EP1708558A2

EP1708558A2 - Proteins which confer biotic and abiotic stress resistance in plants

Info

Publication number: EP1708558A2
Application number: EP04787517A
Authority: EP
Inventors: Villoo M. Avestha Gengraine Tech. Pvt.Ltd PATELL
Original assignee: Avestha Gengraine Technologies Pvt Ltd
Current assignee: Avesthagen Ltd
Priority date: 2003-09-24
Filing date: 2004-09-17
Publication date: 2006-10-11
Also published as: WO2005027632A2; WO2005027631A2; EP1708558A4; EP1705987A2; WO2005027631A3

Abstract

The present invention relates to the isolation of a nucleic acid sequences plants, the products of which, confer resistance to various biotic and abiotic stress like wounding, pathogen infection and drought in plants.

Description

PROTEINS WHICH CONFER BIOTIC AND ABIOTIC STRESS RESISTANCE IN PLANTS

Field of Invention

Prior Art

Arabinogalactan proteins (AGPs) are components of Gum Arabic, a gummy exudation originating from the Acacia tree and known to be produced by stress conditions such as heat, drought and wounding (Clarke et al, 1979). AGPs function in several biological process including cell-cell adhesion, pollen-stigma recognition, water retention and disease resistance in crops.

WO9515377 patent application provides plant arabinogalactan proteins (AGPs) and their genes. AGPs were isolated from Nicotiana alata, Nicotiana plumbaginafolia, and Pyrus communis. Amino acid sequences of isolated AGP peptide fragments are presented. Isolated AGP fragments were used to synthesize oligonucleotide probes to prepare oligonucleotide primers for PCR or prepare RNA probes to screen cDNA libraries of N. alata, N. plumbaginafolia, and P. communis. cDNA clones encoding amino acid sequences of isolated AGP fragments were isolated. The invention presents for the first time an intact AGP amino acid sequence derived from a corresponding AGP gene. The instant invention further provides methods useful in obtaining AGP genes encoding an AGP peptide comprising a specific isolated hydroxyproline-rich (OAST-rich) sequence or a specific isolated hydroxyproline-poor sequence.

Beta glucosidase catalyses one of the last steps in the lignin synthesis in plants. (Ellis Brian et al., 1999). The increased level of beta glucosidase significantly increases the efficiency of hydrolysis of cellulose to glucose by cellulase enzymes, thereby enhancing the production of fuel ethanol from cellulose. In spite of a significant amount of research effort, there has not been a means to produce sufficiently high levels of beta glucosidase. Such a process would be a large step forward in the production of fuel alcohol from cellulose (Theresa White & Christopher Hindle, 2000).

Beta glucosidase activity has been found to be 2-4 times higher as compared to the control plants in the presence of pathogens. (David Blanchard & Asim Esen, 2000). With respect to phosphate starvation stress of the eleven genes preferentially expressed in phosphate starved plant cells, beta glucosidase was one of the candidate genes (Mohd. AH Malboobi, 2000).

Beta glucosidase which is produced naturally in fungi is able to break down and release the aromatic substances in wine. Thus, introduction of this enzyme into simple wines can convert them into more expensive products with greater appeal and commanding a higher price. By introducing this gene into wine yeast through genetic engineering, the scientists have succeeded in creating a wine yeast that produces the beta-glucosidase in large quantities. This technology may be utilised to produce wines with richer aroma than would normally be expected. The same gene has also been implanted into tobacco plants to achieve a more aromatic plant (Dr. Oded Shoseyov et al., 2001).

In one embodiment, transformation vectors may be constructed to over-express the coniferin beta- glucosidase enzyme ("sense" orientation). Enhanced lignin synthesis may be achieved by introducing such vectors into plants. Examples of the application of this approach to modify plant phenotypes include U.S. Pat. No. 5,268,526, "Overexpression of Phytochrome in Transgenic Plants", U.S. Pat. No. 4,795,855, "Transformation and Foreign Gene Expression in Woody Species", and U.S. Pat. No. 5,443,974 (over- expression of stearoyl-ACP desaturase gene).

Alternatively, such over-expression vectors may be used to suppress coniferin beta- glucosidase enzyme activity through sense-suppression, as described in U.S. Pat. Nos. 5,034,323 and 5,283,184, both entitled "Genetic Engineering of Novel Plant Phenotypes".

US patent 5,973,228 describes a cDNA molecule encoding coniferin beta-glucosidase is disclosed. This enzyme catalyzes one of the last steps in the synthesis of lignin in plants. Plants having modified lignin content may be produced by transformation with this cDNA (or parts of the cDNA), for example, in either sense or antisense orientation. The invention includes methods of altering-lignin content in plants using this cDNA, as well as transformed plants, such as conifers, having modified lignin content.

US patent 5,997,913 describes a process for expressing extracellular .beta.-glucosidase in a filamentous fungus by expressing a fungal DNA sequence encoding enhanced, deleted or altered .beta.-glucosidase in a recombinant host microorganism is disclosed. Recombinant fungal cellulase compositions containing enhanced, deleted or altered expression of ,beta.-glucosidase is also disclosed.

Dihydroflavonol 4-reductase (DFR)

US patent application 20020120954 describes modified dihydroflavonol 4-reductase (DFR) nucleic acids characterized by its ability to reduce dihydrokaempferol (DHK) preferentially over dihydroquercetin (DHQ), and dihydromyricetin (DHM). The invention also includes plants having at least one cell expressing the modified DFR, characterized by the increased content of pelargonidin-based pigments.

US patent 5,410,096 describes plants not naturally capable of reducing dihydrokaempferol and containing a DNA sequence inserted according to recombinant DNA techniques which DNA sequence encodes a protein with the enzymatic activity of a dihydroflavonol 4-reductase (DFR) with extended substrate specificity for dihydrokaempferol. Furthermore, methods for the production of said plants, recombinant vectors and the use of said plants for the breeding of plants and parts of plants with modified flower colour are described. US patent 5,861,487 relates to a nucleic acid isolate comprising a sequence of nucleotides encoding, or complementary to a sequence encoding, a dihydrokaempferol (DHK) hydroxylating enzyme or derivative or part thereof. The present invention also relates to transgenic plants carrying and expressing the above mentioned nucleic acid material.

US patent 5,283,184 describes the use of recombinant DNA methods for genetically altering plants, and more particularly, to improved means for altering plant phenotypes, such as color patterns and color intensity of flowers and other plant parts.

WO0105984 patent application relates to methods for enzymatically manipulating the synthesis of flavonoids. It further relates to materials for use in, and resulting from, such methods.

WO03031622 patent application describes nucleic acids and nucleic acid fragments encoding amino acid sequences for flavonoid biosynthetic enzymes in plants, and the use thereof for the modification of flavonoid biosynthesis in plants.

WO9937794 patent application describes a method for manipulating the production of flavonoids other than anthocyanins in plants by manipulating gene activity in the flavonoid biosynthetic pathway by expressing two or more genes encoding transcription factors for flavonoid biosynthesis.

Metallothionein

Studies have revealed that metallothioneins are ubiquitous proteins that bind cations of transition metals (Kaegi, 1991). Although detoxification of heavy metals has been the most relevant role of metallothioneins, their function can be related to a number of different processes, both in animal and plant species. In Arabidopsis, they are induced by copper sulphate excess (Zhou & Goldsborugh, 1994).

Plant metallothionein-like proteins have been shown to bind cadmium and copper, which suggest they play a role in detoxification of metal ions (Evans et al., 1992).

Pathogenesis related protein

WO0032048 patent application provides novel methods for improving plant quality and yield in the presence of pathogens. The method increases the levels of pathogenesis- related proteins, such as PR1 , p enylalanine ammonia lyase, or plant cell wall proteins such as hydroxyproline-rich glycoproteins, in a plant by contacting the plant with a plant systemic inducer and a reactive oxygen species wherein the amount of the reactive oxygen species is sufficient to increase the amount of the pathogenesis-related protein above the level induced by the plant systemic inducer in the absence of the reactive oxygen species. A preferred reactive oxygen species is peracetic acid; a preferred plant systemic inducer is salicylic acid. WO8902437 patent application describe regulatory sequences of the pathogenesis- related (PR) protein genes of the PR-1 group, specifically an inducible PR-1 promoter and, preferably, PR-1 secretion signal sequences, and genetic constructs containing the regulatory sequences. This invention also relates to the construction and use of a recombinant DNA molecule that includes a PR protein gene, and its regulatory sequences, including the promoter and secretion signal sequences. More specifically, the invention concerns the use of the recombinant DNA molecule that includes a PR-protein gene operably linked to PR protein regulatory sequences that upon expression in a plant will enhance the hypersensitive response of the plant to an invading pathogen.

EP0392225 patent application relates to chimeric DNA constructs useful for producing transgenic disease-resistant plants and to genetic engineering of plants to produce the phenotype of disease resistance. In particular it relates to constitutive expression in transgenic plants of DNA sequences which encode pathogenesis-related proteins (PRPs) A further objective of the present invention are transgenic plants constitutively expressing induced levels of plant PRPs or substantially homologous proteins, providing an enhanced disease-resistant phenotype with respect to wild-type plants. Another object of the present invention is to provide transgenic plants constitutively transcribing sense or antisense mRNA strands of DNA sequences encoding plant PRPs or transcribing sense or antisense mRNA strands of DNA sequences substantially homologous to genomic or cDNA sequences encoding plant PRPs, such transgenic plants thus having an enhanced disease-resistant phenotype with respect to wild-type plants.

WO9220800 patent application describes methods of combating fungal disease and fungicidal compositions are provided in which a P14 protein, preferably PI 4a, PI 4b, PI 4c, P14d, P14e or P14f, or a fungicidally active analogue thereof is used as active ingredient. The P14 proteins may be obtained from plant material but the proteins and analogues are preferably prepared by use of recombinant DNA technology. DNA sequences coding for the P14 proteins and analogues, vectors, containing the DNA sequences and host cells transformed with the DNA sequences, as well as processes for production of the protein by culturing the transformed host cells, are provided. In particular transformed plant cells and. plants are provided having resistance to fungal disease.

Plant diseases caused by viral, bacterial, fungal and other pathogens are responsible for enormous economic loss. The ability of a plant to stop invasion of a pathogen depends on the presence of performed barriers. A distinct class of PR1 proteins, called intracellular proteins is expressed during wounding (Warner et al., 1992), osmotic stress (Iturriage et al., 1994) and pathogen colonisation (Chang & Hadwiger, 1990). IPR proteins are classified under PR1 since their function is not known but they share a low homology to them.

The levels of TSI-1 transcripts increased as the concentration of SA was increased and were maximal at 10 mM SA after 48 hours. TSI-1 was not expressed in the control and an extremely faint signal was obtained in leaves treated with low concentration of SA. Upon infecting with Fusarium oxysporum, high intensity signals were obtained in the fungal infected leaves after exposure for 24 hours but no signal was detected in the control lane after exposure for 2 days. The TSI-1 was not expressed constitutively but induced during fungal infection (C.S. Sree Vidya et al., 1999).

Thionin

Thionins are small, basic, cysteine rich proteins, which, may function as defence molecules against an array of plant pathogens (Florack & Stiekema, 1994; Broekaert et al., 1995). These genes appear to be expressed in response to pathogens and to be developmentally regulated. Several accumulate in reproductive tissue (Gu et al., 1992; Milligan & Gasser, 1995; Meyer et al., 1996).

Thionin gene in a plant exhibiting resistance to at least one disease such as a disease caused by a plant pathogenic bacterium, i.e., a bacterium causing bacterial leaf blight of rice or bacterium causing bacterial seedling blight of rice or a plant pathogenic filamentous fungus i.e., a fungus causing late blight of potato has been reported (Honkura Ryosos et al., 1993).

EP0902089 patent application relates to a transgenic plant which exhibits resistance to at least one disease. In particular, the present invention relates to a transgenic plant which comprises an expression cassette including a thionin gene and being capable of expressing the thionin gene, and which exhibits resistance to at least one disease.

EP1101771 patent application describes two cDNA clones, designated to PepDef (pepper defensin protein gene) and PepThi (pepper thionin-like protein gene) and individual component; thereof including its coding region and its gene product; modification thereto; application of said gene, coding region and modification thereto; DNA construct,_, vectors and transformed plants each comprising the gene or part thereof

Summary of the Invention

Detailed description of Figures

Seq ID. No. 1 - 5' EST sequence of Beta glucosidase

Seq ID. No. 2 - 5' EST sequence of Dihydroflavanol-4- Reductase

Seq ID. No. 3 - 5' EST sequence of Diacylglycerol kinase

Seq ID. No. 4 - 5' EST sequence of Pathogenesis related protein

Seq ID. No. 5 - 5' EST sequence of fruit ripening protein

Seq ID. No. 6 - 5' EST sequence of sucrose synthase

Seq ID. No. 7 - 5' EST sequence of Cytochrome P-450 type protein

Seq ID. No. 8 - 5' EST sequence of Metallothionein protein

Seq ID. No. 9 - 5' EST sequence of mutation induced recessive alleles Seq ID. No.10-5' EST sequence of Arabinogalactan

Seq ID. No.11-5' EST sequence of Cytokinin oxidase

Seq ID. No.12 - 5' EST sequence of Glutamate decarboxylase

Seq ID. No.13-5' EST sequence of Leucine rich repeat

Seq ID. No.14 - 5' EST sequence of Thionin

AGPs function in several biological processes including plant development, cell-cell adhesion, pollen-stigma recognition, water retention, and disease resistance. AGPs may serve as glues or provide nutrients for growing pollen tubes. It has been suggested [Fincher et al. (1983) supra] that AGP proteins may interact with lectins or other proteins in the extracellular spaces and may be involved in the cellular response to extracellular oligosaccharide signal molecules [Norman et al. (1990) Planta 181 :365-373]. Since AGPs interact with Yariv antigens and flavonol glycosides [Jermyn (1978) J. Plant Physiol. 5:563-571], they have been thought to have lectin-like properties. The molecular structure of AGPs has been proposed [Randall et al. (1989) Food Hydrocolloids 3:65-75] to resemble a type of block copolymer wherein carbohydrate blocks are covalently linked to a central polypeptide chain, thus explaining its ability to sterically stabilize emulsions and dispersions.

The process of obtaining an AGP clone has been found to be complex and problematic. Two of the problems associated with the AGPs and their genes are - (1) the very high redundancy associated with the characteristic amino acid sequence of an AGP peptide, i.e., a high hydroxyproline content and regions containing a high content of hydroxyproline, alanine, serine and threonine (OAST) and (2) the GC-richness of corresponding oligonucleotides leading to problems with the specificity of hybridisation, indistinct and imprecise alignment during nucleic acid hybridisation, for e.g., the PCR technique, which has resulted in a lack of success in the ability to obtain an AGP clone. This results in the amplification of incorrect sequences. Plants are also known to contain a variety of glycine rich proteins, which are also encoded by GC-rich DNA.

Two approaches to the isolation of the AGPs from plant extracts have been used in previous studies. One approach consists of classical fractionation of plant extracts [Fincher et al. (1974) Aust. J. Biol. Sci. 27:117-132; Aspinall (1969) Adv. Carbohydrate Chem. 24:333-379]. A convenient initial fractionation of extracts is treatment to saturation with (NH4)2 S04, which does not usually precipitate AGPs. Subsequent ion- exchange and affinity chromatography can be used to isolate the AGPs.

Another approach to the isolation of AGPs from plant extracts is precipitation with a class of dyes prepared by coupling diazotized 4-aminophenyl glycosides to phloroglucinol [Jermyn et al. (1975), supra]. These dyes were first prepared by Yariv et al. (1962) Biochem. J. 85:383-388) as precipitating antigens for antibodies to glycoside determinants, and the .beta.-glycosyl artificial carbohydrate antigen was shown to precipitate an arabinose-and-galactose-containing polymer from soya bean, jack bean and maize [Yariv et al. (1967) Biochem. J. 105:lc-2c]. Since then, this precipitation reaction has been widely used to isolate AGPs from extracts of seeds of every taxonomic group of flowering plants, as well as leaf extracts and callus-culture filtrates [Jermyn & Yeow (1975) Aust. J. Plant Physiol. 2:501-531; Anderson et al. (1977), supra; and review by Clarke et al. (1979), Phytochemistry 18:521-540].

The most striking feature of arabinogalactan is that of all the sugars in the furanose conformation. Galactofuronase is rarely found in nature and not at all in mammals, therefore targeting its synthesis and incorporation in to the wall should yield agents which are highly specific for mycobacteria, and not toxic. The arabinogalactan is linked to the 6-positoin of the muramic acids forming a fairly typical bacterial peptidoglycan layer via a unique diglycosylphosphoryl bridge, L-rhamnose-(l->3)-D-N- acetylglucosamine-(l ->P).

The introduction of immunochemistry has made a great impact on AGP research and has revitalised interest in the functional significance of AGP and AGP regulation during growth and development. It has already been postulated that AGPs as a soluble and diffusible component of the extracellular matrix and of the plasma membrane, play a role as messangers in cell-cell interactions during differentiation.

The usefulness of the present invention lies in the fact that the emergence of the AGP gene in rice is significant since the AGP's synthesis can be targeted and incorporated into the cell wall and in turn yield agents which are highly specific for the tuberculosis mycobacteria and not harmful to the patient. Any such agent with low toxicity will exhibit improved pharmacokinetic properties in comparisin with the drugs presently available. It is also an important tool in the food processing industry.

Beta-glucosidase

Beta-Glucosidases occurs in all three domains (Eukarya, Archaea, and Bacteria) of living organisms and play a key role in many biological processes, which make them a suitable target for protein engineering to address the problems of biomass production in agriculture and forestry, as well as biomass conversion in biotechnology. The following selected cases illustrate the importance of enzyme in plants.

1. Defense: Plants are anchored to the soil and generally cannot hide or escape from pests and environmental stresses. Consequently, plants have evolved defense mechanisms against pests based on storing and releasing toxic chemicals. These defense chemicals are typically β-glucosides in monocots and dicots and β-glucosinolates in certain dicots. β- glucosidic substrates and β-glucosidase are stored in different subcellular or tissue compartments. Damage to cells and tissues by pests brings the enzyme and substrate together, leading to the hydrolysis of substrate and release of bitter and toxic aglycones and their breakdown products (e.g., thiocyanates, isothiocyanates, nitriles, terpeno^'id alkaloids, saponins, hydroxymates, benzaldehydes, HCN). These substances then deter herbivores and inhibit the entry, growth and spread of phytopathogens, serving as a built- in pest control system. For example, it has been shown that environmental stresses ranging from chewing insects, nematodes and phosphate starvation to cold as well as specific treatments (e.g., jasmonic acid) induces β-glucosidase genes in Arabidopsis thaliana while NaCl suppresses. In a recent study of the transcriptional profile of Arabidopsis thaliana during systemic acquired resistance (SAR), it is showed that transcription of the β-glucosidase gene psr3.1 was elevated nearly 8-fold within 48 hours after infection with the oomycete Peronospora parasitica. Another Arabidopsis thaliana β-glucosidase gene (T209.120) has been shown to be associated with growth arrest and senescence in cell cultures and mature plants and is also reported to be cold inducible. Although the precise role of these genes in stress, defense and senescence response is not known, two (psr3.1 and F 19K6.15) encode β-glucosidases with a C-terminal ER retention signal and may be involved in signal transduction by activating another protein or nonprotein component in the ER via deglycosylation. These genes are potential targets for engineering enhanced crop protection and reducing or eliminating the need for costly and environmentally undesirable pesticides.

2. Food Processing and Quality Enhancement: There are several hundred different β-glucosidic flavor precursor identified from plants whose aglycones are products of mevalonate or shikimate pathways. Obviously, there are β-glucosidases in source plant tissues that hydrolyze these flavor precursors. Thus, in each case, there is need for isolating and characterrizing the specific enzyme that hydrolyzes a β-glucoside whose aglycone moiety is of interest to food quality and processing. Such biochemical data are crucial to making practical decisions as to whether or not enzymes from host plants or other sources should be added to drinks and beverages before, during or after processing to enhance flavor, aroma and other quality factors. Likewise, such data are essential for targeting enzymes with desirable properties for overproduction in transgenic microbial or plant hosts and improvement of their catalytic properties and stability for specific uses by genetic engineering.

Another aspect of β-glucosidases that pertain to food processing and quality is that edible portions of some plants contain compartmentalized β-glucosidase-β-glucoside systems that produce toxic aglycones and/or HCN when tissue is macerated during preparation or by chewing. This is exemplified by cassava roots and leaves, lima beans and flax seed. Of these, cassava is a food staple in tropical regions of Africa, Asia and South America, consumption reaches about 1 kg/per capita/day in some parts of Africa (e.g., Congo). It contains the cyanogenic β-glucoside linamarin and the corresponding A-glucosidase linamarase. When consumed raw, cyanide poisoning can occur depending on the amount ingested, where symptoms are difficulty in breathing, paralysis, convulsion, coma and even death. Cooking inactivates the enzyme and eliminates the possibility of cyanogenesis. Similar symptoms can arise when bitter almonds are eaten and ingested without roasting.

The myrosinase-glucosinolate (or β-thioglucosidase-β-thioglucoside) system, which occurs in cruciferous vegetables (e.g., mustard, cabbage, kale, broccoli, rapeseed, horseradish, etc.), has also importance for food quality and processing because the aglycone moiety and its breakdown products from enzymatic hydrolysis of glucosinolates are responsible for bitter, pungent taste and aroma associated with these vegetables, as well as the processed foods and relishes that include them (10). The distinct flavor associated with glucosinolates comes primarily from isothiocyanates and is believed to have evolved to serve as a repellent against microorganisms and herbivores. Glucosinolates and their breakdown products may impart undesirable flavors to milk, meat and eggs when farm animals graze on cruciferous plants or when their feed includes seed meals from such plants. Besides off-odors and flavors in foods of animal origin associated with glucosinolates, direct ingestion of large amount of cruciferous vegetables is thought to cause endemic goiter in humans, as well as toxicity in laboratory animals. Similarly, claims have been made on anti-carcinogenic effects of glucosinolates and their breakdown products in humans. Although the precise mechanism of action is not clear, studies on rodents showed that raw or cooked cruciferous vegetables (e.g., cabbage, broccoli, cauliflower and turnip) increased aryl hydrocarbon hydroxylase activity (11).

3. Biomass Conversion: Polysaccharides, specifically cellulose, are the most abundant substances in the biosphere (~5xιo¹⁰ tons produced/year) and are potential renewable sources of chemicals and fuels. Moreover, about 40% of typical municipal garbage includes newspaper and other paper products. Hydrolysis of cellulose using inorganic acids and high temperature is not ecologically sound and economically feasible. An enzyme (cellulase) complex, secreted by cellulolytic organisms, can hydrolyze cellulose to glucose, thus presenting itself as a suitable model for industrial processes that need to be developed (12). The complex includes three enzymes: an endoglucanase, an exoglucanase (cellobiohydrolase) and a β-glucosidase. The rate-limiting step in cellulose degradation is the one that is catalyzed by A-glucosidase, which hydrolyzes cellobiose and other small cellodextrins to glucose. Therefore, cellulosic biomass degradation and cellulose conversion to glucose at industrial scale by using microorganisms or isolated cellulase complex hinge upon increasing the rate of enzymatic reactions and overcoming product inhibition. Some plant β-glucosidases are specifically implicated in cellulose or cell wall metabolism during germination and growth as they hydrolyze cellobiose, as well as other disaccharides and oligosaccharides resulting from cell wall catabolism (13,14). Such β-glucosidases are potential targets for engineering to use in the degradation of cellulosic biomass by the cellulase complex.

4. Lignin Biosynthesis and Paper Quality: Lignin is the second most abundant substance in the biosphere and its major precursor, coniferyl alcohol, is derived from coniferin (4-O-coniferyl glucoside) after hydrolysis by β-glucosidase (15), suggesting that some plant β-glucosidase isoforms are involved in lignin biosynthesis. This makes the enzyme a suitable target for improving wood strength and quality for paper production.

5. Growth and Development: There is circumstantial evidence that β-glucosidases are involved in growth and development by releasing active hormones from phytohormone glucoconjugates, another potential function for some plant β-glucosidases. If indeed this function could be unequivocally shown, it opens the door to engineering the enzyme for regulating and improving plant growth and development to enhance productivity.

6. Secondary Plant Metabolism: Many secondary plant biochemical pathways (e.g., phenyl propanoid metabolism) use β-glucosides as precursors, intermediates or synthesize them as end products. Some of these substances may be glycosylated to enhance solubility or may be deglycosylated as part of degradation pathway. Very little is known about the fate and function of many β-glucosides. These compounds must have a function and be hydrolyzed by β-glucosidases exhibiting unique substrate specificities and tissue and cellular localization, providing further clues to the existence of a 44- member multigene family encoding β-glucosidase in Arabidopsis thaliana.

Aromatic substances are produced in the green tissues of plants and are conveyed from there to the flowers or fruits of the plants. At the proper moment, the enzyme beta glucosidase is released, which serves to break the bonding between the molecules of sugar and the aromatic substances, thus releasing the latter into the air. In flowers this occurs when the flower is ready for pollination, with the smell serving to attract insects that will bear pollen to other plants. In fruits, the aroma is meant to attract animals, which will spread its seeds. However in many vegetables and fruits, there are aromatic substances that remain bound and thus are not released at all. The introduction of the beta glucosidase enzyme can enable these plants to reach their full aromatic potential (Dr. Oded Shoseyov et al., 2001).

Beta glucosidase is of potential value in plant biotechnology programmes for modulation of the lignin content in plants which would help in the production of fuel alcohol from cellulose. The technology could be utilised to increase the aroma in wine as well as in vegetables and fruits.

DFR

The flower industry strives to develop new and different varieties of flowering plants. An effective way to create such novel varieties is through the manipulation of flower colour and classical breeding techniques have been used with some success to produce a wide range of colours for most of the commercial varieties of flowers. This approach has been limited, however, by the constraints of a particular species' gene pool and for this reason it is rare for a single species to have a full spectrum of coloured varieties.

Flower colour is predominantly due to two types of pigment: flavonoids and carotenoids. Flavonoids contribute to a range of colours from yellow to red to blue. Carotenoids impart an orange or yellow tinge and are commonly the only pigment in yellow or orange flowers. The flavonoid molecules which make the major contribution to flower colour are the anthocyanins which are glycosylated derivatives of cyanidin, delphinidin, petunidin, peonidin, malvidin and pelargonidin, and are localised in the vacuole. The different anthocyanins can produce marked differences in colour. Flower colour is also influenced by co-pigmentation with colourless flavonoids, metal complexation, glycosylation, acylation, methylation and vacuolar pH (Forkmann, 1991).

Anthocyanins are classes of pigments that determine flower color and plant pigmentation in angiosperm plants. Among anthocyanins, pelargonidin-based pigments confer bric- red/orange color to plants, while cyanidin- and delphinidin-based pigments confer red and violet color each (Holton, et al. Plant Cell 7:1071-1083 (1995); Tanaka, et al. Plant Cell Physiol. 39: 1119-1126 (1998)). Different ratio of these pigments confers a wide range of flower color. Many anthocyanin bio synthetic genes have been identified. One of key enzyme in the biosynthetic pathway is dihydroflavonol 4-reductase (DFR). The enzyme converts dihydroflavonols (dihydrokaempferol (DHK), dihydroquercetin (DHQ), and dihydromyricetin (DHM)) to leucocyanidins. The leucocyanidins are subsequently converted to anthocyanins by other enzymes. The conversion of DHK to DHQ and DHM are catalyzed by flavonoid 3'-hydroxylase (F3Η) and flavonoid 3',5'-hydroxylase (F3'5'H) Since DFRs in most plants can convert all three dihydroflavonols to leucocyanidins, the ratio of three classes of anthocyanin pigments are mainly determined by the activity of F3Η and F3'5Η (Holton, et al. Plant Cell 7:1071-1083 (1995). Dihydroflavanol 4-reductase ("DFR") reduces Dihydroflavanol to leuco-anthocyanin, which is then further converted into anthocyanin via anthocyanidin. This has been found to be one of the regulatory enzymes in the flavonoid biosynthetic pathway.

The individual or simultaneous enhancement or otherwise manipulation of CHI, CHS, CHR, DFR, LCR, F3'5'H, F3H, F3'H, PAL and/or VR or like gene activities in plants has significant consequences for a range of applications in, for example, plant production and plant protection. For example, it has applications in increasing plant tolerance and plant defense to biotic stresses such as viruses, micro-organisms, insects and fungal pathogens; in improving plant forage quality, for example by disrupting protein foam and in conferring protection from rumen pasture bloat ; in reducing digestion rates in the rumen and reducing parasitic load ; in the production of plant compounds leading to health benefits, such as isoflavonoids, which have been linked to anticancer benefits, and stilbenes that are believed to contribute to reduced heart disease.

Dihydroflavonol 4-reductase (DFR) is of potential value in plant biotechnology programmes for modulation of colour development and it is an important molecule in protecting the plant against Ultra Violet rays and pathogens. It is also a potent molecule for color development in plants especially in flowers and therefore has an important role in the floral industry.

Metallothionein

The discharge of heavy metals into the environment due to agricultural, industrial, and military operations, and the effect of this pollution on the ecosystem are of great concern. Recent research in the area of heavy metal removal from wastewaters and sediments has focused on the development of novel materials with increased affinity, capacity, and selectivity for target metals. One of the most selective heavy metal binding molecules is the family of small molecular weight proteins called metallothioneins (phytochelatins in plants). These proteins are cysteine-rich and bind heavy metals, such as cadmium, with very high affinity. Recently, the expression of the N. crassa metallothionein gene in E. coli and the ability of this recombinant E. coli (NCP) to sequester cadmium from solutions has been reported (Pazirandeh et al. Appl. Microbio. Biotechnol., (1995) 43:1112-1117, the entirety of which is incorporated herein by reference for all purposes; M. Pazirandeh, (1996) Biochem. and Molec. Biol. Intl., Vol. 39, NO. 4:789-795 the entirety of which is incorporated herein by reference for all purposes). These results demonstrated the ability of the recombinant E. coli expressing the metallothionein gene to sequester cadmium from solutions rapidly and with high selectivity. Although these results were encouraging, the development of a bacterial-based heavy metal biosorbent requires the bacteria to be immobilized, reusable, and able to sequester heavy metals from waste waters which are often contaminated with solvents and other toxic compounds (eg. polyaromatic hydrocarbons).

Metallothioneins are ubiquitous proteins that bind cations of transition metals. These peptides contain numerous cysteine residues that form the metal binding sites. According to their primary structure, metallothioneins can be classified in three classes. Class I metallothioneins, represented mainly by mammalian proteins, contain a number of cysteine residues clustered at conserved positions near the N- and C-terminus. Two types are considered within this class. Type 1 metallothioneins have several C-X-C motifs (X is an amino acid other than cysteine), while type 2 includes those metallothioneins with C- X-C motifs and also C-C and C-X-X-C motifs. Class II metallothioneins have cysteines that are not clustered. Metallothioneins of Class III are non-gene encoded peptides, which are also known as either cadystins or phytochelatins in fungi and plants, respectively.

Although detoxification of heavy metals has been the most relevant role of metallothioneins, their function can be related to a number of different processes both in animal and plant species. In animals, metallothioneins gene expression responds to hormones, second messengers, high levels of trace metals and other stress conditions. Unlike in animals, there are just a few genes or cDNAs of plant metallothioneins, though this number is increasing rapidly. Plant metallothionein genes are also induced by different factors. Arabidopsis MT1 and MT2 are induced by copper sulphate excess and IDS-1 from barley responds to iron deficiency. Abscisic acid induces Ec in wheat, and LSC54 is induced by leaf senescence, heat-shock, and starvation in Brassica napus.

The exact function of MTs in plants is still unclear. There is some variation in the cysteine arrangements of the proteins that, along with the amino acids inside the protein, may be the determinants of which metal the MT binds to. (Lacoste, 2001) Despite the lack of knowledge to the true nature of the function of MTs, type 2 MTs have been shown to be the primary determinant of the level of metal tolerance in the plants. Also, overexpression of yeast MT genes have been shown to improve the heavy metal tolerance in plants to which they have been inserted, and plant MT genes have increased the heavy metal tolerance of MT-deficient yeast. (Lacoste, 2001) The actual function of the MTs is thought to be one of several hypothesized but as of yet unproven theories. One theory states that MTs create ion storage pools for free excess heavy metal ions, which are chelated until the plant can use them, if the metals are essential. A second school of thought is that MTs are transport proteins that are responsible for moving excess heavy metals from sites where they have built up to toxic levels to areas of the plant where they are needed, or at least where the ion levels are not toxic.

This metallothionine protein is of potential value on account of its affinity to heavy metals (bioremediation). It could also find its use as an antidote for, harmful heavy metals and harmful substances like free radicals in living beings. The gene can also be used in the production of transgenic plants possessing enhanced heavy metal stress tolerance and the product of which, may be used to produce large quantities of the protein for metal detoxification.

Patho genesis related protein

When a plant is subjected to a stress, e.g. an abiotic stress such as W-irradiation, or a biotic stress caused by pathogenic organisms such as fungi, bacteria, viruses or by insects, the plant defends itself through several mechanisms, including thickening of cell walls by deposition of additional callose or lignin at the site of infection and production of substances that are toxic to the organism. It has also been observed that stressed plants synthesize a number of proteins. These proteins are generally characterized as being acid soluble, protease resistant, relatively small in size, accumulating predominantly in the intercellular space of the plant, and as producing a characteristic pattern in gel electophoresis.

Some of these proteins have been identified as hydrolytic enzymes (e.g. chitinases, ss-1,3 glucanases and proteases), peroxidases and proteinase inhibitors.

The defense of plants to pathogens comprises constitutive barriers present in plants prior to any contact with pathogens or herbivores. Furthermore, exposure to various microorganisms or other forms of stress can lead to the activation of defense mechanisms. Induced resistance depends on the recognition of a pathogen or stress by the plant. This generates a cascade of events, eventually leading to the expression of defense mechanisms, which include physical barriers, metabolites and proteins that interfere with the spread of the invading microorganism. The recognition process can vary in specificity. For instance, in its most extreme form, plants can distinguish subspecies or races of pathogenic organisms. In this case, a corresponding product of a resistance gene in the plant recognizes the product of an avirulence gene of a pathogen, a so-called race- specific elicitor; this results in a race-specific induction of resistance mechanisms. This form of induced resistance is described by the gene-for-gene hypothesis. The same conceptual framework of this hypothesis has been used to explain the situation where a broader resistance is induced in response to several races of a pathogen or even to several species (review by Mitchell-Olds & Bergelson, 2000). Generally, the speed of recognition and ensuing induction of resistance are key determinants in the success of resistance. Disease will occur if the pathogen is faster than the induced response, if no elicitors are produced or if suppressors prevent the plant defense reactions. Induced resistance may be expressed locally in the infected parts as well as in the uninfected parts of the plant. In this case, the initial recognition event also leads to the production of an endogenous systemically translocated signal that has the virtue to activate resistance mechanisms in parts of the plant remotely located from the initial site of interaction. This form of induced resistance is referred to as systemic acquired resistance (SAR) (Sticher et al., 1997; Hunt & Ryals, 1996) or systemic induced resistance (ISR)

Pathogenic infection of a variety of different plant species leads to the synthesis of several host encoded proteins, known as pathogenesis-related proteins (PR proteins), van Loon, Plant Molecular Biol. 4:111-116 (1985). For example, infection of tobacco plants with tobacco mosaic virus (TMW) results in two possible distinct host responses depending on the genetic constitution of the plant cultivar and the virus strain. In a systemic infection, the virus particles are soon found spread all over the plants, resulting in the expression of several disease symptoms. By contrast, in an incompatible reaction, necrotic lesions appear on the leaves around the sites of pathogen entry and the virus particles remain restricted to these areas. This induced defense reaction, called a hypersensitive response, is common among many plant-pathogen interactions. Concomitant with the expression of disease resistance, various biochemical changes are observed in the plants. These changes include increases in the activities of the enzymes involved in the phenylpropanoid pathway and the de novo synthesis of the pathogenesis- related (PR) proteins. Matthews, R.E.F., in "Comprehensive Virology 16," (H. Fraenkel- Conrat and R.R. Wagner, eds.), Plenum Press, NY (1980), pp. 297-359.

PR-proteins were first detected in tobacco cultivars, which exhibited the hypersensitive response upon TMV infection. Gianinazzi et al., CR. Acad. Sci. Paris Ser. D270:2383- 2386 (1970) and van Loon et al., Virology 40:199-211 (1970). As the appearance of these proteins was initially found to be strictly conelated with the plant defense reaction, a possible antiviral function of PR-proteins was postulated. Pierpoint, W.S., Trends in Biochem. 85:5-7 (1983). Since then, proteins with physical properties similar to PR- proteins have been described in many other plant species after infection with viruses, viroids, fungi, as well as bacteria, van Loon (1985), supra. In addition, PR-proteins can be induced artificially in healthy plants by the application of chemicals, such as acetylsalicylic acid, polyacrylic acid, and ethylene. White, R.F., Virology 99:410-412 (1979). Interestingly, these proteins can accumulate under various alternate environmental conditions: they have been in tobacco plants at the onset of flowering (Fraser, R.S.S., Physiol. Plant Pathol. 19:69-76 (1981)) and in tobacco callus cultures (Antoniw et al., Phytopath Z. 101:179-184 (1981).

PR-proteins have several features in common. They are soluble in acidic buffers, have low molecular weights ranging from 10 kD to 20 kD, occur in the intercellular spaces of the leaves, and are resistant to proteases, van Loon (1985), supra. In TMV infected tobacco plants, ten different PR-proteins have been described. The best characterized of these is the PR-1 group which consists of three members, PR- la, PR- lb, and PR-lc. Biochemical, serological, and genetic evidence suggest that these proteins, although closely related, are encoded by separate genes. Antoniw et al., J. Gen. Virol. 47: 79-87 (1980); Matsuoka et al., J. Gen. Virol. 65:2209-2215 (1984); Antoniw et al, Plant Mol. Biol. 4:55-60 (1985); Ahl et al., Phytopathology 72:80-85 (1982); and Gianinazzi et al., Neth. J. Plant Pathol. 89:275-281 (1983).

Pathogenesis-related proteins (PRPs) are plant proteins induced following infection by a pathogen. It is believed that these proteins may have a role in providing systemic acquired resistance to the plant. These plant proteins are induced in large amounts in response to infection by various pathogens, including viruses, bacteria and fungi.

The gene is of potential value in plant biotechnology programmes, by assisting in increasing plant resistance to pathogens.

Thionin

Drought and soil salinity, in both, dry land as well as irrigated agricultural settings are the most important factors limiting modern agricultural production system (Cushman & Bohnert, 2000). Plants, being sessile organisms, have developed a sophisticated and complex set of adaptive responses allowing them to withstand abiotic and environmental insults. This complexity has stymied traditional selection based breeding approaches to isolate germ plasm with improved salinity or drought tolerance (Flowers & Yeo, 1995). Genetic and molecular studies have determined that many gene products contribute to salinity and drought tolerance (Hasegawa et al., 2000). Major classes of salt or drought tolerant determinants include osmo-protectants, ion carriers and channels, transporters and symporters, water channels, reactive oxygen scavengers, heat shock proteins, various stress proteins of unknown functions, transcriptional activators and signalling moleculars (Hasegawa et al., 2000; Cushman, 2001).

Genome sequencing and cDNA clone analysis promise to rapidly isolate and identify all the genes of the 'osmome', 'xerome', or, the 'thermome', the complement essential for tolerance to osmotic dessication and heat or cold stress tolerance respectively (Cushman & Bohnert, 2000).

Thionins are highly abundant polypeptides with anti fungal activities. These polypeptides are located in cell walls of leaf cells and the synthesis of thionine mRNA was increased after fungal attack (Bohlmann et al., 1988). Expression of the a-thionin gene from barley in transgenic tobacco has been shown to confer resistance to bacterial pathogens (Carmona et al., 1993). The in vitro toxicity against plant pathogenic bacteria and fungi indicates the role of thionin in the resistance of plants to the said bacteria and fungi. The induction of the thionin gene in response to salt stress identifies this gene with the osmome of the complement essential for tolerance towards osmotic dessication. The usefulness of the emergence of the thionin gene under salt stress lies in the fact that the rice plant harbouring one or several anti fungal disease genes can control the fungal diseases, thereby minimising the use of chemical fungicides.

Thionins are small, highly basic, Cys-rich proteins that show antimicrobial activity and seem to have a role in plant defense against fungi and bacteria. The overexpression of the THI2.I thionin in Arabidopsis enhanced resistance to a phytopathogenic fungus (Epple et al., 1997). The overexpression of alpha -hordothionin in tobacco also enhanced resistance to a phytopathogenic bacterium (Carmona et al., 1993). In addition, during barley and powdery mildew interactions, the accumulation of thionins was higher in the incompatible interaction than in the compatible one (Ebrahim-Nesbat et al., 1993).

The thionins contain a signal sequence, the thionin domain and an acid polypeptide domain as well as the conserved Cys residues (Bohlmann et al., 1994). A new class of Cys-rich antimicrobial protein, gamma -thionin, has a similar size (5 kD) and the same number of disulfide bridges as thionins. However, since gamma -thionins do not have significant sequence homologies with thionins, they have been described as plant defensins (Terras et al., 1995). Both defensin and thionin genes in Arabidopsis are inducible via a salicylic acid-independent pathway different from that for PR proteins (Epple et al., 1995; Penninckx et al., 1996).

Procedure 1. mRNA purification was performed by first, isolating high quality total RNA from 6 day old RASI seedlings and, subsequently by isolating mRNA from total RNA using oligo (dT) cellulose in a filter syringe by making use of a double purification method. 2. mRNA was converted into first and second strand cDNA followed by Sal I adapter addition, Not I digestion, cDNA vector ligation and transformation to obtain the cDNA library. 3. The superscript ™ plasmid system with Gateway ™ for cDNA cloning and synthesis was employed throughout. 4. The clones obtained were picked, digested using Not I and Sal I enzymes, to obtain the inserts and these were further sequenced and checked for homology. 5. The sequencing of the selected clones was done on ABI Prism, 377, DNA Sequencer (Perkin Elmer).

The instant invention provides a source of AGP that is not dependent upon its isolation from plant exudates, e.g., gum arabic, guar gum, etc. The availability of natural sources of AGP-containing gums, e.g., from trees, roots, seeds, seaweed, microbes, etc., present problems associated with harvesting, climate, man-power, fermentation, isolation, purity, and high costs. The production of AGPs using recombinant gene technology ensures (a) a method of supplying AGP that is independent of harvesting or fermentation requirements and problems, (b) that enables high levels of quality control, (c) that provides a supply of substantially pure AGP product, (d) that permits an overproduction of AGP in a host cell, and (e) that can be adapted to produce a specifically engineered AGP having desired properties. Thus, this invention provides a means for supplying the functions and utilities of plant gums, e.g., gum arabic, etc., without the need for finding renewable but shrinking natural sources of plant gums. These functions find wide applications as thickening, gelling, emulsifying, dispersing, suspending, stabilizing, encapsulating, flocculating, film-forming, sizing, adhesive, binding and/or coating agents, and/or as lubricants, water- retention agents, and coagulants

Claims

We claim 1. Nucleic acid sequences Seq ID. No. 1 to 15, isolated from plants, the protein products of which, confer biotic and abiotic stress tolerance in plants.

2. Nucleic acid sequences Seq ID. No. 1, the protein products of which can be engineered for regulating and improving plant growth, enhanced aroma and flavour and pesticide resistance.

3. Nucleic acid sequences Seq ID. No. 2, the protein products of which can be used . for altering any one or more characteristics in a plant, which characteristics are selected from: colour in fruits, flowers, fibre and\or seeds; resistance to pathogenesis, environmental and\or biotic stresses; flavour; palatability; astringency; nutritive value; ease of processing into feedstuffs and beverages for human and animal consumption.

4. Nucleic acid sequences Seq ID. No. 3, the protein products of which act as intercellular messengers in signal transduction pathway.

5. Nucleic acid sequences Seq ID. No. 4, the protein products of which confers increased tolerance to pathogenic infections, thereby providing novel methods for improving plant quality and yield in the presence of pathogen.

6. Nucleic acid sequences Seq ID. No. 5, the protein products of which, help in producing fruit-bearing plants, wherein the fruits exhibit a delayed ripening phenotype.

7. Nucleic acid sequences Seq ID. No. 6, the protein products of which, generates transgenic plants that confer flood tolerance.

8. Nucleic acid sequences Seq ID. No. 7, the protein products of which, better emergence characteristics when grown in darkness and improved seedling growth when grown under low-light levels.

9. Nucleic acid sequences Seq ID. No. 8, the protein products of which, can be used for bioremediation.

10. Nucleic acid sequences Seq ID. No. 9, the protein products of which, confer increased tolerance to pathogenic infections, thereby providing novel methods for improving plant quality and yield in the presence of pathogen.

11. Nucleic acid sequences Seq ID. No. 10, the protein products of which, play an important role as messangers in cell-cell interactions during differentiation. ( AGP).

12. Nucleic acid sequences Seq ID. No. 11, the protein products of which confer maintenance of an optimal level of cytokinins for growth and/or resetting a cytokinin signaling system to a basal level.

13. Nucleic acid sequences Seq ID. No. 12, the protein products of which help in generating a transgenic plants having enhanced resistance to pests.

14. Nucleic acid sequences Seq ID. No. 13, the protein products of which confer increased tolerance to environmental stress conditions such as drought, salinity, ultra violet radiation, heat and cold.

15. Nucleic acid sequences Seq ID. No. 13, the protein products of which, increased tolerance to environmental stress.