AU2490601A - Plants having enhanced nitrogen assimilation/metabolism - Google Patents

Description

P/00/011 Regulation 3.2

AUSTRALIA

Patents Act 1990

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Plants having enhanced nitrogen assimilation/metabolism The following statement is a full description of this invention, including the best method of performing it known to us: Freehills Carter Smith BeadleMELC601066013.7 CD/01066001.7 PLANTS HAVING ENHANCED NITROGEN ASSIMILATION/METABOLISM This application is a divisional of Australian patent application 15868/97, the entire disclosure of which is incorporated herein by reference.

Field of the Invention The present invention relates to genetic engineering of plants to display enhanced agronomic characteristics and, in particular, the genetic engineering of plants to display enhanced agronomic characteristics by enhancing the nitrogen assimilatory and metabolism capacities of the plants.

Background of the Invention 10 A. Nitrogen Assimilation and Metabolism In many ecosystems, both natural and agricultural, the primary productivity 0 of plants is limited by the three primary nutrients: nitrogen, phosphorous and potassium. The most important of these three limiting nutrients is usually nitrogen.

Nitrogen sources are often the major components in fertilizers (Hageman and Lambert, 1988, In. Corn and Corn Improvement, 3rd ed., Sprague Dudley, American Society of Agronomy, pp. 431-461). Since nitrogen is usually the ratelimiting element in plant growth, most field crops have a fundamental dependence on inorganic nitrogenous fertilizer. The nitrogen source in fertilizer is usually ammonium nitrate, potassium nitrate, or urea. A significant percentage of the 20 costs associated with crop production result from necessary fertilizer applications.

°lille However, it is known that most of the nitrogen applied is rapidly depleted by soil microorganisms, leaching, and other factors, rather than being taken up by the plants.

Nitrogen is taken up by plants primarily as either nitrate (NO3) or ammonium (NH 4 Some plants are able to utilize the atmospheric N 2 pool through a symbiotic association with N 2 -fixing bacteria or ascomycetes. In well aerated, non-acidic soils, plants take up N0 3 which is converted to NH 4 In acidic soils, NH 4 is the predominate form of inorganic nitrogen present and can be taken up directly by plants. NH 4 is then converted to glutamine and glutamate by the enzymes glutamine synthetase (GS) and glutamate synthase WO 97/30163 PCT/CA97/00100 -2- (GOGAT). The glutamine and glutamate can be converted into a variety of amino acids, as shown in Figure 1.

Although some nitrate and ammonia can be detected in the transporting vessels (xylem and phloem), the majority of nitrogen is first assimilated into organic form amino acids) which are then transported within the plant. Glutamine, asparagine and aspartate appear to be important in determining a plant's ability to take up nitrogen, since they represent the major long-distance nitrogen transport compounds in plants and are abundant in phloem sap. Aside from their common roles as nitrogen carriers, these amino acids have somewhat different roles in plant nitrogen metabolism. Glutamine is more metabolically active and can directly donate its amide nitrogen to a large number of substrates. Because of this reactivity, glutamine is generally not used by plants to store nitrogen. By contrast, asparagine is a more efficient compound for nitrogen transport and storage because of its higher N:C ratio. Asparagine is also more stable than glutamine and can accumulate to higher levels in vacuoles. Indeed, in plants that have high nitrogen 15 assimilatory capacities, asparagine appears to play a dominant role in the transport and metabolism of nitrogen (Lam et al, 1995, Plant Cell 7: 887-898). Because of its relative S stability, asparagine does not directly participate in nitrogen metabolism, but must be first hydrolysed by the enzyme asparaginase (ANS) to produce aspartate and ammonia which then can be utilized in the synthesis of amino acids and proteins.

20 However, in addition to aspartate and asparagine, a number of other amino acids can act as storage compounds. The total amount of free amino acids has been shown to change with specific stresses, both biotic and abiotic, different fertilizer regimes and other factors (Bohnert et al., 1995, Plant Cell 7:1099-1111). For example, during drought stress many plants maintain their turgor by osmotic adjustment (Turner, 1979, Stress Physiology in Crop Plants, pp. 181-194). Osmot ijustme'-t, i.e. a ne ncrease in solutes leading to a lowering of osmotic potential, is one of the n. mechaniisms whi -oy crops can adapt to limited water availability (Turner, ibid; Morgan, 1984, Annu Rev Plant Physiol 35: 299-319). The solutes that accumulate during osmotic adjustment include sugars, organic acids and amino acids, such as alanine, aspartate, proline and glycine betaine (Good and Zaplachinski, 1994, Physiol Plant 90: 9- 14; Hanson and Hitz, 1982, Annu Rev Plant Physiol 33: 163-203; Jones and Turner, 1978, Plant WO 97/30163 PCT/CA97/00100 -3- Physiol 61: 122-126). Corn, cotton, soybean and wheat have all demonstrated osmotic adjustment during drought (Morgan, ibid.). One of the best characterized osmoregulatory responses is the accumulation of proline (Hanson and Hitz, ibid.). In some tissues, proline levels increase as much as 100-fold in response to osmotic stress (Voetberg and Sharp, 1991, Plant Physiol 96: 1125-1130). The accumulation of proline results from an increased flux of glutamate to pyrroline-5-carboxylate and proline in the proline biosynthetic pathway, as well as decreased rates of proline catabolism (Rhodes et al., 1986, Plant Physiol 82:890-903; Stewart et al., 1977, Plant Physiol. 59:930-932). The concentrations ofalanine and aspartate have been shown to increase 3.6 and 4.1-fold, respectively, during drought stress in Brassica napus leaves, whereas glutamate levels increased 5.5-fold (Good and Maclagan, 1993, Can J Plant Sci 73: 525-529).

Alanine levels declined after rewatering of the plants whereas aspartate levels remained high.

Pyruvate levels showed a similar pattern, increasing 2.2-fold after 4 days of drought, followed by the return to control levels upon rehydration. However, 2-oxoglutarate levels remained relatively constant during drought stress and rehydration. One of the factors that can determine the value of 15 a specific amino acid as an osmoprotectant may be its use as a carbon or nitrogen storage compound.

Alanine is one of the common amino acids in plants. In Brassica leaves under normal conditions, alanine and aspartate concentrations are roughly equal and have been found to be twice that of asparagine concentrations. In comparison, glutamate levels were double that of 20 alanine or aspartate (Good and Zaplachinski, ibid.). Alanine is synthesized by the enzyme alanine i aminotransferase (AlaAT) from pyruvate and glutamate in a reversible reaction (Goodwin and Mercer, 1983, Introduction to Plant Biochemistry 2nd Ed., Pergamon Press, New York, pp.

341-343), as shown in Figure 2. In addition to drought, alanine is an amino acid that is known to increase under other specific environmental conditions such as anaerobic stress (Muench and Good, 1994, Plant Mol. Biol. 24:417-427; Vanlerberge et al., 1993, Plant Physiol. 95:655-658).

Alanine levels are known to increase substantially in root tissue under anaerobic stress. As an example, in barley roots alanine levels increase 20 fold after 24 hours of anaerobic stress. The alanine aminotransferase gene has also been shown to be induced by light in broom millet and when plants are recovering from nitrogen stress (Son et al., 1992, Arch Biochem Biophys 289: 262-266). Vanlerberge et al. (1993) have shown that in nitrogen starved anaerobic algae, the WO 97/30163 PCT/CA97/00100 -4addition of nitrogen in the form of ammonia resulted in 93% of an N, label being incorporated directly into alanine. Thus, alanine appears to be an important amino acid in stress response in plants.

The nitrate transporter genes (Tsay et al. 1993. Cell 72:705; Unkles et al. 1991 PNAS 88:204), nitrate reductase (NR) and nitrite reductase (NiR) (Crawford, 1995, Plant Cell 7:859-868; Cheng et al, 1988, EMBO J 7:3309-3314) have been cloned and.studied, as have many of the genes encoding enzymes involved in plant nitrogen assimilation and metabolism.

Glutamine synthetase (GS) and glutamate synthetase (GOGAT) have been cloned (Lam et al., ibid.; Zehnacker et al., 1992, Planta 187:266-274; Peterman and Goodman, 1991, Mol. Gen.

Genet. 230:145-154) as have asparaginase (ANS) and aspartate aminotransferase (AspAT) (Lam et al., ibid; Udvardi and Kahn, 1991, Mol. Gen. Genet. 231:97-105). An asparagine synthetase (AS) gene has been cloned from pea (Tsai and Coruzzi, 1990, EMBO J 9:323-332). Glutamate dehydrogenase has been cloned from maize (Sakakibara et al., 1995, Plant Cell Physiol.

36(5):789-797. Alanine aminotransferase has been cloned by Son et al. (1993, Plant Mol. Biol.

15 20:705-713) and by Muench and Good, (1994 Plant Mol. Biol. 24:417-427). Among the plant nitrogen assimilation and utilization genes, the most extensively studied are the glutamine synthetase and asparagine synthetase genes.

In plants, genetic engineering of nitrogen assimilation processes has yielded varied results. Numerous studies examining constitutive overexpression of glutamine synthetase (GS) 20 have failed to report any positive effect of its overexpression on plant growth. These studies include, for example: Eckes et al. (1989, Molec. Gen. Genet 217:263-268) using transgenic tobacco plants overexpressing alfalfa GS; Hemon et al. (1990, Plant Mol. Biol. 15:895-904) using transgenic tobacco plants overexpressing bean GS in the cytoplasm or mitochondria; and Hirel et al. (1992, Plant Mol. Biol. 20:207-218) using transgenic tobacco plants overexpressing soybean GS. One study, by Temple et al (1993, Mol. Gen. Genet. 236:315-325), has reported increases in total soluble protein content in transgenic tobacco plants overexpressing an alfalfa GS gene and similar increases in total soluble protein content in transgenic tobacco plants expressing antisense RNA to a GS gene.

There has been a report that plants engineered to constitutively overexpress an alfalfa GS gene grow more rapidly than control, wild-type plants (Eckes et al., 1988, Australian WO 97/30163 PCT/CA97/00100 published patent application no. 17321/88). Another report (Coruzzi and Brears 1994, WO 95/09911) introduced GS, GOGAT and AS constructs under the control of the constitutive promoter Cauliflower Mosaic Virus 35S (CaMV35S). This document showed that the transgenic plants had increased fresh weight and growth advantage over controls.

B. Turgor Responsive Promoters Maintenance of normal growth and function in plants is dependent on a relatively high intracellular water content. Drought, low temperature and high salinity are all environmental stresses that alter cellular water balance and significantly limit plant growth and crop yield (Morgan, ibid.). Many physiological processes change in response to conditions that reduce 10 cellular water potential, including photosynthesis, stomatal opening and leaf, stem and root growth (Hanson and Hitz, ibid.) Along with physiological responses, metabolic changes can also occur during water loss. One of the most notable changes is in the synthesis and accumulation of low molecular weight, osmotically active compounds, as noted above.

Changes in gene expression also occur during osmotic stress. A number of genes have recently been described that are induced by drought (reviewed by Skiver and Mundy, 1990, Plant Cell 2:503-512).

Summary of the Invention In one aspect, this invention involves a genetic construct which comprises a nitrogen assimilation/metabolism pathway enzyme coding sequence operably associated with an inducible promoter. Such a genetic construct acts to confer to a plant or plant cell, into which it is introduced, enhanced agronomic characteristics.

Such genetic constructs can be inserted into plant transformation vectors and/or introduced to plant cells. Transformed plants can be produced which contain the genetic construct of the present invention.

In accordance with a broad aspect of the present invention, there is provided a genetic construct adapted for expression in a plant system comprising a nitrogen assimilation/metabolism enzyme coding sequence operably associated with an inducible promoter WO 97/30163 PCT/CA97/00100 -6- In accordance with another broad aspect of the present invention, there is provided a method for producing a plant comprising: transforming a plant cell by introducing a genetic construct having a nitrogen assimilation/metabolism enzyme coding sequence operably associated with an inducible promoter; and regenerating the plant cell to a plant.

The promoter is selected to be inducible and preferably inducible in response to a condition where it would be desirable to cause the plant to have enhanced nitrogen uptake, assimilation or use capabilities. For example, suitable promoters include, but are not limited to, those which are: induced by application of sources of nitrogen; stress inducible; wound inducible or induced by application of other chemicals. Transgenic plants containing the genetic construct of the present invention exhibit enhanced agronomic characteristics over control plants or plants having constitutively over-expressed nitrogen assimilation/metabolism genes. The agronomic characteristic which is enhanced over prior art plants can include enhanced stress tolerance and/or more efficient nitrogen uptake, storage or metabolism allowing the plants of the present invention to be cultivated with lower nitrogen fertilizer input and in nitrogen starved conditions or allowing 15 faster growth, greater vegetative and/or reproductive yield.

Plant cells, vectors and plants including the genetic construct are also provided according to the present invention.

In accordance with another broad aspect of the present invention, there is provided a genetic construct adapted for expression in a plant system comprising a coding region for 20 alanine aminotransferae enzyme operably associated with a promoter element from Brassica turgor gene-26.

Plant cells, vectors and plants including the genetic construct are also provided according to the present invention.

CD/01066001.7 6A More specifically, the present invention provides a genetic construct adapted for expression in a plant system comprising a nitrogen assimilation/metabolism enzyme coding sequence operably associated with an inducible promoter from Brassica turgor gene-26, or an active modified form thereof.

Plant cells, vectors and plants including the genetic construct are also provided according to the present invention.

Methods of using the genetic constructs are also provided according to the present invention.

10 As used herein, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude other S additives, components, integers or steps.

Brief Description of the Drawings Figure 1: Major pathways of nitrogen assimilation and metabolism in plants.

(Adapted from Lam et al., 1995, Plant Cell 7:889 where Arabidopsis is used as a model system). Some of the enzymes of the nitrogen assimilation and amide amino acid metabolism pathways are shown. Different isoenzymes are known for some of these enzymes which may play different WO 97/30163 PCT/CA97/00100 -7roles under different environmental and tissue conditions. Nitrogen assimilation occurs primarily through the activities of glutamine synthetase (GS) and glutamate synthase (GOGAT). While not indicated as such, aspartate aminotransferase also catalyses the reverse reaction. The roles of glutamate dehydrogenase (GDH) are postulated, as indicated by the dashed lines.

Figure 2: Pathway for alanine biosynthesis by the enzyme alanine aminotransferase (AlaAT) (From Goodwin and Mercer, 1983).

Figure 3: DNA sequence of the Brassica napus btg-26 promoter.

Figure 4A: Northern blot analysis of btg-26 expression during droughting. Total RNA (10 mg) from leaf tissue taken from control plants having 97% relative water content (97% RWC) and plants dehydrated to the RWC's indicated, was fractionated on a 1.2% agarose formaldehyde gel and probed with btg-26 genomic DNA.

Figure 4B: Quantitative analysis of btg-26 induction. Each time point represents the mean induction determined from three independent slot blots and two Northern blots. All blots were reprobed with a cyclophilin cDNA control to correct for loading error. Induction is b* i 15 determined relative to the level of expression in fully hydrated plants Figure 4C: Northern blot analysis of btg-26 expression during cold acclimation and heat shock. Total RNA (10 mg) from leaf tissue taken from control plants or plants exposed to 4 0 C for one or four days or exposed to 40 0 C for two or four hours. The RNA was fractionated on a 1.2% agarose formaldehyde gel and probed with btg-26 genomic DNA.

Figure 4D: Northern blot analysis of btg-26 expression during salinity stress.

Total RNA (10 mg) from leaf tissue taken from control plants or plants exposed to salinity stress by watering with 50mM NaCI (S50), 150mM NaCI (S150) or 450mM NaCI (S450) for one or four days. The RNA was fractionated on a 1.2% agarose formaldehyde gel and probed with btg-26 genomic DNA.

Figure 4E: Northern blot analysis of btg-26 expression during exposure to abscisic acid (ABA). Total RNA (10 mg) from leaf tissue taken from plants soaked for one day in a solution containing either 0 yM or 100 ,M ABA The RNA was fractionated on a 1.2% agarose formaldehyde gel and probed with btg-26 genomic DNA.

Figure 5: Nucleotide and deduced amino acid sequence of the AlaAT cDNA from barley.

WO 97/30163 PCT/CA97/00100 -8- Figure 6: Plasmid construct Figures 7A to 7C: Plasmid constructs containing the AlaAT coding region and the CaMV, btg-26 and trg-31 promoters that were used for the transformation of Brassica napus plants.

Figure 8: Plasmid construct pCGN 1547 used in producing the overexpressed/AlaAT or stress inducible/AlaAT transformants.

Figure 9: Brassica napus plants grown under nitrogen starved conditions for three weeks followed by drought for 3 days. The plants are identified as A, B and C, as follows: Plant A is a control, wild-type plant; Plant B contains a CaMV/AlaAT construct; and Plant C contains a btg-26/AlaAT construct.

S.

Detailed Description of the Invention 0* In one aspect, the present invention is directed to a genetic construct having a coding sequence of a nitrogen assimilation/metabolism enzyme operably linked to a inducible promoter. The promoter sequence is preferably inducible under conditions where it is desirable 15 for plants to take-up, store and/or use nitrogen. Such a gene can be in purified or isolated form or introduced to an expression cassette, cloning or transformation vector for use in the transformation of plant cells or plants.

Transcription of DNA into messanger RNA (mRNA) is regulated by a region of the gene known as the promoter. The promoter sequences useful in the present invention are 20 those which are inducible and, preferably, those which regulate gene expression in response to a condition in which it would be desirable to cause a plant to assimilate and/or metabolize nitrogen.

Many inducible promoters are known in the art and for example, include, but are not limited to, promoters induced upon the addition of nitrogen or when a plant is under nitrogen stress, those induced by specific environmental conditions, such as drought stress, osmotic stress, wound stress, heat stress, anaerobic stress, and salt stress; or promoters induced by addition of chemical agents, such as for example auxins.

When used to transform plants, such genetic constructs can provide transformed plants with enhanced agronomic characteristics and which it is postulated can assimilate and/or WO 97/30163 WO 9730163PCT/CA97/00100 -9use nitrogen when it is most needed or beneficial. For example, where the promoter is a stress inducible promoter such as, for example, the inducible promoters 26g from Pisum sativum (Guerrero and Mullet, 1988, Plant Physiol, 88:401-408; Guerrero et al., 1990, Plant Mol Biol 11-26), trg-3 I from tobacco (Guerrero and Crossland, 1993, Plant Mol Biol 21:929-935) or btg-26 from Brassica napus as discussed in Example I and shown in Figure 3 and SEQ ID NO: 1, the plant is induced to produce the gene product upon application of a suitable stress to drive the promoter. Such a plant thereby can have increased stress tolerance,'such as by enhanced osmoregulation.

As another example, where the promoter is induced by the presence of nitrate, such as, for example, the nitrate reductase promoter (Cheng et al, 1988, ibid.; Cheng et al, 199 1, .Plant Phsyiol. 96:275-279), the plant will be induced to assimilate and/or use nitrogen upon application of a nitrogenous fertilizer. Alternately, or in addition, the promoter can be inducible under nitrogen stress conditions (Son D. et al, 1992, Plant Cell Physio, 33: 507-509) so that a plant's ability to take up and use nitrogen efficiently is increased under conditions of low nitrogen. The promoter can also be induced by an exogenously applied chemical such as, for example, copper (Met et al., 1993, PNAS 90:4567) or abscisic acid (Marcotte et al, 1989, Plant Cell 1:969-976). These chemicals can be included in formulations for nitrogenous fertilizer. The application of such fertilizer formulations to plants transformed to contain a nitrogen *assimilation/metabolism gene under the control of a promoter induced by the chemical in the fertilizer will induce the activity of the nitrogen assimilation/metabolism gene. It is postulated V4000that transformed plants containing such genetic constructs can utilize more efficiently fertilizer .input over wild type plants by rapidly taking up the nitrogen in the fertilizer and storing it at the time of application, to thereby reduce the amounts of nitrogenous fertilizer which are lost to leaching, etc. This will permit the amount of nitrogenous fertilizer required to be applied to a crop to be reduced over those amounts presently used, while obtaining crop yields comparable to those obtained using normal cultivation techniques and plants.

The inducible promoter useful in the present invention can be homologous or heterologous to the plant to which it is to be introduced. Multiple copies of the promoters can be used. The promoters may be modified, if desired, to alter their expression characteristics, for example, their expression levels and/or tissue specificity. In a preferred embodiment, the WO 97/30163 PCT/CA97/00100 inducible promoter is selected, or ligated to another sequence, or otherwise modified, to exhibit root specific activity. As an example, a root specific promoter is the GS 15 as described by Hirel et al, 1992, Plant Mol Biol. 20:207-218. The selected wild-type or modified inducible promoter can be used as described herein.

The coding regions of interest are those for enzymes active in the assimilation and/or metabolism of nitrogen and, preferably, those which are active in the assimilation of ammonia into amino acids or those which use the formed amino acids in biosynthetic reactions.

Referring to Figure 1, these enzymes include, but are not limited to, glutamine synthetase (GS), asparagine synthetase glutamate synthase (also known as glutamate 2: oxogluturate amino transferase and GOGAT), asparaginase (ANS), glutamate dehydrogenase (GDH), aspartate aminotransferase (AspAT) and alanine aminotransferase (AlaAT) (activity shown in Figure 2).

Sequence information for these enzymes is known, for example, as follows: GS from soybean (Hirel et al, ibid); AS from pea (Tsai, F-Y and Coruzzi, G.M. 1990, EMBO J. 9: 232-332); 1 GOGAT from tobacco (Zehnacker, C. et al, 1992. Planta 187:226-274); ANS (Casado, A. et al.

1995 Plant Physiol. 108:1321); AspAT from alfalfa (Udvardi, M. And Kahn, M. 1991. Mol. Gen.

Genet. 231:97-105). Other coding regions for nitrogen assimilation/metabolism enzymes which are of interest are those for nitrogen transporters (Tsay et al. 1993. Cell 72:705 and Unkles et al.

1991 PNAS 88:204) and/or nitrate reductase (Vincentz, M. And Caboche, M. 1991 EMBO J.

10:1027-1035).

S 20 The nitrogen assimilation/metabolism enzyme coding regions can be used in their wild-type forms or can be altered or modified in any suitable way to achieve a desired improvement. These coding region can be obtained from any source and can be homologous or heterologous to the plant cell into which it is to be introduced. Preferably, the coding region is heterologous to the promoter to which it is linked, in that it is not linked to an unmodified, inducible promoter to which the coding region is naturally linked.

The genetic construct can be modified in any suitable way to provide for expression in plants. The genetic construct is adapted to be transcribable and translatable in a plant system, and, for example, contains all of the necessary 5' and 3' non-translated regions including poly-adenylation sequences, start sites and termination sites which allow the coding sequence to be transcribed to mRNA (messenger ribonucleic acid) and the mRNA to be translated WO 97/30163 PCT/CA97/00100 -11 in the plant system. These sequences and elements can be obtained from any source. The gene construct can be introduced to a plant transformation or cloning vector. The vectors will, as is known, contain sequences suitable for inclusion in such vectors, such as one or more bacterial or plant-expressible selectable or screenable markers, for example a Kanamycin resistance gene (NPTII), [-glucuronidase (GUS) genes and/or sequences required for replication and transformation such as, for example the right and, optionally, the left T-DNA borders, where the vector is to be used in an Agrobacterium-mediated transformation system.

Examples of cloning or transformation vectors are for example plasmids, cosmids and/or viral DNA or RNA. Methods for preparation of such genetic constructs and vectors are well known in the art.

The gene construct of the present invention can be introduced to a plant cell by any useful method. A large number of processes are available and are well known to deliver genes to plant cells. One of the best known processes involves the use of Agrobacterium or similar soil bacteria as a means for introduction of the genetic construct into the plant. In this o. o 0. 15 process, target tissues are co-cultivated with Agrobacterium which inserts the gene of interest to the plant genome. Such methods are well known in the art as illustrated by US Patent 4,940,838 of Schilperoort et al., Horsch et al. 1985, Science 227:1229-1231, Fisher and Guiltinan, 1995, Plant Mol. Biol. Reporter 13:278, and Bayley et al., 1992, Theoret. Applied Genet 83:645.

Alternative gene transfer and transformation methods useful in the present invention include, but 000000 20 are not limited to liposomes, electroporation or chemical-mediated uptake of free DNA, targeted 0 microprojectiles and microinjection. These methods are well documented in the prior art.

Cells that have been transformed with the gene construct of the present invention can be regenerated into differentiated plants using standard nutrient media supplemented with shoot-inducing or root-inducing hormones, using methods known to those skilled in the art.

Suitable plants for the practice of the present invention include, but are not limited to, canola, com, rice, tobacco, soybean, cotton, alfalfa, tomato, wheat and potato. Many other plants can also be advantageously engineered with the gene of the present invention. Transformed plants according to the present invention can be used for breeding, crop production and the like.

While the genetic construct of the present invention can include any promoter which is inducible, a particularly preferred promoter is the btg-26 which was found to be inducible WO 97/30163 PCT/CA97/00100 -12by stresses such as heat shock, drought, salinity and ABA concentration. The btg-26 promoter element is as depicted in Figure 3 and SEQ ID NO: 1.

Nucleotide sequences homologous to the btg-26 promoter described herein are those nucleic acid sequences which are capable of hybridizing to the nucleic acid sequence depicted in Figure 3 (SEQ ID NO: 1) in standard hybridization assays or are homologous by sequence analysis (at least 45% of the nucleotides are identical to the sequence presented herein) using the BLAST programs ofAltschul et al (1989, J. Mol. Biol. 215:403-410). Homologous nucleotide sequences refer to nucleotide sequences including, but not limited to, promoter elements from other plant species or genetically engineered derivatives of the promoter element according to the present invention. The promoter element can be engineered to alter its activity, for example its level of expression. Such engineered promoters are useful in the present invention.

The following examples further demonstrate several preferred embodiments of this invention. While the examples illustrate the invention, they are not intended to limit the invention.

EXAMPLES

S* Example 1: Isolation and characterization of osmotic stress-induced promoter o. A Brassica napus (cv. Bridger) genomic DNA library (Clontech, Palo Alto, California) was screened using standard techniques (Ausubel et al., 1989, Current Protocols in Molecular Biology, Wiley, Wiley, with the Pisum sativum 26g cDNA (complementary deoxyribonucleic acid) clone (Guerrero et al., ibid), 3 P-labelled with a Random Primer Kit (Boehringer Mannheim, Laval, Quebec). A 4.4 kb Sail fragment containing the entire btg-26 gene was subcloned into the commercially available pT7T3-19U vector (Pharmacia Canada, Inc., Baie d'Urf6, Quebec, Canada) for further analyses.

WO 97/30163 PCT/CA97/00100 -13- Identification of an osmotic stress-induced promoter in Brassica napus Several genes activated during drought stress have been isolated and characterized from different plant species. Most of these represent later-responding, ABA-inducible genes (reviewed by Skiver and Mundy, ibid.). Recently, however, an ABA-independent, cycloheximide-independent transcript, 26g, was reported in Pisum sativum (Guerrero and Mullet, ibid; Guerrero et al., ibid). Because this gene does not require protein synthesis for activation, it is postulated that it represents an early factor in the drought signal transduction pathway. To isolate an osmotic stress induced promoter from Brassica napus, the cDNA clone representing the P.

sativum 26g gene (Guerrero et al., ibid) was used. Total RNA was isolated from the third leaf of 10 whole plants that had been either watered continuously or dehydrated for four days. Using low stringency hybridization, RNA blot analysis identified a single 1.75 kb transcript that is greatly induced in droughted plants. To determine if this mRNA represents a single copy gene in B.

napus, genomic DNA was digested with EcoRI, HindIII or BglII and analyzed by DNA blot hybridization using the P. sativum 26g cDNA. A single band was identified in each lane. It was 15 concluded that this transcript represents a single copy, drought-induced gene in B. napus. This gene is referred to as btg-26 (Brassica turgor gene 26).

"Structure of btg-26 gene To isolate the btg-26 gene, a B. napus genomic DNA library in EMBL-3 (Clontech, Palo Alto, California) was screened with the P. sativum 26g cDNA. From 40,000 plaques analyzed, a single positive clone was identified with an insert size of approximately 16 kb. A 4.4 kb Sail fragment containing the entire gene was subcloned. The promoter sequence of the btg-26 gene was determined by identification of the mRNA start site using primer extension (Ausubel, ibid.) and is shown in Figure 3 and SEQ ID NO:1. In Figure 3, the transcription start site is bolded, underlined and indicated by The TATA box and CAAT box are in bold and double underlined. Postulated functional regions are underlined. The sequence of the btg-26 promoter, coding region and 3' region has been presented in Stroeher et al, (1995, Plant Mol. Biol.

27:541-551).

WO 97/30163 PCT/CA97/00100 -14- Expression analysis of btg-26 Induction of btg-26 expression during droughting was examined by RNA blot analysis. Potted B. napus plants were naturally dehydrated by withholding water for various lengths of time. Whole leaves were used either to determine relative water content (RWC) of individual plants or to isolate total RNA. As shown in Figures 4A and 4B, btg-26 expression is induced rapidly during water loss, reaching a six-fold increase over expression in fully hydrated plants at 81% RWC, increasing to eleven-fold induction at 63% RWC. Further decreases in RWC were associated with a decrease in total amount of btg-26 transcript. At 30% RWC expression was only 3.5-fold over fully hydrated levels.

•10 Because other physiological stresses alter intracellular water content, btg-26 expression was examined in B. napus plants exposed to cold, heat shock and salt stress. RNA blot analysis indicated that there was no change in btg-26 expression when plants were transferred from normal growth conditions to 4*C for one day. However, plants left at 4*C for four days showed a five-fold induction in btg-26 mRNA. A similar increase was seen when plants were shifted to 40°C for two or four hours. These results are shown in Figure 4C and demonstrate that expression of btg-26 is induced during temperature stress. To examine the effect of salt stress, plants were watered to capacity one day or four days with 50 mM, 150 mM, or 450 mM NaCI The level of btg-26 expression was not affected by 50 mM NaCI regardless of length of exposure.

The plants watered with 150 mM NaCl showed a two-fold increase in btg-26 mRNA after four days. Exposure to 450 mM NaCI caused the most notable induction, twelve-fold after one day, S:.dropping to four-fold after four days. Refer to Figure 4D for Northern blots showing these results.

Finally, many drought-inducible genes are also ABA responsive. To examine the role of ABA in btg-26 expression, total RNA was isolated from individual leaves treated with or without ABA. In these experiments, leaves were cut at the petiole and placed in a solution of 0 bsM, 50 M.M or 100 pM ABA (mixed isomers, Sigma), 0.02% Tween-20 and pH 5.5 for 24 hours.

As shown in Figure 4E, btg-26 expression was induced 2.5-fold when leaves were exposed to 100 AM ABA. However, when leaves were exposed to 50 ptM ABA, no induction of expression was WO 97/30163 PCT/CA97/00100 observed. These results indicate that btg-26 is ABA responsive, but that this responsiveness is concentration dependent.

Example 2: Creation of stress-induced nitrogen assimilation constructs This step involved the production of either constitutive or stress-induced AlaAT constructs and the introduction of them into Brassica napus using Agrobacterium mediated genetic transformation. The approach of introducing specific sense or antisense cDNA constructs into plants to modify specific metabolic pathways has been used in a number of species and to modify a number of different pathways. (See Stitt Sonnewald 1995 for a review; Ann. Rev. of Plant Physiol. and Plant Mol. Biol. 46:341-368). The AlaAT cDNA was introduced under the 10 control of three different promoters. The CaMV355 promoter which has been shown to be a strong constitutive promoter in a number of different plant species; the btg-26 promoter described in Example 1 and the trg-31 promoter which was isolated from tobacco by Guerrero and Crossland (ibid.). The CaMV promoter induces the constitutive overexpression of AlaAT whereas btg-26 and trg-31 should induce over expression of AlaAT only under conditions of 15 specific stresses, including drought stress.

Example 3: Plasmid constructs The barley AlaAT cDNA clone 3A (As shown in Figure 5 and SEQ ID NO:2 and Muench and Good, ibid) was cloned into the pT7T3-19U vector (Pharmacia Canada) and used for site directed mutagenesis using two specific primers. Primer I introduced a BamH1 restriction site between nucleotides 48-53, while primer 2 was used to introduce a second BamHI restriction site between nucleotides 1558-1563 (See Figure The 1510 bp fragment was then cloned into the vector p25 (Figure 6) which had been cut with BamH1. p 25 contains the double CaMV35S (Ca2) promoter, which has been shown to give high constitutive levels of expression, and the nopaline synthase (NOS) terminator inserted into the Kpnl and Pstl site of pUC19 with a BamH Xbal and Pvul polylinker between the CaMV and NOS region of the plasmid. The resulting plasmid was called pCa2/AlaAT/NOS, and is shown in Figure 7A.

WO 97/30163 PCT/CA97/00100 -16- The plasmids pbtg-26/AlaAT/NOS (Figure 7B) and ptrg-31/AlaAT/NOS (Figure 7C) were created as follows. The trg-31 promoter was subcloned as a 3.0 kb Xbal/BamHl fragment into the Xbal/BamHI site of pCa2/AlaAT/NOS which had been digested with Xbal/BamH1 to release only the Ca2 promoter, resulting in a 3 kb promoter fragment inserted in front of the AlaAT coding region. pbtg-26/AlaAT/NOS was created by inserting a BamHl site at nucleotides +9 to +14 (see Figure 3) and subcloning the 330 bp Kpnl/BamHl fragment (-320 to in Figure 3) into the Kpnl/BamHl site ofpCa2/AlaAT/NOS which had been digested to release the Ca2 promoter. Plasmid constructs pbtg-26/AlaAT/NOS and ptrg-31/AlaAT/NOS are shown in Figures 7B and 7C, respectively.

10 Example 4: Transformation and analysis of Brassica napus plants with AlaAT constructs.

Once the three plasmids, as shown in Figures 7A, 7B and 7C, containing the AlaAT gene had been confirmed by restriction analysis and sequencing they were subcloned into the transformation vector pCGN 1547 (Figure pCGN1547 is an Agrobacterium binary vector 15 developed by McBride and Summerfelt (1990, Plant Mol. Biol. 14:269-276). pCGN1547 contains the neomycin phosphotransferase II (NPTII) gene which encodes Kanamycin resistance.

These constructs were then introduced into the Agrobacterium strain EHA 101 (readily available) by electroporation using the protocol of Moloney et al. (1989, Plant Cell Reports 8:238-242).

Confirmation that the Agrobacterium had been transformed with the pCGN 1547 vector 20 containing the specific construct was made by polymerase chain reaction (PCR).

Transgenic Brassica plants Westar) were produced using the well established cotyledon transformation and regeneration protocols as described by Moloney et al. (ibid.).

Kanamycin resistant plantlets were transferred to soil and then grown. The initial generation, or primary transformants, were referred to as the TO generation and were allowed to self. Each subsequent generation was bagged to ensure selfing and referred to as the TI, T2 generation, respectively. All putative TO transgenic plants were tested for the insertion of the Agrobacterium construct using PCR primers that amplify the NPTII gene and by testing for NPTII activity as described by Moloney et al (ibid.).

WO 97/30163 PCT/CA97/00100 -17- Example 5: Analysis of transformed Brassica plants containing the AlaAT constructs Transgenic plants were assayed for AlaAT activity. Extractions were carried out on ice as described previously (Good and Crosby, 1989, Plant Physiol 90:1305-1309). Leaf tissue was weighed and ground with sand using a mortar and pestle in extraction buffer containing 0.1 M Tris-HCl (pH 10 mM dithiothreitol, 15% glycerol and 10% PVPP. The extract was clarified by centrifugation at 6,000 rpm and the supematant was assayed for enzyme activity.

AlaAT assays were performed in the alanine to pyruvate direction as described previously (Good and Crosby, ibid) using alanine to start the reaction.

After transformation 20 Ca2/AlaAT/NOS, 24 btg-26/AlaAT/NOS and 21 trg- 10 31/AlaAT/NOS plants were produced which appeared to be transformed, based on the 9 •amplification of an NPTII PCR product and NPT activity. AlaAT activity was measured, using the method described above, in the leaf tissue of several of these transformants. As can be seen from Table 1, the btg-26/AlaAT/NOS plants had AlaAT activity levels that ranged from 1.63 to i 3.89 times that of the wild-type, control plants. Ca2/AlaAT/NOS plants had activity levels that 15 ranged from 1.51 to 2.95 times that of wild-type, control plants. Western blots confirmed that the transgenic plants had elevated levels of AlaAT, based on the cross reactivity of a band with the barley AlaAT antibody.

9 9 WO 97/30163 WO 9730163PCTICA97OO 100 -18- Table 1. Alanine aminotransferase (AlaAT) activity in primary transtormants big-26/AlaAT/NQS transformant #4 3.89x transformant #5 1 .63x transformant #7 1 .93x transformant #8 1 .98x transformant #18 1 .63x C£a2/laAINQS transformant #1 1 .51x transformant #2 2.77x transforrnant #6 1.61x transformant #7 2.95x transformant #9 2.14x transformant #12 1 .91x transformant #13 1.77x *Enzyme activity is expressed relative to wild-type controls 20 Example 6: Growth of primary transforniants under normal conditions TI seed f-rm the primary transformants of the groups Ca2/AlaAT and btg- 26/AlaAT were grown along with control, wild-type plants under normal conditions including planting at a 1 cm depth in 13 cm diameter plastic pots containing a soil and fertilizer mixture as described by Good and Maclagan (ibid.). These pots were placed in growth chambers under the following conditions: i) 16 h of 265 MMcl M- 2 S-1 provided by VITA-LITE U.14.0. fluorescent tubes, ii) day and night temperatures of 2C and 15 0 C respectively, iii) relative humidity of 97% and iv) daily watering with 1/2 strength Hoagland's solution. The only difference observed between the plants was that the btg-26/AlaAT plants had thicker stems when compared to the controls and Ca2/A~laAT plants. No significant differences were observed between the three WO 97/30163 PCT/CA97/00100 -19groups in terms of growth rate, plant or leaf size or leaf senescence at identical time points, time to maturity, seed size or seed yield.

Example 7: Growth of primary transformants under nitrogen-starved/drought conditions TI seed from the primary transformants of the Ca2/AlaAT and btg-26/AlaAT groups were grown along with control, wild-type plants for four weeks under normal conditions (as noted above) and then subjected to nitrogen starvation, by watering with only water for three weeks, followed by drought for 3 days. Figure 9 shows representative plants from the three groups after the treatment at an identical time point. Plant A is a control, wild-type plant; Plant B is a Ca2/AlaAT transformed plant; and Plant C is a btg-26/AlaAT plant It can be seen that plant 10 C (btg-26) clearly has a faster growth rate than plants A (control) and B (Ca2/AlaAT). In addition, senescing leaves (indicated by arrows) are present on plants A and B while plant C has no senescing leaves. In summary, the following were observed in the treated btg-26/AlaAT plants when compared to the treated Ca2/AlaAT and control plants: faster growth rate; larger plants at similar time points, less senescence in the lower leaves; earlier maturity; thicker stems; larger 15 seeds; and higher seed yields.

Example 8: Growth of secondary transformants under low nitrogen and well fertilized conditions T2 seed from the TI transformants of the btg26/AlaAT/NOS plants and Ca2/AlaAT/NOS primary transformants were tested to ensure that the original TI plant was homozygous by using PCR (Polymerase Chain Reaction). 20bp primers specific to T-DNA were used to ensure that the plant was transgenic. DNA from the plants was amplified in the presence of primers so that in a plant containing the insert, an appropriate sized band was amplified and in plants lacking the insert a band was not amplified. T2 seed from homozygous TI transgenics were then used as described below.

WO 97/30163 PCTICA97/00100 T2 seeds from btg26/AlaAT(btg) and Ca2/AlaAT (CaMV) plants and control seeds according to Example 6 and then watered with 0-20-20 fertilizer (low nitrogen) or 20-20-20 fertilizer (well fertilized) in the soil mixture being used up in a period of approximately four weeks. Plants were allowed to grow for approximately five weeks. After the fourth week, the plants rapidly increased in size. During this time leaf area was measured using a leaf area meter.

At the end of five weeks plants were harvested and the following measurements were taken: 1) leaf area (in the first, through fifth and, if possible, sixth and seventh leaves); 2) stem diameter; 3) total shoot fresh weight; and, 4) total shoot dry weight A comparitive construct was prepared, according to Example 3, by substituting the AlaAT coding region with a GUS coding region to produce a plasmid construct pbtg- 26/GUS/NOS. Brassica plants were transformed with this construct according to Examples 4 and These plants were grown as described above in this Example.

The results from three btg-26/AlaAT lines (btgl, btg2, btg3) and one control wildtype population can be seen in Tables 2-5. The results from the btg-26/GUS plants are included in Tables 4 and 5. There was no significant difference between control and btg-26/AlaAT transgenics in leaf area or stem diameter when the plants were grown under well fertilized conditions. In contrast, under low nitrogen conditions, the transgenics outperformed the control plants dramatically. Transgenics btgl, btg2 and btg3 outperformed the controls by 30%, 55.8% and 52%, respectively, for the fourth leaf area and 78%, 70% and 86% for the fifth leaf area (Table These are the two largest leaves on the plants so that overall there was a substantial increase in biomass (Table The two lower leaves (second and third) were substantially fully S* developed by the time the nitrogen in the soil was depleted. Thus, there appears to be little difference in these leaves between the controls and the transgenics.

The plants transformed with the CaMV35S promoted constructs had growth characteristics similar to those of the controls.

WO 97/30163 PCT/CA97/00100 -21 Table 2: Average Leaf Area Stem Diameter (Well Fertilized) Y Leaf Area (cm 2 Stem me i Leaf Area (cm 2 3rd 4th 5th G~th Stem If-m\

S

0e*O

S

S. S

S

@5e S. S

S.

btg-1 74.25 131.01 140.94 136.82 na na 0.83 _n 6 5 6 4 0 0 6 standard deviation 17.691 34.927 39.151 17.076 na na 0.078 standard error 7.222 15.620 15.983 8.538 na na 0.032 btg-2 72.87 130.55 160.48 150.41 63.61 na 0.80 _n 5 6 6 6 1 0 6 standard deviation 15.383 22.957 35.945 31.418 na na 0.081 standard error 6.879 9.372 14.674 12.826 na na 0.033 btg-3 52.65 106.77 139.71 150.61 141.51 78.99 0.92 1 6 6 6 3 1 6 standard deviation na 23.121 30.240 23.935 24.543 na 0.093 standard error na 9.439 12.345 9.772 14.170 na 0.038 Control 71.00 135.15 173.75 159.90 120.55 55.85 0.76 6 6 6 6 1 1 6 standard deviation 23.431 40.715 39.048 43.124 na na 0.087 rstandard error 9.566 16.622 15.941 17.6051 na na 0.035 Table 3: Average Leaf Area Stem Diameter (Low Nitrogen) Leaf Area (cm 2 Stem line 2nd 3rd 4th 5th 6th (cm) btg-1 85.71 116.91 155.87 167.12 134.86 0.88 n 2 5 5 5 1 6 standard deviation 2.804 43.552 56.714 42.272 na 0.124 standard error 1.982 19.477 25.363 18.904 0.000 0.051 btg-2 85.12 143.68 187.63 159.73 na 0.87 n_ 6 6 6 6 0 6 standard deviation 17.857 35.962 55.162 48.791 na 0.132 standard error 7.290 14.682 22.520 19.919 na 0.054 btg-3 61.09 117.75 183.78 175.12 na 1.00 n 3 4 6 6 2 6 standard deviation 10.714 28.463 25.477 39.245 33.705 0.124 standard error 6.186 14.232 10.401 16.022 23.833 0.051 Control 70.28 110.02 120.41 94.02 na 0.69 n 6 6 6 6 0 6 standard deviation 16.700 23.685 24.108 11.851 na 0.097 standard error 6.818 9.669 9.842 4.838 na 0.040 S. *S 5 0

S

S

000e 0* WO 97/30163 WO 9730163PCTICA97/00 100 22 Table 4: Average Fresh Dry Weight (Well Fertilized) line FW DW FW IWDW btg-2 34.04 4.50 7.96 n 5 standard error 3.012 0.700 0.676 standard deviation 6.735 1.566 1.513 btg-3 46.93 6.55 7.04 n 6 6 6 standard error 7.167 0.798 0.277 standard deviation 17.555 1.956 0.6791 Control 39.98 5.14 8.02 n 7 7 7 standard error 5.847 0.794 0.664 standard deviation 15.469 2.100 1.758' btg-26IGUS 46.55 6.01 7.76 n 6- 6 6 standard error 3.9871 0.521 0.227 standard deviationi 9.7651 1.275 0.555 Table 5: Average Fresh Dry Weight (Low Nitogn) line FW g)DW(g FW IDW btg-2 37.08 4.73 7.76 n 6 6 6 standard error 8.228 0.957 0.643 standard deviation 20.155 2.343 1.574 btg-3 43.73 6.30 6.92 n 5 5 standard error 7.819 1.025 0.525 standard deviaion 17.483 2.292 1.175 Control 22.24 3.63 6.16' n 6 6 6 standard error 1.666 0.224 0.385 standard deviation 4.080 0.549 0.942 btg-26JGUS 29.25 3.7 8.09 nI 71 71 7 standard errorr 4.2501 0.4111- 1.4321 WO 97/30163 PCT/CA97/00100 -23- Example 9: Transformation and analysis of tobacco (Nicotianan tabacum) In this Example, a genetic construct according to the present invention is used to transform tobacco.

The three plasmids, as shown in Figure 7A, 7B and 7C containing the AlaAT gene were subcloned as described in Example 3 and inserted into the Agrobacterium strain EHA101 by electroporation. PCR was used to confirm that the Agrobacterium was transformed (Example 4).

Transgenic tobacco plants are produced using the well established whole leaf transformation protocol as described by Fisher and Guiltinan (1995; Plant Mol. Biol. Reporter 13:278) and are selected on Kanamycin. The initial generation, or primary transformants are 10 selfed to produce TI generation and are tested to confirm insertion of the construct according to Example 4.

Transgenic plants are tested for AlaAT activity as described in Example The TI seed is tested to ensure homozygousity of the T1 plants as in Example 8.

T2 seed from selfed homozygous T1 plants is grown as in Example 8 using the well fertilized 15 protocol and nitrogen starved protocol of Example 8.

Example 10: Transformation and analysis of cotton (Gossypium hirsutum) In this Example, a genetic construct according to the present invention is used to transform cotton.

S. The three plasmids, as shown in Figures 7A, 7B and 7C containing the AlaAT gene subcloned as described in Example 3 are inserted into the Agrobacterium strain EHA 101 by electroporation. Transformation of the Agrobacterium was confirmed by PCR.

Transgenic cotton plants are produced containing the genetic constructs of Example 3 using a whole leaf transformation protocol as described by Bayley et al. (1992; Theoret. Applied Genet 83:645). TO, T1 and T2 plants are produced and confirmed for transformation and homozygousity as in Example 9. Plants are grown and compared to control plants.

WO 97/30163 PCT/CA97/00100 -24- Example 11: Transformation and analysis of maize (Zea mays) In this Example, a genetic construct according to the present invention is used to transform maize.

For transformation of maize, a 340 bp promoter fragment from the rab28 gene of rice is used (Pla et al. Plant Mol. Biol. 21:259). The rab28 gene from maize has been shown to be responsive to both osmotic stress and drought. This was inserted as a blunt ended fragment into where the Kpnl/BamHl btg-26 promoter had been inserted in the AlaAT construct of Figure 7B.

This resulted in a construct prab28/AlaAT/NOS. The CaMV promoter construct of Figure 7A was also used.

10 These fragments are then separately subcloned into the monocot transformation vector pSB 131 available from Clonetech (Palo Alto, Calif.). This vector is similar to many of the commonly used dicot vectors such as pBI121 in that is carries the gene of interest along with a selectable marker. The selectable marker of the vector is the BAR gene which codes for Basta resistance (Ishida et al., 1996, Nature Biotechnology 14:745). This plasmid containing the 15 rab28/AlaAT construct is then introduced into the Agrobacterium strain LBA4404 (readily available) by electroporation. PCR is used to confirm that Agrobacterium is transformed.

Transgenic plants are produced by infection of immature embryos for maize S: inbred lines and selection is made on Basta as described by Ishida et al (iid). Transgenic plants are tested for insertion of the gene using PCR and then for AlaAT activity as described in Example TO, T1, T2 plants are produced and confirmed for transformation and homozygousity as in Example 9. T2 plants are grown and compared with controls.

Example 12: Transformation and analysis of rice (Oryza sativa) In this Example, a genetic construct according to the present invention is used to transform rice.

The plant transformation vector and Agrobacterium strain as in Example 11 are used to obtain transgenic rice plants by infection of calli which had been derived from rice WO 97/30163 PCT/CA97/00100 scutellum tissue. (Hirei et al. 1994 Plant Journal 6:271). Transgenic plants are tested for insertion of the gene using PCR and then for AlaAT activity as described in Example TO, TI, T2 plants are produced and confirmed for transformation and homozygousity as in Example 9. T2 plants are grown and compared with controls.

Example 13: Tetracycline inducible promoter In this example, a tetracycline inducible promoter is used with a nitrogen assimilation/metabolism gene.

A tetracycline inducible promoter system is commercially available from Clonetech (Palo Alto, California) in the following manner. The TnlO (a transposable element 10 from bacteria encoding tetracycline resistance) encoded Tetracycline repressor (TetR) is combined e with the CaMV promoter so that under normal conditions this gene construct is tightly repressed.

With the addition of tetracycline, there is a 200 to 500 fold induction of the promoter and the corresponding gene (Roeder et al. 1994; Mol. Gen. Genet. 243:32). The AlaAT cDNA is cloned into this construct such that the Tetracycline represser gene is driving the AlaAT cDNA.

15 Therefore in the absence of tetracycline AlaAT will not be expressed whereas in the presence of doxycycline the gene is turned on.

This construct is used to transform canola, tobacco and rice as described in Examples 3, 4, 9 and 12. The plants are then tested for the insertion of the gene using PCR and for the induction of AlaAT activity in the presence of doxycycline.

S 20 Transgenic plants are grown and after about two weeks, one group of each plant species has doxycycline included in the watering media such that the AlaAT gene is induced.

This is tried at different time periods increasing by one week intervals and compared to the control group.

Example 14: Copper inducible promoter In this example, a copper inducible promoter is used with a nitrogen assimilation/metabolism gene.

WO 97/30163 PCT/CA97/00100 -26- A copper inducible promoter by Met et al. (1993 PNAS 90:4567) can be turned on and off in an inducible manner by the addition of copper to the media. The AlaAT cDNA is cloned into a vector under control of this copper inducible promoter. Therefore in the presence of copper alanine aminotransferase will be expressed.

This construct is used to transform canola, tobacco and rice according to Examples 3, 4, 9 and 12. The plants are then tested for the insertion of the gene using PCR and for the induction of AlaAT gene activity in the presence of copper.

Example 15: Nitrogen inducible promoter In this example, a nitrogen inducible promoter is used with a nitrogen 10 assimilation/metabolism gene.

The nitrate reductase (NR) gene from Arabidopsis (Wilkinson Crawford 1991; Plant Cell Physiol. 3:461-471) has been shown to be induced under conditions where plants are shifted from a nitrogen-starved condition to a nitrogen-rich environment (Cheng et al. 1991; Plant Physiol. 96:275). The NR promoter (Wilkinson Crawford, ibid) is cloned as a 1 kb blunt ended 15 fragment into where the Kpnl/BamH1 btg-26 promoter had been inserted in the btg-26/AlaAT construct (Figure 7B). This results in the construct pNR/AlaAT/NOS.

S* This construct is used to transform canola and tobacco as described in Examples 3, 4 and 9. The plants are then tested for the insertion of the gene using PCR.

Transgenic plants are grown and treated to nitrogen-starved conditions followed S 20 by application of a source of nitrate. Transgenic plants are compared with controls.

Example 16: Aspartate aminotransferase with inducible promoter The aspartate aminotransferase (AspAT) coding region from alfalfa (Udvardi, M.

and Kahn, ibid) is cloned between the BamH1/BamH1 sites using the construct of Figure 7B to replace the AlaAT coding region. This results in a btg-26/AspAT/NOS construct.

This construct is used to transform canola and tobacco as described in Examples 4, and 9. The plants are then tested for the insertion of the gene using PCR.

WO 97/30163 PCT/CA97/00100 -27- Transgenic plants are grown and treated to nitrogen starved conditions according to Example 8. Transgenic plants are compared with controls.

Each patent, patent application and publication mentioned hereinbefore is herein individually incorporated by reference.

It will be apparent that many other changes may be made to the illustrative embodiments, while falling within the scope of the invention and it is intended that all such changes be covered by the claims appended hereto.

o* eo WO 97/30163 PCTCA97OO100 -28 SEQUENCE LISTING GENERAL INFORMATION: (iii) NUMB~ER OF SEQUENCES: 2 INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 365 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GTCGACCTGC AGGTCAACGG ATCCTAATCG GGGTATATCC CGACCCGGAA AAAGAAACGT :AGGACACGTG ACAAAACTTC ATATGATCCG AGTGAATCAA GCCAAAAGGG GGATTGACAC 120 AACAGCTCAG C"ITTCGTTTT CGGTCCAATC GCTGTI'CCAA CT=ACTAC AAGTCGTACA 180 .*CGTCTCTCTC TCTCTCTCTC TCTCTCACTC ACTTCCTCTT ATAAAGACTC TCTGATCAAA 240 CGTATAATCG GAAAACTCCA TTCITTMATA CCATCGATAA TACTAAGAGA GGTGATTGAT 300 *TCTTTAATCA CTGTTGATA TCCTTAACTI' TGATCCATTT ACTCTGT TCA ATC-ATITG 360 TAGAG 365- INFORMATION FOR SEQ, ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 1701 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLoOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: GGCCACAAAA CCGCGGAAAG AGATAGACGG ACAGCTAGAG GCGTCGGA.AG ATACTCGCTG SUBSTITT SHEET (RULE 26) WO 97130163 PCTICA97/00100 -29- CTCTGCCGCC CCCTTCGTCT TAGTTGATCT CGCCATGGCT GCCACCGTCG CCGTGGACAA

V.

CCTGrAACCCC

TCAGCGCTTG

CTATTGTAAC

GGTTCTTGCC

CAGTGCTGAT

AGGAGCATAC

CGCTTCACGA

TCCTGGGGTG

CCCGATTCCT

CCCATACTAT

ACTTGAAGAT

AAATCCAACT

PAATGAGGGT

CAAGAAATrC

CCCTCTAGTA

TGGTTACTT

ATCAGTGAAC

ACCAAAGGCT

ATCTrAG3C

TTGCAACGAG

AATTGAGGCT

CGAGTCGACT

GCACTTCAGG

CACGGTGTTC

GATTACATAC

TCTCTCTCTC

ATGTCATCCT

AAGGTTAA

CAGGAACAGC

ATI'GGGAACC

CTTGTGATC

TCTATTTCTC

AGCCATAGCC

GATGGAT1'CC

CACCTGATGA

CAGTACCCCT

CTCAATGAAT

GCTCGGTCAA

GGACAGGTAC

CTrGTTCTTC

CACTCTTTCA

TCATATCAAT

GAGATTACTG

CTATGCTCCA

AGTGATGAAT

CGTCGTGCGA

GCTG=AAGGAG

GCTAAAGCTG

GGAATCGTCG

TGCACGATCC

CATGAGGCGT

AACCCTCATG

TCTCTCTrCTG

TAAAAAAAAA

AATGTGAGTA

TAAAGACTCA

CACAATCTCT

ATCCAGACCT

GAGCAAAGCA

AGGGTATTAA

CTGCTAATGC

TGCAATTACT

TGTACTCGGC

CGACGGGCTG

GAGGCATCAA

7rGCTGAAGA

TAGCTGATGA

AGAAGATAGT

CTGTTTCTAA

GCTTCAGI'GC

ATATCACTGG

CALTACGCTTC

AGGCATTGGA

CAATGTACGT

CTAACA.AAGC

TTGTCCCTIGG

TTCCGCAGGA

TCATGTCAGA

GGGT1TTrCGT

ACAGCATCCT

TGCTGTGCGT

ACCAGGGTCT

TGGTCAGCAA

G=~CAAAGA

GATTCTTGCC

AGGATCGT

TGATGACATT

GATAAGGAAT

TTCCATAGCT

GGGTrrGGAA

CGTTAGGGCT

AAACCAATAT

GGTATACCAA

GAGATC=TG

GGGATATTAT

TCCAGTAAGA

CCAGATCCI-

ATACAAGGCA

GrCATGCATTC

GTTCCCTCAA

ACCTGATGC:A

ATCAGGA7"TT

GGATAAGATC

GTATCGTGAC

AGGCGTrCTT

CCTCTAGATG

GGAGAGATTG

CrACCTITG

CCAGTTACAT

GAGGAAATCA

ATGATACCTG

GATGCAATTG

TTTCTCACAG

GAGAAAGATG

CTTCATGGCG

ACCTCTG-ATG

TTGGTGGTTA

GACATAGTGA

GAGAACATCT

GGATACGGCG

GGTGALGTGTG

GAGCAGATCT

GCTAGTCTTG

GAAAAAGATG

AATAAACTTG

ATCTGTCTGC

'ITCTATGCTC

GGCCAGGT'C

CCGGCAGTCA

TAAAC7TGGTG

GGTTT"IGCCC

AGACAAAATA

TCATCCATGC

ATGAGATCCT

TCTTCAGGGA

AAACATTGTT

GAAGAGCAAC

CTTCTGC7GAT

ATGGAGCAAG

GCATTCTTGT

GAGCTCTTGT

TTAAGAAGCA

TCAATCCAGG

AGTTCTGC.AA

ATGTTGACAA

AGGAGGATCT

GTAAAAGAGG

ACAAAATAGC

TCATGAACCC

GAATCCTCGC

AGGGAATTrAC

CACAGAAGGC

TTCGTCTCCT

CTGGCACATG

TCTCCCGCTT

CAACATGTGG

CCCCCCCCCT

AAGCAAAGCC

180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1701 SUBSTIru1-E SHEET (RULE 26)

Claims (19)

1. A genetic construct adapted for expression in a plant system comprising a nitrogen assimilation/metabolism enzyme coding sequence operably associated with an inducible promoter from Brassica turgor gene-26, or an active modified form thereof.

2. A genetic construct according to claim 1 wherein the coding sequence is for an enzyme selected from the group comprising glutamine synthetase, asparagine synthetase, glutamate 2-oxoglutarate aminotransferase, asparaginase, glutamate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, nitrate transporter genes and nitrate reductase or active modified forms thereof. oo

3. A cloning or expression vector comprising the genetic construct of claim 1 or 2.

4. A plant transformation vector comprising the genetic construct of claim 1 or 2.

5. A plant cell transformed with the genetic construct of claim 1 or 2.

6. The plant cell of claim 5, wherein the cell is from canola.

7. The plant cell of claim 5, wherein the cell is from a plant selected from the group comprising corn, rice, tobacco, soybean, cotton, alfalfa, tomato, wheat and potato.

8. A plant regenerated from the plant cell of any one of claims 5-7.

9. A method for producing a plant comprising: transforming a plant cell by introducing a genetic construct having a nitrogen assimilation/metabolism enzyme coding sequence operably associated with an inducible promoter from Brassica turgor gene-26, or an active modified form thereof; and CD/01066001.7 31 regenerating the plant cell to a plant.

A genetic construct adapted for expression in a plant system comprising a coding sequence for alanine aminotransferase enzyme operably associated with a promoter element from Brassica turgor gene-26.

11. A cloning or expression vector comprising the genetic construct of claim

12. A plant transformation vector comprising the genetic construct of claim

13. A plant cell transformed with the genetic construct of claim *a*g 10

14. The plant cell of claim 13, wherein the cell is from canola.

15. The plant cell of claim 13, wherein the cell is from a plant selected from the group comprising corn, rice, tobacco, soybean, cotton, alfalfa, tomato, wheat and potato.

16. A plant regenerated from the plant cell of any one of claims 13-15.

17. A plant comprising a genetic construct adapted for expression in a plant system including a coding sequence for alanine aminotransferase enzyme operably associated with a promoter element from Brassica turgor gene-26.

18. A method for producing a plant comprising: transforming a plant cell by introducing a genetic construct adapted for expression in a plant system including a coding sequence for alanine aminotransferase enzyme operably associated with a promoter element from Brassica turgor gene-26; and regenerating the plant cell to a plant. CD/01066001.7 32

19. A genetic construct according to claim 1 or 10, substantially as hereinbefore described with reference to any one of the examples. A method according to claim 9 or 18, substantially as hereinbefore described with reference to any one of the examples. THE GOVERNORS OF THE UNIVERSITY OF ALBERTA By their Registered Patent Attorneys Freehills Carter Smith Beadle 7 March 2001 09 o..o 9...i oo 99 9o o999 o9.. @9* o* @9*9 S *99 9 S 9 900 0