AU4716499A - Water stress or salt stress tolerant transgenic cereal plants - Google Patents

Description

WO 99/66785 PCTIUS99/14336 WATER STRESS OR SALT STRESS TOLERANT TRANSGENIC CEREAL PLANTS This application claims the benefit of U.S. Provisional Patent Application 5 Serial No. 60/090.561. filed June 24. 1998. FIELD OF THE INVENTION The present invention relates to transgenic cereal plants which are transformed with a nucleic acid encoding an enzyme for proline biosynthesis that confers water stress or salt stress tolerance to the plant and a method of increasing or conferring 10 water stress or salt stress tolerance to a cereal plant. BACKGROUND OF THE INVENTION Environmental stresses. such as drought. increased salinity of soil. and extreme temperature. are major factors in limiting plant growth and productivity. The 15 worldwide loss in yield of three major cereal crops. rice. maize (corn). and wheat due to water stress (drought) has been estimated to be over ten billion dollars annually. Salt stress and drought stress are the two most important abiotic stresses. Of the 4.870 million hectares of agricultural land in the world. 930 million (19% of total) are salt-affected areas (FAO Quarterly Bulletin of Statistics, Vol. 9 /4 (1996)). Moderate levels of salt 20 content in the soil (such as 50 mM) cause a substantial decrease in the yield of crops. High levels of salt in the soil (higher than 100 or 150 mM) are not at all suitable for planting most cereal crops. Approximately 5.2% of the agricultural lands are under drought stress (FAO Quarterly Bulletin of Statistics. Vol. 9 3/4 (1996)). and the loss of crop yield is also very significant. 25 In practical terms, rice is the most important crop because a high percentage of the world's population depends on it for their staple food. Together with wheat and corn. these three cereal crops constitute the major source of food and calories to feed the people. With an increase in population and a decrease in arable land. there is a real possibility of a food shortage by the year 2030. Therefore. it is essential to fully 30 utilize plant biotechnology to improve plants and produce more food. Breeding of stress-tolerant crop cultivars represents a promising strategy to tackle these problems (Epstein et al.. "Saline Culture of Crops: A Genetic Approach.' WO 99/66785 PCTIUS99/14336 Science. 210:399-404 (1980)). However, conventional breeding is a slow process for generating crop varieties with improved tolerance to stress conditions. Limited germplasm resources for stress tolerance and incompatibility in crosses between distantly related plant species are additional problems encountered in conventional breeding. 5 Recent progress in plant genetic transformation and availability of potentially useful genes characterized from different sources make it possible to generate stress-tolerant crops using transgenic approaches (Tarczynski et al.. "Stress Protection of Transgenic Tobacco by Production of the Osmolyte Mannitol."' Science, 259:508-510 (1993); Pilon Smits et al.. "Improved Performance of Transgenic Fructan-Accumulating Tobacco 10 Under Drought Stress." Physiol. Plant. 107:125-130 (1995)). Transformation of cereal plants with agronomically useful genes that increase tolerance to abiotic stress is one important way to minimize yield loss. For example. it would be highly desirable to produce transgenic rice plants that can give reasonable yield when grown in marginal or waste lands that contain relatively high levels of salt. such as 100-150 mM. in the soil. 15 Characterization and cloning of plant genes that confer stress tolerance remains a challenge. Genetic studies revealed that tolerance to drought and salinity in some crop varieties is principally due to additive gene effects (Akbar et al.. "Breeding For Soil Stress.~ In Prouress in Rainfed Lowland Rice. International Rice Research Institute. manila. Philippines, pp. 263-272 (1986): Akbar et al.. "Genetics of Salt Tolerance in 20 Rice." In Rice Genetics. International Rice Research Institute. Manila. Philippines, pp. 399-409 (1986)). However. the underlying molecular mechanism for the tolerance has never been revealed. Physiological and biochemical responses to high levels of ionic or nonionic solutes and decreased water potential have been studied in a variety of plants. Based on accumulated experimental observations and theoretical consideration. one 25 suggested mechanism that may underlie the adaptation or tolerance of plants to osmotic stresses is the accumulation of compatible. low molecular weight osmolytes such as sugar alcohols, special amino acids, and glycine betaine (Greenway et al.. "Mechanisms of Salt Tolerance in Nonhalophytes." Annu. Rev. Plant Physiol., 31: 149-190 (1980): Yancey et al.. -Livina With Water Stress: Evolution of Osmolvte System." Science. 217: 1214-1222 30 (1982)). In particular, proline level is known to increase in a number of plants and bacteria under drought or salt stress. Recently. a transgenic study has demonstrated that accumulation of the sugar alcohol mannitol in transgenic tobacco conferred protection against salt stress (Tarczvnski et al.. "Stress Protection of Transzenic Tobacco by WO 99/66785 PCT/US99/14336 Production of the Osmolyte Mannitoli Science. 2159:508-510 (1993)). Two recent studies using a transgenic approach have demonstrated that metabolic engineering of the clveine betaine biosynthesis pathway is not only possible but also may eventually lead to production of stress-tolerant plants (Holmstrom et al.. *Production of the Escherichia coli 5 Betaine-Aldehyde Dehydrogenase. An Enzyme Required for the Synthesis of the Osmoprotectant Glycine Betaine. in Transgenic Plants." Plant J., 6:749-758 (1994): Rathinasabapathi et al.. "Metabolic Engineering of Glycine Betaine Synthesis: Plant Betaine Aldehyde Dehydrogenases Lacking Typical Transit Peptides are Targeted to Tobacco Chloroplasts Where they Confer Betaine Aldehyde Resistance.~ Planta. 10 193:155-162 (1994)). In addition to metabolic changes and accumulation of low molecular weight compounds. a large set of genes is transcriptionally activated which leads to accumulation of new proteins in vegetative tissue of plants under osmotic stress conditions. including the late embryogenesis abundant (L EA) family. dehvdrines. and 15 COR47 (Skriver et al., "Gene Expression in Response to Abscisic Acid and Osmotic Stress." Plant Cell. 2:503-512 (1990): Chandler et al.. "Gene Expression Regulated by Abscisic Acid and its Relation to Stress Tolerance." Annu. Rev; Plant Physiol. Plant Mol. Biol.. 45:113-141 (1994)). The expression levels of a number of genes have been reported to be correlated with desiccation. salt. or cold tolerance of different plant 20 varieties of the same species. It is generally assumed that stress-induced proteins might play a role in tolerance. but the functions of many stress-responsive genes are unknown. Elucidating the function of these stress-responsive genes and enzymes involved in the biosynthesis of stress-induced osmolvtes will not only advance the understanding of plant adaptation and tolerance to environmental stresses. but also may 25 provide important information for designing new strategies for crop improvement (Chandler et al.. "Gene Expression Regulated by Abscisic Acid and its Relation to Stress Tolerance." Annu. Rev. Plant Phvsiol. Plant Mol. Biol.. 45:113-141 (1994)). Several genes that encode key enzymes involved in the biosynthesis of specific osmolytes (such as mannitol. proline. or gly'cine betaine) have been introduced 30 into tobacco cells. The regenerated transgenic tobacco plants showed partial tolerance to drought and to salt stress (Tarczynski et al.. "Stress Protection of Transgenic Tobacco bv Production of Osmotic Mannitol," Science. 259:508-5 10 (1993): Kishor et al.. Overexpression of A -pyrroline-5-carboxylate Synthetase Increases Proline Production WO 99/66785 PCT/US99/14336 -4 and Confers Osmotolerance in Transgenic Plants." Plant Phvsiol.. 108:1387-1394 (1995): Lilius et al.. "Enhanced NaCl stress tolerance in transgenic tobacco expressing bacterial choline dehydrogenase? Biotech.. 14:177-180 (1966)). However. only transgenic tobacco was used for these studies. and similar work on producing stress-tolerant cereal 5 crop plants has not been carried out. It is not clear whether these genes in transgenic cereal plants will enhance salt or drought tolerance since the physiology of dicot plants. such as tobacco. is very different from monocots. such as cereal plants. Thus. only experimentation using cereal crop plants can provide the answer. The present invention is directed to overcoming the above-noted 10 deficiencies in the prior art. SUMMARY OF THE INVENTION The present invention relates to a transgenic cereal plant transformed with a nucleic acid encoding an enzyme for proline biosynthesis that confers water stress or 15 salt stress tolerance to the plant. The present invention also relates to a cereal plant cell or protoplast transformed with a nucleic acid encoding an enzyme for proline biosynthesis that confers water stress or salt stress tolerance on a cereal plant regenerated from said cereal plant cell or protoplast. 20 Another aspect of the present invention is a method of conferrinL, water stress or salt stress tolerance to a cereal plant including transforming a cereal plant cell or protoplast with a nucleic acid encoding an enzyme for proline biosynthesis. The present invention also relates to a method of increasing tolerance of a cereal plant to water stress or salt stress conditions. the method including increasing 25 levels of an enzyme for proline biosynthesis in the cereal plant. The present invention allows the production of cereal plants with increased tolerance to water stress (drought) and salt stress. Thus. an enzyme for proline biosynthesis can be used as a molecular tool for genetic crop improvement by conferring stress tolerance. 30 WO 99/66785 PCTIUS99/14336 DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a transgenic cereal plant transformed with a nucleic acid encoding an enzyme for proline biosynthesis that confers water stress or salt stress tolerance to the plant. 5 Suitable nucleic acids encoding an enzyme for proline biosynthesis include the P5CS gene of mothbean and a feedback-inhibition insensitive mutant. P5CS-129A. of the P5CS gene. The sequence of the P5CS gene can be found in Kishor et al.. "Overexpression of A'-pyrroline-5-carboxylate Synthetase Increases Proline Production and Confers Osmotolerance in Transgenic Plants." Plant Phvsiol.. 108:1387-1394 (1995). 10 which is hereby incorporated by reference. and the sequence of the P5CS-129A mutant gene can be found in Zhang et al.. "Removal of Feedback Inhibition of P5CS in Plants." J. Biol. Chem.. 270:20491-20496 (1995). which is hereby incorporated by reference. Cereal which can be transformed in accordance with the subject invention are members of the family Gramineae (also known as Poaceace). and include rice (genus 15 Oryza). wheat. maize (corn). barley. oat. sorghum. and millet. Preferably, the cereal is rice. wheat. or corn, and most preferably the cereal is rice. Many species of cereals can be transformed. and within each species the numerous subspecies and varieties can be transformed. For example. within the rice species is subspecies Indica rice (OryZa saliva ssp. Indica). which includes the varieties IR36. IR64. IR72. Pokkali. Nona Bokra. 20 KDML 105, Suponburi 60. Suponburi 90. Basmati 385. and Pusa Basmati 1. Another rice subspecies is Japonica. which includes Nipponbere. Kenfeng and Tainung 67. Examples of suitable maize varieties include A 188. B73, VA22. L6. L9. KI. 509. 5922, 482, HNP. and IGES. Examples of suitable wheat varieties include Pavon. Anza. Chris. Coker 983. FLA301. FLA302. Fremont and Hunter. 25 Having identified the cereal plant of interest. plant cells suitable for transformation include immature embryos. calli. suspension cells. and protoplasts. It is particularly preferred to use suspension cells and immature embryos. These cereal plant cells are transformed with a nucleic acid. which could be RNA or DNA and which is preferably cDNA. encoding an enzyme for proline 30 biosynthesis. The nucleic acid can be biologically isolated or synthetic. In the followin. Examples. a key enzyme for proline biosynthesis. A'-pyrroline-5-carboxylate synthase (P5CS). is encoded by the P5CS gene of mothbean. However. other genes encoding an WO 99/66785 PCT/US99/14336 -6 enzyme for proline biosynthesis. including a feedback-inhibition insensitive mutant of the P5CS scene. P5CS-129A. can also be utilized. Transformation of plant cells can be accomplished by using a plasmid. The plasmid is used to introduce the nucleic acid encoding an enzyme for proline 5 biosynthesis into the plant cell. Accordingly. a plasmid preferably includes DNA encoding an enzyme for proline biosynthesis inserted into a unique restriction endonuclease cleavage site. Heterologous DNA. as used herein, refers to DNA not normally present in the particular host cell transformed by the plasmid. DNA is inserted into the vector using standard cloning procedures readily known in the art. This generally 10 involves the use of restriction enzymes and DNA ligases. as described by Sambrook et al.. Molecular Cloning: A Laboratory Manual. 2d edition. Cold Spring Harbor Laboratory Press. Cold Spring Harbor. New York (1 989). which is hereby incorporated by reference. The resulting plasmid which includes nucleic acid encoding an enzyme for proline biosynthesis can then be used to transform a host cell. such as an Agrobacteriun 15 and/or a plant cell. (See generally. Plant Molecular Biology Manual. 2nd Edition. Gelvin et al.. Eds.. Kluwer Academic Press. Dordrecht. Netherlands (1994). which is hereby incorporated by reference). For plant transformation. the plasmid preferably also includes a selectable marker for plant transformation. Commonly used plant selectable markers include the 20 hygromycin phosphotransferase (hpt) gene. the phosphinothricin acetyl transferase gene (bar). the 5-enolpyruvylshi kimate-3 -phosphate synthase gene (EPSPS). neomycin 3'-0 phosphotransferase gene (np/ Il). or acetolactate synthase gene (ALS). Information on these selectable markers can be found in Bowen. "Markers for Plant Gene Transfer" in Transeenic Plants. Kung et al.. Eds.. Vol. 1. pp. 89-123. Academic Press. NY (1993). 25 which is hereby incorporated by reference. The plasmid preferably also includes suitable promoters for expression of the nucleic acid encoding an enzyme for proline biosynthesis and for expression of the marker gene. The cauliflower mosaic virus 35S promoter is commonly used for plant transformation. as well as the rice actin 1 gene promoter. In plasmid pJS102 used in the 30 following Examples. the nucleic acid encoding an enzyme for proline biosynthesis is under the control of the constitutive rice actin I gene promoter and the marker gene (har) is under control of the cauliflower mosaic virus 35S promoter. Other promoters useful for plant transformation with an enzyme for prol ine biosynthesis include those from the WO 99/66785 PCT/US99/14336 -7 genes encoding ubiquitin and proteinase inhibitor II (PINII). as well as stress-induced promoters (such as the HVAI gene promoter of barley. an abscisic acid (ABA)-inducible promoter. such as ABRCI from barley linked to a rice Act-100 minimal promoter. and a HVA22 intron). 5 The plasmid designated pIS 112 has been deposited pursuant to. and in satisfaction of. the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. with the American Type Culture Collection (ATCC). 10801 University Boulevard. Manassas. VA 20110-2209. under ATCC Accession No. on June 17. 1999. 10 For plant transformation. the plasmid also preferably includes a nucleic acid molecule encoding a 3' terminator such as that from the 3' non-coding region of genes encoding a proteinase inhibitor. actin. or nopaline synthase (nos). Other suitable plasmids for use in the subject invention can be constructed. For example. genes encoding an enzyme for proline biosynthesis other than the P5CS 15 gene of mothbean could be ligated into plasmid JS109 after use of restriction enzymes to remove the P5CS gene. Other promoters could replace the actin I gene promoter present in plasmid JS 102. Alternatively. other plasmids in general containing genes encoding an enzyme for proline biosynthesis under the control of a suitable promoter. with suitable selectable markers. can be readily constructed using techniques well known in the art. 20 Havin- identified the plasmid. one technique of transforming cereal plant cells with a gene which encodes for an enzyme for proline biosynthesis is by contacting the plant cell with an inoculum of an Agrobaceri-um bacteria transformed with the plasmid comprising the gene that encodes for the enzyme for proline biosynthesis. Generally. this procedure involves inoculating the plant cells with a suspension of the 25 transformed bacteria and incubating the cells for 48 to 72 hours on regeneration medium without antibiotics at 25-28'C. Bacteria from the genus Agrohacieriun can be utilized to transform plant cells. Suitable species include Agrobaceriuim tumef/aciens and Agrohaceriumn rhizogenes. Agrobactieriun ineaciens (e.g.. strains LBA4404 or EHA105) is 30 particularly useful due to its well-known ability to transform plants. In inoculating the cells of cereal plants with .-igrobaiceriun according to the subject invention. the bacteria must be transformed with a vector which includes a gene encoding for an enzyme for proline biosynthesis.

WO 99/66785 PCT/US99/14336 Plasmids. suitable for incorporation in Agrobacteriumn. which include a gene encoding for an enzyme for proline biosynthesis. contain an origin of replication for replication in the bacterium Escherichia coli, an origin of replication for replication in the bacterium Agrohacterium Iunefaciens. T-DNA right border sequences for transfer of 5 genes to plants. and marker genes for selection of transformed plant cells. Particularly preferred is the vector pBI 121 which contains a low-copy RK2 origin of replication, the neomycin phosphotransferase (nptil) marker gene with a nopaline synthase (NOS) promoter and a NOS 3' polyadenylation signal. T-DNA plasmid vector pBIl21 is available from Clonetech Laboratories. Inc.. 4030 Fabian Way. Palo Alto. California 10 94303. A gene encoding for an enzyme for proline biosynthesis is inserted into the vector to replace the beta-glucuronidase (GUS) gene. Typically. Agrobacteriuim spp. are transformed with a plasmid by direct uptake of plasmid DNA after chemical and heat treatment. as described by Holsters et al. "Transfection and Transformation of Agrobacerium tume/aciens." Mol. Gen. Genet.. 15 163:181-187 (1978). which is hereby incorporated by reference: by direct uptake of plasmid DNA after electroporation. as described by Shen et al.. "Efficient Transformation of Agrobacteriun spp. by High Voltage Electroporation."' Nucleic Acids Research, 17:8385 (1989). which is hereby incorporated by reference: by triparental conjugational transfer of plasmids from Escherichia coli to Agrobacteium mediated by a Tra+ help 20 strain as described by Ditta et al.. "Broad Host Range DNA Cloning System for Gram negative Bacteria: Construction of a Gene Bank of Rhizobiwun mneliloli." Proc. Natl. Acad. Sci. USA. 77:7347-7351 (1981). which is hereby incorporated by reference: or by direct conjugational transfer from Escherichia coli to Agroacteriumz as described by Simon et al.. A Broad Host Range Mobilization System for in vivo Genetic Engineering: 25 Transposon Mutagenesis in Gram-negative Bacteria." Biotechnology. 1:784-791 (1982). which is hereby incorporated by reference. Another method for introduction of a plasmid containing nucleic acid encoding an enzyme for proline biosynthesis into a plant cell is by transformation of the plant cell nucleus, such as by particle bombardment. As used throughout this application. 30 particle bombardment (also known as biolistic transformation) of the host cell can be accomplished in one of several ways. The first involves propelling inert or biologically active particles at cells. This technique is disclosed in U.S. Patent Nos. 4.945.050 5.036.006. and 5.100.792. all to Sanford et al.. which are hereby incorporated by WO 99/66785 PCT/US99/14336 -9 reference. Generally, this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and to be incorporated within the interior thereof. When inert particles are utilized, the plasmid can be introduced into the cell by coating the particles with the plasmid 5 containing the heterologous DNA. Alternatively, the target cell can be surrounded by the plasmid so that the plasmid is carried into the cell by the wake of the particle. Biologically active particles (e.g.. dried bacterial cells containing the plasmid and heterologous DNA) can also be propelled into plant cells. A further method for introduction of the plasmid into a plant cell is by 10 transformation of plant cell protoplasts (stable or transient). Plant protoplasts are enclosed only by a plasma membrane and will therefore take up macromolecules like heterologous DNA. These engineered protoplasts can be capable of regenerating whole plants. Suitable methods for introducing heterologous DNA into plant cell protoplasts include electroporation and polyethylene glycol (PEG) transformation. As used 15 throughout this application, electroporation is a transformation method in which. generally. a high concentration of plasmid DNA (containing heterologous DNA) is added to a suspension of host cell protoplasts and the mixture shocked with an electrical field of 200 to 600 V/cm. Following electroporation. transformed cells are identified by growth on appropriate medium containing a selective agent. 20 As used throughout this application. transformation encompasses stable transformation in which the plasmid is integrated into the plant chromosomes. In the Examples which follow, rice has been transformed using biolistic transformation. Other methods of transformation have also been used to successfully transform rice plants. including the protoplast method (for a review, see Cao et al.. 25 "Regeneration of Herbicide Resistant Transgenic Rice Plants Following Microprojectile Mediated Transformation of Suspension Culture Cells." Plant Cell Rep.. 11:586-591 (1992). which is hereby incorporated by reference). and the Agrobacteriun method (Hiei et al.. "Efficient Transformation of Rice (OriyZ saliva L.) Mediated by Agrobacterium and Sequence Analysis of the Boundaries of the T-DNA." The Plant Journal 6:271-282 30 (1994). which is hereby incorporated by reference). Biolistic transformation has also been used to successfully transform maize (for a review. see Mackey et al.. "Transgenic Maize." In Transuenic Plants. Kung et al.. Eds., vol. 2. pp. 21-33 (1993). which is hereby WO 99/66785 PCTIUS99/14336 - 10 incorporated by reference) and wheat (see U.S. Patent No. 5.405.765 to Vasil et al.. which is hereby incorporated by reference). Once a cereal plant cell or protoplast is transformed in accordance with the present invention. it is regenerated to form a transgenic cereal plant. Generally. 5 regeneration is accomplished by culturing transformed cells or protoplasts on medium containing the appropriate growth regulators and nutrients to allow for the initiation of shoot meristems. Appropriate antibiotics are added to the regeneration medium to inhibit the growth of Agrobacteriumn or other contaminants and to select for the development of transformed cells or protoplasts. Following shoot initiation. shoots are allowed to 10 develop in tissue culture and are screened for marker gene activity. In suitable transformation methods. the cereal plant cell to be transformed can be in vitro or in vivo. i.e. the cereal plant cell can be located in a cereal plant. The invention also provides seed produced by the transgenic cereal plant. The invention is also directed to seed. which upon germination, produces the transgenic 15 cereal plant. Also encompassed by the present invention are transgenic cereal plants transformed with fragments of the nucleic acids encoding an enzyme for proline biosynthesis of the present invention. Suitable fragments capable of conferringy water stress or salt stress tolerance to cereal plants can be constructed by using appropriate 20 restriction sites. A fragment refers to a continuous portion of the encoding molecule for an enzyme for proline biosynthesis that is less than the entire molecule. Non-essential nucleotides could be placed at the 5' and/or 3' ends of the fragments (or the full length molecules encoding an enzyme for proline biosynthesis) without affecting the functional properties of the fragment or molecule (i.e. in increasing 25 water stress or salt stress tolerance). For example. the nucleotides encoding an enzyme for proline biosynthesis may be conjugated to a signal (or leader) sequence at the N terminal end (for example) of the enzyme for proline biosynthesis which co translationally or post-translationally directs transfer of the enzyme for proline biosynthesis. The nucleotide sequence may also be altered so that the encoded enzyme is 30 conjugated to a linker or other sequence for ease of synthesis. purification, or identification of the enzyme. The present invention also relates to a cereal plant cell or protoplast transformed with a nucleic acid encoding an enzvme for proline biosynthesis that confers WO 99/66785 PCT/US99/14336 water stress or salt stress tolerance on a cereal plant regenerated from said cereal plant cell or protoplast. Once transformation has occurred. the cereal plant cell or protoplast can be regenerated to form a transgenic cereal plant. Preferably. the nucleic acid encoding an enzyme for proline biosynthesis 5 is controlled by a strong promoter to effect maximum expression of an enzyme for proline biosynthesis. or by a stress-induced promoter to effect induction of the promoter in response to stress conditions. In one embodiment. the transgenic cereal plant cell or protoplast or plant is transformed with the nucleic acid encoding the promoter. such as the rice actin I gene promoter. by providing a plasmid which includes DNA encoding an 10 enzyme for proline biosynthesis and the promoter. The transgenic cereal plant cell or protoplast or plant can also be transformed with a nucleic acid encoding a selectable marker. such as the bar gene. to allow for detection of transformants. and with a nucleic acid encoding the cauliflower mosaic virus 35S promoter to control expression of the har gene. Other selectable 15 markers include genes encoding EPSPS. nptIl. or ALS. Other promoters include those from genes encoding actin 1. ubiquitin. and PINII. These additional nucleic acid sequences can also be provided by the plasmid encoding the enzyme for proline biosynthesis and its promoter. Where appropriate. the various nucleic acids could also be provided by transformation with multiple plasmids. 20 While the nucleotide sequence referred to herein encodes an enzyme for proline biosynthesis. nucleotide identity to previously sequenced enzymes for proline biosynthesis is not required. As should be readily apparent to those skilled in the art. various nucleotide substitutions are possible which are silent mutations (i.e. the amino acid encoded by the particular codon does not change). It is also possible to substitute a 25 nucleotide which alters the amino acid encoded by a particular codon. where the amino acid substituted is a conservative substitution (i.e. amino acid "homology- is conserved). It is also possible to have minor nucleotide and/or amino acid additions. deletions. and/or substitutions in the enzyme for proline biosynthesis nucleotide and/or amino acid sequences which have minimal influence on the properties. secondary structure. and 30 hydrophilic/hydrophobic nature of the encoded enzymes for proline biosynthesis. These variants are encompassed by the nucleic acid encoding an enzyme for proline biosynthesis according to the subject invention.

WO 99/66785 PCT/US99/14336 - 12 The present invention is also directed to a transgenic cereal plant regenerated from the transgenic cereal plant cells or protoplasts. as well as to seed produced by the transgenic cereal plants. Another aspect of the present invention is a method of conferring water 5 stress or salt stress tolerance to a cereal plant including transforming a cereal plant cell or protoplast with a nucleic acid encoding an enzyme for proline biosynthesis. In a preferred embodiment, the method further includes regenerating the transformed cereal plant cell or protoplast to form a transgenic cereal plant. The present invention also includes seed produced by the transgenic cereal plant. 10 The present invention also relates to a method of increasing tolerance of a cereal plant to water stress or salt stress conditions. the method including increasing levels of an enzyme for proline biosynthesis in the cereal plant. In a preferred embodiment, the plasmid is designated pJS102. pJS 107. or pJSI 12 (See Examples 1 and 2). 15 EXAMPLES Example 1 - Construction of the p.JS107 Plasmid for Plant Transformation Plasinid Consiruction pJS 107 was constructed by isolating a 2.4 kb Sal fragment containing 20 mothbean (Vigna aconitifolia L.) P5CS cDNA from the plasmid pUbiP5CS (Hu et aL -A Bifunctional Enzyme (A -pyrroline-5-carboxylate synthetase) Catalyzes the First Two Steps in Pro Biosynthesis in Plants." Proc. Natl. Acad. Sci. USA. 89:9354-9358 (1992). which is hereby incorporated by reference). This DNA fragment was blunted with Klenow DNA polymerase and subcloned into the Smal site of the expression vector 25 pJS 104 (Su et aL "Dehydration-Stress-Regulated Transgene Expression in Stably Transformed Rice Plants." Plant Phvsiol.. 117:913-922 (1998). which is hereby incorporated by reference) to create pJS 107. In pJS 107. the P5CS-coding sequence was downstream of a stress-inducible promoter complex (designated as AIPC-ABA inducible promoter complex). AIPC includes a 49 bp ABA-responsive element from the barley 30 Hva22 gene (Shen et al.. "Functional Dissection Of An Abscisic Acid (ABA)-inducible Gene Reveals Two Independent ABA-Responsive Complexes Each Containing A G-Box And A Novel Cis-Acting Element.* Plant Cell. 7:295-307 (1995). which is hereby WO 99/66785 PCT/US99/14336 - 13 incorporated by reference). a 180 bp minimum rice acting gene promoter (Su et al.. "Deldration-Stress-Regulated Transgene Expression in Stably Transformed Rice Plants." Plant Physiol.. 11 7:913-922 (1998). which is hereby incorporated by reference). and a Hva22 intron (Shen et al.. "Functional Dissection Of An Abscisic Acid (ABA) 5 Inducible Gene Reveals Two Independent ABA-Responsive Complexes Each Containing A G-Box And A Novel Cis-Acting Element." Plant Cell. 7:295-307 (1995). which is hereby incorporated by reference). pJS 107 also contains the bar cassette. which was used for selection of transgenic call and plants in the presence of the herbicide. Bialaphos. 10 Transformation of Rice Cells with a Mothbean P5SS' cDNA (Zhu et al.. Overexpression Of A P5CS Gene And Analysis Of Tolerance To Water And Salt Stress In Transgenic Rice." Plant Science 139:41-48 (1998). which is hereby incorporated by reference) The procedure and media used for the establishment of suspension cells 15 was according to a previously described method (Cao et al.. "Assessment Of Rice Genetic Transformation Techniques, In Rice Biotechnologv. Toenniessen et al.. Eds.. CAB International. Oxon. UK. pp. 175-198 (1991); Cao et al.. "Regeneration Of Herbicide Resistant Transgenic Rice Plants Following Microprojectile-Mediated Transformation Of Suspension Culture Cells." Plant Cell Rep., 11:586-191 (1992). which are hereby 20 incorporated by reference). Dehusked rice seeds (Oryza sativa L. var. Nipponbare) were used for callus induction. Following growth in suspension cultures, pJS 107 was introduced into suspension culture cells by the biolistic method. The cells were cultured and selected in KPR medium (Cao et al., "Assessment Of Rice Genetic Transformation Techniques. In Rice Biotechnologv. Toenniessen et al.. Eds.. CAB International. Oxon. 25 UK, pp. 175-198 (1991). which is hereby incorporated by reference) containing 8 mg per liter Bialaphos. The resistant calli were transferred to MS regeneration medium to regenerate into plants. Plants regenerated from the same resistant callus were regarded as clones of the same line. Regenerated plants were transferred into soil and grown in the greenhouse (32'C dav/22 C night. with supplemental photoperiod of 10 hours). 30 Plasmid pJS 107 (ABRCl/Act-100 promoter/H va 2 2 intron/P5CS cDNA/Pin2 3'//35S promoter/har/Nos 3') was introduced into rice suspension cells using the biolistic-mediated transformation method.

WO 99/66785 PCTIUS99/14336 - 14 Regeneration und Analysis of Transgenic Plants A number of transgenic plants (Orayz sativa L.) were generated. and four lines with relatively low transgene copy number were analyzed. The results are shown in Table 1, below. 5 Table 1. Analysis of Transgenic Rice Plants Transformed With a Mothbean P5CS cDNA. Southern Blot P5CS Proline tg/g Shoot Fresh Plant Copy Number Activity Fresh Leaf Weight (g)b Control 0 27 0.80 N22 5 ++ 45 1.22 N52 3 48 0.82 N60 2.+ 71 1.51 N70 2.++ 68 1.90 10 a P5CS activity was assayed based on the conversion of [ 4 C] glutamate to [1 4 C] proline: TLC separation. b Eight-week-old plants (4-10 per line) were stressed with no water for 6 days, then water I day. Four cycles (28 days). Thus, this data indicated that transgenic rice plants produced an increased 15 level of the P5CS enzyme activity as well as proline content (measured by using a colorimetric method) in leaves. Example 2 - Transformation of Rice Calli with a Mothbean P5CS cDNA and Comparison of an Inducible vs. Constitutive Proline Synthesis in 20 Transgenic Rice Plasmid Construction Three plasmids were constructed. The components of these plasmids are: pJS 102 (with a constitutive promoter): Rice actin I promoter/P5CS cDNA/Pin 2 3'/355S 25 pronoter/har/Nos 3'; piSI 12 (with a stress-inducible promoter): ABRC4/Actl-100 promoter/Hva22 intron/P5CS cDNA/Pin2 3'//35S promoter/har/Nos 3': and pJSI 10 (with a constitutive promoter and all components as in pJSI 12. except that a uidA reporter gene is used in place of the P5CS cDNA in pJS 112). 30 WO 99/66785 PCTIUS99/14336 - 15 Transfirmat ion of Rice Calli with a Mothhean P5'S cDN' A The preparation of rice calli. transformation procedure. and regeneration of plants were similar to those described in Example 1. 5 Analvsis of Transgenic Plants: Growth and Stress Treatments of Plants in Soil Refined and sterilized field soil was used to grow the rice plants in the greenhouse. R2 seeds were germinated in 1/2 MS medium for 7 days. and the 7-day-old seedlings were transplanted into soil in small pots (8x8 inches) with holes in the bottom (4 10 to 6 plants per pot). The pots were kept in flat-bottomed trays containing water. The seedlings were grown for an additional 2 weeks. and within the third week. they were tested for Basta resistance. Two Basta-resistant plants with the same plant height per pot were selected for stress treatments. Stress treatments were carried out as follows. In the first round of stress treatment, water was withheld from the trays for 15 7 days. and. then, the stressed plants were resupplied with water for 2 days. One or three additional rounds of stress treatments were imposed on the plants. For salt stress. 3-week old plants were transferred to trays containing 300 mM NaCl solution for 20 days. The NaCl solution was changed every 3 days to maintain a constant concentration of NaCl in the soil. The pots containing stressed plants were transferred back to trays containing tap 20 water to allow the stressed plants to recover and grow without stress for 10 days. After the 10 days of recovery, a second round of salt stress was imposed by using the same concentration of NaCl solution for 10 days. Liquid fertilizer (Peters Excel. N:P:K = 15:5:15. Scotts Professional Co.) mixed with tap water or NaCl solution was applied to the plants weekly. 25 Growth Perfirmance of Transgenic Plants Under Water Stress Conditions Since there was no significant difference in growth performance between NT plants and uidA plants in seedlings tested, the iLA plants (L3) were chosen as more suitable control plants for the following experiment because they also contained bar and 30 the same promoter cassette as the pcs-transgenic plants. Before initial water stress. all the 3-week-old plants including the L3 control plants. were tested for Basta resistance. Healthy. Basta-resistant plants with similar plant height were selected for analyzing growth performance. Under non-stress WO 99/66785 PCT/US99/14336 -16 conditions in soil. no significant differences were observed between p5cs-containing transgenic plants and SIPC-uidA control plants in their growth performance during the entire period of the experiment. Upon withholding water from the trays. the absolute water content in the soil decreased from 35% to 12% after 7 days water stress. Following 5 2 cycles of the water stress, the leaves of SIPC-uid1 control plants started to turn yellow. and the AcIl-p5cs plants showed low-growth rate, whereas the S/PC-p5cs plants with a stress-inducible promoter showed healthy growth. After 4 cycles of water stress. more severe symptoms. such as leaf chlorosis (in both control and AcIl-p5cs plants) or death of leaf tips (in control plants only), were found. The SIPC-p5cs plants still showed a high 10 rate of growth and less-severe leaf chlorosis. Data in Table 2 (top half) show the average fresh shoot weight and fresh root weight of the plants after 4 cycles of 7 days water stress. The results indicated that under water stress, the SIPC-p5cs plants (L5 and L7). which contained a stress-inducible promoter to drive the p5cs expression, grew much faster as compared to ActI-p5cs plants (LI). which contained a constitutive promoter for driving 15 the p5cs expression. The difference between using a stress-inducible promoter and a constitutive promoter was highly significant (P<0.01: t = 5.88 to 7.64). Growth Performance of Transgenic Rice Plants Under Salt-Stress Conditions To create high soil salinity, 300 mM NaCl solution was added to the trays in which 20 the pots were placed. At an early stage (10 d after the initial stress). the control plants (L3) started to wilt and the leaves began to turn yellow. whereas the p5cs transgenic plants still showed healthy growth. After 20 days of NaCl stress. the Acul-P5CS plants (LI) also started to wilt. Following 10 days of watering to allow recovery and an additional 10 days of 300 mM NaCl stress. more severe damage occurred in both control plants (L3) and 25 Act]-pics plants. On the contrary. the leaves of S/PC-p5cs plants still remained green with a high rate of growth. The average fresh shoot weight and fresh root weight are shown in Table 11 (bottom half). These values indicated that SJPC-p5cs plants (L5 and L7) grew significantly larger (P<0.01: t 6.03 to 7.79) under salt-stress conditions than Act 1-p5cs plants (L ) and control plants (L3). in spite of the finding that the proline level 30 was lower in SIPC-pj5cs plants. Of the two SIPC-p5cs lines. L5 was the better one. In conclusion. stress-inducible transgene expression in p5cs plants shows significant advantages over constitutive expression of the pics-transgene in growth of rice plants under salt- and water-stress conditions.

WO 99/66785 PCTIUS99/14336 -17 Table 2. Growth performance of transgenic plants in soil under water-stress or salt stress conditions Fresh Shoot Wt Fresh Root Wt t Value* in Water-Stress Expt. Rice Line Promoter (mg / plant) (mg / plant) Comparison Shoot Wt Root Wt 10 JS I10 (13) Inducible 300±20 (100) 90±20 (100) LI :L3 9.54 3.21 JSI02 (LI) Constitutive 550±60(183) 130±20(144) L5:L3 14.22 8.05 .JSI12(L5) Inducible 940±100(310) 220±30(224) L7:L3 4.97 6.22 JS112 (L7) Inducible 730±60(243) 170±20(189) LI:L5 7.64 5.88 15 Fresh Shoot Wt Fresh Root Wt t Value* in NaCl-Stress Expt. Iranseenic Line Promoter (mg / plant) (mg / plant) Comparison Shoot Wt Root Wt .S 110 (1.3) Inducible 320±40(100) 70±10(100) 1. :L3 5.68 4.18 20 JS 102 (LI) Constitutive 580±1100(181) 110±20(157) LI:L5 6.03 7.79 JS112 (1.5) Inducible 1030*140 (322) 240±30 (343) L5:L3 11.72 11.92 JS 112 (L7) Inducible 870±150(272) 180±30(257) L7:L3 7.83 7.67 25 * As compared to the t values of Student 's distribution table. tuos (1=)=2.23 and to 0 1 (n= 6 )=3. 17. All values higher than 3.17 are significant. Fresh shoot and root weights are in mg/plant. Nleans±SE represents the averages of 6 plants (Wt). Values in parentheses are the percentages ofp5es-transgenic plants compared to control 30 plants (L3). represented by 100. The spread of data within each set of 6 plants was rather small. For example. the actual values for the fresh shoot wt of six JSI 10 (L3) plants in the water-stress experiment (top half of table) were: 280. 282. 288. 315. 320 and 325: the actual values for the fresh shoot wt of six JS 112 (L5) plants were: 840. 845. 860. 1025. 1045 and 1050. 35 Although the invention has been described in detail for the purpose of illustration, it is understood that such detail is solely for that purpose. and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims.

Claims (32)

1. A transgenic cereal plant transformed with a nucleic acid encoding an enzyme for proline biosynthesis that confers water stress or salt stress tolerance to the plant. 5

2. A transgenic cereal plant according to claim 1. wherein said cereal plant is a rice plant.

3. A transgenic cereal plant according to claim 1. wherein said 10 nucleic acid encoding an enzyme for proline biosynthesis is the P5CS gene of mothbean or a mutant mothbean P5CS gene which is insensitive to feedback inhibition by proline.

4. A transgenic cereal plant according to claim 3. wherein the mutant mothbean P5CS gene is P5CS-129A. 15

5. A transgenic cereal plant according to claim 1. wherein said transgenic cereal plant includes a nucleic acid encoding a promoter. wherein expression of said nucleic acid encoding said enzyme for proline biosynthesis is controlled by said promoter. 20

6. A transgenic cereal plant according to claim 5. wherein said promoter is the rice actin 1 gene promoter.

7. A transgenic cereal plant according to claim 1. wherein said 25 transgenic cereal plant includes a nucleic acid encoding a selectable marker.

8. A seed produced by the transgenic cereal plant of claim 1.

9. A seed, which upon germination. produces the transgenic cereal 30 plant of claim 1. WO 99/66785 PCT/US99/14336 - 19

10. A cereal plant cell or protoplast transformed with a nucleic acid encoding an enzyme for proline biosynthesis that confers water stress or salt stress tolerance on a cereal plant regenerated from said cereal plant cell or protoplast. 5

11. A cereal plant cell or protoplast according to claim 10. wherein said cereal plant cell or protoplast is derived from a rice plant.

12. A cereal plant cell or protoplast according to claim 10, wherein said nucleic acid encoding an enzyme for proline biosynthesis is the P5CS gene of 10 mothbean or a mutant mothbean P5CS gene which is insensitive to feedback inhibition by proline.

13. A cereal plant cell or protoplast according to claim 12. wherein the mutant mothbean P5CS gene is P5CS-129A. 15

14. A cereal plant cell or protoplast according to claim 10. wherein said cereal plant cell or protoplast includes a nucleic acid encoding a promoter. wherein expression of said nucleic acid encoding said enzyme for proline biosynthesis is controlled by said promoter. 20

15. A cereal plant cell or protoplast according to claim 14, wherein said promoter is the rice actin I gene promoter.

16. A cereal plant cell or protoplast according to claim 10. wherein 25 said cereal plant cell or protoplast includes a nucleic acid encoding a selectable marker.

17. A transgenic cereal plant regenerated from the cereal plant cell or protoplast of claim 10. 30

18. A seed produced by the transgenic cereal plant of claim 17.

19. A method of conferring water stress or salt stress tolerance to a cereal plant comprising: WO 99/66785 PCTIUS99/14336 -20 transforming a cereal plant cell or protoplast with a nucleic acid encoding an enzyme for proline biosynthesis under conditions effective to impart water stress or salt stress tolerance to cereal plants. 5

20. A method according to claim 19. wherein said cereal plant cell or protoplast is derived from a rice plant.

21. A method according to claim 19, wherein said nucleic acid encoding an enzyme for proline biosynthesis is the P5CS gene of mothbean or a mutant 10 mothbean P5CS gene which is insensitive to feedback inhibition by proline.

22. A method according to claim 21. wherein the mutant mothbean P5CS gene is P5CS-129A. 15

23. A method according to claim 19, wherein said transforming comprises: propelling particles at said cereal plant cell under conditions effective for the particles to penetrate the cell interior; and introducing a plasmid comprising the nucleic acid encoding an enzyme for 20 proline biosynthesis into the cell interior.

24. A method according to claim 23, wherein the plasmid is associated with the particles, whereby the plasmid is carried into the cell or protoplast interior together with the particles. 25

25. A method according to claim 19, wherein said transforming comprises: contacting tissue of the monocot plant with an inoculum of a bacterium of the genus Agrobaclerium. wherein the bacterium is transformed with a plasmid 30 comprising the gene that increases tolerance to salt stress and drought stress.

26. A method according to claim 19 further comprising: WO 99/66785 PCTIUS99/14336 - 21 regenerating the transformed cereal plant cell or protoplast to form a transgenic cereal plant.

27. A transgenic cereal plant produced by the method of claim 26. 5

28. A seed produced by the transgenic cereal plant of claim 27.

29. A method of increasing tolerance of a cereal plant to water stress or salt stress conditions, said method comprising: 10 increasing levels of an enzyme for proline biosynthesis in said cereal plant.

30. A method according to claim 29, wherein said cereal plant is a rice plant. 15

31. A method according to claim 29. wherein said nucleic acid encoding an enzyme for proline biosynthesis is the P5CS gene of mothbean or a mutant mothbean P5CS gene which is insensitive to feedback inhibition by proline.

32. A method according to claim 31. wherein the mutant mothbean 20 P5CS gene is P5CS-129A.