# Patent

## Title
Plastidial nucleotide sugar epimerases

## Abstract

Isolated polynucleotides and polypeptides and recombinant DNA constructs of plastidial sugar epimerases useful for conferring improved agronomic performance including yield and drought are disclosed. Compositions (such as plants or seeds) having these recombinant DNA constructs, and methods utilizing these recombinant DNA constructs are also disclosed.

## Background
CROSS-REFERENCE
This utility application is a national phase application of application of PCT/CN2011/000852 filed May 16, 2011, which claims priority to CN patent application 201010178405.6 which was filed May 17, 2010, both of which are incorporated herein by reference.
FIELD
The field relates to plant breeding and genetics and, in particular, relates to recombinant DNA constructs useful in plants for conferring tolerance to drought and increase in yield.
BACKGROUND
Abiotic stress is the primary cause of crop loss worldwide, causing average yield losses of more than 50% for major crops (Boyer, J. S. (1982) Science 218:443-448; Bray, E. A. et al. (2000) In Biochemistry and Molecular Biology of Plants, Edited by Buchannan, B. B. et al., Amer. Soc. Plant Biol., pp. 1158-1249). Among the various abiotic stresses, drought is a major factor that limits crop yield worldwide. Exposure of plants to a water-limiting environment during various developmental stages appears to activate various physiological and developmental changes. Understanding of the basic biochemical and molecular mechanism for drought stress perception, transduction and tolerance is a major challenge in biology.
Photosynthetic reactions in higher plants depend on chloroplast thylakoid membrane system. Chloroplast thylakoid assembly and maintenance require a continuous supply of membrane constituents. Galactose-containing glycerolipids are predominant lipid components of photosynthetic membranes in plants, algae, and cyanobacteria. The two most common galactolipids are mono- and digalactosyldiacylglycerol (MGDG and DGDG), which account for about 50 and 25 mol % of total thylakoid lipids, respectively. About 80% of all plant lipids are associated with photosynthetic membranes, and MGDG is considered to be the most abundant membrane lipid on earth. Galactolipids play an important role in the organization of photosynthetic membranes and in their photosynthetic activities.
In plants, MGDG is synthesized in two unique steps: (i) the conversion of UDP-D-glucose (UDP-Glc) into UDP-D-galactose (UDP-Gal) by an UDP-glucose 4-epimerase (UGE), and (ii) the transfer of a galactosyl residue from UDP-Gal to diacylglycerol (DAG) for synthesis of the final product by MGDG synthase (MGD1). MGD1 is localized in the inner chloroplast envelope membrane and uses UDP-Gal as a substrate.
Plants possess a sophisticated sugar biosynthetic machinery comprising families of nucleotide sugars that can be modified at their glycosyl moieties by nucleotide sugar interconversion enzymes to generate different sugars. UDP-glucose 4-epimerase (also UDP-galactose 4-epimerase, UGE; EC 5.1.3.2) catalyzes the interconversion of UDP-Glc and UDP-Gal. UGEs identified from plants lack transmembrane motifs and signal peptides and appear to exist as soluble entities in the cytoplasm. Generally, plant UDP-Glc epimerase enzymes are localized to the cytosol, where their substrates UDP-Glc and UDP-Gal are present at high levels. As a precursor for the synthesis of the galactolipid MGDG in chloroplasts, UDP-Gal is generally thought to be mobilized from the cytosol, because the UDP-Gal concentration is relatively low within plastids and MGDG synthase (MGD1) is associated with the inner envelope membrane
To gain insight into genes controlling photosynthetic activity and carbon assimilation in plants, a rice stunted growth mutant (phd1) with decreased photoassimilate and yield production was identified. A novel chloroplast-localized UDP-Glc epimerase involved in UDP-Gal supply for chloroplast galactolipid biosynthesis during photosynthetic membrane biogenesis is disclosed herein.
SUMMARY
Plastidial UDP glucose epimerase, its homologs and methods of use are disclosed. Transgenic expression of PHD1 increased photosynthetic activity and enhanced growth. Roles of PHD1, homologs, and functional fragments thereof in photosynthetic capability and carbon assimilate homeostasis are discussed herein.
The present disclosure includes:
In one embodiment, a plant comprising in its genome a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory element, wherein said polynucleotide comprises a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding a polypeptide with plastidial epimerase activity, wherein the polypeptide has an amino acid sequence of at least 60%, 80%, 85%, 90%, 95% or 100% sequence identity, based on the Clustal V method of alignment with pairwise alignment default parameters of KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5, when compared to SEQ ID NO: 1-17 (b) a nucleotide sequence encoding a polypeptide with epimerase activity, wherein the nucleotide sequence is hybridizable under stringent conditions with a DNA molecule comprising the full complement of SEQ ID NO: 18-34 (c) a nucleotide sequence encoding a polypeptide with epimerase activity, wherein the nucleotide sequence is derived from SEQ ID NO: 18-34 by alteration of one or more nucleotides by at least one method selected from the group consisting of: deletion, substitution, addition and insertion; (d) a nucleotide sequence encoding a polypeptide wherein the amino acid sequence of the polypeptide comprises SEQ ID NO: 1 and (e) a nucleotide sequence comprising SEQ ID NO: 18 and wherein said plant exhibits increased drought tolerance when compared to a control plant not comprising said recombinant DNA construct. The plant may be a monocot or dicot.
In an embodiment, the PHD1 polypeptide does not have the N-terminal chloroplast transit peptide 1-62 amino acids of SEQ ID NO: 1 or the corresponding equivalent in other PHD1 homologs. For example, a nucleotide molecule substantially lacking the region encoding the chloroplast transit peptide is expressed in a plant cell, for example, in the plastids.
In an embodiment, the chloroplast transit peptide (1-62 amino acids of SEQ ID NO: 1 or a sequence that is substantially similar to the 62-amino acid N-terminal region of SEQ ID NO: 1) is fused to a heterologous peptide for transport of the expressed protein/peptide into the chloroplast.
In another embodiment, a plant comprising in its genome a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory element, wherein said polynucleotide comprises a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding a polypeptide with epimerase activity, wherein the polypeptide has an amino acid sequence of at least 60%, 80%, 85%, 90%, 95% or 100% sequence identity, based on the Clustal V method of alignment with pairwise alignment default parameters of KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5, when compared to SEQ ID NO: 1-17 (b) a nucleotide sequence encoding a polypeptide with epimerase activity, wherein the nucleotide sequence is hybridizable under stringent conditions with a DNA molecule comprising the full complement of SEQ ID NO: 18-34 (c) a nucleotide sequence encoding a polypeptide with epimerase activity, wherein the nucleotide sequence is derived from SEQ ID NO: 18-34 by alteration of one or more nucleotides by at least one method selected from the group consisting of: deletion, substitution, addition and insertion; (d) a nucleotide sequence encoding a polypeptide wherein the amino acid sequence of the polypeptide comprises SEQ ID NO: 1 and (e) a nucleotide sequence comprising SEQ ID NO: 18 and wherein said plant exhibits an increase in yield when compared to a control plant not comprising said recombinant DNA construct. The plant may exhibit said increase in yield when compared, under water limiting conditions, to said control plant not comprising said recombinant DNA construct. The plant may be a monocot or dicot.
In another embodiment, a method of increasing drought tolerance in a plant, comprising: (a) introducing into a regenerable plant cell a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory element, wherein said polynucleotide comprises a nucleotide sequence selected from the group consisting of: (i) a nucleotide sequence encoding a polypeptide with epimerase activity, wherein the polypeptide has an amino acid sequence of at least 60%, 80%, 85%, 90%, 95% or 100% sequence identity, based on the Clustal V method of alignment with pairwise alignment default parameters of KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5, when compared to SEQ ID NO: 1-17 (ii) a nucleotide sequence encoding a polypeptide with epimerase activity, wherein the nucleotide sequence is hybridizable under stringent conditions with a DNA molecule comprising the full complement of SEQ ID NO: 18-34 (iii) a nucleotide sequence encoding a polypeptide with epimerase activity, wherein the nucleotide sequence is derived from SEQ ID NO: 18-34 by alteration of one or more nucleotides by at least one method selected from the group consisting of: deletion, substitution, addition and insertion; (iv) a nucleotide sequence encoding a polypeptide wherein the amino acid sequence of the polypeptide comprises SEQ ID NO: 1 and (v) a nucleotide sequence comprising SEQ ID NO: 18 and (b) regenerating a transgenic plant from the regenerable plant cell after step (a), wherein the transgenic plant comprises in its genome the recombinant DNA construct and exhibits increased drought tolerance when compared to a control plant not comprising the recombinant DNA construct. The method may further comprise: (c) obtaining a progeny plant derived from the transgenic plant, wherein said progeny plant comprises in its genome the recombinant DNA construct and exhibits increased drought tolerance when compared to a control plant not comprising the recombinant DNA construct.
In another embodiment, a method of evaluating drought tolerance in a plant, comprising: (a) obtaining a transgenic plant, wherein the transgenic plant comprises in its genome a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory element, wherein said polynucleotide comprises a nucleotide sequence selected from the group consisting of: (i) a nucleotide sequence encoding a polypeptide with epimerase activity, wherein the polypeptide has an amino acid sequence of at least 60%, 80%, 85%, 90%, 95% or 100% sequence identity, based on the Clustal V method of alignment with pairwise alignment default parameters of KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5, when compared to SEQ ID NO: 1-17 (ii) a nucleotide sequence encoding a polypeptide with epimerase activity, wherein the nucleotide sequence is hybridizable under stringent conditions with a DNA molecule comprising the full complement of SEQ ID NO: 18-34 (iii) a nucleotide sequence encoding a polypeptide with epimerase activity, wherein the nucleotide sequence is derived from SEQ ID NO: 18-34 by alteration of one or more nucleotides by at least one method selected from the group consisting of: deletion, substitution, addition and insertion; (iv) a nucleotide sequence encoding a polypeptide wherein the amino acid sequence of the polypeptide comprises SEQ ID NO: 1 and (v) a nucleotide sequence comprising SEQ ID NO: 18 and (b) obtaining a progeny plant derived from the transgenic plant of (a), wherein the progeny plant comprises in its genome the recombinant DNA construct; and (c) evaluating the progeny plant for drought tolerance compared to a control plant not comprising the recombinant DNA construct.
In another embodiment, a method of determining an alteration of an agronomic characteristic in a plant, comprising: (a) obtaining a transgenic plant, wherein the transgenic plant comprises in its genome a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory element, wherein said polynucleotide comprises a nucleotide sequence selected from the group consisting of: (i) a nucleotide sequence encoding a polypeptide with epimerase activity, wherein the polypeptide has an amino acid sequence of at least 60%, 80%, 85%, 90%, 95% or 100% sequence identity, based on the Clustal V method of alignment with pairwise alignment default parameters of KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5, when compared to SEQ ID NO: 1-17 (ii) a nucleotide sequence encoding a polypeptide with epimerase activity, wherein the nucleotide sequence is hybridizable under stringent conditions with a DNA molecule comprising the full complement of SEQ ID NO: 18-34 (iii) a nucleotide sequence encoding a polypeptide with epimerase activity, wherein the nucleotide sequence is derived from SEQ ID NO: 18-34 by alteration of one or more nucleotides by at least one method selected from the group consisting of: deletion, substitution, addition and insertion; (iv) a nucleotide sequence encoding a polypeptide wherein the amino acid sequence of the polypeptide comprises SEQ ID NO: 1 and (v) a nucleotide sequence comprising SEQ ID NO: 18 and (b) obtaining a progeny plant derived from the transgenic plant of step (a), wherein the progeny plant comprises in its genome the recombinant DNA construct; and (c) determining whether the progeny plant exhibits an alteration of at least one agronomic characteristic when compared to a control plant not comprising the recombinant DNA construct. Said determining step (c) may comprise determining whether the transgenic plant exhibits an alteration of at least one agronomic characteristic when compared, under water limiting conditions, to a control plant not comprising the recombinant DNA construct. Said at least one agronomic trait may be yield and furthermore may be an increase in yield.
In another embodiment, an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding a polypeptide with epimerase activity, wherein the polypeptide has an amino acid sequence of at least 60%, 80%, 85%, 90% or 95% sequence identity, based on the Clustal V method of alignment with pairwise alignment default parameters of KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5, when compared to SEQ ID NO: 1-17 (b) a nucleotide sequence encoding a polypeptide with epimerase activity, wherein the nucleotide sequence is hybridizable under stringent conditions with a DNA molecule comprising the full complement of SEQ ID NO: 18-34 (c) a nucleotide sequence encoding a polypeptide with epimerase activity, wherein the nucleotide sequence is derived from SEQ ID NO: 18-34 by alteration of one or more nucleotides by at least one method selected from the group consisting of: deletion, substitution, addition and insertion; (d) a nucleotide sequence encoding a polypeptide wherein the amino acid sequence of the polypeptide comprises SEQ ID NO: 1 and (e) a nucleotide sequence comprising SEQ ID NO: 18.
In another embodiment, an isolated polynucleotide comprising the full complement of the nucleotide sequence of the disclosure, wherein the full complement and the nucleotide sequence of the disclosure consist of the same number of nucleotides and are 100% complementary.
In another embodiment, a recombinant DNA construct comprising the isolated polynucleotide of the disclosure operably linked to at least one regulatory element.
In another embodiment, a cell comprising the recombinant DNA construct of the disclosure, wherein the cell is selected from the group consisting of a bacterial cell, a yeast cell, and insect cell and a plant cell.
In another embodiment, a plant or a seed comprising the recombinant DNA construct of the disclosure. The plant or seed may be a monocot or a dicot plant or seed.
In another embodiment, a method for isolating a polypeptide encoded by the recombinant DNA construct of the disclosure, wherein the method comprises the following: (a) transforming a cell with the recombinant DNA construct of the disclosure; (b) growing the transformed cell of step (a) under conditions suitable for expression of the recombinant DNA construct; and (c) isolating the polypeptide from the transformed cell of step (b).
In another embodiment, a vector comprising the polynucleotide of the disclosure.
In another embodiment, a method for producing a transgenic plant comprising transforming a plant cell with the recombinant DNA construct of the disclosure and regenerating a transgenic plant from the transformed plant cell.
In another embodiment, the present disclosure includes any of the plants of the present disclosure wherein the plant is selected from the group consisting of: maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, millet, sugarcane, switchgrass, tobacco, potato and sugar beet.
In another embodiment, the present disclosure includes any of the methods of the present disclosure wherein the plant is selected from the group consisting of: maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, millet, sugarcane, switchgrass, tobacco, potato and sugar beet.
In another embodiment, the present disclosure includes seed of any of the plants of the present disclosure, wherein said seed comprises in its genome a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory element, wherein said polynucleotide encodes a polypeptide having an amino acid sequence of at least 60% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 1, and wherein a plant produced from said seed exhibits either an increased drought tolerance, or an increase in yield, or both, when compared to a control plant not comprising said recombinant DNA construct.



BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTING
The disclosure can be more fully understood from the following detailed description and the accompanying drawings and Sequence Listing which form a part of this application.
FIG. 1 shows phd1 mutant phenotypes. (A) Two-week-old seedlings grown on MS media. (B and C) Growth phenotype of 2-month-old (B) and 4-month-old (C) plants grown in a paddy field. (D) The harvested panicles showed a reduced seed-setting ratio for phd1-1. (A-D) wild type (left) and phd1-1 (right). (E-G) Quantification of the agricultural traits of tiller number (E), seed setting ration (F), and grain weight per plant (G). Each bar is the mean±SD from 30 replicate samples. (H, I) Diurnal changes in sucrose (H) and starch (I) content of phd1-1 and wild type. Mature leaves of individual wild type and phd1-1 plants at the anthesis stage were harvested and immediately frozen in liquid nitrogen. Each point is the mean±SD from ten replicate samples.
FIG. 2 shows the molecular identification of PHD1. (A) Structure of the PHD1 gene and its mutation sites in three phd1 alleles. The PHD1 gene consists of nine exons (green boxes) and eight introns (gray lines). Nucleotide insertion and substitutions in the three phd1 alleles are indicated. (B, C) Functional complementation of the phd1 mutant. (B) Phenotypes of wild type, phd1-1, and the complemented line phd1-1+PHD1 plants at the tillering stage. Expression level of PHD1 transcripts was detected by semi-quantitative RT-PCR. (C) Sucrose and starch content in flag leaves of wild type, phd1-1, and the complemented line phd1-1+PHD1 plants at noon of the day at the anthesis stage. Error bars represent SD of eight different individuals. *significant difference between phd1-1 mutant and wild type (P=0.05).
FIG. 3 shows the phylogenetic analysis of PHD1. MEGA4 Neighbor-Joining tree was inferred from the amino add sequences of the PHD1 (Os01g0367100) homologs among green plants. Bootstrap values are based on 1000 replications and are indicated in their respective nodes. The scale bar indicates genetic distance based on branch length. The alignment for the constructed tree is shown in Fig. S3.
FIG. 4 shows PHD1 expression analysis and PHD1 protein subcellular localization. (A) RNA gel blot analysis of the PHD1 gene in roots, culms, flowers, leaf blades, and leaf sheaths just before the anthesis stage. (B-E) PHD1 expression patterns detected by mRNA in situ hybridization. The PHD1 signal was detected at the shoot apical meristem and young leaves (B), leaf mesophyll cells around vascular bundles (C), young inflorescences (D), and axillary buds (E). Bars=150 μm in (B), (C), and (E), and 500 μm in (D). (F) Rice protoplasts transformed with 35S::GFP (lower panel) and 35S::PHD1-GFP (upper panel) plasmids. Chlorophyll autofluorescence (middle); GFP fluorescence (left); merged images (right). Bars=5 μm.
FIG. 5 shows the expression analysis of key genes involved in UDP-Gal biosynthesis and carbohydrate allocation in leaves of phd1-1 plants. (A) The expression of genes involved in the UDP-Gal biosynthesis pathway was upregulated in phd1-1. (B) The expression of starch biosynthesis genes was down-regulated in phd1-1. (C) The expression of sucrose cleavage genes was upregulated in phd1-1. Expression values are displayed as the ratio of expression to rice 18S RNA (mean±SE). All assays were carried out with three biological replicates.
FIG. 6 shows UDP-sugar content and glycolipid composition in phd1 plants. (A) UDP-Glc and UDP-Gal content in leaves of wild type, phd1-1, and PHD1-complemented plants. The values represent the means±SE of six independent experiments. (B) Polar lipid composition in leaves of wild type, phd1-1, and PHD1-complemented plants grown in paddy fields. Glycerolipids were quantified by GC of leaf lipids separated by TLC. The bars show lipid composition in mol % and indicate means±SD of three measurements. UDP-Glc, UDP-glucose; UDP-Gal, UDP-galactose; FW, fresh weight; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; SQDG, sulfoquinovosyldiacylglycerol.
FIG. 7 shows the improved agricultural traits of transgenic rice overexpressing PHD1. (A) Phenotypic differences of wild type, phd1-1, and transgenic line S3 at the grain-filling stage grown in paddy fields. (B) Immunoblot analysis of PHD1 protein expression in wild type, phd1-1, and PHD1 overexpressing transgenic lines (S3, S5, and S8). Tubulin is shown as loading control. Increased accumulation of PHD1 protein was observed in transgenic lines. (C,D) Grain yield per plant (C) and dry weight of vegetative organs after harvesting (D) were increased in transgenic plants. Values are means±SD from at least 30 plants/line, *Significant difference (P<0.05).
FIG. 8 shows a schematic model for the role of PHD1 in the galactolipid biosynthetic pathway for chloroplast membranes. Biosynthetic schemes for two glycoglycerolipids under normal growth conditions are indicated along with the pathway involving PHD1. Glc-1-P, glucose-1-phosphate; UGP, UDP-glucose pyrophosphorylase; DAG, diacylglycerol; UTP, uridine-5′-triphosphate; PPi, pyrophosphate.
FIG. 9 shows the diurnal changes in hexose concentration of phd1-1 and WT. The mature leaves of individual wild type (● black symbols with solid lines) and phd1-1 (∘ empty symbols with broken lines) plants were harvested and immediately frozen in liquid N2. Each point is the mean±standard deviation from ten replicate samples.
FIG. 10 shows PHD1 transcript level in wild type and three allelic phd1 mutants. The equal abundance of RNA among samples was confirmed by RT-PCR detection of ACTIN1 transcripts. phd1-1 to −3, three allelic phd1 mutant lines; S3-1, PHD1 overexpressing transgenic line.
FIG. 11 is a comparison (SEQ ID NOS: 1, 11, 12, 13, 14, 2, 4, 3, 5, 7, 9, 6, 8, 10, 15, 16 and 17, consecutively) of the seventeen conserved regions from PHD1 and the green plant homolog sequences. The sequences were searched using BLASTP and aligned using CLUSTALW. Identical amino acid residues are boxed, and similar residues are shaded. The red bar indicates the conserved motif GXGXXG (NAD+-binding), and catalytic amino acid residues of the active site are boxed in red. PHD1: Os01g0367100.
FIG. 12 shows biochemical function and genetic complement assay of PHD1. (A) UGE activity assay of PHD1 in vitro. Lineweaver-Burk plots of purified recombinant PHD1 UGE activity at 30° C. (▪) and at 37° C. (□). (B) PHD1 can complement a S. cerevisiae gal10 mutant. A yeast gall 0 mutant strain was transformed with plasmids containing PHD1 cDNAs, and grown on either glucose or galactose medium.



The sequence descriptions and Sequence Listing attached hereto comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. §1.821-1.825.
The Sequence Listing contains the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the IUPAC-IUBMB standards described in Nucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219 (No. 2):345-373 (1984) which are herein incorporated by reference. The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.
DETAILED DESCRIPTION
PHD1 encodes a novel rice plastidial UGE involved in the biosynthesis of chloroplast galactolipids. A mutation in PHD1 lead to disturbed carbon assimilation homeostasis and impaired photosynthetic efficiency. PHD1 encodes for an active epimerase that is localized to chloroplasts, and therefore that the UDP-Gal substrate for MGDG biosynthesis can be generated in situ in chloroplasts (FIG. 8). This surprising result provides a genetic and biochemical framework to engineer the novel functional mechanism of this UGE in plastids, and to evaluate the role of galactolipids in photosynthetic activity of plants including rice.
MGD1 is considered to be the major isoform of MGDG synthases mostly important for thylakoid membrane biogenesis. In Arabidopsis, two more MGDG synthases, MGD2 and MGD3, are targeted to the outer chloroplast envelope where substrates can be recruited from the cytosol. MGDG generated by them can move from the outer to the inner envelope and to the thylakoids. Herein, it is shown that compared to wild type, the relative amount of the major galactolipid MGDG in phd1-1 was reduced by 19%, whereas that of DGDG was only slightly decreased by 2.5%. However, slight increases in the mol % amounts of several phospholipids were found to compensate for the approximately 7 mol % of galactolipids lost in the phd1-1 mutant. These results are consistent with the notion that the amounts of glycolipids and phospholipids are reciprocally controlled in plants to maintain proper balance of lipids in the thylakoid membrane.
Most galactolipids are restricted to plastid membranes during normal growth and development, however, DGDG can also be found in extraplastidic membranes following phosphate (Pi) starvation. In addition, x-ray crystallographic analyses of photosynthetic proteins in cyanobacteria revealed that MGDG is associated with the core of the reaction centers of both photosystems I and II (PSI and PSII) indicating that these lipids are required not only as bulk constituents of photosynthetic membranes, but also for the photosynthetic reaction itself. The effective quantum yield of photochemical energy conversion in photosystem II (φPSII) was reduced in the phd1-1 mutant. Seedlings lacking MGDG were shown to have disrupted photosynthetic membranes, leading to a complete impairment of photosynthetic ability and photoautotrophic growth. A reduction of MGDG to 80% of wild type levels in the phd1-1 mutant led to the dramatic phenotype of retarded growth, reduced photosynthetic capability, and decreased photoassimilate accumulation. The stunted growth phenotype of phd1-1 mutants may be due to an insufficient provision of membrane building blocks to support chloroplast proliferation during plant growth, which is also consistent with the reduced size of chloroplasts in mutant plants. These effects may be due to a reduction of the absolute amount of MGDG or a reduced galactolipid to phospholipid ratio in chloroplast membranes.
In plants, starch acts as a depository for reduced carbon produced in leaves during the day, and as a supply of chemical energy and anabolic source molecules during the night. In the phd1-1 mutant, expression levels of starch biosynthesis genes such as AGP, SS, GBSS, and BE, were significantly downregulated in source leaves, leading to a sharp decrease of starch content. However, the reduced starch did not result in increased sucrose levels, because activation of sucrose cleavage genes SuSy1 and INV1/3 resulted in reduced sucrose and increased hexose-phosphate and UDP-Glc levels. Therefore, sucrose as the main transport form of photoassimilate produced in source organs was not able to export efficiently to the sink organs. Moreover, a large amount of UDP-Glc catalyzed by SuSy1 or UGP2 would be converted to UDP-Gal by cytosolic OsUGE1/4 and transported into chloroplast as galactosyl donors of chloroplast glycolipids to compensate for the loss of PHD1 activity in the phd1-1 mutant. In contrast, PHD1 overexpression in rice, which would enhance PHD1 activity in chloroplasts and may increase the relative amount of MGDG and increase the effective quantum yield of photochemical energy conversion in thylakoid membranes, resulted in increased photosynthetic efficiency and growth rate, implicating a key role of PHD1 for the photosynthetic system in plants including rice. These improvements of both biomass production and grain yield have significant economic implications in both traditional crop improvement and bioenergy crop production.
The disclosure of each reference set forth herein is hereby incorporated by reference in its entirety.
As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to “a plant” includes a plurality of such plants, reference to “a cell” includes one or more cells and equivalents thereof known to those skilled in the art, and so forth.
As used herein:
“Epimerase activity” of a polypeptide indicates that peptide is capable of performing the catalysis of UDP-Gal to UDP-Glc.
The terms “monocot” and “monocotyledonous plant” are used interchangeably herein. A monocot of the current disclosure includes the Gramineae.
The terms “dicot” and “dicotyledonous plant” are used interchangeably herein. A dicot of the current disclosure includes the following families: Brassicaceae, Leguminosae, and Solanaceae.
The terms “full complement” and “full-length complement” are used interchangeably herein, and refer to a complement of a given nucleotide sequence, wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary.
“Arabidopsis” and “Arabidopsis thaliana” are used interchangeably herein, unless otherwise indicated.
An “Expressed Sequence Tag” (“EST”) is a DNA sequence derived from a cDNA library and therefore is a sequence which has been transcribed. An EST is typically obtained by a single sequencing pass of a cDNA insert. The sequence of an entire cDNA insert is termed the “Full-Insert Sequence” (“FIS”). A “Contig” sequence is a sequence assembled from two or more sequences that can be selected from, but not limited to, the group consisting of an EST, FIS and PCR sequence. A sequence encoding an entire or functional protein is termed a “Complete Gene Sequence” (“CGS”) and can be derived from an FIS or a contig.
“Agronomic characteristic” is a measurable parameter including but not limited to, greenness, yield, growth rate, biomass, fresh weight at maturation, dry weight at maturation, fruit yield, seed yield, total plant nitrogen content, fruit nitrogen content, seed nitrogen content, nitrogen content in a vegetative tissue, total plant free amino acid content, fruit free amino acid content, seed free amino acid content, free amino acid content in a vegetative tissue, total plant protein content, fruit protein content, seed protein content, protein content in a vegetative tissue, drought tolerance, nitrogen uptake, root lodging, harvest index, stalk lodging, plant height, ear height, ear length, salt tolerance, early seedling vigor and seedling emergence under low temperature stress.
“Transgenic” refers to any cell, cell line, callus, tissue, plant part or plant, the genome of which has been altered by the presence of a heterologous nucleic acid, such as a recombinant DNA construct, including those initial transgenic events as well as those created by sexual crosses or asexual propagation from the initial transgenic event. The term “transgenic” as used herein does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.
“Genome” as it applies to plant cells encompasses not only chromosomal DNA found within the nucleus, but organelle DNA found within subcellular components (e.g., mitochondrial, plastid) of the cell.
“Plant” includes reference to whole plants, plant organs, plant tissues, seeds and plant cells and progeny of same. Plant cells include, without limitation, cells from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores.
“Progeny” comprises any subsequent generation of a plant.
“Transgenic plant” includes reference to a plant which comprises within its genome a heterologous polynucleotide. For example, the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant DNA construct.
“Heterologous” with respect to sequence means 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.
“Polynucleotide”, “nucleic acid sequence”, “nucleotide sequence”, or “nucleic acid fragment” are used interchangeably and is a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. Nucleotides (usually found in their 5′-monophosphate form) are referred to by their single letter designation as follows: “A” for adenylate or deoxyadenylate (for RNA or DNA, respectively), “C” for cytidylate or deoxycytidylate, “G” for guanylate or deoxyguanylate, “U” for uridylate, “T” for deoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines (C or T), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N” for any nucleotide.
“Polypeptide”, “peptide”, “amino acid sequence” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The terms “polypeptide”, “peptide”, “amino acid sequence”, and “protein” are also inclusive of modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation.
“Messenger RNA (mRNA)” refers to the RNA that is without introns and that can be translated into protein by the cell.
“cDNA” refers to a DNA that is complementary to and synthesized from a mRNA template using the enzyme reverse transcriptase. The cDNA can be single-stranded or converted into the double-stranded form using the Klenow fragment of DNA polymerase I.
“Mature” protein refers to a post-translationally processed polypeptide; i.e., one from which any pre- or pro-peptides present in the primary translation product have been removed.
“Precursor” protein refers to the primary product of translation of mRNA; i.e., with pre- and pro-peptides still present. Pre- and pro-peptides may be and are not limited to intracellular localization signals.
“Isolated” refers to materials, such as nucleic acid molecules and/or proteins, which are substantially free or otherwise removed from components that normally accompany or interact with the materials in a naturally occurring environment. Isolated polynucleotides may be purified from a host cell in which they naturally occur. Conventional nucleic acid purification methods known to skilled artisans may be used to obtain isolated polynucleotides. The term also embraces recombinant polynucleotides and chemically synthesized polynucleotides.
“Recombinant” refers to an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques. “Recombinant” also includes reference to a cell or vector, that has been modified by the introduction of a heterologous nucleic acid or a cell derived from a cell so modified, but does not encompass the alteration of the cell or vector by naturally occurring events (e.g., spontaneous mutation, natural transformation/transduction/transposition) such as those occurring without deliberate human intervention.
“Recombinant DNA construct” refers to a combination of nucleic acid fragments that are not normally found together in nature. Accordingly, a recombinant DNA construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that normally found in nature.
The terms “entry clone” and “entry vector” are used interchangeably herein.
“Regulatory sequences” refer to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include, but are not limited to, promoters, translation leader sequences, introns, and polyadenylation recognition sequences. The terms “regulatory sequence” and “regulatory element” are used interchangeably herein.
“Promoter” refers to a nucleic acid fragment capable of controlling transcription of another nucleic acid fragment.
“Promoter functional in a plant” is a promoter capable of controlling transcription in plant cells whether or not its origin is from a plant cell.
“Tissue-specific promoter” and “tissue-preferred promoter” are used interchangeably, and refer to a promoter that is expressed predominantly but not necessarily exclusively in one tissue or organ, but that may also be expressed in one specific cell.
“Developmentally regulated promoter” refers to a promoter whose activity is determined by developmental events.
“Operably linked” refers to the association of nucleic acid fragments in a single fragment so that the function of one is regulated by the other. For example, a promoter is operably linked with a nucleic acid fragment when it is capable of regulating the transcription of that nucleic acid fragment.
“Expression” refers to the production of a functional product. For example, expression of a nucleic acid fragment may refer to transcription of the nucleic acid fragment (e.g., transcription resulting in mRNA or functional RNA) and/or translation of mRNA into a precursor or mature protein.
“Phenotype” means the detectable characteristics of a cell or organism.
“Introduced” in the context of inserting a nucleic acid fragment (e.g., a recombinant DNA construct) into a cell, means “transfection” or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid fragment into a eukaryotic or prokaryotic cell where the nucleic acid fragment may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
A “transformed cell” is any cell into which a nucleic acid fragment (e.g., a recombinant DNA construct) has been introduced.
“Transformation” as used herein refers to both stable transformation and transient transformation.
“Stable transformation” refers to the introduction of a nucleic acid fragment into a genome of a host organism resulting in genetically stable inheritance. Once stably transformed, the nucleic acid fragment is stably integrated in the genome of the host organism and any subsequent generation.
“Transient transformation” refers to the introduction of a nucleic acid fragment into the nucleus, or DNA-containing organelle, of a host organism resulting in gene expression without genetically stable inheritance.
“Allele” is one of several alternative forms of a gene occupying a given locus on a chromosome. When the alleles present at a given locus on a pair of homologous chromosomes in a diploid plant are the same that plant is homozygous at that locus. If the alleles present at a given locus on a pair of homologous chromosomes in a diploid plant differ that plant is heterozygous at that locus. If a transgene is present on one of a pair of homologous chromosomes in a diploid plant that plant is hemizygous at that locus.
A “chloroplast transit peptide” is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the chloroplast or other plastid types present in the cell in which the protein is made. “Chloroplast transit sequence” refers to a nucleotide sequence that encodes a chloroplast transit peptide. A “signal peptide” is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the secretory system (Chrispeels (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the protein is to be directed to a vacuole, a vacuolar targeting signal (supra) can further be added, or if to the endoplasmic reticulum, an endoplasmic reticulum retention signal (supra) may be added. If the protein is to be directed to the nucleus, any signal peptide present should be removed and instead a nuclear localization signal included (Raikhel (1992) Plant Phys. 100:1627-1632). A “mitochondrial signal peptide” is an amino acid sequence which directs a precursor protein into the mitochondria (Zhang and Glaser (2002) Trends Plant Sci 7:14-21).
The percent identity between two amino acid or nucleic acid sequences may be determined by visual inspection and mathematical calculation.
Alternatively, sequence alignments and percent identity calculations may be determined using a variety of comparison methods designed to detect homologous sequences including, but not limited to, the MEGALIGN® program of the LASERGENE® bioinformatics computing suite (DNASTAR® Inc., Madison, Wis.). Unless stated otherwise, multiple alignment of the sequences provided herein were performed using the Clustal V method of alignment (Higgins and Sharp (1989) CABIOS. 5:151 153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments and calculation of percent identity of protein sequences using the Clustal V method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignment of the sequences, using the Clustal V program, it is possible to obtain “percent identity” and “divergence” values by viewing the “sequence distances” table on the same program; unless stated otherwise, percent identities and divergences provided and claimed herein were calculated in this manner.
Alternatively, the percent identity of two protein sequences may be determined by comparing sequence information based on the algorithm of Needleman, S. B. and Wunsch, C. D. (J. Mol. Biol., 48:443-453, 1970) and using the GAP computer program available from the University of Wisconsin Genetics Computer Group (UWGCG). The preferred default parameters for the GAP program include: (1) a scoring matrix, blosum62, as described by Henikoff, S, and Henikoff, J. G. (Proc. Natl. Acad. Sci. USA, 89:10915-10919, 1992); (2) a gap weight of 12; (3) a gap length weight of 4; and (4) no penalty for end gaps.
Other programs used by those skilled in the art of sequence comparison may also be used. The percent identity can be determined by comparing sequence information using, e.g., the BLAST program described by Altschul et al. (Nucl. Acids. Res., 25, p. 3389-3402, 1997). This program is available on the Internet at the web site of the National Center for Biotechnology Information (NCBI) or the DNA Data Bank of Japan (DDBJ). The details of various conditions (parameters) for identity search using the BLAST program are shown on these web sites, and default values are commonly used for search although part of the settings may be changed as appropriate. Alternatively, the percent identity of two amino acid sequences may be determined by using a program such as genetic information processing software GENETYX Ver.7 (Genetyx Corporation, Japan) or using an algorithm such as FASTA. In this case, default values may be used for search.
The percent identity between two nucleic acid sequences can be determined by visual inspection and mathematical calculation, or more preferably, the comparison is done by comparing sequence information using a computer program. An exemplary, preferred computer program is the Genetic Computer Group (GCG®; Madison, Wis.) WISCONSIN PACKAGE® version 10.0 program, “GAP” (Devereux et al., 1984, Nucl. Acids Res., 12:387). In addition to making a comparison between two nucleic acid sequences, this “GAP” program can be used for comparison between two amino acid sequences and between a nucleic acid sequence and an amino acid sequence. The preferred default parameters for the “GAP” program include: (1) the GCG® implementation of a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted amino acid comparison matrix of Gribskov and Burgess, Nucl. Acids Res., 14:6745, 1986, as described by Schwartz and Dayhoff, eds., “Atlas of Polypeptide Sequence and Structure,” National Biomedical Research Foundation, pp. 353-358, 1979, or other comparable comparison matrices; (2) a penalty of 30 for each gap and an additional penalty of 1 for each symbol in each gap for amino acid sequences, or penalty of 50 for each gap and an additional penalty of 3 for each symbol in each gap for nucleotide sequences; (3) no penalty for end gaps; and (4) no maximum penalty for long gaps. Other programs used by those skilled in the art of sequence comparison can also be used, such as, for example, the BLASTN program version 2.2.7, available for use via the National Library of Medicine website, or the WU-BLAST 2.0 algorithm (Advanced Biocomputing, LLC). In addition, the BLAST algorithm uses the BLOSUM62 amino acid scoring matrix, and optional parameters that can be used are as follows: (A) inclusion of a filter to mask segments of the query sequence that have low compositional complexity (as determined by the SEG program of Wootton and Federhen (Computers and Chemistry, 1993); also see Wootton and Federhen, 1996, “Analysis of compositionally biased regions in sequence databases,” Methods Enzymol., 266: 554-71) or segments consisting of short-periodicity internal repeats (as determined by the XNU program of Clayerie and States (Computers and Chemistry, 1993)), and (B) a statistical significance threshold for reporting matches against database sequences, or E-score (the expected probability of matches being found merely by chance, according to the stochastic model of Karlin and Altschul, 1990; if the statistical significance ascribed to a match is greater than this E-score threshold, the match will not be reported); preferred E-score threshold values are 0.5, or in order of increasing preference, 0.25, 0.1, 0.05, 0.01, 0.001, 0.0001, 1e-5, 1e-10, 1e-15, 1e-20, 1e-25, 1e-30, 1e-40, 1e-50, 1e-75, or 1e-100.
Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter “Sambrook”).
Turning now to the embodiments:
Embodiments include isolated polynucleotides and polypeptides, recombinant DNA constructs useful for conferring drought tolerance, compositions (such as plants or seeds) comprising these recombinant DNA constructs, and methods utilizing these recombinant DNA constructs.
Isolated Polynucleotides and Polypeptides:
The present disclosure includes the following isolated polynucleotides and polypeptides:
An isolated polynucleotide comprising: (i) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 1 or (ii) a full complement of the nucleic acid sequence of (i), wherein the full complement and the nucleic acid sequence of (i) consist of the same number of nucleotides and are 100% complementary. Any of the foregoing isolated polynucleotides may be utilized in any recombinant DNA constructs of the present disclosure. The polypeptide is preferably a PHD1 polypeptide.
An isolated polypeptide having an amino acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 1. The polypeptide is preferably a PHD1 polypeptide.
An isolated polypeptide wherein the amino acid sequence is derived from SEQ ID NO: 1 by alteration of one or more amino acids by at least one method selected from the group consisting of: deletion, substitution, addition and insertion; and (c) a polypeptide wherein the amino acid sequence of the polypeptide comprises SEQ ID NO: 1. The polypeptide is preferably a PHD1 polypeptide.
An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide with epimerase activity, wherein the nucleotide sequence is hybridizable under stringent conditions with a DNA molecule comprising the full complement of SEQ ID NO: 18-34.
Site-directed mutagenesis may be accomplished, for example, as follows using a synthetic oligonucleotide primer that is complementary to single-stranded phage DNA to be mutated, except for having a specific mismatch (i.e., a desired mutation). Namely, the above synthetic oligonucleotide is used as a primer to cause synthesis of a complementary strand by phages, and the resulting duplex DNA is then used to transform host cells. The transformed bacterial culture is plated on agar, whereby plaques are allowed to form from phage-containing single cells. As a result, in theory, 50% of new colonies contain phages with the mutation as a single strand, while the remaining 50% have the original sequence. At a temperature which allows hybridization with DNA completely identical to one having the above desired mutation, but not with DNA having the original strand, the resulting plaques are allowed to hybridize with a synthetic probe labeled by kinase treatment. Subsequently, plaques hybridized with the probe are picked up and cultured for collection of their DNA.
The term “under stringent conditions” means that two sequences hybridize under moderately or highly stringent conditions. More specifically, moderately stringent conditions can be readily determined by those having ordinary skill in the art, e.g., depending on the length of DNA. The basic conditions are set forth by Sambrook et al., Molecular Cloning: A Laboratory Manual, third edition, chapters 6 and 7, Cold Spring Harbor Laboratory Press, 2001 and include the use of a prewashing solution for nitrocellulose filters 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization conditions of about 50% formamide, 2×SSC to 6×SSC at about 40-50° C. (or other similar hybridization solutions, such as Stark's solution, in about 50% formamide at about 42° C.) and washing conditions of, for example, about 40-60° C., 0.5-6×SSC, 0.1% SDS. Preferably, moderately stringent conditions include hybridization (and washing) at about 50° C. and 6×SSC. Highly stringent conditions can also be readily determined by those skilled in the art, e.g., depending on the length of DNA.
Generally, such conditions include hybridization and/or washing at higher temperature and/or lower salt concentration (such as hybridization at about 65° C., 6×SSC to 0.2×SSC, preferably 6×SSC, more preferably 2×SSC, most preferably 0.2×SSC), compared to the moderately stringent conditions. For example, highly stringent conditions may include hybridization as defined above, and washing at approximately 65-68° C., 0.2×SSC, 0.1% SDS. SSPE (1×SSPE is 0.15 M NaCl, 10 mM NaH2PO4, and 1.25 mM EDTA, pH 7.4) can be substituted for SSC (1×SSC is 0.15 M NaCl and 15 mM sodium citrate) in the hybridization and washing buffers; washing is performed for 15 minutes after hybridization is completed.
“Suppression DNA construct” is a recombinant DNA construct which when transformed or stably integrated into the genome of the plant, results in “silencing” of a target gene in the plant. The target gene may be endogenous or transgenic to the plant. “Silencing,” as used herein with respect to the target gene, refers generally to the suppression of levels of mRNA or protein/enzyme expressed by the target gene, and/or the level of the enzyme activity or protein functionality. The terms “suppression”, “suppressing” and “silencing”, used interchangeably herein, include lowering, reducing, declining, decreasing, inhibiting, eliminating or preventing. “Silencing” or “gene silencing” does not specify mechanism and is inclusive, and not limited to, anti-sense, cosuppression, viral-suppression, hairpin suppression, stem-loop suppression, RNAi-based approaches, and small RNA-based approaches.
A suppression DNA construct may comprise a region derived from a target gene of interest and may comprise all or part of the nucleic acid sequence of the sense strand (or antisense strand) of the target gene of interest. Depending upon the approach to be utilized, the region may be 100% identical or less than 100% identical (e.g., at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to all or part of the sense strand (or antisense strand) of the gene of interest.
Suppression DNA constructs are well-known in the art, are readily constructed once the target gene of interest is selected, and include, without limitation, cosuppression constructs, antisense constructs, viral-suppression constructs, hairpin suppression constructs, stem-loop suppression constructs, double-stranded RNA-producing constructs, and more generally, RNAi (RNA interference) constructs and small RNA constructs such as sRNA (short interfering RNA) constructs and miRNA (microRNA) constructs.
“Antisense inhibition” refers to the production of antisense RNA transcripts capable of suppressing the expression of the target gene or gene product. “Antisense RNA” refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target isolated nucleic acid fragment (U.S. Pat. No. 5,107,065). The complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, or the coding sequence.
“Cosuppression” refers to the production of sense RNA transcripts capable of suppressing the expression of the target gene or gene product. “Sense” RNA refers to RNA transcript that includes the mRNA and can be translated into protein within a cell or in vitro. Cosuppression constructs in plants have been previously designed by focusing on overexpression of a nucleic acid sequence having homology to a native mRNA, in the sense orientation, which results in the reduction of all RNA having homology to the overexpressed sequence (see Vaucheret et al., Plant J. 16:651-659 (1998); and Gura, Nature 404:804-808 (2000)).
Another variation describes the use of plant viral sequences to direct the suppression of proximal mRNA encoding sequences (PCT Publication No. WO 98/36083 published on Aug. 20, 1998).
Regulatory Sequences:
A recombinant DNA construct of the present disclosure may comprise at least one regulatory sequence. A regulatory sequence may be a promoter.
A number of promoters can be used in recombinant DNA constructs of the present disclosure. The promoters can be selected based on the desired outcome, and may include constitutive, tissue-specific, inducible, or other promoters for expression in the host organism. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”.
High level, constitutive expression of the candidate gene under control of the 35S or UBI promoter may have pleiotropic effects, although candidate gene efficacy may be estimated when driven by a constitutive promoter. Use of tissue-specific and/or stress-specific promoters may eliminate undesirable effects but retain the ability to enhance drought tolerance. This effect has been observed in Arabidopsis (Kasuga et al. (1999) Nature Biotechnol. 17:287-91).
Suitable constitutive promoters for use in a plant host cell 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., Nature 313:810-812 (1985)); rice actin (McElroy et al., Plant Cell 2:163-171 (1990)); ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) and Christensen et al., Plant Mol. Biol. 18:675-689 (1992)); pEMU (Last et al., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten et al., EMBO J. 3:2723-2730 (1984)); ALS promoter (U.S. Pat. No. 5,659,026), and the like. Other constitutive promoters include, for example, those discussed in 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.
In choosing a promoter to use in the methods of the disclosure, it may be desirable to use a tissue-specific or developmentally regulated promoter. A tissue-specific or developmentally regulated promoter is a DNA sequence which regulates the expression of a DNA sequence selectively in the cells/tissues of a plant critical to tassel development, seed set, or both, and limits the expression of such a DNA sequence to the period of tassel development or seed maturation in the plant. Any identifiable promoter may be used in the methods of the present disclosure which causes the desired temporal and spatial expression. A leaf specific promoter is suitable.
Inducible promoters selectively express an operably linked DNA sequence in response to the presence of an endogenous or exogenous stimulus, for example by chemical compounds (chemical inducers) or in response to environmental, hormonal, chemical, and/or developmental signals. Inducible or regulated promoters include, for example, promoters regulated by light, heat, stress, flooding or drought, phytohormones, wounding, or chemicals such as ethanol, jasmonate, salicylic acid, or safeners.
Promoters for use include the following: 1) the stress-inducible RD29A promoter (Kasuga et al. (1999) Nature Biotechnol. 17:287-91); 2) the barley promoter, B22E; expression of B22E is specific to the pedicel in developing maize kernels (“Primary Structure of a Novel Barley Gene Differentially Expressed in Immature Aleurone Layers”. Klemsdal, S. S. et al., Mol. Gen. Genet. 228(1/2):9-16 (1991)); and 3) maize promoter, Zag2 (“Identification and molecular characterization of ZAG1, the maize homolog of the Arabidopsis floral homeotic gene AGAMOUS”, Schmidt, R. J. et al., Plant Cell 5(7):729-737 (1993); “Structural characterization, chromosomal localization and phylogenetic evaluation of two pairs of AGAMOUS-like MADS-box genes from maize”, Theissen et al. Gene 156(2):155-166 (1995); NCBI GenBank Accession No. X80206)). Zag2 transcripts can be detected 5 days prior to pollination to 7 to 8 days after pollination (“DAP”), and directs expression in the carpel of developing female inflorescences and Ciml which is specific to the nucleus of developing maize kernels. Ciml transcript is detected 4 to 5 days before pollination to 6 to 8 DAP. Other useful promoters include any promoter which can be derived from a gene whose expression is maternally associated with developing female florets.
Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments.
Recombinant DNA constructs of the present disclosure may also include other regulatory sequences, including but not limited to, translation leader sequences, introns, and polyadenylation recognition sequences. In another embodiment of the present disclosure, a recombinant DNA construct of the present disclosure further comprises an enhancer or silencer.
An intron sequence can be added to the 5′ untranslated region, the protein-coding region or the 3′ untranslated region to increase the amount of the mature message that accumulates in the cytosol. Inclusion of a spliceable intron in the transcription unit in both plant and animal expression constructs has been shown to increase gene expression at both the mRNA and protein levels up to 1000-fold. Buchman and Berg, Mol. Cell Biol. 8:4395-4405 (1988); Callis et al., Genes Dev. 1:1183-1200 (1987).
Any plant can be selected for the identification of regulatory sequences and PHD1 polypeptide genes to be used in recombinant DNA constructs of the present disclosure. Examples of suitable plant targets for the isolation of genes and regulatory sequences would include but are not limited to alfalfa, apple, apricot, Arabidopsis, artichoke, arugula, asparagus, avocado, banana, barley, beans, beet, blackberry, blueberry, broccoli, brussels sprouts, cabbage, canola, cantaloupe, carrot, cassava, castorbean, cauliflower, celery, cherry, chicory, cilantro, citrus, clementines, clover, coconut, coffee, corn, cotton, cranberry, cucumber, Douglas fir, eggplant, endive, escarole, eucalyptus, fennel, figs, garlic, gourd, grape, grapefruit, honey dew, jicama, kiwifruit, lettuce, leeks, lemon, lime, Loblolly pine, linseed, mango, melon, mushroom, nectarine, nut, oat, oil palm, oil seed rape, okra, olive, onion, orange, an ornamental plant, palm, papaya, parsley, parsnip, pea, peach, peanut, pear, pepper, persimmon, pine, pineapple, plantain, plum, pomegranate, poplar, potato, pumpkin, quince, radiata pine, radicchio, radish, rapeseed, raspberry, rice, rye, sorghum, Southern pine, soybean, spinach, squash, strawberry, sugarbeet, sugarcane, sunflower, sweet potato, sweetgum, tangerine, tea, tobacco, tomato, triticale, turf, turnip, a vine, watermelon, wheat, yams, and zucchini.
Compositions:
A composition of the present disclosure is a plant comprising in its genome any of the recombinant DNA constructs of the present disclosure (such as any of the constructs discussed above). Compositions also include any progeny of the plant, and any seed obtained from the plant or its progeny, wherein the progeny or seed comprises within its genome the recombinant DNA construct. Progeny includes subsequent generations obtained by self-pollination or out-crossing of a plant. Progeny also includes hybrids and inbreds.
In hybrid seed propagated crops, mature transgenic plants can be self-pollinated to produce a homozygous inbred plant. The inbred plant produces seed containing the newly introduced recombinant DNA construct. These seeds can be grown to produce plants that would exhibit an altered agronomic characteristic (e.g., an increased agronomic characteristic optionally under water limiting conditions), or used in a breeding program to produce hybrid seed, which can be grown to produce plants that would exhibit such an altered agronomic characteristic. The seeds may be maize seeds.
The plant may be a monocotyledonous or dicotyledonous plant, for example, a maize, rice or soybean plant, such as a maize hybrid plant or a maize inbred plant. The plant may also be sunflower, sorghum, canola, wheat, alfalfa, cotton, barley, millet, sugarcane, switchgrass, tobacco, potato and sugar beet.
The recombinant DNA construct may be stably integrated into the genome of the plant.
The PHD1 polypeptide may be from Arabidopsis thaliana, Zea mays, Glycine max, Glycine tabacina, Glycine soja or Glycine tomentella. 
At least one agronomic characteristic may be selected from the group consisting of greenness, yield, growth rate, biomass, fresh weight at maturation, dry weight at maturation, fruit yield, seed yield, total plant nitrogen content, fruit nitrogen content, seed nitrogen content, nitrogen content in a vegetative tissue, total plant free amino acid content, fruit free amino acid content, seed free amino acid content, free amino acid content in a vegetative tissue, total plant protein content, fruit protein content, seed protein content, protein content in a vegetative tissue, drought tolerance, nitrogen uptake, root lodging, harvest index, stalk lodging, plant height, ear height, ear length, salt tolerance, early seedling vigor and seedling emergence under low temperature stress. For example, the alteration of at least one agronomic characteristic may be an increase in yield, greenness or biomass.
In any of the foregoing embodiments 1-8 or any other embodiments of the present disclosure, the plant may exhibit the alteration of at least one agronomic characteristic when compared, under water limiting conditions, to a control plant not comprising said recombinant DNA construct.
“Drought” refers to a decrease in water availability to a plant that, especially when prolonged, can cause damage to the plant or prevent its successful growth (e.g., limiting plant growth or seed yield).
“Drought tolerance” is a trait of a plant to survive under drought conditions over prolonged periods of time without exhibiting substantial physiological or physical deterioration.
“Increased drought tolerance” of a plant is measured relative to a reference or control plant, and is a trait of the plant to survive under drought conditions over prolonged periods of time, without exhibiting the same degree of physiological or physical deterioration relative to the reference or control plant grown under similar drought conditions. Typically, when a transgenic plant comprising a recombinant DNA construct in its genome exhibits increased drought tolerance relative to a reference or control plant, the reference or control plant does not comprise in its genome the recombinant DNA construct.
One of ordinary skill in the art is familiar with protocols for simulating drought conditions and for evaluating drought tolerance of plants that have been subjected to simulated or naturally-occurring drought conditions. For example, one can simulate drought conditions by giving plants less water than normally required or no water over a period of time, and one can evaluate drought tolerance by looking for differences in physiological and/or physical condition, including (but not limited to) vigor, growth, size, or root length, or in particular, leaf color or leaf area size. Other techniques for evaluating drought tolerance include measuring chlorophyll fluorescence, photosynthetic rates and gas exchange rates.
A drought stress experiment may involve a chronic stress (i.e., slow dry down) and/or may involve two acute stresses (i.e., abrupt removal of water) separated by a day or two of recovery. Chronic stress may last 8-10 days. Acute stress may last 3-5 days. The following variables may be measured during drought stress and well watered treatments of transgenic plants and relevant control plants:
The variable “% area chg_start chronic-acute2” is a measure of the percent change in total area determined by remote visible spectrum imaging between the first day of chronic stress and the day of the second acute stress
The variable “% area chg_start chronic-end chronic” is a measure of the percent change in total area determined by remote visible spectrum imaging between the first day of chronic stress and the last day of chronic stress.
The variable “% area chg_start chronic-harvest” is a measure of the percent change in total area determined by remote visible spectrum imaging between the first day of chronic stress and the day of harvest.
The variable “% area chg_start chronic-recovery24 hr” is a measure of the percent change in total area determined by remote visible spectrum imaging between the first day of chronic stress and 24 hrs into the recovery (24 hrs after acute stress 2).
The variable “psii_acute1” is a measure of Photosystem II (PSII) efficiency at the end of the first acute stress period. It provides an estimate of the efficiency at which light is absorbed by PSII antennae and is directly related to carbon dioxide assimilation within the leaf.
The variable “psii_acute2” is a measure of Photosystem II (PSII) efficiency at the end of the second acute stress period. It provides an estimate of the efficiency at which light is absorbed by PSII antennae and is directly related to carbon dioxide assimilation within the leaf.
The variable “fv/fm_acute1” is a measure of the optimum quantum yield (Fv/Fm) at the end of the first acute stress−(variable fluorescence difference between the maximum and minimum fluorescence/maximum fluorescence).
The variable “fv/fm_acute2” is a measure of the optimum quantum yield (Fv/Fm) at the end of the second acute stress−(variable flourescence difference between the maximum and minimum fluorescence/maximum fluorescence).
The variable “leaf rolling_harvest” is a measure of the ratio of top image to side image on the day of harvest.
The variable “leaf rolling_recovery24 hr” is a measure of the ratio of top image to side image 24 hours into the recovery.
The variable “Specific Growth Rate (SGR)” represents the change in total plant surface area (as measured by Lemna Tec Instrument) over a single day (Y(t)=Y0*er*t). Y(t)=Y0*er*t is equivalent to % change in Y/Δt where the individual terms are as follows: Y(t)=Total surface area at t; Y0=Initial total surface area (estimated); r=Specific Growth Rate day−1, and t=Days After Planting (“DAP”).
The variable “shoot dry weight” is a measure of the shoot weight 96 hours after being placed into a 104° C. oven.
The variable “shoot fresh weight” is a measure of the shoot weight immediately after being cut from the plant.
The Examples below describe some representative protocols and techniques for simulating drought conditions and/or evaluating drought tolerance.
Methods include but are not limited to the following:
A method for transforming a cell comprising transforming a cell with any of the isolated polynucleotides of the present disclosure. The cell transformed by this method is also included. In particular embodiments, the cell is eukaryotic cell, e.g., a yeast, insect or plant cell, or prokaryotic, e.g., a bacterial cell.
A method for producing a transgenic plant comprising transforming a plant cell with any of the isolated polynucleotides or recombinant DNA constructs of the present disclosure and regenerating a transgenic plant from the transformed plant cell. The disclosure is also directed to the transgenic plant produced by this method, and transgenic seed obtained from this transgenic plant.
A method for isolating a polypeptide of the disclosure from a cell or culture medium of the cell, wherein the cell comprises a recombinant DNA construct comprising a polynucleotide of the disclosure operably linked to at least one regulatory sequence, and wherein the transformed host cell is grown under conditions that are suitable for expression of the recombinant DNA construct.
A method of altering the level of expression of a polypeptide of the disclosure in a host cell comprising: (a) transforming a host cell with a recombinant DNA construct of the present disclosure; and (b) growing the transformed host cell under conditions that are suitable for expression of the recombinant DNA construct wherein expression of the recombinant DNA construct results in production of altered levels of the polypeptide of the disclosure in the transformed host cell.
A method of increasing yield and/or drought tolerance in a plant, comprising: (a) introducing into a regenerable plant cell a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence (for example, a promoter functional in a plant), wherein the polynucleotide encodes a polypeptide having an amino acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 1 and (b) regenerating a transgenic plant from the regenerable plant cell after step (a), wherein the transgenic plant comprises in its genome the recombinant DNA construct and exhibits increased drought tolerance when compared to a control plant not comprising the recombinant DNA construct. The method may further comprise (c) obtaining a progeny plant derived from the transgenic plant, wherein said progeny plant comprises in its genome the recombinant DNA construct and exhibits increased drought tolerance when compared to a control plant not comprising the recombinant DNA construct.
EXAMPLES
The present disclosure is further illustrated in the following Examples, in which parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the disclosure, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the disclosure to adapt it to various usages and conditions. Thus, various modifications of the disclosure in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.
Example 1
Isolation and Characterization of the Phd1 Mutant
Of 480 mutant lines with altered carbohydrate levels in vegetative organs, photoassimilate defective1 (phd1) with both low carbohydrate contents and stunted growth was selected for further characterization (FIG. 1). Compared to wild type, mature leaves of the mutant had low starch levels at all time-points taken during the light/dark cycle (FIG. 11). Scanning electron micrograph of culms demonstrated that fewer starch granules were deposited in parenchyma cells of the phd1 mutants. During the young seedling stage, both the shoots and primary roots of the mutant were shorter and lighter than those of the wild type (FIG. 1A). After internode elongation, the phd1 mutant exhibited a semi-dwarf, less tillering, retarded vegetative growth, later flowering, and less grain-filling phenotype (FIG. 1B-E). In addition, although the grain number per panicle was not altered between the mutant and wild type, the seed-setting ratio of the phd1 mutant was significantly decreased, which finally led to a significant reduction of grain yield (FIG. 1F, G).
Example 2
PHD1 Encodes a Functional Chloroplastic UDP-Glc Epimerase
Genetic analysis indicated that the phd1 phenotype was controlled by a single recessive gene that did not co-segregate with the T-DNA insertion, and hence map-based cloning was carried out. The PHD1 locus was physically delimited to a 72-kb region on the short arm of chromosome 1. This region contains six annotated genes, and sequencing of these genes from phd1-1 identified a single nucleotide transition (G-to-T) in exon 2 of Os01g0367100, leading to a premature translational termination. The identity of Os01g0367100 as PHD1 was confirmed by analysis of two other phd1 alleles isolated from the same genetic screen. A single nucleotide substitution (A-to-T) in exon 7 of phd1-2 and a 13-bp insertion between exon 3 and exon 4 of phd1-3 were found (FIG. 2A). Almost no PHD1 mRNA was detected in any of the three allelic mutants (Figure S2). The phd1 phenotype was fully complemented by transgenic expression of wild type Os01g0367100 in the phd1-1 mutant background (FIG. 2B, C), confirming that the nonsense mutation of Os01g0367100 was responsible for the presumed null mutant phenotype.
PHD1 has similarity to proteins from Thalassiosira pseudonana (XP_002290295), Phaeodactylum tricornutum (XP_002178225), Chlamydomonas reinhardtii (XP_001699105), Micromonas pusilla (EEH60780), Ostreococcus tauri (CAL54696), Physcomitrella patens (XP_001767242), Ricinus communis (XP_002516868), Arabidopsis thaliana (AT2G39080), Populus trichocarpa (XP_002311843), Vitis vinifera (XP_002276706), Zea mays (NP_001131736), and Sorghum bicolor (XP_002457832), incorporated herein by reference, with 27 to 75% amino acid identity (Figure S3). Phylogenetic analysis between PHD1 and its 16 structurally similar proteins indicated that PHD1 is closely related to Sb03g014730 from sorghum and LOC100193101 from maize (FIG. 3). PHD1 homologs/orthologs are only found in the plant kingdom, indicating that these proteins are evolutionally conserved across plant species. Analysis of the conserved domain demonstrated that PHD1 and its homologs contain the consensus WcaG domain, featured in nucleoside-diphosphate sugar epimerases (FIG. 11A). UDP-Glc epimerase (UGE), a sugar epimerase catalyzes the interconversion of UDP-Glc and UDP-Gal. PHD1 and its homologs may function as novel plant specific UGEs.
To understand the PHD1's biochemical function as an UDP-Glc epimerase, the mature PHD1 protein lacking the putative N-terminal 62-aa transit peptide was expressed in E. coli and UGE activity was examined. The result showed that PHD1 could catalyze the conversion of UDP-Gal to UDP-Glc, and curve fitting indicated that UDP-Gal binding followed a simple Michaelis-Menten kinetics with a Km value of 0.84 mM at 30° C. (FIG. 12A). To examine whether PHD1 had UDP-Glc epimerase activity in vivo, the mature PHD1 was expressed from the yeast glyceraldehyde-3-phosphate dehydrogenase promoter to complement the auxotrophic phenotype of a yeast gal10Δ mutant, which cannot grow on a medium containing D-galactose as sole carbon source. The complementation results demonstrated that PHD1 also had UDP-Glc epimerase activity in vivo (FIG. 12B).
RNA gel blot analysis revealed that PHD1 was present in all green tissues, with highest abundance in leaf blades and sheaths, then flowers and culms, but only at very low levels in roots (FIG. 4A). mRNA in situ hybridization revealed that PHD1 was expressed predominantly in leaf primodia and shoot apical meristems (FIG. 4B), the mesophyll cells surrounding the vascular bundles of young leaves (FIG. 4C), inflorescence primodia (FIG. 4D), and axillary buds (FIG. 4E). PHD1 encodes a 340 aa protein with a putative 62-aa chloroplast transit peptide at the N-terminus. To confirm chloroplast localization of PHD1, the full-length PHD1 was fused to the green fluorescent protein (GFP) reporter gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter and subsequently transformed into rice shoot protoplasts. FIG. 4F shows that GFP fluorescence co-localized with the red chlorophyll autofluorescence, confirming that PHD1 was a chloroplast-localized protein and that the predicted transit peptide was functional.
Example 3
PHD1 Influences the Homeostasis of Nucleotide Sugars and Carbon Assimilation in Leaves
UDP-Gal is the activated form of galactose in biosynthetic reactions, but a galactose salvage pathway exists in eukaryotic organisms. To assess expression of genes involved in the Leloir salvage pathway, the expression levels of three key genes of this pathway, GalM, GalK, and GalT, were analyzed in both phd1-1 and wild type. The expression of all three genes was significantly upregulated in the phd1-1 mutant, suggesting an activation of the whole salvage pathway (FIG. 5A). β-Lactase is involved in the generation of free β-D-Gal from polysaccharide breakdown, and UDP-Glc pyrophosphorylase (UGP) catalyzes the formation of UDP-Glc from Glc-1-P. The expression levels of genes encoding β-lactase and UGP3 were also upregulated in phd1-1. Surprisingly, the expression levels of OsUGE1 and OsUGE4 encoding for putative cytoplasmic isoforms of UGE in rice were more than two-fold higher in phd1-1 than in wild type plants, indicating an upregulation of de novo UDP-Gal biosynthesis in the cytoplasm. These results suggested that PHD1 is likely responsible for a majority of the UGE function in chloroplasts, and appears to be involved in the generation of UDP-Gal from UDP-Glc to supply building blocks for galactolipid biosynthesis required for proper chloroplast membrane composition.
To determine whether impairment of UGE activity in phd1-1 had an effect on galactose-containing compounds, the intermediates of galactose metabolism were analyzed. The amount of UDP-Gal was found to be slightly higher in leaves of phd1-1 than in wild type, but the UDP-Glc amount was significantly higher (FIG. 6A). Hence, the ratio of UDP-Glc to UDP-Gal in phd1-1 was also higher than in wild type leaves. These results suggested that PHD1 dysfunction may trigger the accumulation of substrates and disturb the balance of interconversion between the two sugar nucleotides.
Because the phd1-1 mutant exhibited a significant decrease of carbon assimilate levels, it was determined whether transcript levels of several key genes involved in the synthesis, transport, and cleavage of starch and sucrose were altered in mature leaves of wild type and phd1-1 plants. Interestingly, while the expression of genes in starch biosynthesis such as AGPL2, SSI, SSIIIa, GBSS, BE, and BT1, was suppressed in the phd1-1 mutant (FIG. 5B), expression levels of genes participating in sucrose cleavage, such as INV1/3 and SuSy1, were all increased (FIG. 5C). Meanwhile, the GPT gene encoding a glucose-6-phosphate/phosphate translocator was upregulated in phd1-1, indicating an enhanced export of hexose-phosphates from chloroplasts to the cytosol. In addition, increased expression level of UGP2, a gene involved in UDP-Glc synthesis, was correlated with increased UDP-Glc accumulation and a higher UDP-Glc/UDP-Gal ration in the phd1-1 mutant.
Example 4
PHD1 Dysfunction Affects the Photosynthetic Membrane System
Chloroplast membranes contain high levels of glycolipids, and UDP-Gal is a dominant substrate for glycolipid biosynthesis. To examine the effect of PHD1 dysfunction on membrane lipid homeostasis, the composition of total lipids extracted from phd1-1, wild type, and PHD1-complemented plants was analyzed (FIG. 6B). In the phd1-1 mutant, the mol % amount of MGDG was reduced by 19% compared to wild type and the complemented plants, indicating that PHD1 is involved in MGDG biosynthesis. In contrast, only a slight decrease (2.5%) in DGDG content was observed in the phd1-1 mutant, demonstrating that PHD1 may not be required for DGDG synthesis and suggesting that the UDP-Gal substrate for DGDG formation was presumably supplied from the cytosol. Reduced abundance of MGDG in phd1-1 was accompanied by an increased abundance of other major membrane lipids such as phosphatidylcholine (PC), phosphoinositol (PI), and phosphatidylglycerol (PG), while the mol % levels of sulfoquinovosyldiacylglycerol (SQDG) and phosphatidyl ethanolamine (PE) were only slightly but not significantly higher in the phd1-1 mutant (FIG. 6B). Because PC, PI, and PG are also components of thylakoid membranes, these results demonstrated that the mutant thylakoid membranes had an increased mol % amount of phospholipids.
Noninvasive chlorophyll fluorescence measurements indicated that the maximum quantum yields for photosystem II photochemistry (Fv/Fm) were similar for phd1-1 and wild type (Table 1). The effective quantum yield of photochemical energy conversion in photosystem II (φPSII) was slightly but significantly reduced in the mutant (Table 1). Pigment content was also reduced in the phd1-1 mutant (Table 1). Interestingly, in 2-month-old plants chloroplasts were significantly smaller in phd1-1 mutant than wild type plants (wild type, 5.0±0.4 μm; phd1-1, 3.0±0.5 μm), and starch grains were also either absent or reduced in size and/or number in the mutant. These data indicated that a reduced amount of galactolipids in chloroplasts might reduce photosynthetic capability of higher plants.







TABLE 1







Pigment content (mg·g−1 fresh weight) and photosynthetic


parameters of wild type, phd1-1, and the PHD1-complemented plants.











Wild type
phd1-1
phd1-1 + PHD1














Chlorophyll a
2.50 ± 0.34
1.86 ± 0.36*
2.48 ± 0.37


Chlorophyll b
0.96 ± 0.13
0.67 ± 0.12*
0.93 ± 0.18


Chlorophyll a + b
3.46 ± 0.42
2.53 ± 0.43*
3.41 ± 0.52


Chlorophyll a/b
2.62 ± 0.37
2.77 ± 0.41 
2.66 ± 0.55


Carotenoids
0.33 ± 0.04
0.28 ± 0.05 
0.34 ± 0.07


Fv/Fm
0.84 ± 0.01
0.79 ± 0.01 
0.83 ± 0.02


φPSII
0.72 ± 0.01
0.58 ± 0.02*
0.70 ± 0.02





Samples were collected from fully-expanded leaves of 4-month-old plants grown in paddy fields.


Values represent means (±SD) of six to ten independent determinations.


*Significant difference between mutant and wild type (P < 0.05).






Example 5
Expression of PHD1 Increases Growth Rate and Grain Yield
It was investigated whether biomass and grain yield could be improved by PHD1 overexpression. Transgenic rice plants overexpressing PHD1 showed a significant increase in tillering (branching) and photosynthetic rate when grown in paddy fields (FIG. 7A, Table 2). The growth rate of transgenic plants accelerated at the seedling stage and dry material accumulation was enhanced 12.5% to 22.4% at the mature stage compared to non-transgenic plants (FIG. 7D, Table 2). In addition, compared to non-transgenic control plants, grain yield per plant of transgenic lines S3, S5, and S8 increased 10.7, 15.5, and 18.3%, respectively (FIG. 7C). These results demonstrated that PHD1 overexpression in rice is positively correlated with an increase in biomass production and grain yield.







TABLE 2







Characterization of biomass and photosynthetic rate of PHD1-overexpressing


plants and wild type (Nipponbare).












WT
S3
S5
S8















Shoot height (cm)a
15.32 ± 0.15
18.64 ± 0.18*
19.21 ± 0.33*
19.64 ± 0.37*


Shoot mass (mg)a
54.73 ± 1.02
63.45 ± 1.13*
66.55 ± 1.17*
65.91 ± 1.20*


NPR (500 μmol · m−2 · s−1)b
 8.31 ± 0.24
 9.65 ± 0.53*
10.12 ± 0.37*
10.39 ± 0.59*


NPR (2000 μmol · m−2 · s−1)b
14.50 ± 0.99
15.78 ± 0.86 
16.12 ± 1.28 
16.36 ± 1.36 





S3, S5, and S8 represent three independent PHD1-overexpressing transgenic lines.


a20-day-old rice seedlings;


bNPR, rate of net photosynthesis (μmol · CO2 · m−2 · s−1) measured at the heading stage;


Values are means ± SD from at least 30 plants/line,


*Significant difference (P < 0.05).













TABLE 3







Oligonucleotides (SEQ ID NOS: 35-52,


consecutive) used for galactose 


metabolism analysis.










Name
Sequence (5′-3′)







β-lactase2
GTCGTGCCATGACATCTACCA




CTGCTTTATTGCCTCACTTGC



 



GalM4
CGTCGTGCTTCCTGACTCCA




CCTCCACCAACATGCTCCTTC



 



GalK2
ACATAACCTACCGAAGAAGAGTGG




TCACAGCCTGAAGCACATAAAA



 



GalT
GGATACGGCACTGGATCTTGG




TTGAATGGAGGGTCGTTGAGC



 



OsUGE1
TACTGCTCCGATACTTCAACCC




CCATCCGCTAGATCAACAACAT



 



OsUGE2
CCAAGACGCCCTGGTGATGC




TTCGCTTTCCAGTTGAGTTCCTTC



 



OsUGE3
TCGCTACTCCTGACATTGGTT




TGATCGCCCTAATTCTGCTC



 



OsUGE4
TGGAACAGGAAAGGGAACATC




TCGTGGACCAATAACCAAAGG



 



UGP3
GCCAGAACAAACCCATCAAAC




GTAACTCCAGAGCCGAACCAG

















TABLE 4







Oligonucleotides (SEQ ID NOS: 53-76,


consecutive) used for carbohydrate


metabolism analysis.










Name
Sequence (5′-3′)







AGPL2
ATAATCTCCGATGGCTGTTC




TCCAGACCTTATGTAGTATCCC



 



SSI
GGGCCTTCATGGATCAACC




CCGCTTCAAGCATCCTCATC



 



SSIIIa
GCCTGCCCTGGACTACATTG




GCAAACATATGTACACGGTTCTGG



 



GBSSI
AACGTGGCTGCTCCTTGAA




TTGGCAATAAGCCACACACA



 



BEI
TGGCCATGGAAGAGTTGGC




CAGAAGCAACTGCTCCACC



 



BTI-1
GAAGTCCTTGAGCCGTCCTG




AAGTCCCTTGATGCCCTCCT



 



GPT1
AGAAGGGATCCAGATGAAGAA




AACAAGAAACGAGCAACATAGA



 



GPT2
GCCTCCATCATCATCTTCCA




ATTGTTACATCCCGAGCACC



 



UGP2
GCCAGAACAAACCCATCAAAC




GTAACTCCAGAGCCGAACCAG



 



SuSy1
GCTTCCACATTGACCCATAC




CTTGAGGGCATACAGCATCT



 



INV1
CACGACGCAGTGATCTGAGG




GATGAAACGCAGGGAATACG



 



INV3
GACATCGTCAAGAGGGTCG




CCATCCATGATCCATCATCC










Example 9
Electroporation of Agrobacterium tumefaciens LBA4404
Electroporation competent cells (40 μL), such as Agrobacterium tumefaciens LBA4404 containing PHP10523 (“pSB1”; Komari et al., Plant J. 10:165-174 (1996); NCBI General Identifier No. 59797027), are thawed on ice (20-30 min). PHP10523 contains VIR genes for T-DNA transfer, an Agrobacterium low copy number plasmid origin of replication, a tetracycline resistance gene, and a Cos site for in vivo DNA bimolecular recombination. Meanwhile the electroporation cuvette is chilled on ice. The electroporator settings are adjusted to 2.1 kV. A DNA aliquot (0.5 μL parental DNA at a concentration of 0.2 μg-1.0 μg in low salt buffer or twice distilled H2O) is mixed with the thawed Agrobacterium tumefaciens LBA4404 cells while still on ice. The mixture is transferred to the bottom of electroporation cuvette and kept at rest on ice for 1-2 min. The cells are electroporated (Eppendorf electroporator 2510) by pushing the “pulse” button twice (ideally achieving a 4.0 millisecond pulse). Subsequently, 0.5 mL of room temperature 2×YT medium (or SOC medium) are added to the cuvette and transferred to a 15 mL snap-cap tube (e.g., FALCONTM tube). The cells are incubated at 28-30° C., 200-250 rpm for 3 h.
Aliquots of 250 μL are spread onto plates containing YM medium and 50 μg/mL spectinomycin and incubated three days at 28-30° C. To increase the number of transformants one of two optional steps can be performed:
Option 1: Overlay plates with 30 μL of 15 mg/mL rifampicin. LBA4404 has a chromosomal resistance gene for rifampicin. This additional selection eliminates some contaminating colonies observed when using poorer preparations of LBA4404 competent cells.
Option 2: Perform two replicates of the electroporation to compensate for poorer electrocompetent cells.
Identification of Transformants:
Four independent colonies are picked and streaked on plates containing AB minimal medium and 50 μg/mL spectinomycin for isolation of single colonies. The plates are incubated at 28° C. for two to three days. A single colony for each putative co-integrate is picked and inoculated with 4 mL of 10 g/L bactopeptone, 10 g/L yeast extract, 5 g/L sodium chloride and 50 mg/L spectinomycin. The mixture is incubated for 24 h at 28° C. with shaking. Plasmid DNA from 4 mL of culture is isolated using QIAGEN® Miniprep and an optional Buffer PB wash. The DNA is eluted in 30 μL. Aliquots of 2 μL are used to electroporate 20 μL of DH10b+20 μL of twice distilled H2O as per above. Optionally a 15 μL aliquot can be used to transform 75-100 μL of INVITROGENTM Library Efficiency DH5α. The cells are spread on plates containing LB medium and 50 μg/mL spectinomycin and incubated at 37° C. overnight.
Three to four independent colonies are picked for each putative co-integrate and inoculated 4 mL of 2×YT medium (10 g/L bactopeptone, 10 g/L yeast extract, 5 g/L sodium chloride) with 50 μg/mL spectinomycin. The cells are incubated at 37° C. overnight with shaking. Next, isolate the plasmid DNA from 4 mL of culture using QIAPREP® Miniprep with optional Buffer PB wash (elute in 50 μL). Use 8 μL for digestion with SalI (using parental DNA and PHP10523 as controls). Three more digestions using restriction enzymes BamHI, EcoRI, and HindIII are performed for 4 plasmids that represent 2 putative co-integrates with correct SalI digestion pattern (using parental DNA and PHP10523 as controls). Electronic gels are recommended for comparison.
Example 11
Transformation of Maize Using Agrobacterium 
Agrobacterium-mediated transformation of maize is performed essentially as described by Zhao et al. in Meth. Mol. Biol. 318:315-323 (2006) (see also Zhao et al., Mol. Breed. 8:323-333 (2001) and U.S. Pat. No. 5,981,840 issued Nov. 9, 1999, incorporated herein by reference). The transformation process involves bacterium inoculation, co-cultivation, resting, selection and plant regeneration.
1. Immature Embryo Preparation:
Immature maize embryos are dissected from caryopses and placed in a 2 mL microtube containing 2 mL PHI-A medium.
2. Agrobacterium Infection and Co-Cultivation of Immature Embryos:
2.1 Infection Step:
PHI-A medium of (1) is removed with 1 mL micropipettor, and 1 mL of Agrobacterium suspension is added. The tube is gently inverted to mix. The mixture is incubated for 5 min at room temperature.
2.2 Co-culture Step:
The Agrobacterium suspension is removed from the infection step with a 1 mL micropipettor. Using a sterile spatula the embryos are scraped from the tube and transferred to a plate of PHI-B medium in a 100×15 mm Petri dish. The embryos are oriented with the embryonic axis down on the surface of the medium. Plates with the embryos are cultured at 20° C., in darkness, for three days. L-Cysteine can be used in the co-cultivation phase. With the standard binary vector, the co-cultivation medium supplied with 100-400 mg/L L-cysteine is critical for recovering stable transgenic events.
3. Selection of Putative Transgenic Events:
To each plate of PHI-D medium in a 100×15 mm Petri dish, 10 embryos are transferred, maintaining orientation and the dishes are sealed with PARAFILM®. The plates are incubated in darkness at 28° C. Actively growing putative events, as pale yellow embryonic tissue, are expected to be visible in six to eight weeks. Embryos that produce no events may be brown and necrotic, and little friable tissue growth is evident. Putative transgenic embryonic tissue is subcultured to fresh PHI-D plates at two-three week intervals, depending on growth rate. The events are recorded.
4. Regeneration of T0 Plants:
Embryonic tissue propagated on PHI-D medium is subcultured to PHI-E medium (somatic embryo maturation medium), in 100×25 mm Petri dishes and incubated at 28° C., in darkness, until somatic embryos mature, for about ten to eighteen days. Individual, matured somatic embryos with well-defined scutellum and coleoptile are transferred to PHI-F embryo germination medium and incubated at 28° C. in the light (about 80 μE from cool white or equivalent fluorescent lamps). In seven to ten days, regenerated plants, about 10 cm tall, are potted in horticultural mix and hardened-off using standard horticultural methods.
Media for Plant Transformation:

    
    
        1. PHI-A: 4 g/L CHU basal salts, 1.0 mL/L 1000× Eriksson's vitamin mix, 0.5 mg/L thiamin HCl, 1.5 mg/L 2,4-D, 0.69 g/L L-proline, 68.5 g/L sucrose, 36 g/L glucose, pH 5.2. Add 100 μM acetosyringone (filter-sterilized).
        2. PHI-B: PHI-A without glucose, increase 2,4-D to 2 mg/L, reduce sucrose to 30 g/L and supplemented with 0.85 mg/L silver nitrate (filter-sterilized), 3.0 g/L GELRITE®, 100 μM acetosyringone (filter-sterilized), pH 5.8.
        3. PHI-C: PHI-B without GELRITE® and acetosyringonee, reduce 2,4-D to 1.5 mg/L and supplemented with 8.0 g/L agar, 0.5 g/L 2-[N-morpholino]ethane-sulfonic acid (MES) buffer, 100 mg/L carbenicillin (filter-sterilized).
        4. PHI-D: PHI-C supplemented with 3 mg/L bialaphos (filter-sterilized).
        5. PHI-E: 4.3 g/L of Murashige and Skoog (MS) salts, (Gibco, BRL 11117-074), 0.5 mg/L nicotinic acid, 0.1 mg/L thiamine HCl, 0.5 mg/L pyridoxine HCl, 2.0 mg/L glycine, 0.1 g/L myo-inositol, 0.5 mg/L zeatin (Sigma, Cat. No. Z-0164), 1 mg/L indole acetic acid (IAA), 26.4 μg/L abscisic acid (ABA), 60 g/L sucrose, 3 mg/L bialaphos (filter-sterilized), 100 mg/L carbenicillin (filter-sterilized), 8 g/L agar, pH 5.6.
        6. PHI-F: PHI-E without zeatin, IAA, ABA; reduce sucrose to 40 g/L; replacing agar with 1.5 g/L GELRITE®; pH 5.6.
    
    


Plants can be regenerated from the transgenic callus by first transferring clusters of tissue to N6 medium supplemented with 0.2 mg per liter of 2,4-D. After two weeks the tissue can be transferred to regeneration medium (Fromm et al., Bio/Technology 8:833-839 (1990)).
Transgenic T0 plants can be regenerated and their phenotype determined. T1 seed can be collected. T1 plants, and/or their progeny, can be grown and their phenotype determined.
Example 15
Yield Analysis of Maize Lines Transformed with PHD1 Gene
A recombinant DNA construct containing PHD1 gene can be introduced into an elite maize inbred line either by direct transformation or introgression from a separately transformed line.
Transgenic plants, either inbred or hybrid, can undergo more vigorous field-based experiments to study yield enhancement and/or stability under well-watered and water-limiting conditions.
Subsequent yield analysis can be done to determine whether plants that contain the validated drought tolerant lead gene have an improvement in yield performance under water-limiting conditions, when compared to the control plants that do not contain the validated drought tolerant lead gene. Specifically, drought conditions can be imposed during the flowering and/or grain fill period for plants that contain the validated drought tolerant lead gene and the control plants. Reduction in yield can be measured for both. Plants containing the validated drought tolerant lead gene have less yield loss relative to the control plants, for example, at least 25% less yield loss, under water limiting conditions, or would have increased yield relative to the control plants under water non-limiting conditions.
The above method may be used to select transgenic plants with increased yield, under water-limiting conditions and/or well-watered conditions, when compared to a control plant not comprising said recombinant DNA construct.

## Claims

1. A method of increasing the production of UDP-galactose in situ in the chloroplast for mono-galactosyldiacylglycerol (MGDG) biosynthesis in a plant, the method comprising expressing from a DNA construct comprising a nucleic acid encoding a chloroplast UDP-glucose epimerase under the control of at least one regulatory promoter element in the plant, wherein the chloroplast UDP-glucose epimerase comprises a chloroplast transit peptide, and wherein the UDP-glucose epimerase is a monocot polypeptide comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 1.

2. The method of claim 1, wherein the plant is selected from the group consisting of maize, rice, sugarcane, and wheat.

3. The method of claim 1, wherein the carbon partitioning in the plant is altered.

4. The method of claim 1, wherein the photosynthetic efficiency of the plant is increased.

5. The method of claim 1, wherein the drought tolerance of the plant is increased.

6. A method of increasing yield of a plant, the method comprising expressing a recombinant chloroplast UDP-glucose epimerase operably linked to at least one regulatory element, wherein the chloroplast UDP-glucose epimerase is localized in the chloroplast, and wherein the UDP-glucose epimerase is a monocot polypeptide comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 1.

7. The method of claim 6, wherein the plant is selected from the group consisting of maize, rice, sugarcane, and wheat.

8. The method of claim 6, wherein the carbon partitioning in the plant is altered.

9. The method of claim 6, wherein the photosynthetic efficiency of the plant is increased.

10. The method of claim 6, wherein the drought tolerance of the plant is increased.

11. The method of claim 1, wherein the UDP-glucose epimerase is a monocot polypeptide comprising an amino acid sequence that is at least 98% identical to SEQ ID NO: 1.

12. The method of claim 6, wherein the UDP-glucose epimerase is a monocot polypeptide comprising an amino acid sequence that is at least 98% identical to SEQ ID NO: 1.