# Patent

## Title
Enzymatic methods for modulating the levels of organic sulfur compounds in plants

## Abstract

Methods for modulating levels of at least one organic sulfur compound in plants are provided. Also provided are plants and seeds produced by the methods. The methods comprise stably transforming a plant with a DNA construct encoding a cystathionine gamma synthase enzyme, a serine acetyl transferase enzyme and/or other sulfur assimilating enzymes capable of altering the level of at least one organic sulfur compound.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser. No. 10/108,739, filed Mar. 29, 2002, now U.S. Pat. No. 7,038,109 issued May 2, 2006, and which claims the benefit of U.S. Provisional Application No. 60/367,333, Mar. 29, 2001, all of which are hereby incorporated by reference.


FIELD OF THE INVENTION
The invention relates to the genetic manipulation of plants, particularly to enzymatic methods for altering sulfur metabolism in plants and plant seeds.
BACKGROUND OF THE INVENTION
Sulfur in its reduced form plays an important role in plant metabolism, being involved in the biosynthesis of a wide range of primary and secondary sulfur-containing metabolites. In plants, sulfur metabolism includes the uptake of sulfate from the environment, assimilation into organic compounds, and channeling into proteins and secondary substances.
Plants and microorganisms are able to reduce sulfate to sulfide for synthesis of the thiol group of cysteine. The first step is the activation of sulfate by ATP sulfurylase, forming 5′-adenylylsulfate (APS). APS reductase acts upon APS to generate sulfite, and sulfite reductase converts this sulfite to hydrogen sulfide. This hydrogen sulfide provides the thiol group to cysteine, while the carbon portion of cysteine comes from the serine branch of the pathway. In this branch, serine is converted to O-acetylserine by serine acetyltransferase. Cysteine synthase then catalyzes the reaction of O-acetylserine and hydrogen sulfide to form cysteine.
Cystathionine gamma synthase catalyzes the reaction between O-phosphohomoserine and cysteine, wherein the cysteine donates a thiol group to O-phosphohomoserine, thereby forming cystathionine.
Methionine and sulfur-containing vitamins such as biotin or thiamine are essential in human nutrition. Sulfur-mediated functions include electron transport in Fe/S-clusters, structural and regulatory roles via protein disulfide bridges, and catalytic centers. Additionally, secondary sulfur compounds include signaling molecules, anti-carcinogens and atmospheric compounds. See Hell (1997) Planta 202:138.
Often plant protein is deficient in the sulfur amino acids, especially methionine, as well as other essential amino acids such as lysine and tryptophan. As a result, diets must be supplemented with these amino acids in order to provide a balanced diet. A goal of plant breeding has been to increase the amount of sulfur amino acids present in the seed.
A number of methods have been described for increasing sulfur amino acid content of plants. Some of these methods provide for the overexpression of a high methionine seed storage protein, which entails overexpressing the seed storage protein in a transformed plant. Other methods have attempted seed specific expression of synthetic enzymes in the methionine pathway. Still other methods have focused on enzymatic modification of amino acids and capturing these amino acids in transgenic seed storage proteins. However, these methods have met with limited success. There is therefore a need for an effective and direct method of producing significant levels of the sulfur amino acids in plants and plant seeds.
SUMMARY OF THE INVENTION
An object of the present invention is to provide methods for increasing the nutritional value of plants.
Another object of the present invention is to provide plants and plant parts having increased nutritional value.
Another object of the present invention is to provide plants and plant parts having increased levels of organic sulfur compounds.
Another object of the present invention is to provide plants and plant parts having increased levels of methionine.
Another object of the present invention is to provide plants and plant parts having increased levels of cysteine.
In accordance with the present invention, methods for modulating the level of at least one organic sulfur compound in plants are provided. Also provided are plants, plant tissues, plant seeds and plant cells produced by the methods. The methods comprise stably transforming a plant with a DNA construct encoding a cystathionine gamma synthase and/or serine acetyl transferase enzyme.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 sets forth the biosynthesis for the organic sulfur compounds cysteine and methionine with the proposed modifications of the invention.
FIG. 2 sets forth the sulfur amino acid level, on a percent dry weight basis, of certain cystathionine gamma synthase transformed lines.
FIG. 3 sets forth the sulfur amino acid level, on a mol percent level, of certain cystathionine gamma synthase transformed lines.
FIG. 4 sets forth the sulfur amino acid level, on a percent dry weight basis, of certain serine acetyl transferase transformed lines.
FIG. 5 sets for the sulfur amino acid level, on a mol percent level, of certain serine acetyl transferase transformed lines.
FIG. 6 sets for the sulfur amino acid level, on a percent dry weight basis, of the segregating seed (T2) of certain serine acetyl transferase transformed lines.
FIG. 7 sets for the sulfur amino acid level, on a mol percent level dry weight basis, of the segregating seed (T2) of certain serine acetyl transferase transformed lines.



DETAILED DESCRIPTION OF THE INVENTION
In accordance with the subject invention, compositions and methods for modulating the biosynthesis of organic sulfur compounds in plants, particularly sulfur amino acids, more particularly cysteine and methionine, are provided. The methods involve transforming a monoctyledonous plant with one or more nucleic acid(s) encoding cystathionine gamma synthase or serine acetyl transferase. The plant may also comprise one or more additional nucleic acid(s) selected from nucleic acids encoding enzymes involved in amino acid biosynthesis and sulfate reduction.
Previous attempts to modulate the biosynthesis of organic sulfur compounds through enzymatic modification has focused upon the enzymatic modification occurring in the plant seed. This is because the plant seed is the desired source for the modulated organic sulfur compound. Surprisingly, Applicants have discovered that modulated expression of cystathionine gamma synthase and/or serine acetyltransferase need not occur in a monocot plant seed in order to modulate the biosynthesis of organic sulfur compounds in the plant seed. Through non-seed specific enzymatic modulation of cystathionine gamma synthase or serine acetyl transferase, Applicants have increased the levels of methionine in a monocot seed beyond those achieved through seed specific enzyme modification. Perhaps most surprisingly, such increase in the seed was accomplished without transgenic modification of the plant to increase the expression of a seed storage protein containing the desired amino acids.
By “organic sulfur compounds” is intended a compounds such as cysteine, cystathionine, methionine, glutathione, dimethylsulfoniopropionate (DMSP), SMM (Vitamin U), biotin, SAM, Thiamine pyrophosphate (Vitamin B-1), Coenzyme A and sulfur containing phytoalexins.
Sulfate reduction occurs in both roots and shoots of plants. Most of the sulfur transported in the xylem to the leaves is in non-reduced SO42−. Some transport back to roots and other parts of the plant occurs through phloem, and both free SO42− and organic sulfur compounds are transported. In leaves, the process of sulfate reduction occurs in chloroplasts. In roots, most or all of the process occurs in proplastids.
Therefore, a preferred embodiment of the present invention is to provide cystathionine gamma synthase and/or serine acetyl transferase expression in the non-seed tissues of the plant where sulfate reduction is most active. This can be accomplished through tissue specific expression of these enzymes in non-seed tissue, such as leaves, roots or shoots or by constitutive expression of these enzymes. Constitutive expression will result in expression in both seed and non-seed tissue. Other methods can be used for increasing the activity of the cystathionine gamma synthase and/or serine acetyl transferase, such as protein engineering or DNA shuffling.
Other nucleic acids encoding enzymes involved in sulfate reduction or organic sulfur compound biosynthesis, such as cysteine and methionine biosynthesis, can be utilized to shunt the pathway in particular directions. For example, APS kinase can be down-regulated to increase APS concentration for APS reductase. In the same manner, an increase in methionine can be utilized as a sulfur source for downstream sulfur containing compounds. For example, glutathione is produced by the two step reaction of glutamate+cysteine+ATP→gamma-glutamylcysteine+ADP+Pi, followed by gamma-glutamylcysteine+glycine+ATP→glutathione+ADP+Pi, which reaction is catalyzed by glutathione synthetase. Another example is the series of reactions converting methionine to dimethylsulfoniopropionate (DMSP); the reactions converting methionine to vitamins and co-vitamins such as biotin, SMM (Vitamin U), SAM, Thiamine pyrophosphate (Vitamin B-1) and Coenzyme A; and the series of reactions converting methionine to sulfur containing phytoalexins that serve a pathogen defense function.
Also, antisense constructs for cystathionine gamma synthase and/or serine acetyl transferase can be utilized to direct biosynthesis into a particular product or to stop biosynthesis for the build-up of a particular compound. For example, an antisense construct for cystathionine gamma synthase can be used to shunt reduced sulfur away from methionine production.
Any means for producing a plant comprising the cystathionine gamma synthase or serine acetyl transferase are encompassed by the present invention. Additional nucleic acid(s) of interest can also be used to transform a plant at the same time as the cystathionine gamma synthase and/or serine acetyl transferase (cotransformation). The additional nucleic acid(s) of interest may code for other enzymes in the methionine pathway that will further modify cysteine or methionine levels or downstream sulfur containing compounds such as sulfur containing vitamins. The additional nucleic acid can also be introduced into a plant that has already been transformed with the cystathionine gamma synthase and/or serine acetyl transferase nucleic acid. Alternatively, transformed plants, one expressing the cystathionine gamma synthase and/or serine acetyl transferase and one expressing the additional nucleic acid, can be crossed to bring the nucleic acids together in the same plant. Subsequent crosses or transformations can bring additional sequences together in the plant.
Enzymes involved in cysteine and methionine biosynthesis are known in the art. See, for example, aspartokinase (Masakazu et al. (1992) “Mutant Aspartokinase Gene,” Japan Patent 1994062866-A 1 Mar. 8, 1994, Accession No. E06825; Omori et al. (1993) J. Bacteriol. 175(3):785-794; Accession No. X60821; Moriya et al. (1995) Japan Patent 1997070291-A 13 Mar. 18, 1997; Accession No. E12770); aspartate semialdehyde dehydrogenase (Calzada, F. R. A., Direct Submission, Centro Nacional de Investigaciones Cientificas, Avenida 25 esq. 158 reparto Cubanacan, Playa Ciudad de la Habana, Codigo Postal 6990, CUBA (1997), Accession No. Y15281; Daniel et al. (1993) J. Mol. Biol. 232 (2):468-483; Accession No. Z22554; Chen et al. (1993) J. Biol. Chem.; Accession No. Z22554; Accession No. U90239; Brakhage et al. (1990) Biochimie 72(10):725-734; Accession No. Z75208; Gothel et al. (1997) Eur. J. Biochem. 244 (1):59-65; Accession No. Z75208); homoserine kinase (See number two under aspartokinase, Accession No. X60821; Nakabachi et al. (1997) Insect Biochem. Mol. Biol. 27:1057-1062; Accession No. AB004856; Ryoichi et al. (1986) Japan Patent 1987232392-A 12 Oct. 1987 (JP1986076298); Accession No. E01358; Sadao et al., Japan Patent 1993207886-A 4 20 Aug. 1993; Accession No. D14072); threonine synthase (see number two under aspartokinase, Accession No. X6082; Accession No. Z46263; Rognes, S. E., Direct Submission, Oct. 24, 1994, to University of Oslo, Department of Biology, Blindern, 0316 Norway, Accession No. Z46263; Accession No. L41666; Clepet et al. (1992) Mol. Microbiol. 6(21):3109-3119; Accession No. X65033 S50569; Cami, B., Direct Submission, Mar. 11, 1992, Laboratoire de Chimie Bacterienne, Centre Nationale de la Recherche, Scientifique, 31 Chemin I. Aiguier, BP 71 13277 Marseille Cedex, FRANCE, Accession No. X65033 S50569); cystathionine gamma synthase (cystathionine gamma synthase) (Kim and Leustek (1996) “Cloning and analysis of the gene for cystathionine gamma-synthase from Arabidopsis thaliana,” Plant Mol. Biol. 32 (6), 1117-1124, USA, Accession No. AF069317; Locke et al., Direct Submission, Jun. 3, 1997, AG Biotechnology, DuPont AF Products, PO Box 80402, Wilmington, Del. 19880-0402 USA, maize cystathionine gamma synthase, Accession No. AF007785 and AF007786; rice cystathionine gamma synthase (AF076495); potato cystathionine gamma synthase (AF082891, AF082892, AF144102); soybean cystathionine gamma synthase (AF141602); arabidopsis cystathionine gamma synthase (U43709, U83500, X94756, AC027035, AC051630); cystathionine beta lyase (Bork et al. (1997) Plant Physiol. 115:864-864; Accession No. AJ001148; Sienko, M., Direct Submission, Jun. 5, 1995, Marzena Sienko, Genetics, Institute of Biochemistry and Biophysics, Pawinskiego 5a, Warsaw O2-106, POLAND, Accession No. U28383; Ravanel et al. (1995) Plant Mol. Biol. 29 (4):875-882; Accession No. L40511); methionine synthase (Kurvari et al. (1995) Plant Mol. Biol. 29:1235-1252; Accession No. U36197; Ravanel et al. (1998) Proc. Natl. Acad. Sci. USA 95(13):7805-7812; Accession No. U97200; Michalowski et al., Direct Submission, Jan. 12, 1997, Biochemistry, University of Arizona, BioSciences West 513, Tucson, Ariz. 85721 USA, Accession No. U84889; Eichel et al. (1995) Eur. J. Biochem. 230 (3):1053-1058; Accession No. X83499); ATP sulfurylase (Murillo et al. (1995) Arch. Biochem. Biophys. 323(1):195-204; Accession No. U06275; Leustek et al. (1994) Plant Physiol. 105:897-902; Accession No. U05218; Bolchi et al., Direct Submission, Jul. 28, 1997, Scienze Biochimiche, Viale delle Scienze, Parma, PR 43100 ITALY, Accession No. AF016305; Laue et al. (1994) J. Bacteriol. 176:3723-3729; Accession No. L26897; Laeremans et al. Accession No. AJ001223); U.S. patent application Ser. No. 09/346,408 by DuPont entitled, Sulfate Assimilation Proteins; soybean ATP sulfurylase (BG156200, BG045264, BG041863, BF009916, BF009045, BE807651, BE804451, BE556220, BE475509, BE473960, BE210444, BE191366, AW458276, AW309791, AW278474, AW277946, AW277743, AW234410), tomato ATP sulfurylase (BF113119, BF096727), rice ATP sulfurylase (AB015204), brassica juncea ATP sulfurylase (AJ223498), brassica oleracea ATP sulfurylase (AF195511), allium ATP sulfurylase (AF212154), arabidopsis ATP sulfurylase (BE844959, BE039475, AJ012586, U59737, AF198964, AF110407, U59738, AL161539, AP001300, S68202); APS kinase (apk) (Korch et al. (1991) Mol. Gen. Genet. 229(1):96-108; Accession No. S55315; Arz et al. (1994) Biochim. Biophys. Acta 1218 (3):447-452; Accession No. AF044285; Schiffmann et al. “Isolation of cDNA clones encoding adenosine-5′-phophosulfate-kinase (EC2.7.1.25) from Catharanthus roseus (Accession No. AF044285) and an isoform (akn2) from Arabidopsis (Accession No. AF043351)(PGR98-116),” Plant Physiol. 117 (3):1125 (1998); Accession No. AF044285; Jain et al. (1994) Plant Physiol. 105:771-772; Accession No. U05238; Lee et al. (1998) Biochem. Biophys. Res. Commun. 247:171-175; Accession No. U05238); APS reductase (Speich et al. (1994) Microbiology 140 (Pt6): 1273-1284; Accession No. Z69372; Setya et al. (1996) Proc. Natl. Acad. Sci. USA 93(23):13383-1338; Accession No. U56921; Bick et al. (1998) Proc. Natl. Acad. Sci. USA 95(14):8404-8409; DuPont patent application, Genes Encoding Sulfate Assimilation Proteins, PCT publication number WO00/04161); PAPS reductase (Krone et al. (1991) Mol. Gen. Genet. 225(2):314-319; Accession No. Y07525; Krone et al. (1990) FEBS Lett. 260 (1):6-9; Accession No. Y07525; Gutierrez-Marcos et al. (1996) Proc. Natl. Acad. Sci. USA 93:13377-13382; Accession No. U53865; Schwenn, J. D., Direct Submission, Jul. 2, 1993, Ruhr-University-Bochum, Fac. Biology, Biochemistry of Plants, Universitaetsstr. 150, D44780 Bochum, GERMANY, Accession No. Z23169; see number five under ATP sulfurylase, Accession No. AJ001223; Bussey et al. (1997) Nature 387 (6632 Suppl.):103-105; Accession No. U25840 U00094); sulfite reductase (Accession No. Y07525; Accession No. Z23169; Hipp et al. (1997) Microbiology 143 (Pt 9):2891-2902; Accession No. U84760; Pott et al. (1998) Microbiology 144 (Pt 7):1881-1894; Accession No. U84760; Bork et al. (1998) Gene 212 (1):147-153; Accession No. Y10157; Mbeguie-A-Mbeguie et al. Accession No. AF071890; Bruehl et al. (1996) Biochim. Biophys. Acta 1295:119-124; Accession No. Z49217; Hummerjohann et al. (1998) Microbiology 144 (Pt 5):1375-1386; Accession No. AF026066; serine acetyltransferase (Accession No. X80938; Accession No. D88529 and D88530; Saito et al. (1995) J. Biol. Chem. 270 (27):16321-16326; Accession No. D49535; DuPont patent application, Genes Encoding Sulfate Assimilation Proteins, PCT publication number WO 00/04167; see also arabidopsis serine acetyl transferase (U22964, L78443, X8288, L78444, AF112303, U30298, L42212, U22964, L78443, X82888, Z34888 and X80938), soybean serine acetyl transferase (BF041806, BE802695, AW234818, AI965408, AI495784, AI437954), tomato serine acetyl transferase (BF176520, BF098353), allium serine acetyl transferase (AF212156, AB040502) and cotton serine acetyl transferase (A1725434); cysteine synthase (Hesse et al. (1998) “Isolation of cDNAs encoding cytosolic (Accession No. AF044172) and plastidic (Accession No. AF044173) cysteine synthase isoforms from Solanum tuberosum (PGR98-057),” Plant Physiol. 116:1604, Accession No. AF044173; Brander et al. (1995) Plant Physiol. 108:1748-1748; Accession No. X85803; Topczewski et al. (1997) Curr. Genet. 31 (4):348-356; Accession No. U19395); gamma glutamylcysteine synthase (Powles et al. (1996) Microbiology 142 (Pt 9):2543-2548; Accession No. U81808 L75931; Accession No. AL031018; EU Arabidopsis sequencing project, Direct Submission, Jul. 3, 1998, at the Max-Planck-Institut fuer Biochemie, Am Klopferspitz 18a, D-82152 Martinsried, FRG, Accession No. AL031018); glutathione synthetase (Okumura et al. (1997) Microbiology 143 (Pt 9):2883-2890; Accession No. D88540; Inoue et al. (1998) Biochim. Biophys. Acta 1395 (3):315-320; Accession No. Y13804; Accession No. Y10984; Accession No. U22359).
Variants and functional fragments, including shufflents, of the above enzymes, specifically including cystathionine gamma synthase and serine acetyl transferase, may be utilized. It is only required that the enzymes have an activity sufficient to modulate the level of a particular organic sulfur compound in a plant. Variants can be produced by methods known in the art. Variant proteins include those proteins derived from the native protein by deletion (so-called truncation), addition, or substitution of one or more amino acids at one or more sites in the native protein. Additional sequences for use in the invention may be obtained by screening DNA libraries or sequence databases of plants or other species. Some of the RNA partial sequences and precursors listed above may be used to screen DNA libraries to obtain full length genomic or cDNA sequences.
Amino acid sequence variants of the polypeptide can be prepared by mutations in the cloned DNA sequence encoding the native protein of interest. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York); Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods Enzymol. 154:367-382; Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.); U.S. Pat. No. 4,873,192; and the references cited therein; herein incorporated by reference. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), herein incorporated by reference. Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be preferred.
The cystathionine gamma synthase and serine acetyl transferase nucleic acids, as well as any additional genes of interest, can be optimized for enhanced expression in plants of interest. See, for example, EPA0359472; WO91/16432; Perlak et al. (1991) Proc. Natl. Acad. Sci. USA 88:3324-3328; and Murray et al. (1989) Nucleic Acids Res. 17:477-498. In this manner, the nucleic acids can be synthesized utilizing plant-preferred codons. See, for example, Murray et al. (1989) Nucleic Acids Res. 17:477-498, the disclosure of which is incorporated herein by reference. In this manner, synthetic nucleic acids can also be made based on the distribution of codons a particular host uses for a particular amino acid.
Another method for obtaining modified enzymes that can alter the level of at least one organic sulfur compound is by sequence shuffling. Sequence shuffling is described in PCT publication No. 96/19256. See also, Zhang et al. (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509. Libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo.
The terms cystathionine gamma synthase nucleic acid and serine acetyl transferase nucleic acid used in this application refer to all nucleic acids described herein or obtainable by the methods described herein that, when utilized in the present invention, alter the level of an organic sulfur compound.
In some instances, the enzymes of interest are natively expressed in the plant. However, by transformation with heterologous promoters, expression levels or patterns can be altered. See, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.). and Innis et al. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York).
The nucleic acids can be combined with constitutive or non-seed tissue-specific promoters for expression of the metabolite of interest. Such constitutive promoters include, for example, the core promoter of the Rsyn7 (copending U.S. patent application Ser. No. 08/661,601), the core CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. patent application Ser. No. 08/409,297), and the like. Other constitutive promoters include, for example, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; and 5,608,142.
Non-seed tissue specific promoters include, for example Yamamoto et al. (1997) Plant J. 12(2)255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol. Gen Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2):157-168; Rinehart et al. (1996) Plant Physiol. 112(3):1331-1341; Van Camp et al. (1996) Plant Physiol. 112(2):525-535; Canevascini et al. (1996) Plant Physiol. 12(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196; Orozco et al. (1993) Plant Mol. Biol. 23(6):1129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J. 4(3):495-505. All of such promoters can be modified, if necessary, for weak expression.
Leaf-preferred promoters are known in the art. See, for example, Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al. (1994) Plant Physiol. 105:357-67; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al. (1993) Plant Mol. Biol. 23(6):1129-1138; and Matsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590.
Root-preferred promoters are known and can be selected from the many available from the literature or isolated de novo from various compatible species. See, for example, Hire et al. (1992) Plant Mol. Biol. 20(2): 207-218 (soybean root-preferred glutamine synthetase gene); Keller and Baumgartner (1991) Plant Cell 3(10):1051-1061 (root-preferred control element in the GRP 1.8 gene of French bean); Sanger et al. (1990) Plant Mol. Biol. 14(3):433-443 (root-preferred promoter of the mannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao et al. (1991) Plant Cell 3(1):11-22 (full-length cDNA clone encoding cytosolic glutamine synthetase (GS), which is expressed in roots and root nodules of soybean). See also Bogusz et al. (1990) Plant Cell 2(7):633-641, where two root-preferred promoters isolated from hemoglobin genes from the nitrogen-fixing nonlegume Parasponia andersonii and the related non-nitrogen-fixing nonlegume Trema tomentosa are described. The promoters of these genes were linked to a β-glucuronidase reporter gene and introduced into both the nonlegume Nicotiana tabacum and the legume Lotus corniculatus, and in both instances root-preferred promoter activity was preserved. Leach and Aoyagi (1991) describe their analysis of the promoters of the highly expressed roIC and roID root-inducing genes of Agrobacterium rhizogenes (see Plant Science (Limerick) 79(1):69-76). They concluded that enhancer and tissue-preferred DNA determinants are dissociated in those promoters. Teeri et al. (1989) used gene fusion to lacZ to show that the Agrobacterium T-DNA gene encoding octopine synthase is especially active in the epidermis of the root tip and that the TR2′ gene is root specific in the intact plant and stimulated by wounding in leaf tissue, an especially desirable combination of characteristics for use with an insecticidal or larvicidal gene (see EMBO J. 8(2):343-350). The TR1′ gene, fused to nptll (neomycin phosphotransferase 11) showed similar characteristics. Additional root-preferred promoters include the VfENOD-GRP3 gene promoter (Kuster et al. (1995) Plant Mol. Biol. 29(4):759-772); and rolB promoter (Capana et al. (1994) Plant Mol. Biol. 25(4):681-691. See also U.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,110,732; and 5,023,179. Tissue preferred and specific promoters also include the tissue specific and tissue preferred promoters listed in PCT application publication number WO 00/36124, Page 35, Table A, which table is hereby incorporated by reference.
“Seed-specific” promoters, such as globulin 1, glutelin 2, cruciferin, napin, B-conglycinin, phaseolin, as well as other seed or endosperm specific promoters are not appropriate for use in the present invention.
The promoter may be native or analogous or foreign or heterologous to the plant host. Additionally, the promoter may be a synthetic sequence. By foreign is intended that the transcriptional initiation region is not found in the native plant into which the transcriptional initiation region is introduced. Although both monocot and dicot promoters are described herein, monocot promoters are preferred for use in the present invention.
Expression cassettes will comprise a promoter linked to the coding sequence or antisense sequence of the nucleotide of interest. Such an expression cassette is generally provided with a plurality of restriction sites for insertion of the sequence to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes.
The transcriptional cassette will include in the 5′-to-3′ direction of transcription, a transcriptional and translational initiation region, a DNA sequence of interest, and a transcriptional and translational termination region functional in plants. The termination region may be native with the transcriptional initiation region, may be native with the DNA sequence of interest, or may be derived from another source. Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell. 2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; Joshi et al. (1987) Nucleic Acids Res. 15:9627-9639.
In preparing the expression cassette, the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved.
Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell targeted for transformation. Suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606, Agrobacterium-mediated transformation (Townsend et al., U.S. Pat. No. 5,563,055); direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic particle acceleration (see, for example, Sanford et al., U.S. Pat. No. 4,945,050; Tomes et al. (1995) “Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment,” in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); and McCabe et al. (1988) Biotechnology 6:923-926). Also see Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); Tomes, U.S. Pat. No. 5,240,855; Buising et al., U.S. Pat. Nos. 5,322,783 and 5,324,646; Tomes et al. (1995) “Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment,” in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg (Springer-Verlag, Berlin) (maize); Klein et al. (1988) Plant Physiol. 91:440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839 (maize); De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, New York), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418; and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566 (whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens); all of which are herein incorporated by reference.
The biosynthesis of organic sulfur compounds can be altered in accordance with the present invention in any monocot plant of interest. Of particular interest are plants useful for human and domestic animal food. Such plants include forages and seed crop plants such as cereal crops. Of particular interest are plants where the seed is produced in high amounts, or the seed or a seed part is edible. Seeds of interest include the grain seeds such as wheat, barley, rice, corn, rye, millet and sorghum. Especially preferred plants are corn, wheat and rice.
The modified plant may be grown into plants in accordance with conventional ways. See, for example, McCormick et al. (1986) Plant Cell. Reports 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that the subject phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure the desired phenotype or other property has been achieved.
The following examples are offered by way of illustration and not by way of limitation.
EXAMPLES
Example 1
Transformation and Regeneration of Transgenic Maize Plants with Cystathionine Gamma Synthase
Immature maize embryos from greenhouse donor plants are bombarded with a plasmid containing cystathionine gamma synthase nucleotide sequence (Accession No. AF007786, SEQ ID NO: 5) operably linked to a ubiquitin promoter (U.S. Pat. Nos. 5,510,474 and 5,614,399) that has been optimized for maize codon preference and a pin II terminator (An et. al. 1989), plus the selectable marker gene PAT (Wohlleben et al. (1988) Gene 70:25-37) that confers resistance to the herbicide Bialaphos.
Transformation of Maize
Freshly isolated immature embryos of maize, about 10 days after pollination (DAP), are cultured for 4-5 days before transforming DNA is delivered via particle bombardment. The preferred genotype for transformation is the highly transformable genotype Hi-II (Armstrong, C. L., 1991, Development and Availability of Germplasm with High Type II Culture Formation Response, Maize Genetics Cooperation Newsletter, 65:92-93). An F1 hybrid created by crossing with a Hi-II with an elite inbred may also be used. After DNA delivery, the embryos are cultured on medium containing toxic levels of herbicide. Only those cells which receive the herbicide-resistance gene, and the linked gene(s), grow on selective medium. Transgenic events so selected are propagated and regenerated to whole plants, produce seed, and transmit transgenes to progeny.
Particle Gun Terminology and Use
The PDS-1000 Biolistics particle bombardment device is used to transform maize. The operation of this device is detailed in the operating instructions available from the manufacturer (Bio-Rad Laboratories, Hercules, Calif.).
The macrocarrier flight distance is fixed in the instrument at 1⁄4′′ (0.25′′). While the rupture disk-macrocarrier gap distance is adjustable, the device is operated at the factory recommended distance of 1⁄8′′ (0.125′′).
Preparation of Particles
The transforming DNA is associated with either tungsten or gold particles. Prior to association with the transforming DNA, the tungsten particles are prepared essentially as described by Tomes et al. (U.S. Pat. No. 5,990,387).
The preferred method utilizes gold particles. Gold particles are prepared as follows. Sixty mg of 0.6μ gold particles (Bio-Rad) are placed in 2.0 mL Sarstedt tube. The particles are washed three times in absolute ethanol (100%). Each ethanol wash involves adding one mL of absolute ethanol to the tube, sonicating the tube briefly, vortexing the tube on high for one minute, centrifuging the tube to pellet the particles and discarding the supernatant. The particles are then washed two times in sterile deionized water. Each wash involves adding one mL of sterile deionized water to the tube, sonicating the tube briefly, vortexing the tube on high for one minute, centrifuging the tube to pellet the particles and discarding the supernatant. Following the ethanol and water wash steps, one mL of sterile deionized water is added to the tube and the tube is sonicated. Aliquots (250 μL) of the particle-containing suspension are removed to siliconized 1.5 mL tubes and combined with 750 μL sterile deionized water.
Association of Particles with Transforming DNA
The transforming DNA is associated with the prepared tungsten or gold particles by precipitation in a solution comprising CaCl2 and spermidine as follows. A tube containing tungsten or gold particles prepared as described above is sonicated for 3 seconds at setting 2.5 in a water bath probe, Branson Sonicator #450 (Branson Ultrasonics Corp., Danbury Conn.). Ten μL plasmid DNA (1 μg plasmid total) in TE buffer is added to the tube and mixed for 5 seconds. Next, 100 μL 2.5 M CaCl2 and 10 μL 0.1 M spermidine are added. The tube is then shaken on a vortexer for 10 minutes followed by centrifugation for 30 seconds at 10,000 rpm. The supernatant is removed and discarded, and 500 μL absolute ethanol is added. The tube is then sonicated at setting 2.5 for 3 seconds, centrifuged for 30 seconds at 10,000 rpm and the supernatant removed. To the tube, 105 μL of absolute ethanol is added. The tube is sonicated for 3 seconds at setting 2.5 before placing a 10 μL aliquot onto the center of a macrocarrier.
Preparation of Target Tissue
Ears of Hi-II or Hi-II X elite inbred are sampled in planta to assess the developmental stage of the embryos. When immature embryos first become opaque, about 9-12 days post-pollination, the ears are harvested for embryo dissection. The embryos are approximately 1.5-1.8 mm long from coleoptilar to coleorhizal end. Immature embryos are the target tissue for transient and stable transformation experiments.
The ears are surface sterilized in 50% (v/v) Clorox bleach+0.5% (v/v) Micro detergent for 20 minutes, and then rinsed twice with sterile water. The immature embryos are excised from the caryopsis and placed embryo axis side down (scutellum side up) onto transformation support medium.
Embryos are cultured on 560L medium for 4-5 days in darkness at 28° C. At this time, a small amount of incipient embryogenic tissue can be observed at the coleorhizal end of the scutellum, but there is no production of subculturable tissue.
Delivery of DNA
As preparation for bombardment, the 4 day pre-cultured embryos are transferred to 561Y medium, which contains elevated sucrose, and are incubated in darkness at 28° C. for 4 hours. The embryos are arranged, 10 embryos per plate, in a 2 cm target area. The embryos are angled with their coleorhizal end pointing up toward the macrocarrier at approximately a 30° angle. This orientation of the cultured embryos enhances exposure of the preferred cell targets to the path of particles propelled by the particle gun.
Plates of embryos are bombarded at shelf 2 from the bottom of the device, 650 PSI rupture disk, and a chamber vacuum of 28 mm Hg.
The bombarded plates are incubated in darkness at 28° C. for two days. After the two-day bombardment recovery period, the embryos are transferred to Petri dishes containing 560 R medium. This latter medium is comprised of those components which typically are used to initiate and promote embryogenic tissue from maize embryos, and contains 2% sucrose, and 3 ppm bialaphos as a selective agent. The plates are incubated in darkness at 28° C. for 4-6 weeks, or until growth of putatively transformed events are observed. 560 R culture medium does not support the growth of untransformed tissue derived from the bombarded embryos. Therefore, only putatively transformed tissue, resistant to bialaphos as a consequence of expressing the resistance transgene, are competent to grow.
Putatively transformed events are identified first by their growth under selective conditions and individually subcultured to fresh 560 R medium for propagation. Samples of each event are assayed for their transgenic nature by PCR reaction using primer sets designed to specifically amplify sequences in the inserted gene(s).
Regeneration of T0 Plants
Transformed, selection-resistant embryogenic tissue is transferred to 288 J medium to initiate plant regeneration. Following somatic embryo maturation (2-4 weeks), well-developed somatic embryos are transferred to medium for germination (272 V) and transferred to a lighted culture room. Approximately 7-10 days later, developing plantlets are transferred to 272 V hormone-free medium in tubes for 7-10 days until plantlets are well established. Plants are then transferred to inserts in flats (equivalent to 2.5′′ pot) containing potting soil and grown for 1 week in a growth chamber, subsequently grown an additional 1-2 weeks in the greenhouse, then transferred to 1.6 gallon pots and grown to maturity. Plants are monitored and scored for altered cystathionine gamma synthase and/or phenotype such as increased sulfur compounds.
Media Recipes
Medium 288 J contains the following ingredients: 950.000 ml of D-I H2O; 4.300 g of MS Salts; 0.100 g of Myo-Inositol; 5.000 ml of MS Vitamins Stock Solution (No. 36J); 1.000 ml of Zeatin.5 mg/ml; 60.000 g of Sucrose; 3.000 g of Gelrite, which is added after Q.S. to volume; 2.000 ml of IAA 0.5 mg/ml #; 1.000 ml of 0.1 Mm ABA #; 3.000 ml of Bialaphos 1 mg/ml #; and 2.000 ml of Agribio Carbenicillin 50 mg/ml #. Directions are: dissolve ingredients in polished D-I H2O in sequence; adjust to pH 5.6; Q.S. to volume with polished D-I H2O after adjusting pH; sterilize and cool to 60° C. Add 3.5 g/L of Gelrite for cell biology. Ingredients designated with # are added after sterilizing and cooling to temperature.
Medium 272 V contains the following ingredients: 950.000 ml of D-I H2O; 4.300 g of MS Salts; 0.100 g of Myo-Inositol; 5.000 ml of MS Vitamins Stock Solution; 40.000 g of Sucrose; and 6.000 g of Bactoagar, which is added after Q.S. to volume. Directions are: dissolve ingredients in polished D-I H2O in sequence; adjust to pH 5.6; Q.S. to volume with polished D-I H2O after adjusting pH; and sterilize and cool to 60° C.
Medium 560 L contains the following ingredients: 950.000 ml of D-I Water, Filtered; 4.000 g of CHU (N6) Basal Salts (SIGMA C-1416); 1.000 ml of Eriksson's Vitamin Mix (1000× SIGMA-1511); 1.250 ml of Thiamine.HCL 0.4 mg/ml; 30.000 g of Sucrose; 4.000 ml of 2,4-D 0.5 mg/ml; 3.000 g of Gelrite, which is added after Q.S. to volume; and 0.425 ml of Silver Nitrate 2 mg/ml #. Directions are: dissolve ingredients in D-I H2O in sequence; adjust to pH 5.8 with KOH; bring up to volume with D-I H2O; sterilize and cool to room temp. Total volume (L)=1.00. Ingredients designated with # are added after sterilizing and cooling to temperature.
Medium 560 R contains the following ingredients: 950.000 ml D-I Water, Filtered; 4.000 g of CHU (N6) Basal Salts (SIGMA C-1416); 1.000 ml Eriksson's Vitamin Mix (1000× SIGMA-1511); 1.250 ml of Thiamine.HCL 0.4 mg/ml; 30.000 g Sucrose; 4.000 ml of 2,4-D 0.5 mg/ml; 3.000 g of Gelrite, which is added after Q.S. to volume; 0.425 ml of Silver Nitrate 2 mg/ml #; and 3.000 ml of Bialaphos 1 mg/ml #. Directions are: dissolve ingredients in D-I H2O in sequence; adjust to pH 5.8 with KOH; bring up to volume with D-I H2O; sterilize and cool to room temp. Total volume (L)=1.00. Ingredients designated with # are added after sterilizing and cooling to temperature.
Medium 561 Y contains the following ingredients: 950.000 ml of D-I Water, Filtered; 4.000 g of CHU (N6) Basal Salts (SIGMA C-1416); 1.000 ml of Eriksson's Vitamin Mix (1000× SIGMA-1511); 1.250 ml of Thiamine.HCL 0.4 mg/ml; 190.000 g of Sucrose; 2.000 ml of 2,4-D 0.5 mg/ml; 2.880 g of L-Proline; 2.000 g of Gelrite, which is added after Q.S. to volume; and 4.250 ml of Silver Nitrate 2 mg/ml #. Directions are: dissolve ingredients in D-I H2O in sequence; adjust to pH 5.8 with KOH; bring up to volume with D-I H2O; sterilize and cool to room temp. Autoclave less time because of increased sucrose. Total volume (L)=1.00. Ingredients designated with # are added after sterilizing and cooling to temperature.
Example 2
Agrobacterium-Mediated Transformation of Maize with Cystathionine Gamma Synthase
For Agrobacterium-mediated transformation of maize with a cystathionine gamma synthase nucleotide sequence, a maize cDNA for cystathionine gamma synthase (Accession No. AF007786) is fused to a maize optimized ubiquitin promoter (U.S. Pat. Nos. 5,510,474 and 5,614,399) that has been optimized for maize codon preference and a pin 11 terminator sequence (An, et al., 1989). The cystathionine gamma synthase cassette, also containing a CaMV35S-bialaphos selectable marker element, is cloned into a binary vector and introduced into Agrobacterium. 
Transformation of Maize Mediated by Agrobacterium 
Freshly isolated immature embryos of maize, about 10 days after pollination (DAP), are incubated with the Agrobacterium. The preferred genotype for transformation is the highly transformable genotype Hi-II (Armstrong, C. L., 1991, Development and Availability of Germplasm with High Type II Culture Formation Response, Maize Genetics Cooperation Newsletter, 65:92-93). An F1 hybrid created by crossing with an Hi-II with an elite inbred may also be used. After Agrobacterium treatment of immature embryos, the embryos are cultured on medium containing toxic levels of herbicide. Only those cells which receive the herbicide-resistance gene, and the linked gene(s), grow on selective medium. Transgenic events so selected are propagated and regenerated to whole plants, produce seed, and transmit transgenes to progeny.
Preparation of Agrobacterium 
The engineered Agrobacterium tumefaciens LBA4404 is constructed as per U.S. Pat. No. 5,591,616 to contain the linked gene(s) and the selectable marker gene. Typically either BAR (D'Halluin et al. (1992) Methods Enzymol. 216:415-426) or PAT (Wohlleben et al. (1988) Gene 70:25-37) may be used.
To use the engineered vector in plant transformation, a master plate of single bacterial colonies is first prepared by inoculating the bacteria on minimal AB medium and then incubating the bacteria plate inverted at 28° C. in darkness for about 3 days. A working plate is then prepared by selecting a single colony from the plate of minimal A medium and streaking it across a plate of YP medium. The YP-medium bacterial plate is then incubated inverted at 28° C. in darkness for 1-2 days.
Agrobacterium for plant transfection and co-cultivation is prepared 1 day prior to transformation. Into 30 ml of minimal A medium in a flask is placed 50 μg/ml spectinomycin (or appropriate bacterial antibiotic depending on marker in co-integrate), 100 μM acetosyringone, and about a 1⁄8 loopful of Agrobacterium from a 1 to 2-day-old working plate. The Agrobacterium is then grown at 28° C. at 200 rpm in darkness overnight (about 14 hours). In mid-log phase, the Agrobacterium is harvested and resuspended at 3 to 5×108 CFU/ml in 561Q medium+100 μM acetosyringone using standard microbial techniques and standard curves.
Immature Embryo Preparation
Nine to ten days after controlled pollination of a corn plant, developing immature embryos are opaque and 1-1.5 mm long and are the appropriate size for Agro-infection. The husked ears are sterilized in 50% commercial bleach and 1 drop Tween for 30 minutes, and then rinsed twice with sterile water. The immature embryos are aseptically removed from the caryopsis and placed into 2 ml of sterile holding solution comprising of 561Q+100 μM acetosyringone.
Agrobacterium Infection and Co-Cultivation of Embryos
Holding solution is decanted from excised immature embryos and replaced with prepared Agrobacterium. Following gentle mixing and incubation for about 5 minutes, the Agrobacterium is decanted from the immature embryos. Immature embryos are then moved to a plate of 562P medium, scutellum surface upwards, and incubated at 20° C. for 3 days in darkness followed by incubation at 28° C. for 3 days in darkness on medium 562P+100 mg/ml carbenecillin (see U.S. Pat. No. 5,981,840).
Selection of Transgenic Events
Following incubation, the immature embryos are transferred to 5630 medium for selection of events. The transforming DNA possesses a herbicide-resistance gene, in this example the PAT gene, which confers resistance to bialaphos. At 10- to 14-day intervals, embryos are transferred to 5630 medium. Actively growing putative transgenic embryogenic tissue are visible in 6-8 weeks.
Regeneration of T0 Plants
Transgenic embryogenic tissue is transferred to 288W medium and incubated at 28° C. in darkness until somatic embryos matured, or about 10 to 18 days. Individual matured somatic embryos with well-defined scutellum and coleoptile are transferred to 272 embryo germination medium and incubated at 28° C. in the light. After shoots and roots emerge, individual plants are potted in soil and hardened-off using typical horticultural methods.
Confirmation of Transformation
Putative transgenic events are subjected to analysis to confirm their transgenic nature.
Events are tested for the presence of the cystathionine gamma synthase by PCR amplification. Additionally, T0 plants are painted with bialaphos herbicide. The subsequent lack of a herbicide-injury lesion indicates the presence and action of the herbicide resistance gene. The plants are monitored and scored for altered cystathionine gamma synthase expression and/or phenotype such as increased organic sulfur compounds.
Media Recipes
Medium 561 Q contains the following ingredients: 950.000 ml of D-I Water, Filtered; 4.000 g of Chu (N6) Basal Salts (Sigma C-1416); 1.000 ml of Eriksson's Vitamin Mix (1000× Sigma-1511); 1.250 ml of Thiamine.HCL.4 mg/ml; 3.000 ml of 2,4-D 0.5 mg/ml (No. 2A); 0.690 g of L-proline; 68.500 g of Sucrose; and 36.000 g of Glucose. Directions are: dissolve ingredients in polished D-I H2O in sequence; adjust pH to 5.2 w/KOH; Q.S. to volume with polished D-I H2O after adjusting pH; and filter sterilize (do not autoclave).
Medium 562 P contains the following ingredients: 950.000 ml of D-I Water, Filtered; 4.000 g of Chu (N6) Basal Salts (Sigma C-1416); 1.000 ml of Eriksson's Vitamin Mix (1000× Sigma-1511); 1.250 ml of Thiamine.HCL.4 mg/ml; 4.000 ml of 2,4-D 0.5 mg/ml; 0.690 g of L-proline; 30.000 g of Sucrose; 3.000 g of Gelrite, which is added after Q.S. to volume; 0.425 ml of Silver Nitrate 2 mg/ml #; and 1.000 ml of Aceto Syringone 100 mM #. Directions are: dissolve ingredients in polished D-I H2O in sequence; adjust pH to 5.8 w/KOH; Q.S. to volume with polished D-I H2O after adjusting pH; and sterilize and cool to 60° C. Ingredients designated with a # are added after sterilizing and cooling to temperature.
Medium 563 O contains the following ingredients: 950.000 ml of D-I Water, Filtered; 4.000 g of Chu (N6) Basal Salts (Sigma C-1416); 1.000 ml of Eriksson's Vitamin Mix (1000× Sigma-1511); 1.250 ml of Thiamine.HCL.4 mg/ml; 30.000 g of Sucrose; 3.000 ml of 2,4-D 0.5 mg/ml (No. 2A); 0.690 g of L-proline; 0.500 g of Mes Buffer; 8.000 g of Agar (Sigma A-7049, Purified), which is added after Q.S. to volume; 0.425 ml of Silver Nitrate 2 mg/ml #; 3.000 ml of Bialaphos 1 mg/ml #; and 2.000 ml of Agribio Carbenicillin 50 mg/ml #. Directions are: dissolve ingredients in polished D-I H2O in sequence; adjust to pH 5.8 w/koh; Q.S. to volume with polished D-I H2O after adjusting pH; sterilize and cool to 60° C. Ingredients designated with a # are added after sterilizing and cooling to temperature.
Medium 288 W contains the following ingredients: 950.000 ml of D-I H2O; 4.300 g of MS Salts; 0.100 g of Myo-Inositol; 5.000 ml of MS Vitamins Stock Solution (No. 36J); 1.000 ml of Zeatin.5 mg/ml; 60.000 g of Sucrose; 8.000 g of Agar (Sigma A-7049, Purified), which is added after Q.S. to volume; 2.000 ml of IAA 0.5 mg/ml #; 1.000 ml of 0.1 Mm ABA #; 3.000 ml of Bialaphos 1 mg/ml #; and 2.000 ml of Agribio Carbenicillin 50 mg/ml #. Directions are: dissolve ingredients in polished D-I H2O in sequence; adjust to pH 5.6; Q.S. to volume with polished D-I H2O after adjusting pH; sterilize and cool to 60° C. Add 3.5 g/L of Gelrite for cell biology. Ingredients designated with a # are added after sterilizing and cooling to temperature.
Medium 272 contains the following ingredients: 950.000 ml of D-I H2O; 4.300 g of MS Salts; 0.100 g of Myo-Inositol; 5.000 of MS Vitamins Stock Solution; 40.000 g of Sucrose; and 1.500 g of Gelrite, which is added after Q.S. to volume. Directions are: dissolve ingredients in polished D-I H2O in sequence; adjust to pH 5.6; Q.S. to volume with polished D-I H2O after adjusting pH; and sterilize and cool to 60° C.
Medium minimal A contains the following ingredients: 950.000 ml of D-1 H2O; 10.500 g of potassium phosphate dibasic K2HPO4; 4.500 g of potassium phosphate monobasic KH2PO4; 1.000 g of ammonium sulfate; 0.500 g of sodium citrate dihydrate; 10.000 ml of sucrose 20% solution #; and 1.000 ml of 1M magnesium sulfate #. Directions are: dissolve ingredients in polished D-I H2O in sequence; Q.S. to volume with D-I H2O; sterilize and cool to 60° C. Ingredients designated with a # are added after sterilizing and cooling to temperature.
Medium minimal AB contains the following ingredients: 850.000 ml of D-I H2O; 50.000 ml of stock solution 800A; 9 g of Phytagar which is added after Q.S. to volume; 50.000 ml of stock solution 800B #; 5.000 g of glucose #; and 2.000 ml of spectinomycin 50/mg/ml stock #. Directions are: dissolve ingredients in polished D-I H2O in sequence; Q.S. to volume with polished D-I H2O less 100 ml per liter; sterilize and cool to 60° C. Ingredients designated with a # are added after sterilizing and cooling to temperature. Stock solution 800A contains the following ingredients: 950.000 ml of D-I H2O; 60.000 g of potassium phosphate dibasic K2HPO4; and 20.000 g of sodium phos. monobasic, hydrous. Directions are: dissolve ingredients in polished D-I H2O in sequence; adjust pH to 7.0 w/koh; Q.S. to volume with polished D-I H2O after adjusting pH; and sterilize and cool to 60° C. Stock solution 800B contains the following ingredients: 950.000 ml of D-I H2O; 20.000 g of ammonium chloride; 6.000 g of magnesium sulfate 7-H2O, MgSO4, 7H2O; 3.000 g of potassium chloride; 0.200 g of calcium chloride (anhydrate); and 0.050 g of ferrous sulfate 7-hydrate. Directions are: dissolve ingredients in polished D-I H2O in sequence; Q.S. to volume with polished D-I H2O; and sterilize and cool to 60° C.
Medium minimal YP contains the following ingredients: 950.000 ml of D-I H2O; 5.000 g of yeast extract (Difco); 10.000 g of peptone (Difco); 5.000 g of sodium chloride; 15.000 g of bacto-agar, which is added after Q.S. to volume; and 1.000 ml of spectinomycin 50 mg/ml stock #. Directions are: dissolve ingredients in polished D-I H2O in sequence; adjust pH to 6.8 w/koh; Q.S. to volume with polished D-I H2O after adjusting pH; sterilize and cool to 60° C. Ingredients designated with a # are added after sterilizing and cooling to temperature.
Results
Following two generations of selfing, plants homozygous for the cystathionine gamma synthase construct and null segregates derived from more than 40 events are analyzed. As can be seen in FIG. 2, forty seeds from each of three events homozygous for cystathionine gamma synthase (6337, 6326, 6311; shown by shaded bars on FIGS. 2 and 3) and null segregants (C1-C3; shown by open bars on FIGS. 2 and 3) are analyzed for total sulfur content of amino acids. (Beckman Instruments 6300 analyzer—amino acids detected with ninhydrin.) Events homozygous for cystathionine gamma synthase show a statistically significant increase in levels of cysteine and methionine over the null segregates. The cysteine and methionine levels in the homozygous events greatly surpass any increase previously achieved through enzymatic modification with a 38% increase in cysteine on a dry weight basis and a 49% increased in methionine on a dry weight basis (FIG. 2).
As can be seen in FIG. 3, when expressed on a mol percent basis, the data shows that the concentration of cysteine has increased by 9% and methionine has increased by 20%, each of which is statistically significant at a p value of less than 0.05. Over expression of cystathionine gamma synthase in transgenic lines was also confirmed by western blot analysis. Thus, Applicants show that constitutive expression of cystathionine gamma synthase leads to substantial and unexpected increases in total seed cysteine and methionine levels.
Example 3
Transformation and Regeneration of Transgenic Maize Callus with Serine Acetyl Transferase
Immature maize embryos from greenhouse donor plants are bombarded with a plasmid containing serine acetyl transferase nucleotide sequence (Seq. ID No. 1) operably linked to a ubiquitin promoter (U.S. Pat. Nos. 5,510,474 and 5,614,399) that has been optimized for maize codon preference and a pin II terminator (An et. al. 1989), plus a plasmid containing the selectable marker gene PAT (Wohlleben et al. (1988) Gene 70:25-37) that confers resistance to the herbicide Bialaphos. Transformation is performed according to the procedure described in Example 1.
Example 4
Agrobacterium-Mediated Transformation of Maize with Serine Acetyl Transferase
For Agrobacterium-mediated transformation of maize with a serine acetyl transferase nucleotide sequence, a maize cDNA for serine acetyl transferase (Sequence ID No. 1) is fused to a ubiquitin promoter sequence (U.S. Pat. Nos. 5,510,474 and 5,617,399) that has been optimized for maize codon preference and a pin 11 terminator sequence (An, et al., 1989). The serine acetyl transferase cassette, also contains a CaMV35S-bialaphos selectable marker element, is cloned into a binary vector and introduced into Agrobacterium. Transformation is performed according to the procedure described in Example 2.
Example 5
Agrobacterium-Mediated Transformation of Maize with Serine Acetyl Transferase and ATP Sulfurylase
Transgenic plants are constructed by Applicants according to the method described in Example 4, but with an expression cassette containing both the serine acetyl transferase sequence described in Example 4 and an ATP Sulfurylase sequence (Sequence ID No. 3) linked to a ubiquitin promoter sequence (U.S. Pat. Nos. 5,510,474 and 5,617,399) that has been optimized for maize codon preference and a Pin II terminator sequence (An, et al., 1989).
Results
More than twenty events producing T1 seed are analyzed for overexpression of both genes by western blot analysis. Twenty to forty seeds from each of two events show strong expression of both ATP Sulfurylase and serine acetyl transferase, and two events showing weak or undetectable expression are analyzed for total sulfur amino acid content (Beckman Instruments 6300 analyzer—amino acids detected with ninhydrin). These results are shown in FIGS. 4 and 5. Samples 3770 and 3768 (represented by the shaded bars in FIGS. 4 and 5) showed strong expression of ATP Sulfurylase and serine acetyl transferase, while samples 3778 and 3753 (represented by the open bars in FIGS. 4 and 5) showed weak expression of ATP Sulfurylase and serine acetyl transferase. Segregating seed from event 3768 is propagated, selfed, and the plants are analyzed for the presence of the construct by their resistance to herbicide. T2 (second generation) seed was harvested from both resistant and susceptible plants. Sulfur amino acids were analyzed for total sulfur amino acid content using a Beckman Instruments 6300 analyzer, with the amino acids detected by ninhydrin. Results are shown in FIGS. 6 and 7 with the herbicide resistant lines (+ construct) represented by shaded bars and the herbicide susceptible lines (− construct) represented by open bars.
The cysteine and methionine levels in the plants with serine acetyl transferase and ATP Sulfurylase greatly surpassed any increase previously achieved through enzymatic modification with a 20% average increase in cysteine on a dry weight basis and a 97% average increase in methionine on a dry weight basis (FIG. 6). As can be seen in FIG. 7, when expressed on a mol percent basis, the data shows that the concentration of cysteine has increased by an average of 19% and methionine has increased by an average of 63%. Although the construct used by Applicants contains both serine acetyl transferase and ATP Sulfurylase, Applicant believes that the increase in the cysteine and methionine levels of the transformed plants is due to the serine acetyl transferase rather than the ATP Sulfurylase. Thus, the preferred embodiment is the construct taught herein that comprises only the serine acetyl transferase nucleic acid and not the ATP Sulfurylase nucleic acid. However, the data demonstrates that a construct with both the serine acetyl transferase nucleic acid and the ATP Sulfurylase nucleic acid may be used to modulate the biosynthesis of at least one organic sulfur compound. Similarly, only the ATP Sulfurylase nucleic acid and not the serine acetyl transferase nucleic may also be used to modulate the biosynthesis of at least one organic sulfur compound. Such constructs, and plants with such constructs, may be produced by the methods taught in the Examples described herein.
Although Applicant has taught examples using specific enzymes that it believes to be preferred for use in the present invention, Applicant has more generally taught that, to increase sulfur amino acids and organic sulfur compounds via enzymatic modification in the seed of a monocot plant, it is sufficient to express such enzymes in the non-seed tissue of the monocot plant. The plant will then translocate these sulfur amino acids and organic sulfur compounds to the seed, where the seed, without any genetic modification, will accumulate such amino acids and compounds. Applicant reasonably believes that any one or more of the enzymes along the sulfate/serine to cysteine pathways (FIG. 1) will function in the manner as taught herein. In such case, a construct may be produced in the manner described herein but using such enzyme or enzymes. One or more enzymes in the sulfate to cysteine and/or the serine to cysteine pathways are referred to herein as “sulfur assimilating enzymes.” Such term also includes additional enzymes that may activate or deactivate such enzymes, such as kinases that activate or inactivate such enzymes by phosphorylation.
In summary, Applicant has shown that constitutive or non-seed expression of one or more enzymes in the cysteine biosynthetic pathway leads to substantial and unexpected increases in total seed cysteine and methionine levels. This increase in cysteine and methionine can be utilized as a sulfur source for the production of other organic sulfur compounds. Subsequent production of the other organic sulfur compounds from this increased sulfur source may occur in either the seed, leaf, root, stem or other plant tissue.
All publications, patents and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications, patents and patent applications are herein incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

## Claims

1. A method for modulating biosynthesis of at least one organic sulfur compound in the seed of a maize plant, the method comprising stably transforming the plant with a DNA construct comprising a nucleic acid encoding a plant cystathionine gamma synthase and having the sequence set forth in SEQ ID NO:5, wherein the nucleic acid is operably linked to a constitutive preferred promoter that drives expression in the plant and wherein the level of the at least one organic sulfur compound is altered.

2. The method of claim 1, wherein the organic sulfur compound is cysteine or methionine.

3. A maize plant with seed having increased levels of at least one organic sulfur compound, the plant having stably transformed into its genome a DNA construct comprising a nucleic acid encoding a plant cystathionine gamma synthase and having the sequence set forth in SEQ ID NO:5, wherein the nucleic acid is operably linked to a constitutive promoter that drives expression in the plant.

4. The plant of claim 3, wherein the organic sulfur compound is cysteine or methionine.

5. Seed of the plant of claim 4, wherein the seed comprises the DNA construct.