003/0217393) and rd29a (Yamaguchi-Shinozaki, et al., (1993) Mol. Gen. Genetics 236:331-340).
Stress-insensitive promoters can also be used in the methods of the invention. This class of promoters, as well as representative examples, are further described elsewhere herein.
Nitrogen-responsive promoters can also be used in the methods of the invention. Such promoters include, but are not limited to, the 22 kDa Zein promoter (Spena, et al., (1982) EMBO J 1:1589-1594 and Muller, et al., (1995) J. Plant Physiol 145:606-613); the 19 kDa zein promoter (Pedersen, et al., (1982) Cell 29:1019-1025); the 14 kDa zein promoter (Pedersen, et al., (1986) J. Biol. Chem. 261:6279-6284), the b-32 promoter (Lohmer, et al., (1991) EMBO J 10:617-624); and the nitrite reductase (NiR) promoter (Rastogi, et al., (1997) Plant Mol Biol. 34(3):465-76 and Sander, et al., (1995) Plant Mol Biol. 27(1):165-77). For a review of consensus sequences found in nitrogen-induced promoters, see for example, Muller, et al., (1997) The Plant Journal 12:281-291.
Chemically-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. Depending upon the objective, the promoter may be a chemically-inducible promoter, where application of the chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-1a promoter, which is activated by salicylic acid. Other chemical-regulated promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena, et al., (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 and McNellis, et al., (1998) Plant J. 14(2):247-257) and tetracycline-inducible and tetracycline-repressible promoters (see, for example, Gatz, et al., (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat. Nos. 5,814,618 and 5,789,156), herein incorporated by reference.
A promoter induced by cytokinin, such as the ZmCkx1-2 promoter (U.S. Pat. No. 6,921,815, and pending U.S. patent application Ser. No. 11/074,144), may also be used in the methods and compositions of the invention. Such a construct would amplify biosynthesis of cytokinin occurring in developmental stages and/or tissues of interest. Other cytokinin-inducible promoters are described in pending U.S. patent application Ser. No. 11/094,917 and 60/627,394, all hereby incorporated by reference.
Additional inducible promoters include heat shock promoters, such as Gmhsp17.5-E (soybean) (Czarnecka, et al., (1989) Mol Cell Biol. 9(8):3457-3463); APX1 gene promoter (Arabidopsis) (Storozhenko, et al., (1998) Plant Physiol. 118(3):1005-1014): Ha hsp17.7 G4 (Helianthus annuus) (Almoguera, et al., (2002) Plant Physiol. 129(1):333-341; and Maize Hsp70 (Rochester, et al., (1986) EMBO J. 5:451-8.
The methods of the invention involve introducing a polypeptide or polynucleotide into a plant. “Introducing” is intended to mean presenting to the plant the polynucleotide or polypeptide in such a manner that the sequence gains access to the interior of a cell of the plant. The methods of the invention do not depend on a particular method for introducing a sequence into a plant, only that the polynucleotide or polypeptides gains access to the interior of at least one cell of the plant. Methods for introducing polynucleotides or polypeptides into plants are known in the art and include, but are not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.
“Stable transformation” is intended to mean that the nucleotide construct of interest introduced into a plant integrates into the genome of the plant and is capable of being inherited by the progeny thereof. “Transient transformation” is intended to mean that a sequence is introduced into the plant and is only temporarily expressed or present in the plant.
Transformation protocols as well as protocols for introducing polypeptides or polynucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing polypeptides and polynucleotides into plant cells include microinjection (Crossway, et al., (1986) Biotechniques 4:320-334), electroporation (Riggs, et al., (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606), Agrobacterium-mediated transformation (U.S. Pat. No. 5,563,055 and U.S. Pat. No. 5,981,840), direct gene transfer (Paszkowski, et al., (1984) EMBO J. 3:2717-2722), and ballistic particle acceleration (see, for example, U.S. Pat. No. 4,945,050; 5,879,918; 5,886,244 and 5,932,782; Tomes, et al., (1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); McCabe, et al., (1988) Biotechnology 6:923-926); and Lec1 transformation (WO 00/28058). Also see, Weissinger, et al., (1988) Ann. Rev. Genet. 22:421-477; Sanford, et al., (1987) Particulate Science and Technology 5:27-37 (onion); Christou, et al., (1988) Plant Physiol. 87:671-674 (soybean); McCabe, et al., (1988) Bio/Technology 6:923-926 (soybean); Finer and McMullen, (1991) In Vitro Cell Dev. Biol. 27P:175-182 (soybean); Singh, et al., (1998) Theor. Appl. Genet. 96:319-324 (soybean); Datta, et al., (1990) Biotechnology 8:736-740 (rice); Hoque, et al., (2005) Plant Cell Tissue & Organ Culture 82(1):45-55 (rice); Sreekala, et al., (2005) Plant Cell Reports 24(2):86-94 (rice); Klein, et al., (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein, et al., (1988) Biotechnology 6:559-563 (maize); U.S. Pat. Nos. 5,240,855; 5,322,783 and 5,324,646; Klein, et al., (1988) Plant Physiol. 91:440-444 (maize); Fromm, et al., (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren, et al., (1984) Nature (London) 311:763-764; U.S. Pat. No. 5,736,369 (cereals); Bytebier, et al., (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet, et al., (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman, et al., (Longman, N.Y.), pp. 197-209 (pollen); Kaeppler, et al., (1990) Plant Cell Reports 9:415-418 and Kaeppler, et al., (1992) Theor. Appl. Genet. 84:560-566 (whisker-mediated transformation); D'Halluin, et al., (1992) Plant Cell 4:1495-1505 (electroporation); Li, et al., (1993) Plant Cell Reports 12:250-255 and Christou and Ford, (1995) Annals of Botany 75:407-413 (rice); Osjoda, et al., (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens); all of which are herein incorporated by reference.
In specific embodiments, the IPT sequences or the IPT promoter sequences of the invention can be provided to a plant using a variety of transient transformation methods. Such transient transformation methods include, but are not limited to, the introduction of the IPT protein or IPT promoter or variants and fragments thereof directly into the plant or the introduction of an IPT transcript into the plant. Such methods include, for example, microinjection or particle bombardment. See, for example, Crossway, et al., (1986) Mol Gen. Genet. 202:179-185; Nomura, et al., (1986) Plant Sci. 44:53-58; Hepler, et al., (1994) Proc. Natl. Acad. Sci. 91:2176-2180 and Hush, et al., (1994) The Journal of Cell Science 107:775-784, all of which are herein incorporated by reference. Alternatively, the IPT polynucleotide or the IPT promoter can be transiently transformed into the plant using techniques known in the art. Such techniques include viral vector system and the precipitation of the polynucleotide in a manner that precludes subsequent release of the DNA. Thus, the transcription from the particle-bound DNA can occur, but the frequency with which it is released to become integrated into the genome is greatly reduced. Such methods include the use of particles coated with polyethyenlimine (PEI; Sigma #P3143).
In other embodiments, the polynucleotide of the invention may be introduced into plants by contacting plants with a virus or viral nucleic acids. Generally, such methods involve incorporating a nucleotide construct of the invention within a viral DNA or RNA molecule. It is recognized that an IPT polynucleotide of the invention may be initially synthesized as part of a viral polyprotein, which later may be processed by proteolysis in vivo or in vitro to produce the desired recombinant protein. Further, it is recognized that promoters useful for the invention also encompass promoters utilized for transcription by viral RNA polymerases. Methods for introducing polynucleotides into plants and expressing a protein encoded therein, involving viral DNA or RNA molecules, are known in the art. See, for example, U.S. Pat. Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367, 5,316,931, and Porta, et al., (1996) Molecular Biotechnology 5:209-221; herein incorporated by reference.
Methods are known in the art for the targeted insertion of a polynucleotide at a specific location in the plant genome. In one embodiment, the insertion of the polynucleotide at a desired genomic location is achieved using a site-specific recombination system. See, for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, and WO99/25853, and U.S. Pat. Nos. 6,187,994; 6,552,248; 6,624,297; 6,331,661; 6,262,341; 6,541,231; 6,664,108; 6,300,545; 6,528,700 and 6,911,575, all of which are herein incorporated by reference. Briefly, the polynucleotide of the invention can be contained in a transfer cassette flanked by two non-recombinogenic recombination sites. The transfer cassette is introduced into a plant having stably incorporated into its genome a target site which is flanked by two non-recombinogenic recombination sites that correspond to the sites of the transfer cassette. An appropriate recombinase is provided and the transfer cassette is integrated at the target site. The polynucleotide of interest is thereby integrated at a specific chromosomal position in the plant genome.
The cells that have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick, et al., (1986) Plant Cell Reports 5:81-84. These plants may then be grown, and pollinated with either the same transformed strain or different strains, and the resulting progeny having expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure that expression of the desired phenotypic characteristic has been achieved. In this manner, the present invention provides transformed seed (also referred to as “transgenic seed”) having a polynucleotide of the invention, for example, an expression cassette of the invention, stably incorporated into their genome.
Pedigree breeding starts with the crossing of two genotypes, such as an elite line of interest and one other inbred line having one or more desirable characteristics (i.e., having stably incorporated a polynucleotide of the invention, having a modulated activity and/or level of the polypeptide of the invention, etc) which complements the elite line of interest. If the two original parents do not provide all the desired characteristics, other sources can be included in the breeding population. In the pedigree method, superior plants are selfed and selected in successive filial generations. In the succeeding filial generations the heterozygous condition gives way to homogeneous lines as a result of self-pollination and selection. Typically in the pedigree method of breeding, five or more successive filial generations of selfing and selection are practiced: F1→F2; F2→F3; F3→F4; F4→F5, etc. After a sufficient amount of inbreeding, successive filial generations will serve to increase seed of the developed inbred. In specific embodiments, the inbred line comprises homozygous alleles at about 95% or more of its loci.
In addition to being used to create a backcross conversion, backcrossing can also be used in combination with pedigree breeding to modify an elite line of interest and a hybrid that is made using the modified elite line. Backcrossing can be used to transfer one or more specifically desirable traits from one line, the donor parent, to an inbred called the recurrent parent, which has overall good agronomic characteristics yet lacks that desirable trait or traits. However, the same procedure can be used to move the progeny toward the genotype of the recurrent parent but at the same time retain many components of the non-recurrent parent by stopping the backcrossing at an early stage and proceeding with selfing and selection. For example, an F1, such as a commercial hybrid, is created. This commercial hybrid may be backcrossed to one of its parent lines to create a BC1 or BC2. Progeny are selfed and selected so that the newly developed inbred has many of the attributes of the recurrent parent and yet several of the desired attributes of the non-recurrent parent. This approach leverages the value and strengths of the recurrent parent for use in new hybrids and breeding.
Therefore, an embodiment of this invention is a method of making a backcross conversion of a maize inbred line of interest, comprising the steps of crossing a plant of a maize inbred line of interest with a donor plant comprising a mutant gene or transgene conferring a desired trait (i.e., a modulation in the level of cytokinin (an increase or a decrease) or any plant phenotype resulting from the modulated cytokinin level (such plant phenotypes are discussed elsewhere herein)), selecting an F1 progeny plant comprising the mutant gene or transgene conferring the desired trait, and backcrossing the selected F1 progeny plant to a plant of the maize inbred line of interest. This method may further comprise the step of obtaining a molecular marker profile of the maize inbred line of interest and using the molecular marker profile to select for a progeny plant with the desired trait and the molecular marker profile of the inbred line of interest. In the same manner, this method may be used to produce F1 hybrid seed by adding a final step of crossing the desired trait conversion of the maize inbred line of interest with a different maize plant to make F1 hybrid maize seed comprising a mutant gene or transgene conferring the desired trait.
Recurrent selection is a method used in a plant breeding program to improve a population of plants. The method entails individual plants cross pollinating with each other to form progeny. The progeny are grown and the superior progeny selected by any number of selection methods, which include individual plant, half-sib progeny, full-sib progeny, selfed progeny and toperossing. The selected progeny are cross-pollinated with each other to form progeny for another population. This population is planted and again superior plants are selected to cross pollinate with each other. Recurrent selection is a cyclical process and therefore can be repeated as many times as desired. The objective of recurrent selection is to improve the traits of a population. The improved population can then be used as a source of breeding material to obtain inbred lines to be used in hybrids or used as parents for a synthetic cultivar. A synthetic cultivar is the resultant progeny formed by the intercrossing of several selected inbreds.
Mass selection is a useful technique when used in conjunction with molecular marker enhanced selection. In mass selection seeds from individuals are selected based on phenotype and/or genotype. These selected seeds are then bulked and used to grow the next generation. Bulk selection requires growing a population of plants in a bulk plot, allowing the plants to self-pollinate, harvesting the seed in bulk and then using a sample of the seed harvested in bulk to plant the next generation. Instead of self pollination, directed pollination could be used as part of the breeding program.
Mutation breeding is one of many methods that could be used to introduce new traits into an elite line. Mutations that occur spontaneously or are artificially induced can be useful sources of variability for a plant breeder. The goal of artificial mutagenesis is to increase the rate of mutation for a desired characteristic. Mutation rates can be increased by many different means including temperature, long-term seed storage, tissue culture conditions, radiation such as X-rays, Gamma rays (e.g., cobalt 60 or cesium 137), neutrons, (product of nuclear fission by uranium 235 in an atomic reactor), Beta radiation (emitted from radioisotopes such as phosphorus 32 or carbon 14), or ultraviolet radiation (preferably from 2500 to 2900 nm), or chemical mutagens (such as base analogues (5-bromo-uracil), related compounds (8-ethoxy caffeine), antibiotics (streptonigrin), alkylating agents (sulfur mustards, nitrogen mustards, epoxides, ethylenamines, sulfates, sulfonates, sulfones, lactones), azide, hydroxylamine, nitrous acid, or acridines. Once a desired trait is observed through mutagenesis the trait may then be incorporated into existing germplasm by traditional breeding techniques, such as backcrossing. Details of mutation breeding can be found in “Principals of Cultivar Development,” Fehr, 1993 Macmillan Publishing Company, the disclosure of which is incorporated herein by reference. In addition, mutations created in other lines may be used to produce a backcross conversion of elite lines that comprises such mutations.
The present invention may be used for transformation of any plant species, including, but not limited to, monocots and dicots. Examples of plant species of interest include, but are not limited to, corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, and conifers.
Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), and chrysanthemum. 
Conifers that may be employed in practicing the present invention include, for example, pines such as loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firs such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as Western red cedar (Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis). In specific embodiments, plants of the present invention are crop plants (for example, corn, alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, etc.). In other embodiments, corn and soybean plants are optimal, and in yet other embodiments corn plants are optimal.
Other plants of interest include grain plants that provide seeds of interest, oil-seed plants, and leguminous plants. Seeds of interest include grain seeds, such as maize, wheat, barley, rice, sorghum, rye, etc. Oil-seed plants include cotton, soybean, safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants include beans and peas. Beans include guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, etc.
Typically, an intermediate host cell will be used in the practice of this invention to increase the copy number of the cloning vector. With an increased copy number, the vector containing the nucleic acid of interest can be isolated in significant quantities for introduction into the desired plant cells. In one embodiment, plant promoters that do not cause expression of the polypeptide in bacteria are employed.
Prokaryotes most frequently are represented by various strains of E. coli; however, other microbial strains may also be used. Commonly used prokaryotic control sequences which are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding sequences, include such commonly used promoters as the beta lactamase (penicillinase) and lactose (lac) promoter systems (Chang, et al., (1977) Nature 198:1056), the tryptophan (trp) promoter system (Goeddel, et al., (1980) Nucleic Acids Res. 8:4057) and the lambda derived P L promoter and N-gene ribosome binding site (Shimatake, et al., (1981) Nature 292:128). The inclusion of selection markers in DNA vectors transfected in E coli. is also useful. Examples of such markers include genes specifying resistance to ampicillin, tetracycline, or chloramphenicol.
The vector is selected to allow introduction into the appropriate host cell. Bacterial vectors are typically of plasmid or phage origin. Appropriate bacterial cells are infected with phage vector particles or transfected with naked phage vector DNA. If a plasmid vector is used, the bacterial cells are transfected with the plasmid vector DNA. Expression systems for expressing a protein of the present invention are available using Bacillus sp. and Salmonella (Palva, et al., (1983) Gene 22:229-235); Mosbach, et al., (1983) Nature 302:543-545).
A variety of eukaryotic expression systems such as yeast, insect cell lines, plant and mammalian cells, are known to those of skill in the art. As explained briefly below, a polynucleotide of the present invention can be expressed in these eukaryotic systems. In some embodiments, transformed/transfected plant cells, as discussed infra, are employed as expression systems for production of the proteins of the instant invention.
Synthesis of heterologous polynucleotides in yeast is well known (Sherman, et al., (1982) Methods in Yeast Genetics, Cold Spring Harbor Laboratory). Two widely utilized yeasts for production of eukaryotic proteins are Saccharomyces cerevisiae and Pichia pastoris. Vectors, strains and protocols for expression in Saccharomyces and Pichia are known in the art and available from commercial suppliers (e.g., Invitrogen). Suitable vectors usually have expression control sequences, such as promoters, including 3-phosphoglycerate kinase or alcohol oxidase, and an origin of replication, termination sequences and the like as desired. A protein of the present invention, once expressed, can be isolated from yeast by lysing the cells and applying standard protein isolation techniques to the lists. The monitoring of the purification process can be accomplished by using Western blot techniques or radioimmunoassay or other standard immunoassay techniques.
The sequences of the present invention can also be ligated to various expression vectors for use in transfecting cell cultures of, for instance, mammalian, insect, or plant origin. Illustrative cell cultures useful for the production of the peptides are mammalian cells. A number of suitable host cell lines capable of expressing intact proteins have been developed in the art, and include the HEK293, BHK21, and CHO cell lines. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter (e.g., the CMV promoter, a HSV tk promoter or pgk (phosphoglycerate kinase) promoter), an enhancer (Queen, et al., (1986) Immunol. Rev. 89:49), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., an SV40 large T Ag poly A addition site), and transcriptional terminator sequences. Other animal cells useful for production of proteins of the present invention are available, for instance, from the American Type Culture Collection.
Appropriate vectors for expressing proteins of the present invention in insect cells are usually derived from the SF9 baculovirus. Suitable insect cell lines include mosquito larvae, silkworm, armyworm, moth and Drosophila cell lines such as a Schneider cell line (See, Schneider (1987) J. Embryol. Exp. Morphol. 27:353-365).
As with yeast, when higher animal or plant host cells are employed, polyadenylation or transcription terminator sequences are typically incorporated into the vector. An example of a terminator sequence is the polyadenylation sequence from the bovine growth hormone gene. Sequences for accurate splicing of the transcript may also be included. An example of a splicing sequence is the VP1 intron from SV40 (Sprague, et al., (1983) J. ViroL 45:773-781). Additionally, gene sequences to control replication in the host cell may be incorporated into the vector such as those found in bovine papilloma virus type-vectors (Saveria-Campo (1985) DNA Cloning Vol. II a Practical Approach, D. M. Glover, Ed., IRL Press, Arlington, Va., pp. 213-238).
Animal and lower eukaryotic (e.g., yeast) host cells are competent or rendered competent for transfection by various means. There are several well-known methods of introducing DNA into animal cells. These include: calcium phosphate precipitation, fusion of the recipient cells with bacterial protoplasts containing the DNA, treatment of the recipient cells with liposomes containing the DNA, DEAE dextrin, electroporation, biolistics, and micro-injection of the DNA directly into the cells. The transfected cells are cultured by means well known in the art (Kuchler, (1997) Biochemical Methods in Cell Culture and Virology, Dowden, Hutchinson and Ross, Inc.).
II. Modulating the Concentration and/or Activity of an Isopentenyl Transferase Polypeptide
A method for modulating the concentration and/or activity of the polypeptide of the present invention in a plant is provided. In general, concentration and/or activity of the IPT polypeptide is increased or reduced by at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% or more, relative to a native control plant, plant part or cell which does not comprise the introduced sequence. Modulation of the concentration and/or activity may occur at one or more stages of development. In specific embodiments, the polypeptides of the present invention are modulated in monocots, such as maize.
The expression level of the IPT polypeptide may be measured directly, for example, by assaying for the level of the IPT polypeptide in the plant, or indirectly, for example, by measuring the cytokinin synthesis activity in the plant. Methods for assaying for cytokinin synthesis activity are described elsewhere herein.
In specific embodiments, the polypeptide or the polynucleotide of the invention is introduced into the plant cell. Subsequently, a plant cell having the introduced sequence of the invention is selected using methods known to those of skill in the art such as, but not limited to, Southern blot analysis, DNA sequencing, PCR analysis or phenotypic analysis. A plant or plant part altered or modified by the foregoing embodiments is grown under plant forming conditions for a time sufficient to modulate the concentration and/or activity of polypeptides of the present invention in the plant. Plant forming conditions are well known in the art and discussed briefly elsewhere herein.
It is also recognized that the level and/or activity of the polypeptide may be modulated by employing a polynucleotide that is not capable of directing, in a transformed plant, the expression of a protein or an RNA. For example, the polynucleotides of the invention may be used to design polynucleotide constructs that can be employed in methods for altering or mutating a genomic nucleotide sequence in an organism. Such polynucleotide constructs include, but are not limited to, RNA:DNA vectors, RNA:DNA mutational vectors, RNA:DNA repair vectors, mixed-duplex oligonucleotides, self-complementary RNA:DNA oligonucleotides and recombinogenic oligonucleobases. Such nucleotide constructs and methods of use are known in the art. See, U.S. Pat. Nos. 5,565,350; 5,731,181; 5,756,325; 5,760,012; 5,795,972 and 5,871,984; all of which are herein incorporated by reference. See also, WO 98/49350, WO 99/07865, WO 99/25821, and Beetham, et al., (1999) Proc. Natl. Acad. Sci. USA 96:8774-8778; herein incorporated by reference.
It is therefore recognized that methods of the present invention do not depend on the incorporation of the entire polynucleotide into the genome, only that the plant or cell thereof is altered as a result of the introduction of the polynucleotide into a cell. In one embodiment of the invention, the genome may be altered following the introduction of a polynucleotide into a cell. For example, the polynucleotide, or any part thereof, may incorporate into the genome of the plant. Alterations to the genome include, but are not limited to, additions, deletions, and substitutions of nucleotides into the genome. While the methods of the present invention do not depend on additions, deletions, and substitutions of any particular number of nucleotides, it is recognized that such additions, deletions, or substitutions comprise at least one nucleotide.
It is further recognized that modulating the level and/or activity of the IPT sequence can be performed to elicit the effects of the sequence only during certain developmental stages and to switch the effect off in other stages where expression is no longer desirable. Control of the IPT expression can be obtained via the use of inducible or tissue-preferred promoters. Alternatively, the gene could be inverted or deleted using site-specific recombinases, transposons or recombination systems, which would also turn on or off expression of the IPT sequence.
A “subject plant or plant cell” is one in which genetic alteration, such as transformation, has been effected as to a gene of interest, or is a plant or plant cell which is descended from a plant or cell so altered and which comprises the alteration. A “control” or “control plant” or “control plant cell” provides a reference point for measuring changes in phenotype of the subject plant or plant cell.
A control plant or plant cell may comprise, for example: (a) a wild-type plant or cell, i.e., of the same genotype as the starting material for the genetic alteration which resulted in the subject plant or cell; (b) a plant or plant cell of the same genotype as the starting material but which has been transformed with a null construct (i.e., with a construct which has no known effect on the trait of interest, such as a construct comprising a marker gene); (c) a plant or plant cell which is a non-transformed segregant among progeny of a subject plant or plant cell; (d) a plant or plant cell genetically identical to the subject plant or plant cell but which is not exposed to conditions or stimuli that would induce expression of the gene of interest; or (e) the subject plant or plant cell itself, under conditions in which the gene of interest is not expressed.
In the present case, for example, changes in cytokinin levels, including changes in absolute amounts of cytokinin, cytokinin ratios, cytokinin activity, or cytokinin distribution, or changes in plant or plant cell phenotype, such as flowering time, seed set, branching, senescence, stress tolerance or root mass, could be measured by comparing a subject plant or plant cell to a control plant or plant cell.
A. Increasing the Activity and/or Concentration of an Isopentenyl Transferase Polypeptide
Methods are provided to increase the activity and/or concentration of the IPT polypeptide of the invention. An increase in the concentration and/or activity of the IPT polypeptide of the invention can be achieved by providing to the plant an IPT polypeptide. As discussed elsewhere herein, many methods are known in the art for providing a polypeptide to a plant including, but not limited to, direct introduction of the polypeptide into the plant, and introducing into the plant (transiently or stably) a polynucleotide construct encoding a polypeptide having cytokinin synthesis activity. It is also recognized that the methods of the invention may employ a polynucleotide that is not capable of directing, in the transformed plant, the expression of a protein or an RNA. Thus, the level and/or activity of an IPT polypeptide may be increased by altering the gene encoding the IPT polypeptide or its promoter. See, e.g., Kmiec, U.S. Pat. No. 5,565,350; Zarling, et al., PCT/US93/03868. Therefore mutagenized plants that carry mutations in IPT genes, where the mutations increase expression of the IPT gene or increase the cytokinin synthesis activity of the encoded IPT polypeptide are provided. As described elsewhere herein, methods to assay for an increase in protein concentration or an increase in cytokinin synthesis activity are known.
B. Reducing the Activity and/or Concentration of an Isopentenyl Transferase Polypeptide
Methods are provided to reduce or eliminate the activity and/or concentration of the IPT polypeptide by transforming a plant cell with an expression cassette that expresses a polynucleotide that inhibits the expression of the IPT polypeptide. The polynucleotide may inhibit the expression of an IPT polypeptide directly, by preventing translation of the IPT polypeptide messenger RNA, or indirectly, by encoding a molecule that inhibits the transcription or translation of an IPT polypeptide gene encoding an IPT polypeptide. Methods for inhibiting or eliminating the expression of a gene in a plant are well known in the art, and any such method may be used in the present invention to inhibit the expression of the IPT polypeptides.
In accordance with the present invention, the expression of an IPT polypeptide is inhibited if the level of the IPT polypeptide is statistically lower than the level of the same IPT polypeptide in a plant that has not been genetically modified or mutagenized to inhibit the expression of that IPT polypeptide. In particular embodiments of the invention, the protein level of the IPT polypeptide in a modified plant according to the invention is less than 95%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10% or less than 5% of the protein level of the same IPT polypeptide in a plant that is not a mutant or that has not been genetically modified to inhibit the expression of that IPT polypeptide. The expression level of the IPT polypeptide may be measured directly, for example, by assaying for the level of the IPT polypeptide expressed in the cell or plant or indirectly, for example, by measuring the cytokinin synthesis activity in the cell or plant. Methods for determining the cytokinin synthesis activity of the IPT polypeptide are described elsewhere herein.
In other embodiments of the invention, the activity of one or more IPT polypeptides is reduced or eliminated by transforming a plant cell with an expression cassette comprising a polynucleotide encoding a polypeptide that inhibits the activity of one or more IPT polypeptides. The cytokinin synthesis activity of an IPT polypeptide is inhibited according to the present invention if the cytokinin synthesis activity of the IPT polypeptide is statistically lower than the cytokinin synthesis activity of the same IPT polypeptide in a plant that has not been genetically modified to inhibit the cytokinin synthesis activity of that IPT polypeptide. In particular embodiments of the invention, the cytokinin synthesis activity of the IPT polypeptide in a modified plant according to the invention is less than 95%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10% or less than 5% of the cytokinin synthesis activity of the same IPT polypeptide in a plant that that has not been genetically modified to inhibit the expression of that IPT polypeptide. The cytokinin synthesis activity of an IPT polypeptide is “eliminated” according to the invention when it is not detectable by the assay methods described elsewhere herein. Methods of determining the cytokinin synthesis activity of an IPT polypeptide are described elsewhere herein.
In other embodiments, the activity of an IPT polypeptide may be reduced or eliminated by disrupting the gene encoding the IPT polypeptide. The invention encompasses mutagenized plants that carry mutations in IPT genes, where the mutations reduce expression of the IPT gene or inhibit the cytokinin synthesis activity of the encoded IPT polypeptide.
Thus, many methods may be used to reduce or eliminate the activity of an IPT polypeptide. More than one method may be used to reduce the activity of a single IPT polypeptide. In addition, combinations of methods may be employed to reduce or eliminate the activity of two or more different IPT polypeptides.
Non-limiting examples of methods of reducing or eliminating the expression of an IPT polypeptide are given below.
1. Polynucleotide-Based Methods
In some embodiments of the present invention, a plant cell is transformed with an expression cassette that is capable of expressing a polynucleotide that inhibits the expression of an IPT sequence. The term “expression” as used herein refers to the biosynthesis of a gene product, including the transcription and/or translation of said gene product. For example, for the purposes of the present invention, an expression cassette capable of expressing a polynucleotide that inhibits the expression of at least one IPT sequence is an expression cassette capable of producing an RNA molecule that inhibits the transcription and/or translation of at least one IPT polypeptide. The “expression” or “production” of a protein or polypeptide from a DNA molecule refers to the transcription and translation of the coding sequence to produce the protein or polypeptide, while the “expression” or “production” of a protein or polypeptide from an RNA molecule refers to the translation of the RNA coding sequence to produce the protein or polypeptide.
Examples of polynucleotides that inhibit the expression of an IPT sequence are given below.
i. Sense Suppression/Cosuppression
In some embodiments of the invention, inhibition of the expression of an IPT polypeptide may be obtained by sense suppression or cosuppression. For cosuppression, an expression cassette is designed to express an RNA molecule corresponding to all or part of a messenger RNA encoding an IPT polypeptide in the “sense” orientation. Over expression of the RNA molecule can result in reduced expression of the native gene. Accordingly, multiple plant lines transformed with the cosuppression expression cassette are screened to identify those that show the greatest inhibition of IPT polypeptide expression.
The polynucleotide used for cosuppression may correspond to all or part of the sequence encoding the IPT polypeptide, all or part of the 5′ and/or 3′ untranslated region of an IPT polypeptide transcript, or all or part of both the coding sequence and the untranslated regions of a transcript encoding an IPT polypeptide. In some embodiments where the polynucleotide comprises all or part of the coding region for the IPT polypeptide, the expression cassette is designed to eliminate the start codon of the polynucleotide so that no protein product will be transcribed.
Cosuppression may be used to inhibit the expression of plant genes to produce plants having undetectable protein levels for the proteins encoded by these genes. See, for example, Broin, et al., (2002) Plant Cell 14:1417-1432. Cosuppression may also be used to inhibit the expression of multiple proteins in the same plant. See, for example, U.S. Pat. No. 5,942,657. Methods for using cosuppression to inhibit the expression of endogenous genes in plants are described in Flavell, et al., (1994) Proc. Natl. Acad. Sci. USA 91:3490-3496; Jorgensen, et al., (1996) Plant Mol. Biol. 31:957-973; Johansen and Carrington, (2001) Plant Physiol. 126:930-938; Broin, et al., (2002) Plant Cell 14:1417-1432; Stoutjesdijk, et al., (2002) Plant Physiol. 129:1723-1731; Yu, et al., (2003) Phytochemistry 63:753-763 and U.S. Pat. Nos. 5,034,323, 5,283,184 and 5,942,657; each of which is herein incorporated by reference. The efficiency of cosuppression may be increased by including a poly-dT region in the expression cassette at a position 3′ to the sense sequence and 5′ of the polyadenylation signal. See, US Patent Application Publication Number 2002/0048814, herein incorporated by reference. Typically, such a nucleotide sequence has substantial sequence identity to the sequence of the transcript of the endogenous gene, optimally greater than about 65% sequence identity, more optimally greater than about 85% sequence identity, most optimally greater than about 95% sequence identity. See, U.S. Pat. Nos. 5,283,184 and 5,034,323; herein incorporated by reference.
ii. Antisense Suppression
In some embodiments of the invention, inhibition of the expression of the IPT polypeptide may be obtained by antisense suppression. For antisense suppression, the expression cassette is designed to express an RNA molecule complementary to all or part of a messenger RNA encoding the IPT polypeptide. Over expression of the antisense RNA molecule can result in reduced expression of the native gene. Accordingly, multiple plant lines transformed with the antisense suppression expression cassette are screened to identify those that show the greatest inhibition of IPT polypeptide expression.
The polynucleotide for use in antisense suppression may correspond to all or part of the complement of the sequence encoding the IPT polypeptide, all or part of the complement of the 5′ and/or 3′ untranslated region of the IPT polypeptide transcript, or all or part of the complement of both the coding sequence and the untranslated regions of a transcript encoding the IPT polypeptide. In addition, the antisense polynucleotide may be fully complementary (i.e., 100% identical to the complement of the target sequence) or partially complementary (i.e., less than 100% identical to the complement of the target sequence) to the target sequence. Antisense suppression may be used to inhibit the expression of multiple proteins in the same plant. See, for example, U.S. Pat. No. 5,942,657. Furthermore, portions of the antisense nucleotides may be used to disrupt the expression of the target gene. Generally, sequences of at least 50 nucleotides, 100 nucleotides, 200 nucleotides, 300, 400, 450, 500, 550 or greater may be used. Methods for using antisense suppression to inhibit the expression of endogenous genes in plants are described, for example, in Liu, et al., (2002) Plant Physiol. 129:1732-1743 and U.S. Pat. Nos. 5,759,829 and 5,942,657, each of which is herein incorporated by reference. Efficiency of antisense suppression may be increased by including a poly-dT region in the expression cassette at a position 3′ to the antisense sequence and 5′ of the polyadenylation signal. See, US Patent Application Publication Number 2002/0048814, herein incorporated by reference.
iii. Double-Stranded RNA Interference
In some embodiments of the invention, inhibition of the expression of an IPT polypeptide may be obtained by double-stranded RNA (dsRNA) interference. For dsRNA interference, a sense RNA molecule like that described above for cosuppression and an antisense RNA molecule that is fully or partially complementary to the sense RNA molecule are expressed in the same cell, resulting in inhibition of the expression of the corresponding endogenous messenger RNA.
Expression of the sense and antisense molecules can be accomplished by designing the expression cassette to comprise both a sense sequence and an antisense sequence. Alternatively, separate expression cassettes may be used for the sense and antisense sequences. Multiple plant lines transformed with the dsRNA interference expression cassette or expression cassettes are then screened to identify plant lines that show the greatest inhibition of IPT polypeptide expression. Methods for using dsRNA interference to inhibit the expression of endogenous plant genes are described in Waterhouse, et al., (1998) Proc. Natl. Acad. Sci. USA 95:13959-13964, Liu, et al., (2002) Plant Physiol. 129:1732-1743, and WO 99/49029, WO 99/53050, WO 99/61631, and WO 00/49035; each of which is herein incorporated by reference.
iv. Hairpin RNA Interference and Intron-Containing Hairpin RNA Interference
In some embodiments of the invention, inhibition of the expression of one or more IPT polypeptides may be obtained by hairpin RNA (hpRNA) interference or intron-containing hairpin RNA (ihpRNA) interference. These methods are highly efficient at inhibiting the expression of endogenous genes. See, Waterhouse and Helliwell, (2003) Nat. Rev. Genet. 4:29-38 and the references cited therein.
For hpRNA interference, the expression cassette is designed to express an RNA molecule that hybridizes with itself to form a hairpin structure that comprises a single-stranded loop region and a base-paired stem. The base-paired stem region comprises a sense sequence corresponding to all or part of the endogenous messenger RNA encoding the gene whose expression is to be inhibited, and an antisense sequence that is fully or partially complementary to the sense sequence. Thus, the base-paired stem region of the molecule generally determines the specificity of the RNA interference. hpRNA molecules are highly efficient at inhibiting the expression of endogenous genes, and the RNA interference they induce is inherited by subsequent generations of plants. See, for example, Chuang and Meyerowitz, (2000) Proc. Natl. Acad. Sci. USA 97:4985-4990; Stoutjesdijk, et al., (2002) Plant Physiol. 129:1723-1731; and Waterhouse and Helliwell, (2003) Nat. Rev. Genet. 4:29-38. Methods for using hpRNA interference to inhibit or silence the expression of genes are described, for example, in Chuang and Meyerowitz, (2000) Proc. Natl. Acad. Sci. USA 97:4985-4990; Stoutjesdijk, et al., (2002) Plant Physiol. 129:1723-1731; Waterhouse and Helliwell, (2003) Nat. Rev. Genet. 4:29-38; Pandolfini, et al., BMC Biotechnology 3:7 and US Patent Application Publication Number 2003/0175965; each of which is herein incorporated by reference. A transient assay for the efficiency of hpRNA constructs to silence gene expression in vivo has been described by Panstruga, et al., (2003) Mol. Biol. Rep. 30:135-140, herein incorporated by reference.
Alternatively, the base-paired stem region may correspond to a portion of a promoter sequence controlling expression of the gene to be inhibited. Transcriptional gene silencing (TGS) may be accomplished through use of hpRNA constructs wherein the inverted repeat of the hairpin shares sequence identity with the promoter region driving expression of a gene to be silenced. Processing of the hpRNA into short RNAs which can interact with the homologous promoter region may trigger degradation or methylation to result in silencing (Aufsatz, et al., (2002) PNAS 99(4):16499-16506; Mette, et al., (2000) EMBO J 19(19):5194-5201).
For ihpRNA, the interfering molecules have the same general structure as for hpRNA, but the RNA molecule additionally comprises an intron that is capable of being spliced in the cell in which the ihpRNA is expressed. The use of an intron minimizes the size of the loop in the hairpin RNA molecule following splicing, and this increases the efficiency of interference. See, for example, Smith, et al., (2000) Nature 407:319-320. In fact, Smith, et al., show 100% suppression of endogenous gene expression using ihpRNA-mediated interference. Methods for using ihpRNA interference to inhibit the expression of endogenous plant genes are described, for example, in Smith, et al., (2000) Nature 407:319-320; Wesley, et al., (2001) Plant J. 27:581-590; Wang and Waterhouse, (2001) Curr. Opin. Plant Biol. 5:146-150; Waterhouse and Helliwell, (2003) Nat. Rev. Genet. 4:29-38; Helliwell and Waterhouse, (2003) Methods 30:289-295, and US Patent Application Publication Number 2003/0180945, each of which is herein incorporated by reference.
The expression cassette for hpRNA interference may also be designed such that the sense sequence and the antisense sequence do not correspond to an endogenous RNA. In this embodiment, the sense and antisense sequence flank a loop sequence that comprises a nucleotide sequence corresponding to all or part of the endogenous messenger RNA of the target gene. Thus, it is the loop region that determines the specificity of the RNA interference. See, for example, WO 02/00904, herein incorporated by reference.
v. Amplicon-Mediated Interference
Amplicon expression cassettes comprise a plant virus-derived sequence that contains all or part of the target gene but generally not all of the genes of the native virus. The viral sequences present in the transcription product of the expression cassette allow the transcription product to direct its own replication. The transcripts produced by the amplicon may be either sense or antisense relative to the target sequence (i.e., the messenger RNA for an IPT polypeptide). Methods of using amplicons to inhibit the expression of endogenous plant genes are described, for example, in Angell and Baulcombe, (1997) EMBO J. 16:3675-3684, Angell and Baulcombe, (1999) Plant J. 20:357-362 and U.S. Pat. No. 6,646,805, each of which is herein incorporated by reference.
vi. Ribozymes
In some embodiments, the polynucleotide expressed by the expression cassette of the invention is catalytic RNA or has ribozyme activity specific for the messenger RNA of an IPT polypeptide. Thus, the polynucleotide causes the degradation of the endogenous messenger RNA, resulting in reduced expression of the IPT polypeptide. This method is described, for example, in U.S. Pat. No. 4,987,071, herein incorporated by reference.
vii. Small Interfering RNA or Micro RNA
In some embodiments of the invention, inhibition of the expression of one or more IPT polypeptides may be obtained by RNA interference by expression of a gene encoding a micro RNA (miRNA). miRNAs are regulatory agents consisting of about 22 ribonucleotides. miRNA are highly efficient at inhibiting the expression of endogenous genes. See, for example Javier, et al., (2003) Nature 425: 257-263, herein incorporated by reference.
For miRNA interference, the expression cassette is designed to express an RNA molecule that is modeled on an endogenous miRNA gene. The miRNA gene encodes an RNA that forms a hairpin structure containing a 22-nucleotide sequence that is complementary to another endogenous gene (target sequence). For suppression of IPT polypeptide expression, the 22-nucleotide sequence is selected from an IPT polypeptide transcript sequence and contains 22 nucleotides encoding said IPT polypeptide sequence in sense orientation and 21 nucleotides of a corresponding antisense sequence that is complementary to the sense sequence. miRNA molecules are highly efficient at inhibiting the expression of endogenous genes, and the RNA interference they induce is inherited by subsequent generations of plants.
2. Polypeptide-Based Inhibition of Gene Expression
In one embodiment, the polynucleotide encodes a zinc finger protein that binds to a gene encoding an IPT polypeptide, resulting in reduced expression of the gene. In particular embodiments, the zinc finger protein binds to a regulatory region of an IPT polypeptide gene. In other embodiments, the zinc finger protein binds to a messenger RNA encoding an IPT polypeptide and prevents its translation. Methods of selecting sites for targeting by zinc finger proteins have been described, for example, in U.S. Pat. No. 6,453,242, and methods for using zinc finger proteins to inhibit the expression of genes in plants are described, for example, in US Patent Application Publication Number 2003/0037355; each of which is herein incorporated by reference.
3. Polypeptide-Based Inhibition of Protein Activity
In some embodiments of the invention, the polynucleotide encodes an antibody that binds to at least one IPT polypeptide, and reduces the cytokinin synthesis activity of the IPT polypeptide. In another embodiment, the binding of the antibody results in increased turnover of the antibody-IPT polypeptide complex by cellular quality control mechanisms. The expression of antibodies in plant cells and the inhibition of molecular pathways by expression and binding of antibodies to proteins in plant cells are well known in the art. See, for example, Conrad and Sonnewald, (2003) Nature Biotech. 21:35-36, incorporated herein by reference.
4. Gene Disruption
In some embodiments of the present invention, the activity of an IPT polypeptide is reduced or eliminated by disrupting the gene encoding the IPT polypeptide. The gene encoding the IPT polypeptide may be disrupted by any method known in the art. For example, in one embodiment, the gene is disrupted by transposon tagging. In another embodiment, the gene is disrupted by mutagenizing plants using random or targeted mutagenesis, and selecting for plants that have reduced IPT activity.
i. Transposon Tagging
In one embodiment of the invention, transposon tagging is used to reduce or eliminate the cytokinin synthesis activity of one or more IPT polypeptides. Transposon tagging comprises inserting a transposon within an endogenous IPT gene to reduce or eliminate expression of the IPT polypeptide. “IPT gene” is intended to mean the gene that encodes an IPT polypeptide according to the invention.
In this embodiment, the expression of one or more IPT polypeptides is reduced or eliminated by inserting a transposon within a regulatory region or coding region of the gene encoding the IPT polypeptide. A transposon that is within an exon, intron, 5′ or 3′ untranslated sequence, a promoter, or any other regulatory sequence of an IPT polypeptide gene may be used to reduce or eliminate the expression and/or activity of the encoded IPT polypeptide.
Methods for the transposon tagging of specific genes in plants are well known in the art. See, for example, Maes, et al., (1999) Trends Plant Sci. 4:90-96; Dharmapuri and Sonti, (1999) FEMS Microbiol. Lett. 179:53-59; Meissner, et al., (2000) Plant J. 22:265-274; Phogat, et al., (2000) J. Biosci. 25:57-63; Walbot, (2000) Curr. Opin. Plant Biol. 2:103-107; Gai, et al., (2000) Nucleic Acids Res. 28:94-96; Fitzmaurice, et al., (1999) Genetics 153:1919-1928). In addition, the TUSC process for selecting Mu insertions in selected genes has been described in Bensen, et al., (1995) Plant Cell 7:75-84; Mena, et al., (1996) Science 274:1537-1540 and U.S. Pat. No. 5,962,764; each of which is herein incorporated by reference.
ii. Mutant Plants with Reduced Activity
Additional methods for decreasing or eliminating the expression of endogenous genes in plants are also known in the art and can be similarly applied to the instant invention. These methods include other forms of mutagenesis, such as ethyl methanesulfonate-induced mutagenesis, deletion mutagenesis, and fast neutron deletion mutagenesis used in a reverse genetics sense (with PCR) to identify plant lines in which the endogenous gene has been deleted. For examples of these methods see Ohshima, et al., (1998) Virology 243:472-481; Okubara, et al., (1994) Genetics 137:867-874 and Quesada, et al., (2000) Genetics 154:421-436; each of which is herein incorporated by reference. In addition, a fast and automatable method for screening for chemically induced mutations, TILLING (Targeting Induced Local Lesions In Genomes), using denaturing HPLC or selective endonuclease digestion of selected PCR products is also applicable to the instant invention. See, McCallum, et al., (2000) Nat. Biotechnol. 18:455-457, herein incorporated by reference.
Mutations that impact gene expression or that interfere with the function (IPT activity) of the encoded protein are well known in the art. Insertional mutations in gene exons usually result in null-mutants. Mutations in conserved residues are particularly effective in inhibiting the cytokinin synthesis activity of the encoded protein. Conserved residues of plant IPT polypeptides suitable for mutagenesis with the goal to eliminate IPT activity have been described. See, for example, FIG. 1. Such mutants can be isolated according to well-known procedures, and mutations in different IPT loci can be stacked by genetic crossing. See, for example, Gruis, et al., (2002) Plant Cell 14:2863-2882.
In another embodiment of this invention, dominant mutants can be used to trigger RNA silencing due to gene inversion and recombination of a duplicated gene locus. See, for example, Kusaba, et al., (2003) Plant Cell 15:1455-1467.
The invention encompasses additional methods for reducing or eliminating the activity of one or more IPT polypeptides. Examples of other methods for altering or mutating a genomic nucleotide sequence in a plant are known in the art and include, but are not limited to, the use of RNA:DNA vectors, RNA:DNA mutational vectors, RNA:DNA repair vectors, mixed-duplex oligonucleotides, self-complementary RNA:DNA oligonucleotides, and recombinogenic oligonucleobases. Such vectors and methods of use are known in the art. See, for example, U.S. Pat. Nos. 5,565,350; 5,731,181; 5,756,325; 5,760,012; 5,795,972 and 5,871,984; each of which are herein incorporated by reference. See also, WO 98/49350, WO 99/07865, WO 99/25821 and Beetham, et al., (1999) Proc. Natl. Acad. Sci. USA 96:8774-8778; each of which is herein incorporated by reference.
III. Modulating Cytokinin Level and/or Activity
As used herein, “cytokinin” refers to a class, or member of the class, of plant-specific hormones that play a central role during the cell cycle and influence numerous developmental programs. Cytokinins comprise an N6-substituted purine derivative. Representative cytokinins include isopentenyladenine (N6-(Δ2-isopentenyl)adenine (hereinafter, iP), zeatin (6-(4-hydroxy-3methylbut-trans-2-enylamino) purine) (hereinafter, Z), and dihydrozeatin (DZ). The free bases and their ribosides (iPR, ZR, and DZR) are believed to be the active compounds. Additional cytokinins are known. See, for example, U.S. Pat. No. 5,211,738 and Keiber, et al., (2002) Cytokinins, The Arabidopsis Book, American Society of Plant Biologists, both of which are herein incorporated by reference.
“Modulating the cytokinin level” includes any statistically significant decrease or increase in cytokinin level and/or activity in the plant when compared to a control plant. For example, modulating the level and/or activity can comprise either an increase or a decrease in overall cytokinin content of about 0.1%, 0.5%, 1%, 3% 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or greater when compared to a control plant or plant part. Alternatively, the modulated level and/or activity of the cytokinin can include about a 0.2 fold, 0.5 fold, 2 fold, 4 fold, 8 fold, 16 fold, 32 fold or greater overall increase or decrease in cytokinin level/activity in the plant or a plant part when compared to a control plant or plant part.
It is further recognized that the modulation of the cytokinin level/activity need not be an overall increase/decrease in cytokinin level and/or activity, but also includes a change in tissue distribution of the cytokinin. Moreover, the modulation of the cytokinin level/activity need not be an overall increase/decrease in cytokinins, but also includes a change in the ratio of various cytokinin derivatives. For example, the ratio of various cytokinin derivatives such as isopentenyladenine-type, zeatin-type, or dihydrozeatin-type cytokinins, and the like, could be altered and thereby modulate the level/activity of the cytokinin of the plant or plant part when compared to a control plant.
Methods for assaying for a modulation in cytokinin level and/or activity are known in the art. For example, representative methods for cytokinin extraction, immunopurification, HPLC separation, and quantification by ELISA methods can be found, for example, in Faiss, et al., (1997) Plant J. 12:401-415. See, also, Werner, et al., (2001) PNAS 98:10487-10492) and Dewitte, et al., (1999) Plant Physiol. 119:111-121. Each of these references are herein incorporated by reference. As discussed elsewhere herein, modulation in cytokinin level and/or activity can further be detected by monitoring for particular plant phenotypes. Such phenotypes are described elsewhere herein.
In specific methods, the level and/or activity of a cytokinin in a plant is increased by increasing the level or activity of the IPT polypeptide in the plant. Methods for increasing the level and/or activity of IPT polypeptides in a plant are discussed elsewhere herein. Briefly, such methods comprise providing an IPT polypeptide of the invention to a plant and thereby increasing the level and/or activity of the IPT polypeptide. In other embodiments, an IPT nucleotide sequence encoding an IPT polypeptide can be provided by introducing into the plant a polynucleotide comprising an IPT nucleotide sequence of the invention, expressing the IPT sequence, and thereby increasing the level and/or activity of a cytokinin in the plant or plant part when compared to a control plant. In some embodiments, the IPT nucleotide construct introduced into the plant is stably incorporated into the genome of the plant.
In other methods, the level and/or activity of cytokinin in a plant is decreased by decreasing the level and/or activity of one or more of the IPT polypeptides in the plant. Such methods are disclosed in detail elsewhere herein. In one such method, an IPT nucleotide sequence is introduced into the plant and expression of the IPT nucleotide sequence decreases the activity of the IPT polypeptide, and thereby decreases the level and/or activity of a cytokinin in the plant or plant part when compared to a control plant or plant part. In other embodiments, the IPT nucleotide construct introduced into the plant is stably incorporated into the genome of the plant.
As discussed above, one of skill will recognize the appropriate promoter to use to modulate the level/activity of a cytokinin in the plant. Exemplary promoters for this embodiment have been disclosed elsewhere herein.
Accordingly, the present invention further provides plants having a modulated level/activity of a cytokinin when compared to the cytokinin level/activity of a control plant. In one embodiment, the plant of the invention has an increased level/activity of the IPT polypeptide of the invention, and thus has an increased level/activity of cytokinin. In other embodiments, the plant of the invention has a reduced or eliminated level of the IPT polypeptide of the invention, and thus has a decreased level/activity of a cytokinin. In certain embodiments, such plants have stably incorporated into their genome a nucleic acid molecule comprising an IPT nucleotide sequence of the invention operably linked to a promoter that drives expression in the plant cell.
IV. Modulating Root Development
Methods for modulating root development in a plant are provided. By “modulating root development” is intended any alteration in the development of the plant root when compared to a control plant. Such alterations in root development include, but are not limited to, alterations in the growth rate of the primary root, the fresh root weight, the extent of lateral and adventitious root formation, the vasculature system, meristem development, or radial expansion.
Methods for modulating root development in a plant are provided. The methods comprise modulating the level and/or activity of the IPT polypeptide in the plant. In one method, an IPT sequence of the invention is provided to the plant. In another method, the IPT nucleotide sequence is provided by introducing into the plant a polynucleotide comprising an IPT nucleotide sequence of the invention (which may be a fragment of a full-length IPT sequence provided), expressing said IPT sequence, and thereby modifying root development. In still other methods, the IPT nucleotide construct introduced into the plant is stably incorporated into the genome of the plant.
In other methods, root development is modulated by decreasing the level or activity of the IPT polypeptide in the plant. Such methods can comprise introducing an IPT nucleotide sequence into the plant and decreasing the activity of the IPT polypeptide. In some methods, the IPT nucleotide construct introduced into the plant is stably incorporated into the genome of the plant. A decrease in cytokinin synthesis activity can result in at least one or more of the following alterations to root development, including, but not limited to, larger root meristems, increased root growth, enhanced radial expansion, an enhanced vasculature system, increased root branching, more adventitious roots and/or an increase in fresh root weight when compared to a control plant.
As used herein, “root growth” encompasses all aspects of growth of the different parts that make up the root system at different stages of its development in both monocotyledonous and dicotyledonous plants. It is to be understood that enhanced root growth can result from enhanced growth of one or more of its parts including the primary root, lateral roots, adventitious roots, etc. Methods of measuring such developmental alterations in the root system are known in the art. See, for example, US Patent Application Publication Number 2003/0074698 and Werner, et al., (2001) PNAS 18:10487-10492, both of which are herein incorporated by reference.
As discussed above, one of skill will recognize the appropriate promoter to use to modulate root development in the plant. Exemplary promoters for this embodiment include constitutive promoters and root-preferred promoters. Exemplary root-preferred promoters have been disclosed elsewhere herein.
Stimulating root growth and increasing root mass by decreasing the activity and/or level of the IPT polypeptide also finds use in improving the standability of a plant. The term “resistance to lodging” or “standability” refers to the ability of a plant to fix itself to the soil. For plants with an erect or semi-erect growth habit, this term also refers to the ability to maintain an upright position under adverse environmental conditions. This trait relates to the size, depth and morphology of the root system. In addition, stimulating root growth and increasing root mass by decreasing the level and/or activity of the IPT polypeptide at appropriate developmental stages also finds use in promoting in vitro propagation of explants.
Increased root biomass and/or altered root architecture may also find use in improving nitrogen-use efficiency of the plant. Such improved efficiency may lead to, for example, an increase in plant biomass and/or seed yield at an existing level of available nitrogen, or maintenance of plant biomass and/or seed yield when available nitrogen is limited. Thus, agronomic and/or environmental benefits may ensue.
Furthermore, higher root biomass production due to a decreased level and/or activity of an IPT polypeptide has an indirect effect on production of compounds produced by root cells or transgenic root cells or cell cultures of said transgenic root cells. One example of an interesting compound produced in root cultures is shikonin, the yield of which can be advantageously enhanced by said methods.
Accordingly, the present invention further provides plants having modulated root development when compared to the root development of a control plant. In some embodiments, the plant of the invention has a decreased level/activity of an IPT polypeptide of the invention and has enhanced root growth and/or root biomass. In certain embodiments, such plants have stably incorporated into their genome a nucleic acid molecule comprising an IPT nucleotide sequence of the invention operably linked to a promoter that drives expression in the plant cell.
V. Modulating Shoot and Leaf Development
Methods are also provided for modulating vegetative tissue growth in plants. In one embodiment, shoot and leaf development in a plant is modulated. By “modulating shoot and/or leaf development” is intended any alteration in the development of the plant shoot and/or leaf when compared to a control plant or plant part. Such alterations in shoot and/or leaf development include, but are not limited to, alterations in shoot meristem development, in leaf number, leaf size, leaf and stem vasculature, internode length, and leaf senescence. As used herein, “leaf development” and “shoot development” encompasses all aspects of growth of the different parts that make up the leaf system and the shoot system, respectively, at different stages of their development, both in monocotyledonous and dicotyledonous plants. Methods for measuring such developmental alterations in the shoot and leaf system are known in the art. See, for example, Werner, et al., (2001) PNAS 98:10487-10492 and US Patent Application Publication Number 2003/0074698, each of which is herein incorporated by reference.
The method for modulating shoot and/or leaf development in a plant comprises modulating the activity and/or level of an IPT polypeptide of the invention. In one embodiment, an IPT sequence of the invention is provided. In other embodiments, the IPT nucleotide sequence can be provided by introducing into the plant a polynucleotide comprising an IPT nucleotide sequence of the invention, expressing the IPT sequence, and thereby modifying shoot and/or leaf development. In other embodiments, the IPT nucleotide construct introduced into the plant is stably incorporated into the genome of the plant.
In specific embodiments, shoot or leaf development is modulated by decreasing the level and/or activity of the IPT polypeptide in the plant. A decrease in IPT activity can result in one or more alterations in shoot and/or leaf development, including, but not limited to, smaller apical meristems, reduced leaf number, reduced leaf surface, reduced vascular tissues, shorter internodes and stunted growth, and accelerated leaf senescence, when compared to a control plant.
As discussed above, one of skill will recognize the appropriate promoter to use to modulate shoot and leaf development of the plant. Exemplary promoters for this embodiment include constitutive promoters, shoot-preferred promoters, shoot meristem-preferred promoters, senescence-activated promoters, stress-induced promoters, root-preferred promoters, nitrogen-induced promoters and leaf-preferred promoters. Exemplary promoters have been disclosed elsewhere herein.
Decreasing cytokinin synthesis activity in a plant generally results in shorter internodes and stunted growth. Thus, the methods of the invention find use in producing dwarf plants. In addition, as discussed above, modulation of cytokinin synthesis activity in the plant modulates both root and shoot growth. Thus, the present invention further provides methods for altering the root/shoot ratio.
Shoot or leaf development can further be modulated by increasing the level and/or activity of the IPT polypeptide in the plant. An increase in IPT activity can result in one or more alterations in shoot and/or leaf development including, but not limited to, increased leaf number, increased leaf surface, increased vascular tissue, increased shoot formation, longer internodes, improved growth, improved plant yield and vigor, and retarded leaf senescence when compared to a control plant.
In one embodiment, the tolerance of a plant to flooding is improved. Flooding is a serious environmental stress that affects plant growth and productivity. Flooding causes premature senescence which results in leaf chlorosis, necrosis, defoliation, cessation of growth and reduced yield. Cytokinins can regulate senescence, and by increasing the level/activity of the IPT polypeptide in the plant, the present invention improves the tolerance of the plant to a variety of environmental stresses, including flooding. Delayed senescence may also advantageously expand the maturity adaptation of crops, improve the shelf-life of potted plants, and extend the vase-life of cut flowers.
In still other embodiments, methods for modulating shoot regeneration in a callus are provided. In this method, increasing the level and/or activity of the IPT polypeptide will increase the level of cytokinins in the plant. Accordingly, lower concentrations of exogenous growth regulators (i.e., cytokinins) or no exogenous cytokinins in the culture medium will be needed to enhance shoot regeneration in callus. Thus, in one embodiment of the invention, the increased level and/or activity of the IPT sequence can be used to overcome the poor shooting potential of certain species that has limited the success and speed of transgene technology for those species. Moreover, multiple shoot induction can be induced for crops where it is economically desirable to produce as many shoots as possible. Accordingly, methods are provided to increase the rate of regeneration for transformation. In specific embodiments, the IPT sequence will be under the control of an inducible promoter (e.g., heat shock promoter, chemically inducible promoter). Additional inducible promoters are known in the art and are discussed elsewhere herein.
Methods for establishing callus from explants are known. For example, roots, stems, buds, and aseptically germinated seedlings are just a few of the sources of tissue that can be used to induce callus formation. Generally, young and actively growing tissues (i.e., young leaves, roots, meristems or other tissues) are used, but are not required. Callus formation is controlled by growth regulating substances present in the medium (auxins and cytokinins). The specific concentrations of plant regulators needed to induce callus formation vary from species to species and can even depend on the source of explant. In some instances, it is advised to use different growth substances (e.g. 2,4-D or NAA) or a combination of them during tests, since some species may not respond to a specific growth regulator. In addition, culture conditions (i.e., light, temperature, etc.) can also influence the establishment of callus. Once established, callus cultures can be used to initiate shoot regeneration. See, for example, Gurel, et al., (2001) Turk J. Bot. 25:25-33; Dodds, et al., (1995). Experiments in Plant Tissue Culture, Cambridge University Press; Gamborg (1995) Plant Cell, Tissue and Organ Culture, eds. G. Phillips; and, US Patent Application Publication Number 2003/0180952, all of which are herein incorporated by reference.
It is further recognized that increasing seed size and/or weight can be accompanied by an increase in the rate of growth of seedlings or an increase in vigor. In addition, modulating the plant's tolerance to stress, as discussed below, along with modulation of root, shoot and leaf development can increase plant yield and vigor. As used herein, the term “vigor” refers to the relative health, productivity, and rate of growth of the plant and/or of certain plant parts, and may be reflected in various developmental attributes, including, but not limited to, concentration of chlorophyll, photosynthetic rate, total biomass, root biomass, grain quality, and/or grain yield. In Zea mays in particular, vigor may also be reflected in ear growth rate, ear size, and/or expansiveness of silk exsertion. Vigor may relate to the ability of a plant to grow rapidly during early development and to the successful establishment, after germination, of a well-developed root system and a well-developed photosynthetic apparatus. Vigor may be determined with reference to different genotypes under similar environmental conditions, or with reference to the same or different genotypes under different environmental conditions.
Accordingly, the present invention further provides plants having modulated shoot and/or leaf development when compared to a control plant. In some embodiments, the plant of the invention has an increased level/activity of the IPT polypeptide of the invention. In other embodiments, the plant of the invention has a decreased level/activity of the IPT polypeptide of the invention.
VI. Modulating Reproductive Tissue Development
Methods for modulating reproductive tissue development are provided. In one embodiment, methods are provided to modulate floral development in a plant. By “modulating floral development” is intended any alteration in a structure of a plant's reproductive tissue as compared to a control plant or plant part. “Modulating floral development” further includes any alteration in the timing of the development of a plant's reproductive tissue (i.e., delayed or accelerated floral development) when compared to a control plant or plant part. Macroscopic alterations may include changes in size, shape, number, or location of reproductive organs, the developmental time period during which these structures form, or the ability to maintain or proceed through the flowering process in times of environmental stress. Microscopic alterations may include changes to the types or shapes of cells that make up the reproductive organs.
The method for modulating floral development in a plant comprises modulating (either increasing or decreasing) the level and/or activity of the IPT polypeptide in a plant. In one method, an IPT sequence of the invention is provided. An IPT nucleotide sequence can be provided by introducing into the plant a polynucleotide comprising an IPT nucleotide sequence of the invention, expressing the IPT sequence, and thereby modifying floral development. In some embodiments, the IPT nucleotide construct introduced into the plant is stably incorporated into the genome of the plant.
As discussed above, one of skill will recognize the appropriate promoter to use to modulate floral development in the plant. Exemplary promoters for this embodiment include constitutive promoters, inducible promoters, shoot-preferred promoters, and inflorescence-preferred promoters (including developing-female-inflorescence-preferred promoters), including those listed elsewhere herein.
In specific methods, floral development is modulated by increasing the level and/or activity of the IPT sequence of the invention. Such methods can comprise introducing an IPT nucleotide sequence into the plant and increasing the activity of the IPT polypeptide. In some methods, the IPT nucleotide construct introduced into the plant is stably incorporated into the genome of the plant. An increase in the level and/or activity of the IPT sequences can result in one or more alterations in floral development including, but not limited to, accelerated flowering, increased number of flowers, and improved seed set when compared to a control plant. In addition, an increase in the level or activity of the IPT sequences can result in the prevention of flower senescence and an alteration in embryo number per kernel. See, Young, et al., (2004) Plant J. 38:910-22. Methods for measuring such developmental alterations in floral development are known in the art. See, for example, Mouradov, et al., (2002) The Plant Cell S111-S130, herein incorporated by reference.
In other methods, floral development is modulated by decreasing the level and/or activity of the IPT sequence of the invention. A decrease in the level and/or activity of the IPT sequence can result in kernel abortion and infertile female inflorescence. Inducing delayed flowering or inhibiting flowering can be used to enhance yield in forage crops such as alfalfa.
Accordingly, the present invention further provides plants having modulated floral development when compared to the floral development of a control plant. Compositions include plants having a decreased level/activity of the IPT polypeptide of the invention and having an altered floral development. Compositions also include plants having an increased level/activity of the IPT polypeptide of the invention wherein the plant maintains or proceeds through the flowering process in times of stress.
VII. Modulating the Stress Tolerance of a Plant
Methods are provided for the use of the IPT sequences of the invention to modify the tolerance of a plant to abiotic stress. Increases in the growth of seedlings or early vigor is often associated with an increase in stress tolerance. For example, faster development of seedlings, including the root system of seedlings upon germination, is critical for survival particularly under adverse conditions such as drought. Promoters that can be used in this method are described elsewhere herein, including low-level constitutive, inducible, or root-preferred promoters, such as root-preferred promoters derived from ZmIPT4 and ZmIPT5 regulatory sequences. Accordingly, in one method of the invention, a plant's tolerance to stress is increased or maintained when compared to a control plant by decreasing the level of IPT activity in the germinating seedling. In other methods, an IPT nucleotide sequence is provided by introducing into the plant a polynucleotide comprising a IPT nucleotide sequence of the invention, expressing the IPT sequence, and thereby increasing the plant's tolerance to stress. In other embodiments, the IPT nucleotide construct introduced into the plant is stably incorporated into the genome of the plant.
Methods are also provided to increase or maintain seed set during abiotic stress episodes. During periods of stress (i.e., drought, salt, heavy metals, temperature, etc.) embryo development is often aborted. In maize, halted embryo development results in aborted kernels on the ear (Cheikh and Jones, (1994) Plant Physiol. 106:45-51; Dietrich, et al., (1995) Plant Physiol Biochem 33:327-336). Preventing this kernel loss will maintain yield. Accordingly, methods are provided to increase the stress resistance in a plant (e.g., during flowering and seed development). Increasing expression of the IPT sequence of the invention can also modulate floral development during periods of stress, and thus methods are provided to maintain or improve the flowering process in plants under stress. The method comprises increasing the level and/or activity of the IPT sequence of the invention. In one method, an IPT nucleotide sequence is introduced into the plant and the level and/or activity of the IPT polypeptide is increased, thereby maintaining or improving the tolerance of the plant under stress conditions. In other methods, the IPT nucleotide construct introduced into the plant is stably incorporated into the genome of the plant. See, for example, WO 00/63401.
Significant yield instability can occur as a result of unfavorable environments during the lag phase of seed development. During this period, seeds undergo dramatic changes in ultra structure, biochemistry, and sensitivity to environmental perturbation, yet demonstrate little change in dry mass accumulation. Two important events that occur during the lag phase are initiation and division of endosperm cells and amyloplasts (which are the sites for starch deposition). It has been demonstrated that during the lag phase (around 10-12 days after pollination (DAP) in maize) a dramatic increase in cytokinin concentration immediately precedes maximum rates of endosperm cell division and amyloplast formation, indicating that this hormone plays a central role in these processes and in what is called the ‘sink strength’ of the developing seed. Cytokinins have been demonstrated to play an important role in establishing seed size, decreasing tip kernel abortion, and increasing seed set during unfavorable environmental conditions. For example, elevated temperatures affect seed formation. Elevated temperatures can inhibit the accumulation of cytokinin, decrease endosperm cell division and amyloplast number, and as a consequence, increase kernel abortion.
Kernel sink capacity in maize is principally a function of the number of endosperm cells and starch granules established during the first 6 to 12 DAP. The final number of endosperm cells and amyloplasts formed is highly correlated with final kernel weight. (Capitanio, et al., (1983); Reddy and Daynard, (1983); Jones, et al., (1985), Engelen-Eigles, et al., (2000)). Hormones, especially cytokinins, have been shown to stimulate cell division, plastid initiation and other processes important in the establishment of kernel sink capacity (Davies, (1987)). Cytokinin levels could for example be manipulated using the ZmIPT2 promoter to drive the expression of the Agrobacterium IPT gene. Similarly, endosperm- and/or pedicel-preferred promoters could be used to increase the level and/or duration of expression of ZmIPT2, which would result in an increase of cytokinin levels which would in turn increase sink strength and kernel yield. Capitano, et al., (1983). Grain weight and its components in maize inbred lines. Maydica 23:365-379. Jones, et al., (1985). Thermal environment during endosperm cell division in maize: effects on number of endosperm cells and starch granules. Crop Science 25:830-834. Jones, et al., (1996). Kernel sink strength capacity in maize: Genotypic and maternal regulation. Crop Science 36:301-306. Davies, (1987). The plant hormones: their nature, occurrences and function. P 1-12. In Davies and Nijhoff (ed.). Plant hormones and their role in plant growth and development. Dordrecht, the Netherlands. Engelen-Eigles, et al., (2000). DNA endoreduplication in maize endosperm cells: the effect of exposure to short-term high temperature. Plant, Cell and Environment 23:657-663.
Methods are therefore provided to increase the activity and/or level of IPT polypeptides in the developing inflorescence, thereby elevating cytokinin levels and allowing developing seed to achieve their full genetic potential for size, minimize seed abortion, and buffer seed set during unfavorable environments. The methods further allow the plant to maintain and/or improve the flowering process during unfavorable environments.
In this embodiment, a variety of promoters could be used to direct the expression of a sequence capable of increasing the level and/or activity of the IPT polypeptide, including but not limited to, constitutive promoters, seed-preferred promoters, developing seed or kernel promoters, meristem-preferred promoters, stress-induced promoters, and inflorescence-preferred (such as developing female inflorescence promoters). In one method, a promoter that is stress insensitive and is expressed in a tissue of the developing seed during the lag phase of development is used. By “insensitive to stress” is intended that the expression level of a sequence operably linked to the promoter is not altered or only minimally altered under stress conditions. By “lag phase” promoter is intended a promoter that is active in the lag phase of seed development. A description of this developmental phase is found elsewhere herein. By “developing seed-preferred” is intended a promoter that allows for enhanced IPT expression within a developing seed. Such promoters that are stress insensitive and are expressed in a tissue of the developing seed during the lag phase of development are known in the art and include Zag2.1 (Theissen, et al., (1995) Gene 156:155-166, Genbank Accession No. X80206), and mzE40 (Zm40) (U.S. Pat. No. 6,403,862 and WO01/2178).
An expression construct may further comprise nucleotide sequences encoding peptide signal sequences in order to effect changes in cytokinin level and/or activity in the mitochondria or chloroplasts. See, for example, Neupert, (1997) Annual Rev. Biochem. 66:863-917; Glaser, et al., (1998) Plant Molecular Biology 38:311-338; Duby, et al., (2001) The Plant J 27(6):539-549.
Methods to assay for an increase in seed set during abiotic stress are known in the art. For example, plants having the increased IPT activity can be monitored under various stress conditions and compared to control plants. For instance, the plant having the increased cytokinin synthesis activity can be subjected to various degrees of stress during flowering and seed set. Under identical conditions, the genetically modified plant having the increased cytokinin synthesis activity will have a higher number of developing kernels than a control plant.
Accordingly, the present invention further provides plants having increased yield or a maintained yield and/or an increased or maintained flowering process during periods of abiotic stress (drought, salt, heavy metals, temperature extremes, etc.). In some embodiments, the plants having an increased or maintained yield during abiotic stress have an increased level/activity of the IPT polypeptide of the invention. In some embodiments, the plant comprises an IPT nucleotide sequence of the invention operably linked to a promoter that drives expression in the plant cell. In some embodiments, such plants have stably incorporated into their genome a nucleic acid molecule comprising an IPT nucleotide sequence of the invention operably linked to a promoter that drives expression in the plant cell.
VIII. Methods of Use for IPT promoter Polynucleotides
The polynucleotides comprising the IPT promoters disclosed in the present invention, as well as variants and fragments thereof, are useful in the genetic manipulation of any host cell, preferably plant cell, when assembled with a DNA construct such that the promoter sequence is operably linked to a nucleotide sequence comprising a polynucleotide of interest. In this manner, the IPT promoter polynucleotides of the invention are provided in expression cassettes along with a heterologous polynucleotide sequence of interest for expression in the host cell of interest. As discussed in Example 2 below, the IPT promoter sequences of the invention are expressed in a variety of tissues and thus the promoter sequences can find use in regulating the temporal and/or the spatial expression of polynucleotides of interest.
Synthetic hybrid promoter regions are known in the art. Such regions comprise upstream promoter elements of one polynucleotide operably linked to the promoter element of another polynucleotide. In an embodiment of the invention, heterologous sequence expression is controlled by a synthetic hybrid promoter comprising the IPT promoter sequences of the invention, or a variant or fragment thereof, operably linked to upstream promoter element(s) from a heterologous promoter. Upstream promoter elements that are involved in the plant defense system have been identified and may be used to generate a synthetic promoter. See, for example, Rushton, et al., (1998) Curr. Opin. Plant Biol. 1:311-315. Alternatively, a synthetic IPT promoter sequence may comprise duplications of the upstream promoter elements found within the IPT promoter sequences.
It is recognized that a promoter sequence of the invention may be used with its native IPT coding sequence. A DNA construct comprising an IPT promoter operably linked with its native IPT gene may be used to transform any plant of interest to bring about a desired phenotypic change, such as modulating cytokinin levels, modulating root, shoot, leaf, floral and embryo development, stress tolerance, and any other phenotype described elsewhere herein.
The promoter nucleotide sequences and methods disclosed herein are useful in regulating expression of any heterologous nucleotide sequence in a host plant in order to vary the phenotype of a plant. Various changes in phenotype are of interest including modifying the fatty acid composition in a plant, altering the amino acid content of a plant, altering a plant's pathogen defense mechanism, and the like. These results can be achieved by providing expression of heterologous products or increased expression of endogenous products in plants. Alternatively, the results can be achieved by providing for a reduction of expression of one or more endogenous products, particularly enzymes or cofactors in the plant. These changes result in a change in phenotype of the transformed plant.
Genes of interest are reflective of the commercial markets and interests of those involved in the development of the crop. Crops and markets of interest change, and as developing nations open up world markets, new crops and technologies will emerge also. In addition, as our understanding of agronomic traits and characteristics such as yield and heterosis increase, the choice of genes for transformation will change accordingly. General categories of genes of interest include, for example, those genes involved in information, such as zinc fingers, those involved in communication, such as kinases, and those involved in housekeeping, such as heat shock proteins. More specific categories of transgenes, for example, include genes encoding important traits for agronomics, insect resistance, disease resistance, herbicide resistance, sterility, grain characteristics, and commercial products. Genes of interest include, generally, those involved in oil, starch, carbohydrate, or nutrient metabolism as well as those affecting kernel size, sucrose loading, and the like.
In one embodiment, sequences of interest improve plant growth and/or crop yields. In more specific embodiments, expression of the nucleotide sequence of interest improves the plant's response to stress induced under high density growth conditions. For example, sequences of interest include agronomically important genes that result in improved primary or lateral root systems. Such genes include, but are not limited to, nutrient/water transporters and growth inducers. Examples of such genes, include but are not limited to, maize plasma membrane H+-ATPase (MHA2) (Frias, et al., (1996) Plant Cell 8:1533-44); AKT1, a component of the potassium uptake apparatus in Arabidopisis, (Spalding, et al., (1999) J Gen Physiol 113:909-18); RML genes which activate cell division cycle in the root apical cells (Cheng, et al., (1995) Plant Physiol 108:881); maize glutamine synthetase genes (Sukanya, et al., (1994) Plant Mol Biol 26:1935-46) and hemoglobin (Duff, et al., (1997) J. Biol. Chem 27:16749-16752, Arredondo-Peter, et al., (1997) Plant Physiol. 115:1259-1266; Arredondo-Peter, et al., (1997) Plant Physiol 114:493-500 and references sited therein). The sequence of interest may also be useful in expressing antisense nucleotide sequences of genes that negatively affect root development.
Additional, agronomically important traits such as oil, starch, and protein content can be genetically altered in addition to using traditional breeding methods. Modifications include increasing content of oleic acid, changing the proportions of saturated and unsaturated oils, increasing levels of lysine and sulfur, providing essential amino acids, and also modification of starch. Hordothionin protein modifications are described in U.S. Pat. Nos. 5,703,049, 5,885,801, 5,885,802 and 5,990,389, herein incorporated by reference. Another example is lysine and/or sulfur rich seed protein encoded by the soybean 2S albumin described in U.S. Pat. No. 5,850,016, and the chymotrypsin inhibitor from barley, described in Williamson, et al., (1987) Eur. J. Biochem. 165:99-106, the disclosures of which are herein incorporated by reference.
Derivatives of the coding sequences can be made by site-directed mutagenesis to increase the level of preselected amino acids in the encoded polypeptide. For example, the gene encoding the barley high lysine polypeptide (BHL) is derived from barley chymotrypsin inhibitor, U.S. patent application Ser. No. 08/740,682, filed Nov. 1, 1996, and WO 98/20133, the disclosures of which are herein incorporated by reference. Other proteins include methionine-rich plant proteins such as from sunflower seed (Lilley, et al., (1989) Proceedings of the World Congress on Vegetable Protein Utilization in Human Foods and Animal Feedstuffs, ed. Applewhite (American Oil Chemists Society, Champaign, Ill.), pp. 497-502; herein incorporated by reference); corn (Pedersen, et al., (1986) J. Biol. Chem. 261:6279; Kirihara, et al., (1988) Gene 71:359; both of which are herein incorporated by reference); and rice (Musumura, et al., (1989) Plant Mol. Biol. 12:123, herein incorporated by reference). Other agronomically important genes encode latex, Floury 2, growth factors, seed storage factors, and transcription factors.
Insect resistance genes may encode resistance to pests that have great yield drag such as rootworm, cutworm, European Corn Borer, and the like. Such genes include, for example, Bacillus thuringiensis toxic protein genes (U.S. Pat. Nos. 5,366,892; 5,747,450; 5,736,514; 5,723,756; 5,593,881 and Geiser, et al., (1986) Gene 48:109); and the like.
Genes encoding disease resistance traits include detoxification genes, such as against fumonosin (U.S. Pat. No. 5,792,931); avirulence (avr) and disease resistance (R) genes (Jones, et al., (1994) Science 266:789; Martin, et al., (1993) Science 262:1432; and Mindrinos, et al., (1994) Cell 78:1089); and the like.
Herbicide resistance traits may include genes coding for resistance to herbicides that act to inhibit the action of acetolactate synthase (ALS), in particular the sulfonylurea-type herbicides (e.g., the acetolactate synthase (ALS) gene containing mutations leading to such resistance, in particular the S4 and/or Hra mutations), genes coding for resistance to herbicides that act to inhibit action of glutamine synthase, such as phosphinothricin or basta (e.g., the bar gene), or other such genes known in the art. The bar gene encodes resistance to the herbicide basta, the nptII gene encodes resistance to the antibiotics kanamycin and geneticin, and the ALS-gene mutants encode resistance to the herbicide chlorsulfuron.
Sterility genes can also be encoded in an expression cassette and provide an alternative to physical emasculation. Examples of genes used in such ways include male tissue-preferred genes and genes with male sterility phenotypes such as DAM, described in U.S. Pat. Nos. 5,750,868; 5,689,051 and 6,281,348. Other genes include kinases and those encoding compounds toxic to either male or female gametophytic development.
The quality of grain is reflected in traits such as levels and types of oils, saturated and unsaturated, quality and quantity of essential amino acids, and levels of cellulose. In corn, modified hordothionin proteins are described in U.S. Pat. Nos. 5,703,049, 5,885,801, 5,885,802 and 5,990,389.
Commercial traits can also be encoded on a gene or genes that could increase for example, starch for ethanol production, or provide expression of proteins. Another important commercial use of transformed plants is the production of polymers and bioplastics such as described in U.S. Pat. No. 5,602,321. Genes such as β-Ketothiolase, PHBase (polyhydroxyburyrate synthase), and acetoacetyl-CoA reductase (see, Schubert, et al., (1988) J. Bacteriol. 170:5837-5847) facilitate expression of polyhyroxyalkanoates (PHAs).
Exogenous products include plant enzymes and products as well as those from other sources including prokaryotes and other eukaryotes. Such products include enzymes, cofactors, hormones, and the like. The level of proteins, particularly modified proteins having improved amino acid distribution to improve the nutrient value of the plant, can be increased. This is achieved by the expression of such proteins having enhanced amino acid content.
IX. Antibody Creation and Use
Antibodies can be raised to a protein of the present invention, including variants and fragments thereof, in both their naturally-occurring and recombinant forms. Many methods of making antibodies are known to persons of skill. A variety of analytic methods are available to generate a hydrophilicity profile of a protein of the present invention. Such methods can be used to guide the artisan in the selection of peptides of the present invention for use in the generation or selection of antibodies which are specifically reactive, under immunogenic conditions, to a protein of the present invention. See, e.g., Janin, (1979) Nature 277:491-492; Wolfenden, et al., (1981) Biochemistry 20:849-855; Kyte and Doolite, (1982) J. Mol Biol. 157:105-132; Rose, et al., (1985) Science 229:834-838. The antibodies can be used to screen expression libraries for particular expression products such as normal or abnormal protein, or altered levels of the same, which may be useful for detecting or diagnosing various conditions related to the presence of the respective antigens. Assays indicating high levels of an IPT protein of the invention, for example, could be useful in detecting plants, or specific plant parts, with elevated cytokinin levels. Usually the antibodies in such a procedure are labeled with a moiety which allows easy detection of presence of antigen/antibody binding.
The following discussion is presented as a general overview of the techniques available; however, one of skill will recognize that many variations upon the following methods are known.
A number of immunogens are used to produce antibodies specifically reactive with a protein of the present invention. Polypeptides encoded by isolated recombinant, synthetic, or native polynucleotides of the present invention are the preferred antigens for the production of monoclonal or polyclonal antibodies. Polypeptides of the present invention are optionally denatured, and optionally reduced, prior to injection into an animal capable of producing antibodies. Either monoclonal or polyclonal antibodies can be generated for subsequent use in immunoassays to measure the presence and quantity of the protein of the present invention. Methods of producing polyclonal antibodies are known to those of skill in the art. In brief, an antigen, preferably a purified protein, a protein coupled to an appropriate carrier (e.g., GST, keyhole limpet hemanocyanin, etc.), or a protein incorporated into an immunization vector such as a recombinant vaccinia virus (see, U.S. Pat. No. 4,722,848) is mixed with an adjuvant and animals are immunized with the mixture. The animal's immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to the protein of interest. When appropriately high titers of antibody to the immunogen are obtained, blood is collected from the animal and antisera are prepared. Specific monoclonal and polyclonal antibodies will usually have an antibody binding site with an affinity constant for its cognate monovalent antigen at least between 106-107, usually at least 108, 109, 1010 and up to about 1011 liters/mole. Further fractionation of the antisera to enrich for antibodies reactive to the protein is performed where desired (See, e.g., Coligan, Current Protocols in Immunology, Wiley/Greene, NY (1991); and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press, NY (1989)).
Antibodies, including binding fragments and single chain recombinant versions thereof, against predetermined fragments of a protein of the present invention are raised by immunizing animals, e.g., with conjugates of the fragments with carrier proteins as described above. Typically, the immunogen of interest is a protein of at least about 5 amino acids, more typically the protein is 10 amino acids in length, often 15 to 20 amino acids in length, and may be longer. The peptides are typically coupled to a carrier protein (e.g., as a fusion protein), or are recombinantly expressed in an immunization vector. Antigenic determinants on peptides to which antibodies bind are typically 3 to 10 amino acids in length.
Monoclonal antibodies are prepared from hybrid cells secreting the desired antibody. Monoclonal antibodies are screened for binding to a protein from which the antigen was derived. Description of techniques for preparing such monoclonal antibodies are found in, e.g., Basic and Clinical Immunology, 4th ed., Stites, et al., Eds., Lange Medical Publications, Los Altos, Calif., and references cited therein; Harlow and Lane, Supra; Goding, Monoclonal Antibodies: Principles and Practice, 2nd ed., Academic Press, New York, N.Y. (1986) and Kohler and Milstein, (1975) Nature 256:495-497. Summarized briefly, this method proceeds by injecting an animal with an antigen comprising a protein of the present invention. The animal is then sacrificed and cells taken from its spleen, which are fused with myeloma cells. The result is a hybrid cell or “hybridoma” that is capable of reproducing in vitro. The population of hybridomas is then screened to isolate individual clones, each of which secretes a single antibody species to the antigen. In this manner, the individual antibody species obtained are the products of immortalized and cloned single B cells generated by the animal in response to a specific site recognized on the antigenic substance.
Other suitable techniques involve selection of libraries of recombinant antibodies in phage or similar vectors (see, e.g., Huse, et al., (1989) Science 246:1275-1281; and Ward, et al., (1989) Nature 341:544-546; and Vaughan, et al., (1996) Nature Biotechnology 14:309-314). Also, recombinant immunoglobulins may be produced. See, Cabilly, U.S. Pat. No. 4,816,567 and Queen, et al., (1989) Proc. Nat'l Acad. Sci. 86:10029-10033.
Antibodies to the polypeptides of the invention are also used for affinity chromatography in isolating proteins of the present invention. Columns are prepared, e.g., with the antibodies linked to a solid support, e.g., particles, such as agarose, SEPHADEX, or the like, where a cell lysate is passed through the column, washed, and treated with increasing concentrations of a mild denaturant, whereby purified proteins are released.
Frequently, the proteins and antibodies of the present invention will be labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal. A wide variety of labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionucleotides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles and the like.
Protein Immunoassays
Means of detecting the proteins of the present invention are not critical aspects of the present invention. In certain examples, the proteins are detected and/or quantified using any of a number of well-recognized immunological binding assays (see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288 and 4,837,168). For a general review of immunoassays, see also, Methods in Cell Biology, Vol. 37: Antibodies in Cell Biology, Asai, Ed., Academic Press, Inc. New York (1993); Basic and Clinical Immunology 7th Edition, Stites & Terr, Eds. (1991). Moreover, the immunoassays of the present invention can be performed in any of several configurations, e.g., those reviewed in Enzyme Immunoassay, Maggio, Ed., CRC Press, Boca Raton, Fla. (1980); Tijan, Practice and Theory of Enzyme Immunoassays, Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers B.V., Amsterdam (1985); Harlow and Lane, supra; Immunoassay: A Practical Guide, Chan, Ed., Academic Press, Orlando, Fla. (1987); Principles and Practice of Immunoassaysm, Price and Newman Eds., Stockton Press, NY (1991); and Non-isotopic Immunoassays, Ngo, Ed., Plenum Press, NY (1988).
Immunological binding assays (or immunoassays) typically utilize a “capture agent” to specifically bind to and often immobilize the analyte (in this case, a protein of the present invention). The capture agent is a moiety that specifically binds to the analyte. In certain embodiments, the capture agent is an antibody that specifically binds a protein of the present invention. The antibody may be produced by any of a number of means known to those of skill in the art as described herein.
Immunoassays also often utilize a labeling agent to specifically bind to and label the binding complex formed by the capture agent and the analyte. The labeling agent may itself be one of the moieties comprising the antibody/analyte complex. Thus, the labeling agent may be a labeled protein of the present invention or a labeled antibody specifically reactive to a protein of the present invention. Alternatively, the labeling agent may be a third moiety, such as another antibody, that specifically binds to the antibody/protein complex.
Throughout the assays, incubation and/or washing steps may be required after each combination of reagents. Incubation steps can vary from about 5 seconds to several hours, often from about 5 minutes to about 24 hours. However, the incubation time will depend upon the assay format, analyte, volume of solution, concentrations and the like. Usually, the assays will be carried out at ambient temperature, although they can be conducted over a range of temperatures, such as 10° C. to 40° C.
While the details of the immunoassays of the present invention may vary with the particular format employed, the method of detecting a protein of the present invention in a biological sample generally comprises the steps of contacting the biological sample with an antibody which specifically reacts, under immunologically reactive conditions, to a protein of the present invention. The antibody is allowed to bind to the protein under immunologically reactive conditions, and the presence of the bound antibody is detected directly or indirectly.
A. Non-Competitive Assay Formats
Immunoassays for detecting proteins of the present invention include competitive and noncompetitive formats. Noncompetitive immunoassays are assays in which the amount of captured analyte (i.e., a protein of the present invention) is directly measured. In one example, the “sandwich” assay, the capture agent (e.g., an antibody specifically reactive, under immunoreactive conditions, to a protein of the present invention) can be bound directly to a solid substrate where it is immobilized. These immobilized antibodies then capture the protein present in the test sample. The protein thus immobilized is then bound by a labeling agent, such as a second antibody bearing a label. Alternatively, the second antibody may lack a label, but it may, in turn, be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived. The second antibody can be modified with a detectable moiety, such as biotin, to which a third labeled molecule can specifically bind, such as enzyme-labeled streptavidin.
B. Competitive Assay Formats
In competitive assays, the amount of analyte present in the sample is measured indirectly by measuring the amount of an added (exogenous) analyte (e.g., a protein of the present invention) displaced (or competed away) from a capture agent (e.g., an antibody specifically reactive, under immunoreactive conditions, to the protein) by the analyte present in the sample. In one competitive assay, a known amount of analyte is added to the sample and the sample is then contacted with a capture agent that specifically binds a protein of the present invention. The amount of protein bound to the capture agent is inversely proportional to the concentration of analyte present in the sample.
In one embodiment, the antibody is immobilized on a solid substrate. The amount of protein bound to the antibody may be determined either by measuring the amount of protein present in a protein/antibody complex, or alternatively by measuring the amount of remaining uncomplexed protein. The amount of protein may be detected by providing a labeled protein.
A hapten inhibition assay is another competitive assay. In this assay a known analyte, such as a protein of the present invention, is immobilized on a solid substrate. A known amount of antibody specifically reactive, under immunoreactive conditions, to the protein is added to the sample, and the sample is then contacted with the immobilized protein. In this case, the amount of antibody bound to the immobilized protein is inversely proportional to the amount of protein present in the sample. Again, the amount of immobilized antibody may be determined by detecting either the immobilized fraction of antibody or the fraction of the antibody that remains in solution. Detection may be direct, where the antibody is labeled, or indirect, by the subsequent addition of a labeled moiety that specifically binds to the antibody, as described above.
C. Generation of Pooled Antisera for Use in Immunoassays
A protein that specifically binds to, or that is specifically immunoreactive with, an antibody generated against a defined antigen is determined in an immunoassay. The immunoassay uses a polyclonal antiserum which is raised to a polypeptide of the present invention (i.e., the antigenic polypeptide). This antiserum is selected to have low cross-reactivity against other proteins, and any such cross-reactivity is removed by immunoabsorbtion prior to use in the immunoassay (e.g., by immunosorbtion of the antisera with a protein of different substrate specificity (e.g., a different enzyme) and/or a protein with the same substrate specificity but of a different form).
In order to produce antisera for use in an immunoassay, a polypeptide of the present invention is isolated as described herein. For example, recombinant protein can be produced in a mammalian or other eukaryotic cell line. An inbred strain of mice is immunized with the protein using a standard adjuvant, such as Freund's adjuvant, and a standard mouse immunization protocol (see, Harlow and Lane, supra). Alternatively, a synthetic polypeptide derived from the sequences disclosed herein and conjugated to a carrier protein is used as an immunogen. Polyclonal sera are collected and titered against the immunogenic polypeptide in an immunoassay, for example, a solid phase immunoassay with the immunogen immobilized on a solid support. Polyclonal antisera with a titer of 104 or greater are selected and tested for their cross reactivity against polypeptides of different forms or substrate specificity, using a competitive binding immunoassay such as the one described in Harlow and Lane, supra, at pages 570-573. Preferably, two or more distinct forms of polypeptides are used in this determination. These distinct types of polypeptides are used as competitors to identify antibodies which are specifically bound by the polypeptide being assayed for. The competitive polypeptides can be produced as recombinant proteins and isolated using standard molecular biology and protein chemistry techniques as described herein.
Immunoassays in the competitive binding format are used for cross-reactivity determinations. For example, the immunogenic polypeptide is immobilized to a solid support. Proteins added to the assay compete with the binding of the antisera to the immobilized antigen. The ability of the above proteins to compete with the binding of the antisera to the immobilized protein is compared to the immunogenic polypeptide. The percent cross-reactivity for the above proteins is calculated, using standard methods. Those antisera with less than 10% cross-reactivity for a distinct form of a polypeptide are selected and pooled. The cross-reacting antibodies are then removed from the pooled antisera by immunoabsorbtion with a distinct form of a polypeptide.
The immunoabsorbed and pooled antisera are then used in a competitive binding immunoassay as described herein to compare a second “target” polypeptide to the immunogenic polypeptide. In order to make this comparison, the two polypeptides are each assayed at a wide range of concentrations and the amount of each polypeptide required to inhibit 50% of the binding of the antisera to the immobilized protein is determined using standard techniques. If the amount of the target polypeptide required is less than twice the amount of the immunogenic polypeptide that is required, then the target polypeptide is said to specifically bind to an antibody generated to the immunogenic protein. As a final determination of specificity, the pooled antisera is fully immunosorbed with the immunogenic polypeptide until no binding to the polypeptide used in the immunosorbtion is detectable. The fully immunosorbed antisera is then tested for reactivity with the test polypeptide. If no reactivity is observed, then the test polypeptide is specifically bound by the antisera elicited by the immunogenic protein.
D. Other Assay Formats
In certain embodiments, Western blot (immunoblot) analysis is used to detect and quantify the presence of protein of the present invention in the sample. The technique generally comprises separating sample proteins by gel electrophoresis on the basis of molecular weight, transferring the separated proteins to a suitable solid support, (such as a nitrocellulose filter, a nylon filter, or derivatized nylon filter), and incubating the sample with the antibodies that specifically bind a protein of the present invention. The antibodies specifically bind to the protein on the solid support. These antibodies may be directly labeled, or may be subsequently detected using labeled antibodies (e.g., labeled sheep anti-mouse antibodies) that specifically bind to the antibodies.
E. Quantification of Proteins.
The proteins of the present invention may be detected and quantified by any of a number of means well known to those of skill in the art. These include analytic biochemical methods such as electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like, and various immunological methods such as fluid or gel precipitin reactions, immunodiffusion (single or double), immunoelectrophoresis, radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, and the like.
F. Reduction of Non-Specific Binding
One of skill will appreciate that it is often desirable to reduce non-specific binding in immunoassays and during analyte purification. Where the assay involves an antigen, antibody, or other capture agent immobilized on a solid substrate, it is desirable to minimize the amount of non-specific binding to the substrate. Means of reducing such non-specific binding are well known to those of skill in the art. Typically, this involves coating the substrate with a proteinaceous composition. In particular, protein compositions such as bovine serum albumin (BSA), nonfat powdered milk, and gelatin are widely used.
G. Immunoassay Labels
The labeling agent can be, e.g., a monoclonal antibody, a polyclonal antibody, a binding protein or complex, or a polymer such as an affinity matrix, carbohydrate or lipid. Detectable labels suitable for use in the present invention include any composition detectable by spectroscopic, radioisotopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Detection may proceed by any known method, such as immunoblotting, Western analysis, gel-mobility shift assays, fluorescent in situ hybridization analysis (FISH), tracking of radioactive or bioluminescent markers, nuclear magnetic resonance, electron paramagnetic resonance, stopped-flow spectroscopy, column chromatography, capillary electrophoresis, or other methods which track a molecule based upon an alteration in size and/or charge. The particular label or detectable group used in the assay is not a critical aspect of the invention. The detectable group can be any material having a detectable physical or chemical property, including magnetic beads, fluorescent dyes, radiolabels, enzymes, and colorimetric labels or colored glass or plastic beads, as discussed for nucleic acid labels, supra. The label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art. As indicated above, a wide variety of labels may be used, with the choice of label depending on the sensitivity required, ease of conjugation of the compound, stability requirements, available instrumentation, and disposal provisions. Means of detecting labels are well known to those of skill in the art.
Non-radioactive labels are often attached by indirect means. Generally, a ligand molecule (e.g., biotin) is covalently bound to the molecule. The ligand then binds to an anti-ligand (e.g., streptavidin) molecule which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound. A number of ligands and anti-ligands can be used.
The molecules can also be conjugated directly to signal-generating compounds, e.g., by conjugation with an enzyme or fluorophore. Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidoreductases, particularly peroxidases. Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc. Chemiluminescent compounds include luciferin, and 2,3-dihydrophthalazinediones, e.g., luminol. For a review of various labeling or signal-producing systems which may be used, see, U.S. Pat. No. 4,391,904, which is incorporated herein by reference.
Some assay formats do not require the use of labeled components. For instance, agglutination assays can be used to detect the presence of the target antibodies. In this case, antigen-coated particles are agglutinated by samples comprising the target antibodies. In this format, none of the components need be labeled and the presence of the target antibody is detected by simple visual inspection.
Assays for Compounds that Modulate Enzymatic Activity or Expression
A catalytically active polypeptide of the present invention may be contacted with a compound in order to determine whether said compound binds to and/or modulates the enzymatic activity of such polypeptide. The polypeptide employed will have at least 20%, 30%, 40%, 50% 60%, 70% or 80% of the specific activity of the native, full-length enzyme of the present invention. Generally, the polypeptide will be present in a range sufficient to determine the effect of the compound, typically about 1 nM to 10 μM. Likewise, the compound being tested will be present in a concentration of from about 1 nM to 10 μM. Those of skill will understand that such factors as enzyme concentration, ligand concentrations (i.e., substrates, products, inhibitors, activators), pH, ionic strength, and temperature will be controlled so as to obtain useful kinetic data and determine the presence or absence of a compound that binds or modulates polypeptide activity. Methods of measuring enzyme kinetics are well known in the art. See, e.g., Segel, Biochemical Calculations, 2nd ed., John Wiley and Sons, New York (1976).
Embodiments of the invention include, but are not limited to, an isolated polypeptide comprising an amino acid sequence of SEQ ID NO:2, 6, 9, 12, 15, 18, 23, 27, 41, 43, 46, 49, 52, 54, 57, 59, 61, 63, 66 or 77; an isolated polynucleotide comprising a nucleotide sequence of SEQ ID NO:1, 3, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 21, 22, 24, 26, 28, 40, 42, 44, 45, 47, 48, 50, 51, 53, 55, 56, 58, 60, 62, 64, 65, 69, 70, 71, 72, 73, 74 or 76; and expression cassettes, vectors and transformed plants comprising same. Further embodiments include, but are not limited to, a plant that is genetically modified at a native genomic locus, said genomic locus encoding a polypeptide of SEQ ID NO: 2, 6, 9, 12, 15, 18, 23, 27, 41, 43, 46, 49, 52, 54, 57, 59, 61, 63, 66 or 77; and methods for modulating expression of a polynucleotide of SEQ ID NO: 1, 3, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 21, 22, 24, 26, 28, 40, 42, 44, 45, 47, 48, 50, 51, 53, 55, 56, 58, 60, 62, 64, 65, 69, 70, 71, 72, 73, 74 or 76. Further embodiments include, but are not limited to, an isolated polynucleotide comprising a nucleotide sequence of SEQ ID NO: 25 or 75; and compositions and methods of use comprising a nucleotide sequence of SEQ ID NO: 25 or 75.
The following examples are offered by way of illustration and not by way of limitation.
EXPERIMENTAL
Example 1
Cloning and Gene Characterization of ZmIPT1
Below we describe the identification and characterization of an IPT polypeptide from maize designated ZmIPT1.
Material and Methods:
A Mo17 BAC library was screened using a 3′-end fragment of the ZmIPT1 cDNA from B73, identified by sequence similarity to Agrobacterium ipt. One of the positive clones was digested by HindIII, and subcloned into pBluescript. Recombinant plasmids were screened using colony screening and a 3′-end fragment as a probe. One positive clone was sequenced. Samples used for RT-PCR were harvested in the field from three individual plants. Five μg of total RNA was used for RT-PCR using ThermoScript RT-PCR System from Invitrogen. The reverse transcribed mixture for PCR used primers designed across an intron-exon-intron junction in order to avoid amplification of genomic DNA.
Results:
A. Deduced Protein Sequence:
Two putative maize ipt ESTs were identified whose deduced amino acid sequences show similarity to the Agrobacterium (data not shown), Arabidopsis and Petunia IPT proteins (FIG. 1). Full-insert sequencing of these two ESTs revealed that they were identical and the corresponding cDNA sequence was called ZmIPT1 (SEQ ID NO: 22).
FIG. 1 provides an amino acid alignment of the ZmIPT1, Arabidopsis, and Petunia cytokinin biosynthetic enzymes. Asterisks indicate amino acids conserved in many cytokinin biosynthetic enzymes. The amino acids designated by the underline indicate a putative ATP/GTP binding site (at about amino acids 84-90). As shown in FIG. 1, the deduced protein sequence of ZmIPT1 contains the exact consensus sequence GxTxxGK[ST]xxxxx[VLI]xxxxxxx[VLI][VLI]xxDxxQx{57,60}[VLI][VLI]xGG[ST] (SEQ ID NO: 32) (where x denotes any amino acid residue, [ ] any one of the amino acids shown in [ ], and x{m,n} m to n amino acid residues in number) that was used by Takei, et al., (2001) J. Biol. Chem. 276:26405-26410 to isolate the Arabidopsis genes. Note that ZmIPT1 also has a putative ATP/GTP binding site at about amino acids 51-58. In addition, the length of ZmIPT1 is very similar to the AtIPT4 and Sho genes. In addition, the specific zinc-finger like motif (CxxCx{12,18}HxxxxxH) (SEQ ID NO:33) found in all tRNA IPTs of eukaryotes (which is necessary to bind tRNA molecules) is absent from ZmIPT1.
The ZmIPT1 sequence shares 21.9% amino acid sequence identity (34.1% similarity) across the full length to Sho (cytokinin biosynthetic protein from Petunia); 10.8% identity (21.2% similarity) across its full length to ipt (Agrobacterium); 24.7% identity (34.8% similarity) across its full length to AtIPT1 (Arabidopsis); 35.6% identity (45.3% similarity) across its full length to AtIPT2 (Arabidopsis); 22.4% identity (34.6% similarity) across its full length to AtIPT3 (Arabidopsis); 20.7% identity (31.6% similarity) across its full length to AtIPT4 (Arabidopsis); 22.7% identity (35.7% similarity) across its full length to AtIPT5 (Arabidopsis); 21.8% identity (36.4% similarity) across its full length to AtIPT6 (Arabidopsis); 23.4% identity (33.1% similarity) across its full length to AtIPT7 (Arabidopsis); 26.3% identity (35.9% similarity) across its full length to AtIPT8 (Arabidopsis); and, 18.9% identity (31.2% similarity) across its full length to AtIPT9 (Arabidopsis).
A variant of the ZmIPT1 sequence is also provided. SEQ ID NOS: 22, 23 and 24 correspond to the nucleotide and amino acid sequence of ZmIPT1 derived from the spliced sequence of the Mo17 genomic clone. SEQ ID NOS: 26, 27 and 28 are variants of the ZmIPT1 sequence derived from sequencing the full-length EST from B73. An alignment of ZmIPT1 and its variant is shown in FIG. 3. These sequences share 98% overall amino acid sequence identity.
B. Gene Structure:
A Mo17 BAC library was screened using probes corresponding to the two ESTs and four identical clones were identified. An 11 kb HindIII fragment from one of the clones was subcloned in pBluescript and sequenced. Alignment with the full-insert sequence of the EST clone revealed the presence of six introns. Interestingly neither the AtIPT4 gene from Arabidopsis nor the Sho gene from Petunia contain introns. The genomic sequence of ZmIPT1 is set forth in SEQ ID NO: 21.
Example 2
Gene Expression of ZmIPT1
One of the identified ZmIPT1 ESTs was from a B73 embryo library, the other one was from a developing root library. In order to gain an impression of the level of expression of ZmIPT1, a search of the Lynx database was performed. A perfect tag was found in the 3′-end of the gene, 231 bp from the poly A tail start. Tissue types, number of library hits, and average ppm are presented in Table 1. Expression was found to be very low in most organs, but higher in seedling and embryo libraries. In embryo libraries, expression was higher at 10 DAP than at later development stages (FIG. 4).







TABLE 1







Number of Lynx libraries containing the


ZmIPT1 tag and average ppm values.











Tissue
# of Lynx
Average



type
libraries
ppm















Seedling
1
29



Ear
5
4



Endosperm
2
8



Embryo
4
10.75



Stalk
1
9



Leaf
2
3



Root
2
2.5










Using RT-PCR, the expression pattern of ZmIPT in various maize organs and on a kernel developmental series was tested. No amplification product could be detected after 20 cycles, confirming the very low expression of the gene. However, after 30 cycles, bands of the appropriate size were amplified (FIG. 5). FIG. 5A shows ZmIPT1 transcripts are present in ovaries of mature plants and leaf, mesocotyl, and roots of seedlings. This distribution indicates a bias in expression of this gene to meristematic-like and rapidly developing tissues. In developing B73 kernels (FIG. 5B), ZmIPT1 transcript is strongly present from 0 to 10 DAP, then decreases beyond 15 DAP. This pattern of expression of ZmIPT1 correlates with the known profile of cytokinin accumulation in developing kernels, which peaks during the lag phase. This accumulation of cytokinin is thought to drive early cell division in endosperm and embryo development.
In a similar manner, the ZmIPT1 tag could only be detected in the cell division zone of leaves, and in leaf discs treated with BA. This distribution of transcripts indicates a bias in expression of ZmIPT1 to meristematic-like and rapidly developing tissues, indicating that maize roots and developing kernels are strong sites for cytokinin synthesis.
Example 3
Isolation and Gene Characterization of ZmIPT2, ZmIPT4, ZmIPT5, ZmIPT6, ZmIPT7, ZmIPT8 and ZmIPT9
The AtIPT1 and AtIPT3 to AtIPT8 protein sequences were blasted against the six possible frames generated by the maize genomic sequences and searched for some degree of similarity. Because rice and maize genomes show a significant degree of synteny, the same method was used against rice genomic database to optimize this search. The rice sequences with an E-score of at least 200 were then used for an additional screen of the GSS maize database. Since at that time, the GSS database had not been assembled into contigs, the sequences obtained which had an E-score of at least 150 were pooled and aligned using Sequencher.
Using this method, eight maize contigs encoding putative CK biosynthetic enzymes were identified (ZmIPT2 to ZmIPT9), six of them showing an open reading frame without introns. The translated proteins corresponding to these putative genes contained 320 to 380 amino acids, which correlates with the expected size for plant IPT proteins. An alignment of the corresponding proteins is presented in FIG. 1. The deduced protein sequences of the new ZmIPT genes (except for ZmIPT8) contain the exact consensus sequence found in IPT proteins from different species. This sequence, GxTxxGK[ST]xxxxx[VLI]xxxxxxx[VLI][VLI]xxDxxQx{57,60}[VLI][VLI]xGG[ST] (where x denotes any amino acid residue, [ ] anyone of the amino acids shown in [ ], and x{m,n} m to n amino acid residues in number) (SEQ ID NO: 32) is also found in ZmIPT1 and was previously used by Kakimoto and Takei to isolate the Arabidopsis IPT genes. Homology with other plant IPT proteins was found to be around 40%.
The amino acid sequence identity and similarity to top BLAST hits across the full length of ZmIPT2, ZmIPT4, ZmIPT5, ZmIPT6, ZmIPT7, ZmIPT8 and ZmIPT9 are provided below in Table 2.












TABLE 2









34394150 (rice)

AtIPT5 (Arabidopsis)














Gene
Similarity
Identity
Similarity
Identity







ZmIPT2
55.15
46.51
53.02
45.30



ZmIPT4
74.05
69.68
58.70
53.73



ZmIPT5
71.99
65.96
60.70
55.59



ZmIPT6
71.86
64.67
59.62
53.53



ZmIPT7
58.63
50.16
59.36
51.94



ZmIPT8
54.71
47.72
46.73
38.89










The maize IPT sequences also have putative ATP/GTP binding sites at about amino acids 17-24 for ZmIPT2, about amino acids 72-79 for ZmIPT4, about amino acids 57-64 for ZmIPT5, about amino acids 55-62 for ZmIPT6, about amino acids 23-30 for ZmIPT7 and about amino acids 83-90 for ZmIPT8.
The polypeptides encoded by ZmIPT polynucleotides share sequence similarity to known proteins. For example, a polypeptide encoded by nucleotides 821 to 3 of ZmIPT9 shares 55% amino acid sequence identity to amino acids 48 to 327 of a tRNA isopentenyltransf erase from Arabidopsis thaliana (GenBank Accession Number BAB59048.1). A polypeptide encoded by nucleotides 821 to 3 of ZmIPT9 shares 55% amino acid sequence identity to amino acids 48 to 327 of a putative IPP transferase from Arabidopsis thaliana (GenBank Accession Number AAK64114.1). A polypeptide encoded by nucleotides 821 to 3 of ZmIPT9 shares 55% amino acid sequence identity to amino acids 48 to 327 of a IPP transferase-like protein from Arabidopsis thaliana (GenBank Accession Number AAM63091.1). A polypeptide encoded by nucleotides 839 to 3 of ZmIPT9 shares 36% amino acid sequence identity to amino acids 28 to 278 of a putative tRNA delta-2-isopentenylpyrophosphate transferase from Arabidopsis thaliana (GenBank Accession Number YP_008242.1). A polypeptide encoded by nucleotides 824 to 3 of ZmIPT9 shares 35% amino acid sequence identity to amino acids 17 to 248 of a tRNA isopentenylpyrophosphate transferase from Streptococcus pneumoniae R6 (GenBank Accession Number NP_358182.1). A polypeptide encoded by nucleotides 818 to 3 of ZmIPT9 shares 34% amino acid sequence identity to amino acids 2 to 231 of a tRNA delta(2)-isopentenylpyrophosphate transferase (GenBank Accession Number Q8CWS7). A polypeptide encoded by nucleotides 818 to 3 of ZmIPT9 shares 34% amino acid sequence identity to amino acids 2 to 231 of a tRNA isopentenylpyrophosphate transferase from Streptococcus pneumoniae R6 (GenBank Accession Number NP_345176.1). A polypeptide encoded by nucleotides 818 to 3 of ZmIPT9 shares 34% amino acid sequence identity to amino acids 31 to 275 of a tRNA delta(2)-isopentenylpyrophosphate transferase from Chlamydophila caviae (GenBank Accession Number AAP05599.1). A polypeptide encoded by nucleotides 818 to 435 of ZmIPT9 shares 48% amino acid sequence identity to amino acids 6 to 133 of a tRNA delta(2)-isopentenylpyrophosphate transferase from Xylella fastidiosa 9a5c (GenBank Accession Number NP_297383.1). A polypeptide encoded by nucleotides 818 to 435 of ZmIPT9 shares 48% amino acid sequence identity to amino acids 6 to 133 of a tRNA delta(2)-isopentenylpyrophosphate transferase from Xylella fastidiosa Dixon (GenBank Accession Number Xylella fastidiosa Dixon).
Example 4
Isolation of ZmIPT2 from Mo17 and B73 Maize Lines and Molecular Characterization of the ZmIPT2 Gene
Material and Methods:
Plant Materials:
Maize (Zea mays) varieties B73 and Mo17 were used in this study. Samples were harvested from field-grown plants at different stages of development and stored at −80° C. Kernel samples were harvested every five days from 0 to 25 DAP and dissected by isolating whole kernels (0 DAP), pedicel, nucellus and pericarp (5 DAP), pedicel, nucellus, endosperm/embryo sac and pericarp (10 DAP), or pedicel, embryo, endosperm and pericarp (15, 20 and 25 DAP). Tissues corresponding to 2 to 4 different ears were pooled. The series of sample harvested every DAP from 0 to 5 (whole kernels), from 6 to 15 and then 20, 27 and 34 DAP (seeds without pedicel) or 20, 25, 30 and 35 DAP (pedicels) were previously used to study the expression pattern of the cytokinin oxidase 1 gene (Ckx1) from corn (Brugière, et al., (2003) supra).
Arabidopsis thaliana ecotype Columbia was used for Arabidopsis transformation studies.
PCR:
ZmIPT2 coding sequence was PCR amplified from B73 and Mo17 genomic DNA. Primers ZmIPT2-5′ (5′-ATCATCAAGACAATGGAGCACGGTG-3′) (SEQ ID NO: 78) and ZmIPT2-3′ (5′-CGTCCGCTAGCTACTTATGCATCAG-3′) (SEQ ID NO: 79) were designed based on the GSS contig sequence (coding sequence is underlined). As part of the Gateway cloning procedure, att-flanked ZmIPT2 fragment was amplified using primers ZmIPT2-5-Gateway (5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTCAATGG-AGCACGGTGCCGTCGCCG-3′) (SEQ ID NO: 80) and ZmIPT2-3-Gateway (5′-GGGGACCACTTTGTACAA-GAAAGCTGGGTCTTATGCATCAGCCACGGCGGTG-3′) (SEQ ID NO: 81).
In each case, a touchdown PCR was performed (GeneAmp PCR System 9700), using the following cycling parameters: 94° C. for 2 min (one cycle), 94° C. for 30 s, 65° C. for 45 s and 72° C. for 1 min 30 s (5 cycles, annealing temperature reduced by 1° C. per cycle), 94° C. for 30 s, 60° C. for 45 s and 72° C. for 1 min 30 s (30 cycles), 72° C. for 7 min, and termination at 4° C. Pfu Ultra Hotstart DNA polymerase (Stratagene) for its very low average error rate (less than 0.5% per 500-bp fragment amplified) was used.
PCR products were loaded on an agarose gel containing ethidium bromide (1:10000, v/v). Bands corresponding to ZmIPT2 gene and att-flanked ZmIPT2 gene were gel extracted using QIAquick PCR purification kit (QIAgen).
DNA and RNA Extraction:
Genomic DNA was extracted from B73 and Mo17 plant samples at V3-4 stage according to Dellaporta, et al., (1983) Plant Mol Biol 1:19-21 and stored at −20° C. Total RNA was prepared using a hot phenol extraction procedure according to Verwoerd, et al., (1989) Nucleic Acid Res 17:2362 and stored at −80° C. The kernel developmental series samples were purified using RNeasy Mini Protocol for RNA Cleanup (QIAgen) and eluted in 50 μl DEPC water. Optical Density (DO) at 260 and 280 nm was used to assess the purity of RNA preps and measure RNA and DNA concentrations.
Southern Blots, Northern Blots, and Hybridization:
For Southern blots, digested genomic or BAC clones DNA were run on 0.8% agarose gel at 110V, stained after migration in a 1:10000 (v/v) ethidium bromide solution in TAE buffer, and transferred as indicated below. For Northern blots, ethidium bromide was added to denatured RNA samples and run at 80 V on 1.5% denaturing agarose gel (Brugière, et al., (2003) Plant Physiol. 132:1228-1240). Blotting was performed using Turbo-blotter (Schleicher and Schuell) according to the manufacturer guidelines. After transfer, nylon membranes (Nytran plus, Schleicher and Schuell) were cross-linked with a Stratalinker (Stratagene) and baked at 80° C. for 30 min. Probes were labeled with [α-32P]-dCTP using random priming (Rediprime II RandomPrime Labelling System, Amersham Biosciences) and purified with Quick Spin Columns (Roche). Hybridizations were carried out at 65° C. for 16 h using ExpressHyb hybridization solution (BD Biosciences) and membranes were washed under stringent conditions (0.1×SSC, 0.1% SDS) as previously described (Brugière, et al., (2003) Plant Physiol. 132:1228-1240). Relative transcript abundance was quantified using a phosphor imager (MD860, Molecular Dynamic) with imaging software (ImageQuant, Molecular Dynamics).
BAC Subcloning:
BAC clones were digested and subcloned in pBluescript SK+. This plasmid includes a multiple cloning site between the lacZ gene and its promoter. The lacZ gene is often used as a reporter gene because it encodes a β-galactosidase, which produces a dark blue precipitate on X-gal1 enzymatic hydrolysis. The bacteria containing a plasmid in which the BAC fragment is inserted in the multiple cloning site and therefore do not synthesize this enzyme will appear white. This allows the selection of colonies containing BAC subclones that can be further screened by PCR or Southern blot.
Results:
Isolation of the ZmIPT2 Gene from Corn Genomic DNA:
Genomic DNA from two different varieties of corn, B73 and Mo17, was extracted and ZmIPT2 coding sequence was amplified by touch-down PCR. The ZmIPT2 gene amplification product was used as a probe for Southern and Northern blot experiments. The ZmIPT2 CDS was cloned in pDONR221 (Invitrogen) and sequenced. The Mo17 sequence was 100% homologous to the GSS contig sequence. The B73 and Mo17 genes were found to be 98.8% homologous at the nucleotide level. The differences between the two genes at the nucleotide level resulted in the modification of 3 amino acids at the protein level (96.1% identity). The nucleotide and amino acid sequences of ZmIPT2 are set forth in SEQ ID NO:3 and 2, and the nucleotide and amino acid sequences of a variant of ZmIPT2 are set forth in SEQ ID NO:76 and 77. An alignment of ZmIPT 2 and the variant of ZmIPT2 shows that the differences in the polypeptides occur at amino acid 125 (A→L), amino acid 138 (Q→R), and amino acid 193 R→H.
Mapping of the Gene on the Maize Genome:
To determine if ZmIPT2 was a single- or multi-copy gene in maize, B73 and Mo17 genomic DNA were digested with HindIII, EcoRI, and EcoRV and run on a gel, which was blotted as described in the Materials and Methods section. The membrane was hybridized with the previously extracted ZmIPT2 genomic fragment as a probe. The picture of the membrane autoradiography is presented in FIG. 6.
The single bands observed for each of these digestions show that ZmIPT2 is most likely a single-copy gene. The size of the corresponding HindIII fragment was later confirmed using BAC clones. To obtain additional information regarding the physical location of the ZmIPT2 gene in the maize genome, the Oat-Maize chromosome addition lines were used (Ananiev, et al., (1997) Proc. Natl. Acad. Sci. 94:3524-3529). A PCR was performed with the ZmIPT2-5′ and ZmIPT2-3′ primers as described in the Materials and Methods section (data not shown). The expected size of the amplification fragment was 995 bp. B73 genomic DNA samples were used as positive controls, while oat genomic DNA and water were used as negative ones.
The data shows that the amplification of the ZmIPT2 sequence could only be seen with the chromosome 2 oat-maize addition line. This finding was verified and the position on chromosome 2 refined using a bioinformatics approach. The ZmIPT2 sequence was first used to screen the B73 and Mo17 public and proprietary Bacterial Artificial Chromosomes (BAC) libraries. Positive clones were identified and used to identify a BAC contig using FPC Contig Viewer. Using this strategy, markers were identified including several MZA markers that were physically mapped on maize chromosome 2, bin 4 (data not shown), which confirmed the experimental mapping of ZmIPT2 gene using the OMA lines.
Example 5
Gene Expression of ZmIPT2, ZmIPT4, ZmIPT5, ZmIPT6 and ZmIPT8
In order to gain an impression of the level of expression of the various ZmIPT sequences, a search of the Lynx database was performed. Tissue types, number of library hits, and average ppm are presented in Tables 3-7.
As shown in table 3, expression of ZmIPT2 was found to be restricted to kernel tissue and to correlate with the start of cytokinin biosynthesis in the kernel as described in Brugière, et al., (2003) Plant Physiol. 132(3):1228-1240. FIG. 9 provides a graphical illustration of the ppm values for ZmIPT2 in the Lynx embryo libraries. Other ZmIPT genes have low expression, consistent with their possible function as cytokinin synthases in other tissues such as root, meristem and endosperm. See table 4-7 below.







TABLE 3







Lynx libraries containing the ZmIPT2 tag and average ppm values












# of Lynx
Average



Tissue type
Libraries
ppm















Endosperm < 10 DAP
2
393



Corn Endosperm > 10 DAP
1
5



Embryo
2
17



Whole Kernels < 10 DAP
2
10



Whole Kernels > 10 DAP
4
11



Ear
2
20



Pericarp
1
5



Pith
1
5

















TABLE 4







Lynx libraries containing the ZmIPT4 tag and average ppm values.












# of Lynx
Average



Tissue type
libraries
PPM















Embryos 15 DAP
1
5



Roots
3
33

















TABLE 5







Lynx libraries containing the ZmIP75 tag and average ppm values.












# of Lynx
Average



Tissue type
libraries
PPM















Roots, V6 or less
9
4.8



Roots, V12-R1
1
91



Seedling
4
3.5



Leaf
5
5.2



Embryo 11 DAP
1
3



Stalk
4
33.25



Sheath or husk
2
1



Tassel spikelet
1
2



Ear 0 DAP
1
7



Rind
2
2.5



Pulvinus
1
7

















TABLE 6







Lynx libraries containing the ZmIPT6 tag and average ppm values.












# of Lynx




Tissue type
libraries
PPM















Roots, V6 or less
7
6



Roots, V12-R1
1
0



Seedling mesocotyl
1
5



Silk or ear shoot
5
6



R1 apical meristem
1
23



V3 leaf base
1
1



Stalk
4
19



Root
2
13

















TABLE 7







Lynx libraries containing the ZmIPT8 tag and average ppm values.












# of Lynx
Average



Tissue type
libraries
PPM















Base of immature ear
1
3



Ear meristem
1
9



Endosperm
1
7



Stalk-rot-resistant inbred
1
12



Stalk-rot-susceptible
1
5



inbred










Example 6
Expression of the ZmIPT2 Gene in Corn Tissues
The expression pattern of ZmIPT2 in different organs at different stages of development was studied in order to provide information regarding its putative function in cytokinin biosynthesis. Based on Lynx data (discussed in Example 5), the expression of ZmIPT2 seemed to be restricted to developing kernels (FIG. 9). To get an overall view of ZmIPT2 expression in corn and verify Lynx data, RNA was extracted from different B73 tissues: leaf, stalk, roots, and whole kernels at 0, 5, 10, 15, 20 and 25 DAP. Forty μg of total RNA from each sample were stained with ethidium bromide and loaded on an agarose gel. The blot obtained was hybridized with a [α-32P]-dCTP labeled ZmIPT2 probe. In a second hybridization, a cyclophilin probe was used as a loading control. After quantification via a phosphor-imager, the ratio of the expression of ZmIPT2 compared to cyclophilin was calculated. Cyclophilin is considered to be constitutively expressed across different organs (Marivet, et al. (1995) Mol Genet Gen 247:222-228. Results are shown in FIG. 7. ZmIPT2 transcripts are detected at low levels in the leaf, stalk and roots, but at higher levels in kernels, where expression is low at 0 DAP, increases from 5 to 10 DAP and decreases from 15 to 25 DAP. This expression profile not only confirms the Lynx data but coincides with the appearance and disappearance of CK in the kernels (See, Example 5).
To obtain a more precise view of the expression pattern of ZmIPT2 in kernels, levels of ZmIPT2 transcripts were measured in 0- to 5-DAP kernels with pedicels; 6- to 34-DAP kernels without pedicels; and pedicels alone, 6 to 42 DAP. See FIG. 8. As previously done, the gel was loaded with 40 μg of total RNA, stained with ethidium bromide, and blotted on a nylon membrane. Membranes were hybridized with a 32P-dCTP labeled ZmIPT2 probe. The results of the Northern experiment with “seed without pedicel” samples indicate that low levels of ZmIPT2 expression are detected between 0 and 4 DAP. The expression increases from 5 to 8 DAP, peaks at 8 DAP, and decreases until 14 DAP. At later stages (15 to 34 DAP), transcript levels seem to increase again. However, it is not clear whether this is the result of diminution of cyclophilin expression. This phenomenon was also observed for Ckx1 expression (Brugière, et al., (2003) Plant Physiol 132:1228-1240). In the Northern experiment with pedicel samples, transcript levels drastically increase from 6 to 10 DAP, peak at 10 DAP, and slowly decrease until 15 DAP. In this experiment again, transcript levels seem to rise at later stages, but it is unclear whether this is due to the diminution of cyclophilin expression. In this experiment, the sample corresponding to “seed without pedicel at 9 DAP” was used as a control to allow the comparison with the other blot. The relative expression at 9 DAP is four times as high as in the control, showing that ZmIPT2 expression in the pedicel is much higher than in the rest of the seed. This difference is consistent with the fact that CK levels are nearly twice as abundant in the pedicel as in the rest of the seed (Brugière, et al., (2003) Plant Physiol 132:1228-1240).
The relative transcript levels were compared to ZR concentrations measured in the same samples (solid line in FIG. 8). Interestingly, ZmIPT2 expression nicely overlaid ZR accumulation in the pedicel, whereas in the rest of the seed, it slightly preceded the peak in ZR, including at later stages.
Taken together, these results indicate that ZmIPT2 is expressed transiently during kernel development both in the pedicel and the rest of the seed, and that its pattern of expression, which parallels ZR levels in the kernel, is consistent with ZmIPT2 role as a CK biosynthetic gene.
The expression of ZmIPT2 in different kernel tissue was also studied in different kernel tissues. In this study, dissected kernel samples from 0 to 25 DAP were used. Samples were collected from the field in Johnston, Iowa, USA. Kernels were dissected into different parts (pedicel, nucellus, endosperm/embryo sac, endosperm, embryo and pericarp) depending on the stage considered. The gel was loaded with 30 μg of purified total RNA, stained with ethidium bromide, and blotted onto a nylon membrane.
The results shown in FIG. 11 confirm that ZmIPT2 transcripts levels in pedicel are more abundant than in the rest of the seed. This is especially true at 15, 20 and 25 DAP where some expression is also seen in the embryo samples. At 10 DAP however, ZmIPT2 transcripts are found in similar amounts in developing endosperm/embryo sac and pedicel. Together with the fact that 1) cytokinins are more abundant in the pedicel than the rest of the seed and that 2) transcript and activity of cytokinin oxidase in this organ is also more abundant than the rest of the seed (Brugière, et al., (2003) Plant Physiol. 132:1228-1240), these results indicate that the pedicel is most likely a major site for CK biosynthesis. Recent data presented on the expression of Arabidopsis IPT genes allows us to hypothesize that the expression of ZmIPT2 could occur in phloem cells where it could be responsible for the synthesis of CK, which would be targeted to the vascular bundles for transport to developing kernels. The presence of ZmIPT2 transcripts in both developing endosperm and embryo is observed at times when cell division is the most active in these tissues. This again supports a role for ZmIPT2 as a CK biosynthetic protein, which catalyzes CK formation in fast dividing/developing tissues such as endosperm at 10 DAP and growing embryo, and could drive sink strength in the pedicel to support kernel growth.
Example 7
Expression and Purification of the ZmIPT2 Polypeptide from E. coli 
Materials and Methods:
Recombinant Protein Purification, Gel Electrophoresis and Western Blot:
BL21-Al (Invitrogen) E. coli harboring the pDEST17-ZmIPT2 (Mo17) plasmid was grown overnight. A 1/200 dilution of this culture was used to inoculate fresh LB medium and bacteria were grown at 37° C. for 2 h before induction with 0.2% L-arabinose and then grown for 2 to 4 h. Bacterial protein extracts were prepared as described by the supplier and run on a 12.5% polyacrylamide gel in denaturing condition. The His-tagged protein was purified from crude protein extracts using a Ni-NTA agarose solution according to the provider's recommendations (Qiagen). After electrophoresis, proteins were either revealed by gel staining with GelCode (Pierce) or blotted onto a polyvinylidene difluoride (PVDF) membrane using an electro-transfer procedure. Western blot was carried out as described previously (Brugière, et al., (1999) Plant Cell 11:1995-2012) using an anti-poly histidine monoclonal antibody developed in mice (Sigma-Aldrich) and an anti-mice IgG antibody conjugated to alkaline phosphatase developed in goat.
Results:
Cloning of the ZmIPT2 Coding Sequence in a Vector Compatible with the Gateway System:
In an effort to characterize the function of the ZmIPT2 protein both in vitro and in vivo, two approaches were used. The first aimed at expressing and purifying a tagged ZmIPT2 protein in E. coli, and the second at transforming Arabidopsis calli with a construct driving the over-expression of the ZmIPT2 gene under the control of the 35S promoter of the cauliflower mosaic virus. For this purpose the Gateway system for molecular cloning was used.
The Gateway technology was used to build both the protein expression vector (E. coli Expression System with Gateway Technology kit, Invitrogen) and Arabidopsis transformation vector (Multisite Gateway Three-Fragment Vector Construction Kit, Invitrogen). The first step was the addition of specific att sequences to the previously extracted ZmIPT2 fragment. This was achieved by amplifying this fragment by PCR with a pair of primers specially designed to flank the gene with the proper att sites.
Once flanked with these specific sites, the new gel-extracted fragment was inserted by recombination into a donor vector (pDONR221). Recombination was catalyzed in vitro by the BP clonase. The vector generated was checked by digestion with multiple restriction enzymes and migration on agarose gel electrophoresis. The sizes of digested fragments matched with the expected length of digestion products for each enzyme. Both Mo17 and B73 ZmIPT2 genes were cloned in individual donor vectors and the inserts sequenced using M13 forward and reverse primers. The Mo17 clone showed a homology of 100% with the GSS contig sequence and was therefore used to build the expression and transformation constructs.
In Vitro Study: Expression of the ZmIPT2 Protein in E. coli: 
A tagged recombinant ZmIPT2 protein was expressed in E. coli. Besides allowing the ZmIPT2 gene to be transcriptionally activated in E. coli via induction of the T7 promoter, this approach also permitted addition of a six-histidine-tag to the N-terminal end of the recombinant ZmIPT2 protein, which could be used for its purification. The expression vector was generated by recombination of the ZmIPT2 coding sequence between pDONR221-ZmIPT2 and pDEST17 (Invitrogen).
The transcriptional fusion of the 6×His-tag with ZmIPT2 was sequenced and the expression vector was used to transform the BL21-Al strain of E. coli. These cells contain an expression system modulated by L-arabinose, which induces the expression of T7 RNA polymerase. Protein extracts collected at different times (2 h and 4 h) after T7 RNA polymerase induction were tested on denaturing polyacrylamide gel electrophoresis (SDS-PAGE). Samples that had not been induced were collected and used as negative controls, as well as GUS protein expression with and without induction. The gel was stained with Coomassie blue, which reveals the presence of proteins.
After electrophoresis, induced and non-induced samples were blotted onto a PVDF membrane. A Western blot was performed to confirm that the induced protein contained a His-tag. For this purpose, we used mice antibodies raised against a poly histidine peptide. These antibodies would only recognize the tagged protein and would be in turn recognized by anti-mice IgG antibodies carrying alkaline phosphatase. The presence of the recombinant protein could therefore be characterized by a reaction transforming a colorless substrate in a purple product that precipitates on the membrane.
Two bands could be observed, one of expected size (approximately 37 kDa) and one of a slightly bigger size (approximately 40 kDa), both of which were induced by L-arabinose. The two bands could be due to the addition of extra-basic residues that would increase the positive charge of the protein therefore altering its migration. It could also be due to the presence of a covalently bound co-factor on the E. coli expressed protein. In order to decipher between these two possibilities trypsic digestions of each purified bands could be analyzed by mass spectrometry. In order to further characterize the two bands it was necessary to partially purify the protein.
Purification of the His-Tagged ZmIPT2 Protein:
The presence of a 6×His tag allowed affinity purification of this protein using a Ni-NTA resin column. The crude extract was loaded on the column and the effluent collected. After several washes of the column, the protein was eluted using a solution containing imidazol that, because of its higher affinity to the column, is able to release the protein from the column. Samples were collected at each step of the purification and run on a gel, which was stained as previously.
The experiment indicated that the protein is expressed in a soluble form since it was shown to be present in both the supernatant and the effluent, but in smaller amounts in the pellet. The protein was eluted in the second and third elution fractions. The amount of ZmIPT2 protein in the fraction corresponding to the second volume of elution was visually estimated to represent 70 to 80% of the total protein.
A Western blot was carried out using the same samples. The result confirmed that although the protein is present in small amount in the pellet, most of it remains in solution (effluent). The strong signal with the effluent indicates that the amount of ZmIPT2 protein in the crude extract exceeded the column capacity.
In addition, the ZmIPT2 protein was expressed using a C-terminal tag which allowed the purification of ZmIPT2 as one single band on SDS-PAGE. The purity of the fractions was close to 100%. Fractions were found to provide DMAPP:ADP and DMAPP:ATP isopentenyltransf erase activity.
Example 8
In Vivo Study of the Over-Expression of the ZmIPT2 Gene in Arabidopsis Calli
Materials and Methods:
In Vitro Culture:
Different media were used for Arabidopsis germination, callus culture and regeneration (Kakimoto, (1998) J. Plant Res. 111:261-265). The media used were as follows:

    
    
        5000×CIM (callus-inducing medium) hormone mix: 2.5 mg/ml 2,4 dichlorophenoxyacetic acid (2,4-D), 0.25 mg/ml kinetin and 5 mg/ml biotin dissolved in dimethyl sulfoxide (DMSO).
        500× vitamin mix: 50 mg/ml myo-inositol, 10 mg/ml thiamine-HCl, 0.5 mg/ml pyridoxine-HCl, and 0.5 mg/ml nicotinic acid.
        GM (germination medium): 1 L of mixture comprising 4.3 g Murashige and Skoog's medium salt base (Sigma), 10 g sucrose, 2 ml 500× vitamin mix, 10 ml 5% 2-(N-morpholino)-ethanesulfonic acid (MES, adjusted to pH 5.7 with KOH), and 3 g Phytagel (Sigma), autoclaved.
        CIM (callus-inducing medium): 1 L of mixture comprising 3.08 g Gamborg's B5 medium salt base (Sigma), 20 g glucose, 2 ml 500× vitamin mix, 10 ml 5% MES (adjusted to pH 5.7 with KOH), and 3 g Phytagel, autoclaved and 200 μl 5000×CIM hormone mix added to it.
        AIM (Agrobacterium infection medium): CIM from which Phytagel is omitted.
        WASHM (washing medium): GM from which Phytagel is omitted, plus 100 mg/l sodium cefotaxime.
    
    


Selection Media for Transformed Calli:

    
    
        GM+IBA (GIBA): GM plus 100 mg/l cefotaxime, 50 mg/l carbenicilin, 3 mg/l Bialaphos and 0.3 mg/l indolebutyric acid (IBA).
        GM+IBA+Z (GIBAZ): GM plus 100 mg/l cefotaxime, 50 mg/l carbenicilin, 3 mg/l Bialaphos, 0.3 mg/l indolebutyric acid (IBA), and 1 mg/l trans-zeatin (tZ).
    
    


In the experiment aimed at testing the effect of auxin to cytokinin ratio on root and shoot regeneration, GM was prepared and different amounts of hormones were added. Twenty-five media containing different combinations of tZ and IBA concentrations, which were set at 0, 100, 300, 1000 and 3000 ng/ml for each hormone, were prepared.
Sterilized Arabidopsis thaliana seeds were germinated on GM medium and grown on continuous light at 23° C. For the above experiment, hypocotyls from 15 day-old seedlings were cut with a scalpel and grown on each of the 25 media for 3 weeks at 23° C. under continuous light. For experiments requiring the use of callus tissue, hypocotyls were grown on CIM for 10 to 12 days in the same conditions.
Arabidopsis Calli Transformation:
Induced calli were soaked in a suspension of Agrobacterium (0.2 OD600) in AIM for 5 minutes. Most of the liquid was removed on filter paper, and calli were placed on CIM culture medium and grown in continuous light at 23° C. for 2 days. Calli were then washed thoroughly in WASHM medium and placed on GIBA or GIBAZ medium and cultured for about 3 weeks.
Cloning:
In order to constitutively express ZmIPT2 in Arabidopsis using the Gateway system, a construct was built in which the gene was placed under the control of the 35S promoter of the cauliflower mosaic virus. A Gateway clone containing the 35S promoter was constructed using the pDONR-P4-P1R plasmid. Once these 3 elements were available, a multisite recombination was performed using the three donor vectors and a fourth vector called destination vector.
The LR clonase allows an organized “three-site” recombination to occur between the plasmids carrying the promoter, gene of interest and terminator, and a binary vector containing the left and right border of the Ti plasmid and the BAR resistance gene. The resulting construct was verified by digestion with restriction enzymes, migration on agarose gel, and comparison of digested fragment sizes with expected digestion products.
The final construct contained the 35S-ZmIPT2-PINII sequence and included the BAR gene. This gene is used as a selection marker for the herbicide resistance it confers to transformed plant cells.
Transformation in Agrobacterium: 
The next step was the transformation of a plasmid containing the 35S-ZmIPT2-PINII construct in Agrobacterium tumefaciens (LBA4044). This plasmid contains the genes required for infection and delivery of the T-DNA to A. thaliana cells (vir genes). After electroporation in the bacteria (Suzuki, (1999) Plant Cell Physiol 39:1258-1268), the two plasmids are able to recombine at their respective COS sites. The result of this recombination is a 48 kb plasmid called “co-integrate”.
Agrobacteria containing this co-integrate were checked using a quality control process. This procedure consists of extracting the co-integrate plasmid and transforming it into E. coli in order to verify it by restriction digestions. This step is necessary to screen for “mis-recombinations” of the two plasmids at the COS sites, which would result in a non-functional construct.
Although many trials were attempted to transform Agrobacterium cells with the 35S-ZmIPT2-PINII construct, no colonies containing the right co-integrate plasmid could be identified. Since the 35S promoter is leaky in Agrobacterium, it was assumed that ZmIPT2 expression could be lethal for Agrobacterium. The lethality of the construct could be the result of an active degradation of an essential compound for Agrobacterium. Such a metabolite could for example be from the isoprenoid biosynthetic pathway, which includes potential substrates of CK biosynthesis, such as 4-hydroxy-3methyl-2-(E)-butenyl diphosphate (HMBPP).
Analysis of microbial genomes combined with biochemical experiments established the existence of two pathways for isoprenoid synthesis, the mevalonate (MVA) and non-mevalonate (1-deoxyxylulose 5-phosphate, DXP or 2-C-methyl-D-erythritol-4-phosphate, MEP) pathways. The DXP pathway has been found to be present in some bacteria and the chloroplasts of plants. The genes encoding the non-mevalonate pathway are present mostly in Gram-positive bacteria. HMBPP is a precursor of the non-mevalonate (MEP) pathway of isoprenoid biosynthesis and was shown to be a possible substrate for AtIPT7 (Takei, et al., (2003) J Plant Res 116:265-9). Analysis of the genomic sequence of A. tumefaciens C58 showed that it encodes the enzymes of the MEP pathway but that those of the MVA pathway are absent (Wood, et al., (2001) Science 294:2317-2323; Goodner, et al., (2001) Science 294:232-2328). Based on these results we believe that HMBPP could be the substrate of the ZmIPT2 protein and that utilization of this compound by the enzyme could prevent the formation of isoprenoid, which would result in the incapacity of the bacteria to grow.
To elude this problem, the same construct was built but this time using the 35S promoter with the ADH1 intron to prevent the expression of ZmIPT2 gene in Agrobacterium. Using this construct, Agrobacteria carrying the right co-integrate were obtained.
Results:
Arabidopsis calli in culture regenerate roots or shoots depending on auxin and cytokinin levels present in the medium. As a proof of concept, Arabidopsis hypocotyls were cultured on media containing increasing levels of auxin and cytokinin. Twenty-five different combinations of tZ and IBA concentrations, at 0, 100, 300, 1000 and 3000 ng/ml for each hormone, were prepared and hypocotyls transferred to the media as described above. After 3 weeks in the culture room, pictures of 2 representative calli were taken for each hormone combination. Results indicated that a higher auxin:cytokinin ratio favored root formation, while a higher cytokinin:auxin ratio favored shoot formation.
This experiment confirmed that root or shoot formation is influenced by the auxin/cytokinin ratio. Auxins have a root-inducing effect whereas cytokinins induce shoot formation. Based on these results, Arabidopsis calli over-expressing a cytokinin biosynthetic gene should not be able to develop roots on a medium containing only auxin. The functionality of this assay to characterize putative cytokinin biosynthetic genes by using the Agrobacterium tumefaciens IPT (tmr) gene has been tested. Specifically, using the Gateway cloning system, two constructs were developed aimed at over-expressing either IPT as a cytokinin biosynthetic enzyme or GUS as a control. Three weeks after transformation of Arabidopsis calli, roots could be observed on calli transformed with the 35S-GUS-PINII construct but not on calli transformed with the 35S-IPT-PINII construct. In order to demonstrate that calli were efficiently transformed, in situ GUS staining was performed. Tissue transformed with 35S-GUS-PINII contained the GUS protein as revealed by the blue color observed after incubation in a solution containing the GUS substrate. These experiments validated the use of a high-throughput assay to test the putative corn CK biosynthetic genes.
The 35S-ADHI-ZmIPT2-PinII construct was transformed into 10 day-old Arabidopsis calli which were transferred onto GM medium containing either auxin or both auxin and cytokinin. Bialaphos was added to select for transformed calli. Clear phenotypes could be observed 3 weeks after transformation. Control and 35S-ADHI-ZmIPT2-PINII calli grew identically on medium containing both auxin and cytokinin. As expected, control calli transformed with the 35S-GUS-PINII construct were able to regenerate roots on medium containing only auxin. On the contrary, calli transformed with the 35S-ADH1-ZmIPT2-PINII construct, like calli transformed with the 35S-IPT-PINII construct, could not form any roots on this medium and some calli were even able to regenerate shoots. Given results of the preliminary experiment described above, this implies that these calli are synthesizing CK due to the expression of the ZmIPT2 gene. In turn this decreases the auxin:cytokinin ratio, which prevents root formation. These results support the conclusion that ZmIPT2 is a cytokinin biosynthetic gene.
Example 9
Isolation and Sequencing of the ZmIPT2 Promoter
To isolate the promoter of the ZmIPT2 gene, a high-throughput Bacterial Artificial Chromosomes (BAC) screening process was used. Five positive clones were isolated by PCR screening based on the ZmIPT2 sequence. To confirm that the gene of interest was present in the bacterial chromosome, the BAC clones were cultured and prepped. The BACs obtained were digested with HindIII and run on an agarose gel, which was used for a Southern blot. The blot was hybridized with a [α-32P]-dCTP labeled ZmIPT2 probe. Methods for the Southern blot are described above in Example 4.
The Southern blot confirmed the presence of the ZmIPT2 sequence on all BAC clones isolated. Once checked, the BACs were subcloned in pBluescript after digestion with BamHI and HindIII. After ligation, chemically competent E. coli were transformed and grown on ampicillin LB medium. Positive clones were then screened by a colony hybridization method. Colonies were transferred onto a nylon membrane, which was hybridized with a [α-32P]-dCTP ZmIPT2 probe to detect the clones containing the ZmIPT2 region on their plasmid. Finally, the colonies selected were prepped and the plasmid was sent for sequencing using 5′OH-oriented primers. This allowed the upstream region of ZmIPT2 up to 1354 bp to be sequenced. A BAC walking strategy was employed which gave 3280 bp of promoter sequence for this gene. The sequence for the ZmIPT2 promoter is set forth in SEQ ID NO: 75. A similar strategy was followed to identify the ZmIPT1 promoter set forth in SEQ ID NO: 25.
Promoter sequences for ZmIPT4 through ZmIPT9, and OsIPT 1 through OsIPT11, may be isolated in a similar manner. Sequences provided herein for ZmIPT4 (SEQ ID NO: 5), ZmIPT5 (SEQ ID NO: 8), ZmIPT6 (SEQ ID NO: 11), ZmIPT7 (SEQ ID NO: 14), ZmIPT8 (SEQ ID NO: 17), and ZmIPT9 (SEQ ID NO: 20), OsIPT1 (SEQ ID NO: 47), OsIPT2 (SEQ ID NO: 44), OsIPT3 (SEQ ID NO: 62), OsIPT4 (SEQ ID NO: 64), OsIPT5 (SEQ ID NO: 50), OsIPT6 (SEQ ID NO: 55), OsIPT7 (SEQ ID NO: 53), OsIPT8 (SEQ ID NO: 40), OsIPT9 (SEQ ID NO: 60), OsIPT10 (SEQ ID NO: 58), and OsIPT11 (SEQ ID NO: 42) include appropriate upstream regions useful for characterization of functional promoter sequence.
Example 10
Assaying for IPT Activity
A. Synthesis of Cytokinin by Maize or Rice IPT Sequences in Bacterial Culture Medium
The ability of an IPT sequence of the invention to synthesize cytokinin is assayed in a bacterial culture medium in which cytokinin is known to be secreted. Enzyme activity in E. coli is measured.
E. coli strain BL21-Al (Invitrogen) containing a T7 promoter::IPT sequence (IPT cloned in pDEST17 (Invitrogen)) is cultured for 4 h at 37° C. and the accumulation of the protein is induced for 12 hours at 20° C. in the presence of 0.2% arabinose. The microorganisms are collected by centrifugation, and after Buffer A (25 mM Tris-HCl, 50 mM KCl, 5 mM β-mercaptoethanol, 1 mM PMSF and 20 μg/ml of leupeptin) is added to an OD600 of 100, the E. coli are disrupted by freezing and thawing. The disrupted E. coli are then centrifuged for 10 minutes at 300,000 g followed by recovery of the supernatants. 10 μl of these supernatants are mixed with Buffer A containing 60 μM DMAPP, 5 μM [3H]AMP (722 GBq/mmol) and 10 MM MgCl2 followed by incubation for 30 minutes at 25° C. Subsequently, 50 mM of Tris-HCl (pH 9) is added to this reaction liquid followed by the addition of calf intestine alkaline phosphatase to a concentration of 2 units/30 μl and incubating for 30 minutes at 37° C. to carry out a dephosphatization reaction. As a result of developing the reaction liquid by C18 reversed-phase thin layer chromatography (mobile phase: 50% methanol) and detecting the reaction products by autoradiography, formation of isopentenyl adenosine is confirmed in the reaction liquids containing extracts of E. coli having T7::IPT sequence.
It is further recognized that 3H-HMBPP (4-hydroxyl-3-methyl-2-(E)-butenyl diphosphate) could also be used as a substrate in the assay described above. See, for example, Krall, et al., (2002) FEBS Letters 527:318-8, herein incorporated by reference.
B. Assay for DMAPP:ATP or ADP or AMP Isopentenyl Transferase Activity
DMAPP:ATP (or ADP or AMP) isopentenyl transferase activity is measured by the method described by Blackwell and Horgan, (1991) FEBS Lett. 16:10-12, with some modifications. The samples to be assayed are crude extracts and purified proteins of E. coli harboring the T7 promoter::IPT sequence. Purified proteins are diluted to appropriate concentrations with dilution buffer (25 mM Tris-HCl, pH 7.5; 5 mM 2-mercaptoethanol; 0.2 mg ml−1 bovine serum albumin). Isopentenylation reactions are started by mixing samples with an equal volume of 2× assay mixture containing 25 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 5 mM 2-mercaptoethanol, 60 μM DMAPP, and 2 μM [2,8-3H]ATP (120 GBq mmol-1), [2,8-3H]ADP (118 GBq mmol-1), [2-3H]AMP (72 GBq mmol-1), or [2,8-3H]adenosine (143 GBq mmol-1). After incubation for an appropriate time, 1⁄2 volume of calf intestine alkaline phosphatase (CIAP) mix [0.5 Tris-HCl (pH 9.0), 10 mM MgCl2, and 1,000 units ml-1 of CIAP (Takara Shuzo Co. Ltd., Otsu, Shiga, Japan)] is added and the mixtures are incubated at 37° C. for 30 min. Then, 700 μl of ethyl acetate is added and the mixtures are vortexed. After centrifugation at 17,000×g for 2 min, the organic phase is recovered and washed twice with water. The organic phase is mixed with ten volumes of scintillant, ACSII (Amersham Pharmacia Biotech, Tokyo, Japan), and radioactivity levels are measured with a liquid scintillation counter. Recovery of [2,8-3H]isopentenyladenosine (iPA) is measured and is used to calculate the amounts of the products formed. The [2,8-3H]iPA is synthesized through isopentenylation of ATP by using purified IPT sequences, followed by CIAP treatment as described above. All assays are performed in duplicate and mean values are used for calculation.
To determine the Km for ATP, purified protein (2 ng ml−1 in dilution buffer) is mixed with the same volume of a 2× assay mixture containing 25 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 5 mM 2-mercaptoethanol, 0.4 mM DMAPP, and ATP (2-502 μM [2,8-3H]ATP, 1.22 MBq ml-1). To determine the Km for DMAPP, purified protein (2 ng ml-1) is mixed with the same volume of a 2× assay mixture containing 25 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 5 mM 2-mercaptoethanol, 0.25-200 μM DMAPP, and 200 μM [2,8-3]ATP (7.07 GBq mmol-1). After the mixture is incubated at 24° C. for 0 min or 4 min, the reaction mixtures are treated with CIAP, and then extracted with ethyl acetate as described above. Values obtained at 0 min are subtracted from those at 4 min, and the resulting differences are taken as enzyme activity.
To confirm that the IPT sequences catalyzed the transfer of the isopentenyl moiety to ATP, ADP or AMP, the reaction products are analyzed by HPLC and mass spectrometry. Briefly, crude extract prepared from IPTG-induced E. coli harboring the pDEST17-IPT plasmid is incubated with Ni-NTA agarose beads. After the beads have been washed thoroughly, they are re-suspended in a solution containing 25 mM Tris-HCl (pH 7.5), 100 mM KCl, and 5 mM 2-mercaptoethanol. The bead pellets are mixed with an equal volume of a 2× assay mixture that contains I mM unlabeled ATP and 1 mM DMAPP, and incubated at 25° C. for 1 h with shaking. After a brief spin, the supernatant is recovered and separated into two portions, and one portion is treated with CIAP as described before. The supernatant with or without treatment with CIAP is mixed with three volumes of acetone. The mixture is incubated at −80° C. for 30 min and centrifuged at 17,000×g for 30 min to remove the proteins. The supernatants are dried under vacuum, and the residues are dissolved in methanol. Aliquots are separated by HPLC with a Chemcobond ODS-W column (Chemco, Osaka, Japan), by using the following program: 20 mM KH2PO4 for 15 min, followed by linear gradient of 0% acetonitrile and 20 mM KH2PO4 to 80% acetonitrile and 4 mM KH2PO4 over 30 min. The fractions are collected and dried under vacuum, and the residues are resuspended in ethanol. After centrifugation to remove any possible salt precipitates, the solutions are subjected to fast atom bombardment mass spectrometry (JMS-SX102 or JEOL MStation, JEOL DATUM LTD., Tokyo, Japan).
C. Assaying for Shoot and Root Regeneration
Transformation of Arabidopsis callus is performed as follows. Selection for transformants is made using 3 mg/L of bialaphos. Arabidopsis seeds are sterilized according to Koncz, et al., (1992) Methods in Arabidopsis Research, Sinapore, River Edge, N.J., World Scientific. Seeds are placed on GM medium and grown in continuous light at 23° C. for 11 days. Hypocotyl segments are cut and placed on CIM for 8 days. Calli are soaked in a suspension of Agrobacterium (0.2 OD600) in AIM for 5 minutes. Most of the liquid is removed on the filter paper, and the Arabidopsis is placed on CIM culture medium and grown in continuous light at 23° C. for 2 days. The calli are washed thoroughly in WASHM medium and placed on GM+IBA or GM+Z+IBA medium and cultured for about 3 weeks. Selection for transformants is made on 3 mg/L of bialaphos.
Media recipes for the transformation protocol discussed above are as follows. 5000×CIM hormone mix comprises 2.5 mg/ml 2,4-D (Sigma Cat. No. D 6679); 0.25 mg/ml kinetin (Sigma Cat no. K 0753); and, 5 mg/ml biotin dissolved in DMSO (Sigma Cat. No. B 3399. 500× vitamin mix comprises 50 g/l myo-inositol (Sigma Cat. No. I 3011); 10 g/l thiamine-HCl (Sigma Cat. No. T 3902); 0.5 g/l pyridoxine-HCl (Sigma Cat. No. P 8666); and, 0.5 g/l nicotinic acid (Sigma Cat. No. N0765). GM (germination medium) (for 1 liter) comprises 4.3 g MS medium salt base (Sigma Cat. No. M 5524); 10 g sucrose (Sigma Cat. No. S 8501); 2 ml 500× vitamin mix; 10 ml 5% MES (adjusted to pH 5.7 with KOH) (Sigma Cat. No. M 2933); and, 3 g Phytagel (Sigma Cat. No. P 8169). The mixture is autoclaved and poured in Petri dishes. CIM (callus inducing medium) comprises 3.08 g Gamborg's B5 medium salt base (Sigma Cat. No. G 5768); 20 g glucose (Sigma Cat. No. G7528); 2 ml 500× vitamin mix; 10 ml 5% MES (adjusted to pH 5.7 with KOH); and, 3 g Phytagel. The mixture is autoclaved, cooled and 200 μl of CIM hormone mix is added. The mixture is then poured into Petri dishes. AIM (Agrobacterium infection medium) comprises CIM without Phytagel. WASHM (washing medium) comprises GM from which Phytagel has been omitted, plus 100 mg/l of sodium cefotaxime. GM+ (selection of transformed calli) comprises GM medium that was autoclaved with the following components add via filter: 1 ml of 100 mg/ml cefotaxime (Sigma Cat. No. C 7039); 1 ml of 50 mg/ml of carbenicilin (Sigma Cat. No. C 3416); and, 3 ml of 1 mg/ml Bialaphos. GM+IBA comprises the addition of 300 μl of 1 mg/ml indolebutyric acid (IBA) (Sigma Cat. No. I 7512) to the GM media described above. GM+IBA+Z comprises the addition of 300 μl of 1 mg/ml IBA and 1 ml of 1 mg/ml trans-Zeatin (Z) (Sigma Cat. No. Z 2753) to the GM media described above.
In order to examine the function of IPT, the maize IPT sequences are first selected and introduced in Arabidopsis calli under the control of the 35S promoter. Calli transformed with a control vector will exhibit normal hormone responses: root formation in the presence of only an auxin and shoot formation in the presence of a cytokinin and an auxin. By contrast, calli transformed with 35S::IPT will regenerate shoots even in the absence of exogenously applied cytokinins or in the presence of a reduced concentration of exogenously applied cytokinins. In addition, modulation in cytokinin synthesis could be assayed for changes in either direction. Representative methods include cytokinin extraction, immunopurification, HPLC separation, and quantification by ELISA methods can be found, for example, in Faiss, et al., (1997) Plant J. 12:401-415. See, also, Werner, et al., (2001) PNAS 98:10487-10492) and Dewitte, et al., (1999) Plant Physiol. 119:111-121.
D. Assaying for DMAPP:tRNA Isopentenyltransferase Activity
Undermodified tRNA is prepared by permanganate-treatment of yeast tRNA (type X, Sigma-Aldrich Japan, Tokyo, Japan) according to the method of Kline, et al., (1969) Biochemistry 8:4361-4371. Twenty microliters of purified protein samples (20 ng (protein ml−1) in dilution buffer is mixed with the same volume of 2× tRNA isopentenyltransferase assay mixture (25 mM Tris-HCl, pH 7.5; 10 mM MgCl2; 5 mM 2-mercaptoethanol; 0.67 μM [1-3H]DMAPP, 555 GBq mmol−1; and 567 A260 units ml−1 undermodified tRNA), and incubated at 25° C. for 30 min. After 160μ of 0.4 M sodium acetate and 500 μl of ethanol is added and allowed to settle on ice for 10 minutes, the tRNA precipitates are recovered by centrifugation (17,000×g for 20 minutes), washed with 80% ETOH, and dissolved in 30 μl of distilled water. These are mixed with ten volumes of ACSII, and radioactivity levels are measured.
Example 11
Maintaining or Increasing Seed Set During Stress
Targeted overexpression of the IPT sequences of the invention to the developing female inflorescence will elevate cytokinin levels and allow developing maize seed to achieve their full genetic potential for size, minimize tip kernel abortion, and buffer seed set during unfavorable environments. Abiotic stress that occurs during kernel development in maize has been shown to cause reduction in cytokinin levels. Under stress conditions, it is likely that cytokinin biosynthesis activity is decreased and cytokinin degradation is increased (Brugière, et al., (2003) Plant Physiol. 132(3):1228-40). Consequently, in one non-limiting method, to maintain cytokinin levels in lag phase kernels, IPT genes could be ligated to control elements that: 1) are stress insensitive; 2) direct expression of structural genes predominantly to the developing kernels; and 3) preferentially drive expression of structural genes during the lag phase of kernel development. Promoters which target expression to related maternal tissues at or around anthesis may also be employed. Alternatively, a constitutive promoter could be employed.
For example, immature maize embryos from greenhouse donor plants are bombarded with a plasmid containing a sequence, chosen from ZmIPT1-9 or OsIPT1-11, operably linked to the Zag2.1 promoter (Schmidt, et al., (1993) Plant Cell 5:729-737) and containing the selectable marker gene BAR (Wohlleben, et al., (1988) Gene 70:25-37), which confers resistance to the herbicide Bialaphos. Alternatively, the selectable marker gene is provided on a separate plasmid. Transformation is performed as follows. Media recipes follow below.
The ears are husked and surface-sterilized in 30% Clorox bleach plus 0.5% Micro detergent for 20 minutes, and rinsed two times with sterile water. The immature embryos are excised and placed embryo axis side down (scutellum side up), 25 embryos per plate, on 560Y medium for 4 hours and then aligned within the 2.5 cm target zone in preparation for bombardment.
A plasmid vector comprising the IPT sequence operably linked to a Zag2.1 promoter is made. This plasmid DNA plus plasmid DNA containing a BAR selectable marker is precipitated onto 1.1 μm (average diameter) tungsten pellets using a CaCl2 precipitation procedure as follows: 100 μl prepared tungsten particles in water; 10 μl (1 μg) DNA in Tris EDTA buffer (1 μg total DNA); 100 μl 2.5 M CaCl2; and, 10 μl 0.1 M spermidine.
Each reagent is added sequentially to the tungsten particle suspension, while maintained on the multitube vortexer. The final mixture is sonicated briefly and allowed to incubate under constant vortexing for 10 minutes. After the precipitation period, the tubes are centrifuged briefly, liquid removed, washed with 500 ml 100% ethanol, and centrifuged for 30 seconds. Again the liquid is removed, and 105 μl 100% ethanol is added to the final tungsten particle pellet. For particle gun bombardment, the tungsten/DNA particles are briefly sonicated and 10 μl spotted onto the center of each macrocarrier and allowed to dry about 2 minutes before bombardment.
The sample plates are bombarded at level #4 in particle gun #HE34-1 or #HE34-2. All samples receive a single shot at 650 PSI, with a total of ten aliquots taken from each tube of prepared particles/DNA.
Following bombardment, the embryos are kept on 560Y medium for 2 days, then transferred to 560R selection medium containing 3 mg/liter Bialaphos, and subcultured every 2 weeks. After approximately 10 weeks of selection, selection-resistant callus clones are transferred to 288J medium to initiate plant regeneration. Following somatic embryo maturation (2-4 weeks), well-developed somatic embryos are transferred to medium for germination and transferred to the lighted culture room. Approximately 7-10 days later, developing plantlets are transferred to 272V hormone-free medium in tubes for 7-10 days until plantlets are well established. Plants are then transferred to inserts in flats (equivalent to 2.5′′ pot) containing potting soil and grown for 1 week in a growth chamber, subsequently grown an additional 1-2 weeks in the greenhouse, then transferred to classic 600 pots (1.6 gallon) and grown to maturity. Plants are monitored and scored for the maintenance or increase of seed set during an abiotic stress episode. In addition, transformants under stress will be monitored for cytokinin levels (as described in Example 5c) and maintenance of kernel growth.
Bombardment medium (560Y) comprises 4.0 g/l N6 basal salts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000× SIGMA-1511), 0.5 mg/l thiamine HCl, 120.0 g/l sucrose, 1.0 mg/l 2,4-D, and 2.88 g/l L-proline (brought to volume with D-I H2O following adjustment to pH 5.8 with KOH); 2.0 g/l Gelrite (added after bringing to volume with D-I H2O); and 8.5 mg/l silver nitrate (added after sterilizing the medium and cooling to room temperature). Selection medium (560R) comprises 4.0 g/l N6 basal salts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000× SIGMA-1511), 0.5 mg/l thiamine HCl, 30.0 g/l sucrose, and 2.0 mg/l 2,4-D (brought to volume with D-I H2O following adjustment to pH 5.8 with KOH); 3.0 g/l Gelrite (added after bringing to volume with D-I H2O); and 0.85 mg/l silver nitrate and 3.0 mg/l bialaphos (both added after sterilizing the medium and cooling to room temperature).
Plant regeneration medium (288J) comprises 4.3 g/l MS salts (GIBCO 11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/l glycine brought to volume with polished D-I H2O) (Murashige and Skoog, (1962) Physiol. Plant. 15:473), 100 mg/l myo-inositol, 0.5 mg/l zeatin, 60 g/l sucrose, and 1.0 ml/l of 0.1 mM abscisic acid (brought to volume with polished D-I H2O after adjusting to pH 5.6); 3.0 g/l Gelrite (added after bringing to volume with D-I H2O); and 1.0 mg/l indoleacetic acid and 3.0 mg/l bialaphos (added after sterilizing the medium and cooling to 60° C.). Hormone-free medium (272V) comprises 4.3 g/l MS salts (GIBCO 11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g/l nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/l glycine brought to volume with polished D-I H2O), 0.1 g/l myo-inositol, and 40.0 g/l sucrose (brought to volume with polished D-I H2O after adjusting pH to 5.6); and 6 g/l bacto-agar (added after bringing to volume with polished D-I H2O), sterilized and cooled to 60° C.
Example 12
Modulating Root Development
For Agrobacterium-mediated transformation of maize with a plasmid designed to achieve post-transcriptional gene silencing (PTGS) with an appropriate promoter, the method of Zhao may be employed (U.S. Pat. No. 5,981,840, and PCT Patent Publication Number WO98/32326, the contents of which are hereby incorporated by reference). Briefly, immature embryos are isolated from maize and the embryos contacted with a suspension of Agrobacterium capable of transferring a DNA construct. Said construct may comprise the CRWAQ81 root-preferred promoter::ADH intron promoter operably linked to a hairpin structure made from the coding sequence of any one of the ZmIPT1-9 or OsIPT1-11 polynucleotides of the invention. Other useful constructs may comprise a hairpin construct targeting the promoter of any one of the ZmIPT1-9 or OsIPT1-11 polynucleotides of the invention. (Aufsatz, et al., (2002) PNAS 99(4):16499-16506; Mette, et al., (2000) EMBO J 19(19):5194-5201) The construct is transferred to at least one cell of at least one of the immature embryos (step 1: the infection step). In this step the immature embryos are immersed in an Agrobacterium suspension for the initiation of inoculation. The embryos are co-cultured for a time with the Agrobacterium (step 2: the co-cultivation step); this may take place on solid medium. Following this co-cultivation period an optional “resting” step is contemplated. In this resting step, the embryos are incubated in the presence of at least one antibiotic known to inhibit the growth of Agrobacterium without the addition of a selective agent for plant transformants (step 3: resting step). Next, inoculated embryos are cultured on medium containing a selective agent; growing, transformed callus is recovered (step 4: the selection step). The callus is then regenerated into plants (step 5: the regeneration step).
Plants are monitored and scored for a modulation in root development. The modulation in root development includes monitoring for enhanced root growth of one or more root parts including the primary root, lateral roots, adventitious roots, etc. Methods of measuring such developmental alterations in the root system are known in the art. See, for example, US Patent Application Publication Number 2003/0074698 and Werner, et al., (2001) PNAS 18:10487-10492, both of which are herein incorporated by reference.
Example 13
Modulating Senescence of a Plant
A DNA construct comprising any of the ZmIPT1-9 or OsIPT1-11 polynucleotides operably linked to a constitutive promoter, a root-preferred promoter, or a senescence-activated promoter, such as SAG12 (Gan, et al., (1995) Science 270:5244, Genbank Accession Number U37336) is introduced into maize plants as outlined in Zhao, et al., (1998) Maize Genetics Corporation Newsletter 72:34-37, herein incorporated by reference.
For example, maize plants comprising the IPT sequence operably linked to the SAG12 promoter are obtained. As a control, a non-cytokinin-related construct is also introduced into maize plants using the transformation method outlined above. The phenotypes of transgenic maize plants having an elevated level of the IPT polypeptide are studied. For example, plants can be monitored for an improved vitality, shelf and vase life, and improved tolerance against infection. Plants could also be monitored for delayed senescence under various environmental stresses including, for example, flooding which normally results in leaf chlorosis, necrosis, defoliation, cessation of growth and reduction in yield.
Example 14
Soybean Embryo Transformation
Soybean embryos are bombarded with a plasmid containing the IPT sequence operably linked to a ubiquitin promoter as follows. To induce somatic embryos, cotyledons, 3-5 mm in length dissected from surface-sterilized, immature seeds of the soybean cultivar A2872, are cultured in the light or dark at 26° C. on an appropriate agar medium for six to ten weeks. Somatic embryos producing secondary embryos are then excised and placed into a suitable liquid medium. After repeated selection for clusters of somatic embryos that multiplied as early, globular-staged embryos, the suspensions are maintained as described below.
Soybean embryogenic suspension cultures can be maintained in 35 ml liquid media on a rotary shaker, 150 rpm, at 26° C. with florescent lights on a 16:8 hour day/night schedule. Cultures are subcultured every two weeks by inoculating approximately 35 mg of tissue into 35 ml of liquid medium.
Soybean embryogenic suspension cultures may then be transformed by the method of particle gun bombardment (Klein, et al., (1987) Nature (London) 327:70-73, U.S. Pat. No. 4,945,050). A Du Pont Biolistic PDS1000/HE instrument (helium retrofit) can be used for these transformations.
A selectable marker gene that can be used to facilitate soybean transformation is a transgene composed of the 35S promoter from Cauliflower Mosaic Virus (Odell, et al., (1985) Nature 313:810-812), the hygromycin phosphotransferase gene from plasmid pJR225 (from E. coli; Gritz, et al, (1983) Gene 25:179-188), and the 3′ region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacterium tumefaciens. The expression cassette comprising the IPT sequence operably linked to the ubiquitin can be isolated as a restriction fragment. This fragment can then be inserted into a unique restriction site of the vector carrying the marker gene.
To 50 μl of a 60 mg/ml 1 μm gold particle suspension is added (in order): 5 μl DNA (1 μg/μl), 20 μl spermidine (0.1 M), and 50 μl CaCl2 (2.5 M). The particle preparation is then agitated for three minutes, spun in a microfuge for 10 seconds and the supernatant removed. The DNA-coated particles are then washed once in 400 μl 70% ethanol and resuspended in 40 μl of anhydrous ethanol. The DNA/particle suspension can be sonicated three times for one second each. Five microliters of the DNA-coated gold particles are then loaded on each macro carrier disk.
Approximately 300-400 mg of a two-week-old suspension culture is placed in an empty 60×15 mm petri dish and the residual liquid removed from the tissue with a pipette. For each transformation experiment, approximately 5-10 plates of tissue are normally bombarded. Membrane rupture pressure is set at 1100 psi, and the chamber is evacuated to a vacuum of 28 inches mercury. The tissue is placed approximately 3.5 inches away from the retaining screen and bombarded three times. Following bombardment, the tissue can be divided in half and placed back into liquid and cultured as described above.
Five to seven days post bombardment, the liquid media may be exchanged with fresh media, and eleven to twelve days post-bombardment with fresh media containing 50 mg/ml hygromycin. This selective media can be refreshed weekly. Seven to eight weeks post-bombardment, green, transformed tissue may be observed growing from untransformed, necrotic embryogenic clusters. Isolated green tissue is removed and inoculated into individual flasks to generate new, clonally propagated, transformed embryogenic suspension cultures. Each new line may be treated as an independent transformation event. These suspensions can then be subcultured and maintained as clusters of immature embryos or regenerated into whole plants by maturation and germination of individual somatic embryos.
Example 15
Sunflower Meristem Tissue Transformation
Sunflower meristem tissues are transformed with an expression cassette containing the IPT sequence operably linked to a ubiquitin promoter as follows (see also, European Patent Number EP 0 486233, herein incorporated by reference, and Malone-Schoneberg, et al., (1994) Plant Science 103:199-207). Mature sunflower seed (Helianthus annuus L.) are dehulled using a single wheat-head thresher. Seeds are surface sterilized for 30 minutes in a 20% Clorox bleach solution with the addition of two drops of Tween 20 per 50 ml of solution. The seeds are rinsed twice with sterile distilled water.
Split embryonic axis explants are prepared by a modification of procedures described by Schrammeijer, et al., (Schrammeijer, et al., (1990) Plant Cell Rep. 9:55-60). Seeds are imbibed in distilled water for 60 minutes following the surface sterilization procedure. The cotyledons of each seed are then broken off, producing a clean fracture at the plane of the embryonic axis. Following excision of the root tip, the explants are bisected longitudinally between the primordial leaves. The two halves are placed, cut surface up, on GBA medium consisting of Murashige and Skoog mineral elements (Murashige, et al., (1962) Physiol. Plant. 15:473-497), Shepard's vitamin additions (Shepard, (1980) in Emergent Techniques for the Genetic Improvement of Crops (University of Minnesota Press, St. Paul, Minn.), 40 mg/l adenine sulfate, 30 g/l sucrose, 0.5 mg/l 6-benzyl-aminopurine (BAP), 0.25 mg/l indole-3-acetic acid (IAA), 0.1 mg/l gibberellic acid (GA3), pH 5.6, and 8 g/l Phytagar.
The explants are subjected to microprojectile bombardment prior to Agrobacterium treatment (Bidney, et al., (1992) Plant Mol. Biol. 18:301-313). Thirty to forty explants are placed in a circle at the center of a 60× 20 mm plate for this treatment. Approximately 4.7 mg of 1.8 mm tungsten microprojectiles are resuspended in 25 ml of sterile TE buffer (10 mM Tris HCl, 1 mM EDTA, pH 8.0) and 1.5 ml aliquots are used per bombardment. Each plate is bombarded twice through a 150 mm nytex screen placed 2 cm above the samples in a PDS 1000® particle acceleration device.
Disarmed Agrobacterium tumefaciens strain EHA105 is used in all transformation experiments. A binary plasmid vector comprising the expression cassette that contains the IPT gene operably linked to the ubiquitin promoter is introduced into Agrobacterium strain EHA105 via freeze-thawing as described by Holsters, et al., (1978) Mol. Gen. Genet. 163:181-187. This plasmid further comprises a kanamycin selectable marker gene (i.e., npt11). Bacteria for plant transformation experiments are grown overnight (28° C. and 100 RPM continuous agitation) in liquid YEP medium (10 gm/l yeast extract, 10 gm/l Bactopeptone, and 5 gm/l NaCl, pH 7.0) with the appropriate antibiotics required for bacterial strain and binary plasmid maintenance. The suspension is used when it reaches an OD600 of about 0.4 to 0.8. The Agrobacterium cells are pelleted and resuspended at a final OD600 of 0.5 in an inoculation medium comprised of 12.5 mM MES pH 5.7, 1 gm/l NH4Cl, and 0.3 gm/l MgSO4.
Freshly bombarded explants are placed in an Agrobacterium suspension, mixed, and left undisturbed for 30 minutes. The explants are then transferred to GBA medium and co-cultivated, cut surface down, at 26° C. and 18-hour days. After three days of co-cultivation, the explants are transferred to 374B (GBA medium lacking growth regulators and a reduced sucrose level of 1%) supplemented with 250 mg/l cefotaxime and 50 mg/l kanamycin sulfate. The explants are cultured for two to five weeks on selection and then transferred to fresh 374B medium lacking kanamycin for one to two weeks of continued development. Explants with differentiating, antibiotic-resistant areas of growth that have not produced shoots suitable for excision are transferred to GBA medium containing 250 mg/l cefotaxime for a second 3-day phytohormone treatment. Leaf samples from green, kanamycin-resistant shoots are assayed for the presence of NPTII by ELISA and for the presence of transgene expression by assaying for cytokinin synthesis activity. Such assays are described elsewhere herein.
NPTII-positive shoots are grafted to Pioneer® hybrid 6440 in vitro-grown sunflower seedling rootstock. Surface sterilized seeds are germinated in 48-0 medium (half-strength Murashige and Skoog salts, 0.5% sucrose, 0.3% gelrite, pH 5.6) and grown under conditions described for explant culture. The upper portion of the seedling is removed, a 1 cm vertical slice is made in the hypocotyl, and the transformed shoot inserted into the cut. The entire area is wrapped with parafilm to secure the shoot. Grafted plants can be transferred to soil following one week of in vitro culture. Grafts in soil are maintained under high humidity conditions followed by a slow acclimatization to the greenhouse environment. Transformed sectors of T0 plants (parental generation) maturing in the greenhouse are identified by NPTII ELISA and/or by cytokinin synthesis activity analysis of leaf extracts while transgenic seeds harvested from NPTII-positive T0 plants are identified by cytokinin synthesis activity analysis of small portions of dry seed cotyledon.
Example 16
Variants of IPT
A. Variant Nucleotide Sequences of ZmIPT1-9 and OsIPT1-11 (SEQ ID NO: 1, 3, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 21, 22, 24, 26, 28, 40, 42, 44, 45, 47, 48, 50, 51, 53, 55, 56, 58, 60, 62, 64, 65, 69, 70, 71, 72, 73, 74 or 76) that do not Alter the Encoded Amino Acid Sequence
The ZmIPT1-9 or OsIPT1-11 nucleotide sequences set forth in SEQ ID NO: 1, 3, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 21, 22, 24, 26, 28, 40, 42, 44, 45, 47, 48, 50, 51, 53, 55, 56, 58, 60, 62, 64, 65, 69, 70, 71, 72, 73, 74 or 76 are used to generate variant nucleotide sequences having the nucleotide sequence of the open reading frame with about 70%, 75%, 80%, 85%, 90% and 95% nucleotide sequence identity when compared to the corresponding starting unaltered ORF nucleotide sequence. These functional variants are generated using a standard codon table. While the nucleotide sequence of the variant is altered, the amino acid sequence encoded by the open reading frame does not change.
B. Variant Amino Acid Sequences of ZmIPT1-9 and OsIPT1-11
Variant amino acid sequences of ZmIPT1-9 and OsIPT1-11 are generated. In this example, one or more amino acids are altered. Specifically, the open reading frame set forth in SEQ ID NO: 1, 3, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 21, 22, 24, 26, 28, 40, 42, 44, 45, 47, 48, 50, 51, 53, 55, 56, 58, 60, 62, 64, 65, 69, 70, 71, 72, 73, 74 or 76 is reviewed to determine the appropriate amino acid alteration. The selection of an amino acid to change is made by consulting a protein alignment with orthologs and other gene family members from various species. See FIG. 1 and/or FIG. 10. An amino acid is selected that is deemed not to be under high selection pressure (not highly conserved) and which is rather easily substituted by an amino acid with similar chemical characteristics (i.e., similar functional side-chain). Assays as outlined in Example 10 may be followed to confirm functionality. Variants having about 70%, 75%, 80%, 85%, 90%, or 95% nucleic acid sequence identity to each of SEQ ID NO: 1, 3, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 21, 22, 24, 26, 28, 40, 42, 44, 45, 47, 48, 50, 51, 53, 55, 56, 58, 60, 62, 64, 65, 69, 70, 71, 72, 73, 74 and 76 are generated using this method.
C. Additional Variant Amino Acid Sequences of ZmIPT1-9 and OsIPT1-11
In this example, artificial protein sequences are created having 80%, 85%, 90% and 95% identity relative to the reference protein sequence. This latter effort requires identifying conserved and variable regions from the alignment set forth in FIG. 1 and/or FIG. 10 and then the judicious application of an amino acid substitutions table. These parts will be discussed in more detail below.
Largely, the determination of which amino acid sequences are altered is made based on the conserved regions among the IPT proteins or among the other IPT polypeptides. See, FIGS. 1 and 10. Based on the sequence alignment, the various regions of the IPT polypeptides that can likely be altered can be determined. It is recognized that conservative substitutions can be made in the conserved regions without altering function. In addition, one of skill will understand that functional variants of the IPT sequence of the invention can have minor non-conserved amino acid alterations in the conserved domain.
Artificial protein sequences are then created that are different from the original in the intervals of 80-85%, 85-90%, 90-95% and 95-100% identity. Midpoints of these intervals are targeted, with liberal latitude of plus or minus 1%, for example. The amino acids substitutions will be effected by a custom Perl script. The substitution table is provided below in Table 8.







TABLE 8







Substitution Table













Strongly Similar
Rank of




Amino
and Optimal
Order to



Acid
Substitution
Change
Comment
















I
L, V
1
50:50 substitution



L
I, V
2
50:50 substitution



V
I, L
3
50:50 substitution



A
G
4



G
A
5



D
E
6



E
D
7



W
Y
8



Y
W
9



S
T
10



T
S
11



K
R
12



R
K
13



N
Q
14



Q
N
15



F
Y
16



M
L
17
First methionine






cannot change



H

Na
No good substitutes



C

Na
No good substitutes



P

Na
No good substitutes










First, any conserved amino acids in the protein that should not be changed are identified and “marked off” for insulation from the substitution. The start methionine will of course be added to this list automatically. Next, the changes are made.
H, C and P are not changed. The changes will occur with isoleucine first, sweeping N-terminal to C-terminal. Then leucine, and so on down the list until the desired target is reached. Interim number substitutions can be made so as not to cause reversal of changes. The list is ordered 1-17, so start with as many isoleucine changes as needed before leucine, and so on down to methionine. Clearly many amino acids will in this manner not need to be changed. L, I and V will involve a 50:50 substitution of the two alternate optimal substitutions.
The variant amino acid sequences are written as output. Perl script is used to calculate the percent identities. Using this procedure, variants of ZmIPT1-9 and OsIPT1-11 are generating having about 82%, 87%, 92% and 97% amino acid identity to the starting unaltered ORF nucleotide sequence of SEQ ID NO: 1, 3, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 21, 22, 24, 26, 28, 40, 42, 44, 45, 47, 48, 50, 51, 53, 55, 56, 58, 60, 62, 64, 65, 69, 70, 71, 72, 73, 74 or 76.
Example 17
Characterization of Rice IPT Sequences
Eleven putative rice ipt sequences were identified which comprise deduced amino acid sequences showing similarity to the Arabidopsis and Petunia IPT proteins. FIG. 10 provides an alignment of the amino acid sequences corresponding to Arabidopsis IPT proteins (AtIPT), the petunia IPT protein (Sho) and rice putative IPT proteins (OsIPT). Asterisks indicate the positions of amino acids conserved in most IPT proteins and following the consensus sequence GxTxxGK[ST]xxxxx[VLI]xxxxxxx[VLI][VLI]xxDxx Qx{57,60}[VLI][VLI]xGG[ST] (SEQ ID NO: 32) (where x denotes any amino acid residue, [ ] any one of the amino acids shown in [ ], and x{m,n} m to n amino acid residues in number) (Takei, et al., (2001) J. Biol. Chem. 276:26405-26410).
The presence of putative ATP/GTP-binding site (P-loop) motif (prosite PS00017: consensus [AG]-x(4)-G-K-[ST]), (SEQ ID NO: 68) is underlined. This domain is found at about amino acids 128-135 of SEQ ID NO: 54; at about amino acids 59-66 of SEQ ID NO: 66: at about amino acids 59-66 of SEQ ID NO: 63; at about amino acids 40-47 of SEQ ID NO: 61; at about amino acids 320-327 of SEQ ID NO: 43; at about amino acids 22-29 of SEQ ID NO: 49; at about amino acids 315-322 of SEQ ID NO: 59; at about amino acids 32-39 of SEQ ID NO: 57; at about amino acids 41-48 of SEQ ID NO: 41; at about amino acids 25-32 of SEQ ID NO: 46; and, at about amino acids 37-44 of SEQ ID NO: 52. The presence of a putative tRNA isopentenyltransferase domain (PF01715) was found at about amino acids 59-348 of SEQ ID NO: 57 and about amino acids 69-352 of SEQ ID NO: 41.
The Align X program was used on default settings to determine the overall amino acid sequence identity for the various rice IPT sequences compared with known Arabidopsis IPT sequences. Table 9 summarizes these results. Table 10 provides polypeptides that share homology to the rice IPT sequences. Such sequences were identified using BLASTP2.2.6













TABLE 9








SEQ
Best
Similarity
Identity



Rice protein
ID NO:
IPT hit
(%)
(%)






















OsIPT7
54
AtIPT1
43.4
32.1



OsIPT6
57
AtIPT9
61
51.9



OsIPT10
59
AtIPT1
28
19.2



OsIPT3
63
AtIPT5
48.3
41.4



OsIPT8
41
AtIPT2
57.9
46.2



OsIPT11
43
AtIPT1
28.6
20.4



OsIPT4
66
AtIPT5
55.6
45.9



OsIPT5
52
AtIPT1
38.4
28.4



OsIPT1
49
AtIPT5
40.4
28.2



OsIPT9
61
AtIPT1
37.1
26.5



OsIPT2
46
AtIPT7
51.6
37.7




















TABLE 10







Approximate





region of rice IPT
% identity of


Rice IPT

sequence sharing
homologous


sequence
Sequence having homology to rice IPT sequence
homology
region.







SEQ ID
NP_917001.1
16-427
73%


NO: 54
cytokinin synthase -like protein



XP_475862.1
121-398 
61%



putative tRNA delta(2)-isopentenylpyrophosphate



transferase



AAT85187.1| unknown protein [Oryza sativa (japonica
121-398 
61%



cultivar-group)] |BACK|



BAB59040.1| adenylate isopentenyltransferase
123-365 
39%



[Arabidopsis thaliana]



BAB59029.1| cytokinin synthase [Arabidopsis thaliana]
123-365 
39%


SEQ ID
NP_914320.1| similar to tRNA isopentenyltransferase
 1-450
80%


NO: 41
[Oryza sativa (japonica cultivar-group)]



BAB59042.1| tRNA isopentenyltransferase
35-441
35%



[Arabidopsis thaliana]



F84676 hypothetical protein At2g27760 [imported] -
35-441
37%



Arabidopsis thaliana



AAS79605.1| putative tRNA
35-442
46%



isopentenylpyrophosphatase [Ipomoea trifida]



AAL87321.1| putative tRNA
156-441 
43%



isopentenylpyrophosphate transferase [Arabidopsis



thaliana]


SEQ ID
XP_476953.1| hypothetical protein [Oryza sativa
 1-252
93%


NO: 43
(japonica cultivar-group)]



XP_475862.1| putative tRNA delta(2)-
314-590 
57%



isopentenylpyrophosphate transferase [Oryza sativa



(japonica cultivar-group)]



AAT85187.1| unknown protein [Oryza sativa (japonica
314-590 
57%



cultivar-group)]



NP_917001.1| cytokinin synthase -like protein [Oryza
315-590 
56%



sativa (japonica cultivar-group)]



BAB59029.1| cytokinin synthase [Arabidopsis
313-590 
38%



thaliana]


SEQ ID
AAT77921.1| putative adenylate isopentenyltransferase
17-288
42%


NO: 46
[Oryza sativa (japonica cultivar-group)]



XP_477138.1| putative cytokinin synthase [Oryza
 7-288
39%



sativa (japonica cultivar-group)]



AAN46854.1| At3g63110/T20O10_210 [Arabidopsis
17-288
37%



thaliana]



emb|CAB87756.1| tRNA isopentenyl transferase-like
17-288
37%



protein [Arabidopsis thaliana]



BAB02782.1| tRNA isopentenyl transferase-like
10-287
37%



protein [Arabidopsis thaliana]


SEQ ID
AAT77921.1| putative adenylate isopentenyltransferase
10-287
43%


NO: 49
[Oryza sativa (japonica cultivar-group)]



XP_477138.1| putative cytokinin synthase [Oryza
14-312
39%



sativa (japonica cultivar-group)]



AAN46854.1| At3g63110/T20O10_210 [Arabidopsis
13-287
40%



thaliana]



CAB87756.1| tRNA isopentenyl transferase-like
13-287
40%



protein [Arabidopsis thaliana]



BAB59032.1| cytokinin synthase [Arabidopsis
14-287
40%



thaliana]


SEQ ID
NP_917001.1| cytokinin synthase -like protein [Oryza
21-251
75%


NO: 52
sativa (japonica cultivar-group)]



XP_475862.1| putative tRNA delta(2)-
30-251
63%



isopentenylpyrophosphate transferase [Oryza sativa



(japonica cultivar-group)]



AAT85187.1| unknown protein [Oryza sativa (japonica
30-251
63%



cultivar-group)]



BAB59040.1| adenylate isopentenyltransferase
32-251
38%



[Arabidopsis thaliana]



BAB59029.1| cytokinin synthase [Arabidopsis
32-251
38%



thaliana]



BAB02956.1| tRNA isopentenyl transferase-like
32-251
36%



protein [Arabidopsis thaliana]


SEQ ID
BAD62118.1| putative tRNA isopentenyltransferase
 1-417
96%


NO: 57
[Oryza sativa (japonica cultivar-group)]



AAK64114.1| putative IPP transferase [Arabidopsis
24-411
58%



thaliana]



AAM63091.1| IPP transferase-like protein
24-411
58%



[Arabidopsis thaliana]



BAB59048.1| tRNA isopentenyltransferase
24-411
58%



[Arabidopsis thaliana]



YP_008242.1| putative tRNA delta-2-
17-346
33%



isopentenylpyrophosphate transferase


SEQ ID
XP_476953.1| hypothetical protein [Oryza sativa
 1-199
91%


NO: 59
(japonica cultivar-group)]



XP_475862.1| putative tRNA delta(2)-
309-585 
55%



isopentenylpyrophosphate transferase [Oryza sativa



AAT85187.1| unknown protein [Oryza sativa (japonica
309-585 
55%



cultivar-group)]



NP_917001.1| cytokinin synthase -like protein [Oryza
310-585 
54%



sativa (japonica cultivar-group)]



BAB59029.1| cytokinin synthase [Arabidopsis
308-585 
37%



thaliana]


SEQ ID
XP_475862.1| putative tRNA delta(2)-
 1-360
83%


NO: 61
isopentenylpyrophosphate transferase [Oryza sativa



(japonica cultivar-group)]



AAT85187.1| unknown protein [Oryza sativa (japonica
 1-360
83%



cultivar-group)]



NP_917001.1| cytokinin synthase -like protein [Oryza
33-312
74%



sativa (japonica cultivar-group)]



BAB59040.1| adenylate isopentenyltransferase
34-312
43%



[Arabidopsis thaliana]



BAB59029.1| cytokinin synthase [Arabidopsis
34-312
43%



thaliana]


SEQ ID
AAT77921.1| putative adenylate isopentenyltransferase
 1-344
71%


NO: 63
[Oryza sativa (japonica cultivar-group)]



XP_477138.1| putative cytokinin synthase [Oryza
35-326
53%



sativa (japonica cultivar-group)]



AAN46854.1| At3g63110/T20O10_210 [Arabidopsis
49-320
43%



thaliana]



CAB87756.1| tRNA isopentenyl transferase-like
49-320
43%



protein [Arabidopsis thaliana]



BAB59041.1| adenylate isopentenyltransferase
51-325
44%



[Arabidopsis thaliana]


SEQ ID
XP_477138.1| putative cytokinin synthase [Oryza
 1-316
78%


NO: 66
sativa (japonica cultivar-group)]



AAT77921.1| putative adenylate isopentenyltransferase
51-314
67%



[Oryza sativa (japonica cultivar-group)]



AAN46854.1| At3g63110/T20O10_210 [Arabidopsis
51-314
51%



thaliana]



CAB87756.1| tRNA isopentenyl transferase-like
51-314
51%



protein [Arabidopsis thaliana]



BAB59041.1| adenylate isopentenyltransferase
51-314
52%



[Arabidopsis thaliana]
















TABLE 11







Summary of Zm and Os IPT Sequences









SEQ ID NO
Description
Type












1
ZmIPT2 full length
DNA


2
ZmIPT2 polypeptide
AA


3
ZmIPT2 coding sequence
DNA


76
ZmIPT2 variant coding sequence
DNA


77
ZmIPT2 variant polypeptide
AA


4
ZmIPT1 duplicate sequence
DNA


5
ZmIPT4 full length
DNA


6
ZmIPT4 polypeptide
AA


7
ZmIPT4 coding sequence
DNA


8
ZmIPT5 full length
DNA


9
ZmIPT5 polypeptide
AA


10
ZmIPT5 coding sequence
DNA


11
ZmIPT6 full length
DNA


12
ZmIPT6 polypeptide
AA


13
ZmIPT6 coding sequence
DNA


14
ZmIPT7 full length
DNA


15
ZmIPT7 polypeptide
AA


16
ZmIPT7 coding sequence
DNA


17
ZmIPT8 full length
DNA


18
ZmIPT8 polypeptide
AA


19
ZmIPT8 coding sequence
DNA


20
ZmIPT9 full length
DNA


21
ZmIPT1 genomic
DNA


22
ZmIPT1 full length
DNA


23
ZmIPT1 polypeptide
AA


24
ZmIPT1 coding sequence
DNA


26
Variant ZmIPT1 full length
DNA


27
Variant ZmIPT1 polypeptide
AA


28
Variant ZmIPT1 coding sequence
DNA


25
ZmIPT1 promoter
DNA


40
OsIPT8 genomic
DNA


41
OsIPT8 polypeptide
AA


71
OsIPT8 coding sequence
DNA


42
OsIPT11 genomic
DNA


43
OsIPT11 polypeptide
AA


74
OsIPT11 coding sequence
DNA


44
OsIPT2 genomic
DNA


45
OsIPT2 coding sequence
DNA


46
OsIPT2 polypeptide
AA


47
OsIPT1 genomic
DNA


48
OsIPT1 coding sequence
DNA


49
OsIPT polypeptide
AA


50
OsIPT5 genomic
DNA


51
OsIPT5 coding sequence
DNA


52
OsIPT5 polypeptide
AA


53
OsIPT7 genomic
DNA


54
OsIPT7 polypeptide
AA


70
OsIPT7 coding sequence
DNA


55
OsIPT6 genomic
DNA


56
OsIPT6 coding sequence
DNA


57
OsIPT6 polypeptide
AA


58
OsIPT10 genomic
DNA


59
OsIPT10 polypeptide
AA


73
OsIPT10 coding sequence
DNA


60
OsIPT9 genomic
DNA


61
OsIPT9 polypeptide
AA


72
OsIPT9 coding sequence
DNA


62
OsIPT3 genomic
DNA


63
OsIPT3 polypeptide
AA


69
OsIPT3 coding sequence
DNA


64
OsIPT4 genomic
DNA


65
OsIPT4 coding sequence
DNA


66
OsIPT4 polypeptide
AA


75
ZmIPT2 promoter
DNA









Example 18
IPT Activity Assay
Assays were conducted to test the ability of a protein encoded by a sequence of the invention to synthesize cytokinin in a bacterial culture medium. The results confirmed that sequences of the invention encode proteins with isopentenyltransferase activity. The reaction catalyzed by Agrobacterium ipt is shown in Akiyoshi, et al., (1984) PNAS 81(19):5994-5998.
The IPT assay protocol was adapted from the following references: Kakimoto, (2001) Identification of plant biosynthetic enzymes as dimethylallyl diphosphate: ATP/ADP isopentenyltransferases, Plant Cell Physiol 42:677-685. Sakakibara and Takei, (2002) Identification of Cytokinin Biosynthesis Genes in Arabidopsis: A Breakthrough for Understanding the Metabolic Pathway and the Regulation in Higher Plants, J. Plant Growth Regul. 21:17-23. Sakano, et al., (2004) Molecular cloning, expression, and characterization of adenylate isopentenyltransferase from hop (Humulus lupulus L.), Phytochemistry 65:2439-2446.
The ZmIPT2 gene was amplified using gene-specific primers with appropriate NdeI and NotI restriction site extensions and cloned into pET28a (N-terminal tag) or pET30b (C-terminal tag) digested by NdeI and NotI. The sequence of the resulting plasmid was verified by sequencing of the His-tag translational fusion with ZmIPT2, and BL21-StarTM E. coli competent cells (InvitrogenTM) were transformed with pET28a-ZmIPT2 and pET30b-ZmIPT2. Similarly, The tzs IPT gene from Agrobacterium tumefaciens was cloned into pET28a to yield a plasmid for transformation of Rosetta2(DE3)pLysS.
Recombinant his-tagged proteins were purified using a TALONTM column (BD Biosciences) according to the instructions provided by the manufacturer. Purified protein samples were used to determine Dimethylallyl diphosphate (DMAPP)::AMP and DMAPP::ATP isopentenyl transferase activities using the following protocol:

    
    
        Each purified protein extract was incubated in a reaction mixture containing 12.5 mM Tris-HCl (pH 7.5), 37.5 mM KCl, 5 mM MgCl2, 1 mM DMAPP and 1 mM AMP or ATP for 2 hours at 30° C. The reaction was stopped by boiling the samples for 5 minutes.
        Half of the reaction mixture was treated with calf intestine alkaline phosphatase (CAIP) by adding one volume of 2×CAIP reaction buffer (0.45M Tris-HCl pH 9, 10 mM MgCl2, 1000 unit of CAIP/ml) and incubating for 1 hour at 37° C.
        The reaction products were separated using reversed phase HPLC (Agilent 1100 system with diode-array-detector) using a C18-ODS2 column (Phenomenex) and a separation protocol using 0.1 M acetic acid pH 3.3 (Buffer A) and acetonitrile (Buffer B) as follows:
        
            100% buffer A for 15 minutes,
            linear gradient from 100% buffer A and 0% buffer B to 20% buffer A and 80% buffer B over 35 minutes.
        
        
        UV absorbance was monitored at 280 nm. Product retention times were compared to standards obtained from Sigma or OlChemlm.
    
    


The recombinant Tzs and ZmIPT2 proteins were first used to determine DMAPP::AMP isopentenyl transferase activity. FIG. 12A and FIG. 12B show HPLC chromatograms obtained for one of the substrates of the reaction, 5′-AMP (Sigma), and the expected product isopentenyladenosine 5′-monophosphate (iPMP) (OlChemlm). The chromatogram obtained with the IPT (tzs) protein shows that almost all 5′-AMP substrate has been converted to iPMP (FIG. 12C). Similarly, the chromatogram obtained with ZmIPT2 purified protein shows that the enzyme is able to convert 5′-AMP to iPMP but with a lower efficiency than does Agrobacterium IPT since not all the 5′-AMP has been converted (FIG. 12D).
Treatment of reaction products with calf intestine alkaline phosphatase (CAIP) and chromatography using HPLC confirmed the identity of the reaction product iPMP. FIGS. 13A and 13B show chromatograms obtained with Adenosine (Ado) (Sigma) and isopentenyladenosine (iPAR) (Sigma). As expected, after dephosphorylation of the product of each reaction, iPMP was transformed to isopentenyladenosine (iPAR) (FIGS. 13C and 13D) whereas remaining 5′-AMP was transformed to Ado (FIG. 13D). This confirms that ZmIPT2 can metabolize 5′-AMP and DMAPP into iPMP.
Determination of DMAPP::ATP activity was carried out using the same reaction buffer but replacing 5′-AMP by 5′-ATP in the reaction mixture. FIG. 14A shows the chromatogram obtained with 5′-ATP. If ZmIPT2 is able to catalyze the transfer of DMAPP onto 5′ATP, the resulting product should create iPTP. The chromatogram of FIG. 14B shows that all 5′-ATP was metabolized into iPTP by ZmIPT2, suggesting that ZmIPT2 uses 5′-ATP with higher efficiency than 5′-AMP. The reaction product was treated with CAIP to ascertain its identity. Such treatment should yield iPAR. After separation by HPLC, the chromatogram was compared to the chromatogram of an iPAR standard (FIG. 14C). After treatment, the reaction product was transformed to iPAR (FIG. 14D) therefore confirming that ZmIPT2 can metabolize 5′-ATP and DMAPP into iPTP. Altogether these results prove that ZmIPT2 is a cytokinin biosynthetic enzyme preferentially using 5′-ATP as a substrate.
Similar experiments established that 5′-ADP is also a suitable substrate for the encoded enzyme. Taken together, these results prove that ZmIPT2 is a cytokinin biosynthetic enzyme preferentially using 5′-ATP as a substrate. Future experiments will determine the kinetic properties for each substrate using a purified ZmIPT2 protein.
Example 19
Detection of the ZmIPT2 Protein in Developing Kernels
In order to study further the expression pattern of the ZmIPT2 protein, polyclonal antibodies were used for a Western blot experiment. Polyclonal antibodies were raised in rabbit against purified N-terminal His-tagged recombinant ZmIPT2 protein. Fifteen micrograms of proteins extracted from whole kernels harvested at different days after pollination (DAP) were run using SDS-PAGE and blotted on a PVDF membrane. ZmIPT2 proteins were detected using the method of Laemmli (Nature 227:680-685, 1970) with anti-ZmIPT2 polyclonal antibodies as primary antibodies and anti-rabbit IgG antibodies raised in goat conjugated to an alkaline phosphatase as secondary antibodies.
FIG. 15 shows that ZmIPT2 protein levels increase from 0 to 10 DAP, peak at 10 DAP, then decrease from 10 DAP to 15 DAP and stay approximately constant thereafter. This is in agreement with Northern blot results showing that expression of the gene peaks around 10 DAP where it is strong in the pedicel and the endosperm. Although total expression of the gene decreases thereafter, expression levels remain high in the pedicel at later stages. ZmIPT2 protein levels in kernels were very high compared to other organs. Results suggest that the cytokinin activity of ZmIPT2 protein in kernels is most likely controlled at the transcriptional level. The Western blot analysis of protein levels in kernels suggests that the antibodies are very specific to ZmIPT2. Antibodies, together with in situ hybridization, will be very useful in determining the precise site of expression of the gene during kernel development.
Example 20
Ectopic Overexpression of ZmIPT2 in Transgenic Arabidopsis 
Previous examples describe the overexpression of ZmIPT2 in Arabidopsis calli. In order to study the effects of the overexpression of ZmIPT2 at the whole plant level, Arabidopsis plants were transformed with an Agrobacterium tumefaciens strain containing a plasmid comprising the construct 35S-Adhl-ZmIPT2-PinII with the bar herbicide resistance gene as a marker. (Thompson, et al., (1987) EMBO J 6(9):2519-2523; White, et al., (1990) Nucleic Acids Res. 18(4):1062). The simplified Arabidopsis transformation protocol (Clough and Bent, (1998) Plant J. 16:735-743) was used. Seeds were sown in flats containing soil and incubated for 2 days at 4° C. to optimize germination. After 10 days in the greenhouse, transformants were selected by spraying the seedlings daily for 5 days with a 1/1000 dilution of FinaleTM herbicide.
After selection, several plants resistant to the herbicide treatment were identified. Some plants appeared small and dark green compared to others. Leaf greenness is linked to cytokinin levels, suggesting that the dark green transformed plants have elevated levels of cytokinin. Some transgenic plants appeared more affected than others, possibly linked to the level of expression of the transgene which is known to be variable depending on position effects related to insertion in the genome.
At an early stage of development, some transgenic plants showed signs of anthocyanin accumulation in leaves compared to non transgenic plants. Some transgenics had highly serrated leaves compared to wild-type Arabidopsis. This phenotype has previously been reported in Arabidopsis plants over-expressing the Agrobacterium ipt gene (van der Graaff, et al., (2001) Plant Growth Regul. 34(3):305-315). High levels of cytokinin are often detrimental to plant growth (van der Graaff, et al., 2001) and some transgenic plants appeared to struggle in their development compared to control plants. Some transgenics appeared to have a decreased apical dominance in inflorescence stems compared to controls, which was previously reported in Arabidopsis and tobacco plants with high levels of cytokinins (van der Graaff, et al., 2001; Crozier, et al., (2000) Biosynthesis of hormones and ellicitor molecules. In Biochemistry and molecular biology of plants, Buchanan, et al., eds (Rockville, Md.: American Society of Plant Biologists), pp. 850-929)
Some transgenics appeared to have a “bushy” phenotype, most likely due to a larger number of leaves resulting from decreased apical dominance. Some plants also had a poor seed set due to the absence of siliques or smaller siliques with few seeds. They also displayed anthocyanin accumulation in leaves and along inflorescence stems. Plants often displayed serrated cauline leaves. The most extreme phenotype was a transgenic plant with a rosette of approximately 5 mm in diameter with very small curly leaves showing signs of anthocyanin accumulation. The plant was able to flower but never yielded seeds. It also displayed an unusual abundance of large trichomes. Curly leaf phenotype was previously described in tobacco with higher cytokinin levels (Crozier, et al., 2000). Thus, over-expression of the protein in Arabidopsis further confirmed the protein's function by creating a range of phenotypes in agreement with previous attempts to over-express the IPT gene in Arabidopsis and tobacco (Van der Graaff, et al., 2001; Crozier, et al., 2000). The phenotypes observed in several independent transgenic plants are consistent with a phenotype of cytokinin accumulation, confirming that ZmIPT2 is a cytokinin biosynthetic enzyme.
Example 21
Determining Gene Function Through Mu Tagging
Gene function can be further confirmed and described by the study of mutants in which transcription and/or translation of the sequence of interest is disrupted. In certain embodiments this is accomplished through use of methods disclosed in U.S. Pat. No. 5,962,764. The Trait Utility System for Corn (TUSC) is a proprietary resource for selecting gene-specific transposon insertions from a saturated collection of maize mutants created using the Mutator transposable element system. For example, effect of the ZmIPT sequences of the invention on traits such as plant sink strength may be investigated. The following methods were applied to identify and characterize a TUSC mutant for ZmIPT2.
A 1495 bp genomic sequence was supplied for TUSC screening to identify germinal Mutator insertions in the maize IPT2 gene (ZmIPT2). This working annotation of the gene contained a 966 bp open reading frame (ORF; nt83-1048) that is uninterrupted by introns.
Primary screening against TUSC DNA Pools was initiated with two ZmIPT2-specific primers (PHN79087 and PHN79088), each in combination with the Mutator terminal inverted repeat (TIR) primer as described in U.S. Pat. No. 5,962,764. Primer sequences are listed below and provided as SEQ ID Nos: 82-84, respectively.









PHN79087
zmIPT2-F
5′> TGTTGTGTGCACAGAATCGAGCGG <3′


 


PHN79088
zmIPT2-R
5′> CGTCCGCTAGCTACTTATGCATCAG <3′


 


PHN9242
MuTIR
5′> AGAGAAGCCAACGCCAWCGCCTCYATTTC




GTC <3′






Primers were validated prior to use by performing gene-specific amplification of ZmIPT2 using B73 genomic DNA and the PHN79087+79088 primer combination. The control amplification product was excised from an agarose gel and used as a 32P-labeled hybridization probe for the TUSC screening.













expected (re.
observed B73


Primer Pair
reference seq) (bp)
gDNA (bp)







79087 + 79088
1033
~1050









Following successive rounds of screening the TUSC DNA template Pools and Individual samples by PCR and ZmIPT2 hybridization, prospective zmIPT2::Mu alleles were tested for their heritability through the germline. This was achieved by repeating the ZmIPT2::Mu PCR assays against DNA template prepared from 5 kernels of selfed (F2) seed from selected Individual TUSC plants. One TUSC family, PV03_13 H-07 (from Pool 26), showed strong positive results in the F2 template assay for both 79087+9242 and 79088+9242 primer combinations. These PCR products were cloned into Topo-TA vector (Invitrogen) for DNA sequence confirmation of the Mu insertion allele of zmIPT2 harbored by family PV03 13 H-07. Each PCR fragment was expected to be homologous to the ZmIPT2 locus, and also share  ̃71 bp of Mutator TIR homology. An expected 9 bp host site duplication, which is created upon insertion of Mu elements into maize genomic DNA, was also an expected outcome of the PV03 13 H-07 ZmIPT2::Mu allele.
As shown in FIG. 16, DNA sequence characterization of each TUSC PCR product exhibits these expected features. The 79087+9242 PCR product contains 600 bp of direct homology to ZmIPT2 from the left flank of the PV03 13 H-07 Mu insertion site. This product contains the PHN79087 PCR primer site. The 79088+9242 PCR product contains 442 bp of DNA sequence identity with ZmIPT2, representing the right flank of the Mu insertion site, and contains the PHN79088 primer site. When trimmed of Mutator TIR sequences and aligned to the zmIPT2 reference sequence, these PCR fragments overlap by 9 bp (nt 624-632 of the 1495 bp ZmIPT2 reference sequence), representing the expected 9 bp host site duplication created upon insertion of Mutator into ZmIPT2.
Thus, TUSC family PV03 13 H-07 contains a heritable Mutator insertion into the coding sequence (ORF) of the ZmIPT2 gene. This allele is expected to produce a null mutation or “knockout” of the ZmIPT2 locus. F2 progeny seed from PV03 13 H-07, which genetically segregates for the ZmIPT2::Mu mutation, known as zmIPT2-H07, was withdrawn from the TUSC seed bank and propagated for phenotypic and biomolecular analyses.
FIG. 16 graphically summarizes this TUSC result, and the corresponding sequence is provided as SEQ ID NO: 85.
As added characterization, BLAST searches of the MuTIR portions of each zmIPT2::Mu PCR product were conducted to ascribe an identity for the Mu element residing at the ZmIPT2 locus. TUSC PCR products amplified with the Mutator PHN9242 primer contain 39 bp of flanking TIR sequence that are specific to the resident element being amplified. BLAST results are consistent with the zmIPT2::Mu element being either a Mu4 or a Mu3 element.







>IPT2_TIR_L


(SEQ ID NO: 86)


GAGATAATTGCCATTATAGAAGAAGAGAGAAGGGGATTCGACGAAATA


GAGGCGATGGCGTTGGCTTCTCT


 


>IPT2_TIR_R


(SEQ ID NO: 87)


AAGCCAACGCCAACGCCTCTATTTCGTCGAATCCCCTTCTCTCTTCTT


CTATAATGGCAATTATCTC






In certain embodiments the nucleic acid constructs of the present invention can be used in combination (“stacked”) with other polynucleotide sequences of interest in order to create plants with a desired phenotype. The polynucleotides of the present invention may be stacked with any gene or combination of genes, and the combinations generated can include multiple copies of any one or more of the polynucleotides of interest. The desired combination may affect one or more traits; that is, certain combinations may be created for modulation of gene expression affecting cytokinin activity. For example, up-regulation of cytokinin synthesis may be combined with down-regulation of cytokinin degradation. Other combinations may be designed to produce plants with a variety of desired traits, such as those previously described.
All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

## Claims

1. A plant comprising a polynucleotide comprising a nucleotide sequence operably linked to a heterologous regulatory sequence, wherein the nucleotide sequence encodes an amino acid sequence that is at least 95% identical to SEQ ID NO: 6, and wherein said amino acid sequence has cytokinin synthesis activity.

2. The plant of claim 1, wherein said regulatory sequence comprises a tissue-preferred promoter, a constitutive promoter, or an inducible promoter.

3. The plant of claim 2, wherein said tissue-preferred promoter is a root-preferred promoter, a leaf-preferred promoter, a shoot-preferred promoter, or an inflorescence-preferred promoter.

4. The plant of claim 1, wherein said regulatory sequence is stress-insensitive or stress-induced and is preferentially expressed in a tissue of the developing seed or related maternal tissue at or about the time of anthesis.

5. A seed of the plant of claim 1, wherein said seed comprises said polynucleotide.

6. The plant of claim 1, wherein said plant is maize, wheat, rice, barley, sorghum, or rye.