WO2003083119A1

WO2003083119A1 - Transgenic plant expressing phosphoenolpyruvate carboxykinase

Info

Publication number: WO2003083119A1
Application number: PCT/GB2003/001474
Authority: WO
Inventors: Ian Graham; Elizabeth Rylott
Original assignee: University of York
Current assignee: University of York
Priority date: 2002-04-03
Filing date: 2003-04-03
Publication date: 2003-10-09
Anticipated expiration: 2004-10-03
Also published as: AU2003224251A1

Abstract

We describe a transgenic plant which has been genetically modified to modulate the activity of phosphoenolpyruvate carboxykinase (PEPCK), the use of PEPCK as a herbicide target; methods to identify agents which inhibit the activity of PEPCK; and including methods to regulate post-germinative plant growth.

Description

Transgenic Plant Expressing Phosphoenolpyruvate Carboxykinase

The invention relates to a transgenic plant which has been genetically modified to modulate the activity of phosphoenolpyruvate carboxykinase (PEPCK), the use of PEPCK as a herbicide target; methods to identify agents which inhibit the activity of PEPCK; and including methods to regulate post-germinative growth.

PEPCK catalyses the first step in the gluconeogenic pathway, the ATP-dependent decarboxylation of OAA to phosphoenolpyruvate (PEP). As well as gluconeogenesis in germinating oilseeds, PEPCK is also involved in a wide range of CAM plants (1) and in the C4 cycle of a specific sub group of C4 grasses (2). It has also been found in a broad range of C3 plant tissues including cauliflower florets (3), turnip tap root (4), grape (5), apple, kiwi fruit and aubergine (6). More recently PEPCK has also been located in developing seeds (7), phloem and trichome tissues, oil and resin ducts (9) and ripening tomato fruit (8) suggesting that it may have additional roles. In trichomes, where the carbohydrate supply may be limited by the epidermis, PEPCK might play an anaplerotic role in the catabolism of organic or amino acids (9) and there is also support for the involvement of PEPCK in pH stability (7,10) and amino acid metabolism (11,12).

Two PEPCK genes have been identified in the Arabidopsis genome. PCK1 on chromosome 4 (Genbank accession no. AL035709) and PCK2 (AL021684) on chromosome 5. They exhibit 80% identity at the cDNA level. PCK1 has 91% identity with a 1509 bp cold-inducible Brassica napus PEP-carboxykinase cDNA from RNA from cold acclimated etiolated seedlings (Genbank accession no. U21745) (13) and 81% identity to a 2408 bp PEPCK cDNA isolated from senescing cucumber cotyledons (14) (Genbank accession no. L31899). PCK1 and PCK2 encode for proteins of 671 and 628 amino acids respectively with 71% identity. PCK1 is the predominant gene expressed during early post-germinative growth. Transcription is known to play a major role in the regulation of the glyoxylate cycle where both ICL and MS are co-ordinately expressed during early post-germinative growth and senescence (15,16). In cucumber, PCK1 expression parallels MS and ICL (14) and more recently co-ordinate expression of genes of the β-oxidation pathway, glyoxylate cycle and gluconeogenesis have all been demonstrated during germination in Arabidopsis spp (17). It should be noted that while the PEPCK expression levels rise to a peak during early-postgerminative growth and thereafter decline to low basal levels in seedlings, the transcriptionally regulated MS and ICL genes are induced during early postgerminative growth but thereafter decline to undetectable levels once seedlings become photosynthetically competent (17) which is incorporated by reference.

There is strong evidence that PEPCK is a key enzyme in the regulation of flux through the gluconeogenic pathway. Firstly, studies in marrow (Cucurbita pepό) have demonstrated that the maximum catalytic activity of PEPCK is higher than, and changes simultaneously with the flux through the gluconeogenic pathway during post-germinative growth (18). Secondly, PEPCK and fructose 1, 6-bisphosphatase (FBPase) are the only enzymes in the conversion of oxaloacetic acid to glucose 6 phosphate during gluconeogenesis that are substantially displaced from equilibrium in vivo (19). Thirdly, studies using the PEPCK inhibitor, 3-mercaptopicolinic acid (3- MPA), demonstrated a high flux control coefficient of between 0.7-1.0 in gluconeogenic marrow cotyledons (20, 18). FBPase has also been shown to regulate the flux through the gluconeogenic pathway (19) and the relative contributions of PEPCK and FBPase in the regulation of gluconeogenesis have yet to be determined.

A number of techniques have been developed in recent years which purport to specifically ablate genes and/or gene products.

A more recent technique to specifically ablate gene function is through the introduction of double stranded RNA, also referred to as inhibitory RNA (RNAi), into a cell which results in the destruction of RNA complementary to the sequence included in the RNAi molecule. The RNAi molecule comprises two complementary strands of RNA (a sense strand and an antisense strand) annealed to each other to form a double stranded RNA molecule. The RNAi molecule is typically derived from exonic or coding sequence of the gene which is to be ablated. Surprisingly, only a few molecules of RNAi are required to block gene expression which implies the mechanism is catalytic. The site of action appears to be nuclear as little if any RNAi is detectable in the cytoplasm of cells indicating that RNAi exerts its effect during mRNA synthesis or processing.

An alternative embodiment of RNAi involves the synthesis of so called "stem loop RNAi" molecules which are synthesised from expression cassettes carried in vectors. The DNA molecule encoding the stem- loop RNA is constructed in two parts, a first part which is derived from a gene the regulation of which is desired. The second part is provided with a DNA sequence which is complementary to the sequence of the first part. The cassette is typically under the control of a promoter which transcribes the DNA into RNA. The complementary nature of the first and second parts of the RNA molecule results in base pairing over at least part of the length of the RNA molecule to form a double stranded hairpin RNA structure or stem-loop. The first and second parts can be provided with a linker sequence. Stem loop RNAi has been successfully used in plants to ablate specific mRNAs and thereby affect the phenotype of the plant , see, Smith et al (2000) Nature 407, 319-320.

We herein disclose PEPCK plants which have been genetically modified such that PEPCK activity is inhibited. In one embodiment the transgenic plants comprise anti- sense constructs. Plant transformants have a range of PEPCK activity from 20 to 80% of wild-type levels. With decreasing PEPCK activity, the ability of seedlings to establish by reaching photosynthetic competency was also decreased. This effect was further enhanced with decreasing light intensity. Seedling establishment could be rescued by the addition of an exogenous supply of sucrose. Whilst soluble sugar levels were lower in the antisense seedlings, storage lipid catabolism during post- germinative growth was not perturbed. According to a first aspect of the invention there is provided a transgenic plant cell characterised in that the genome of said cell is modified such that the activity of phosphoenolpyruvate carboxykinase is reduced when compared to a non-transgenic reference cell of the same species.

In a preferred embodiment of the invention said activity is reduced by at least 10%. Preferably said activity is reduced by between about 10% - 90%. Preferably said activity is reduced by at least 20%, 30%, 40%, 50%, 60%, 70%, or at least 80% when compared to a non-transgenic reference cell of the same species.

In a preferred embodiment of the invention said cell is transformed with a nucleic acid molecule comprising a nucleic acid sequence operably linked to a promoter, said sequence selected from the group consisting of: i) a sequence, or part thereof, as represented in Figure 6 or 7 wherein transcription from said sequence produces an antisense sequence; ii) antisense sequences which hybridise to the sense sequence presented in Figure 6 or 7 and which inhibit the activity of phosphoenolpyruvate carboxykinase.

In an alternative preferred embodiment of the invention said cell is transformed with a nucleic acid molecule comprising an expression cassette which cassette comprises a nucleic acid sequence which encodes at least part of phosphoenolpyruvate carboxykinase wherein said cassette is adapted such that both sense and antisense nucleic acid molecules are transcribed from said cassette.

In a preferred embodiment of the invention said nucleic acid molecule comprises a nucleic acid sequence selected from the group consisting of:

i) a nucleic acid sequence, or part thereof, represented by the sequence in Figure 6 or 7; ii) a nucleic acid sequence, or part thereof, which hybridizes to the sequence in Figure 6 or 7and encodes at least part of a phosphoenolpyruvate carboxykinase polypeptide; and iii) a nucleic acid sequence which is degenerate as a result of the genetic code to the sequences in (i) and (ii).

In a further preferred embodiment of the invention said cassette is provided with at least two promoters adapted to transcribe sense and antisense strands of said nucleic acid molecule.

In a further preferred embodiment of the invention said cassette comprises a nucleic acid molecule wherein said molecule comprises a first part linked to a second part wherein said first and second parts are complementary over at least part of their sequence and further wherein transcription of said nucleic acid molecule produces an RNA molecule which forms a double stranded region by complementary base pairing of said first and second parts.

In a preferred embodiment of the invention said first and second parts are linked by at least one nucleotide base. In a further preferred embodiment of the invention said first and second parts are linked by 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide bases. In a yet further preferred embodiment of the invention said linker is at least 10 nucleotide bases.

In a further preferred embodiment of the invention the length of the RNA molecule or antisense RNA is between 10 nucleotide bases (nb) and lOOOnb. Preferably said RNA molecule or antisense RNA is lOOnb; 200nb; 300nb; 400nb; 500nb; 600nb; 700nb; 800nb; 900nb; or lOOOnb in length. More preferably still said RNA molecule or antisense RNA is at least lOOOnb in length.

More preferably still the length of the RNA molecule or antisense RNA is at least lOnb; 20nb; 30nb; 40nb; 50nb; 60nb; 70nb; 80nb; or 90nb in length. More preferably still said RNA molecule is 21nb in length.

In a further preferred embodiment of the invention said expression cassette is part of a vector.

Preferably the nucleic acid in the vector is operably linked to an appropriate promoter or other regulatory elements for transcription in a host plant cell. The vector may be a bi-functional expression vector which functions in multiple hosts. In the example of nucleic acids according to the invention this may contain its native promoter or other regulatory elements.

By "promoter" is meant a nucleotide sequence upstream from the transcriptional initiation site and which contains all the regulatory regions required for transcription. Suitable promoters include constitutive, tissue-specific, inducible, developmental or other promoters for expression in plant cells comprised in plants depending on design. Such promoters include viral, fungal, bacterial, animal and plant-derived promoters capable of functioning in plant cells.

Constitutive promoters include, for example CaMV 35S promoter (Odell et al (1985) Nature 313, 9810-812); rice actin (McElroy et al (1990) Plant Cell 2: 163-171); ubiquitin (Christian et al . (1989) Plant Mol. Biol. 18 (675-689); pEMU (Last et al (1991) Theor Appl. Genet. 81 : 581-588); MAS (Velten et al (1984) EMBO J. 3. 2723-2730); ALS promoter (U.S. Application Seriel No. 08/409,297), and the like. Other constitutive promoters include those in U.S. Patent Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680, 5,268,463; and 5,608,142.

Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. Depending upon the objective, the promoter may be a chemical-inducible promoter, where application of the chemical induced gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR- la promoter, which is activated by salicylic acid. Other chemical-regulated promoters of interest include steroid- responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena et al (1991) Proc. Natl. Acad. Sci. USA 88: 10421-10425 and McNellie et al. (1998) Plant J. 14(2): 247-257) and tetracycline-inducible and tetracycline- repressible promoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet. 227: 229-237, and US Patent Nos. 5,814,618 and 5,789,156, herein incorporated by reference.

Where enhanced expression in particular tissues is desired, tissue-specific promoters can be utilised. Tissue-specific promoters include those described by Yamamoto et al. (1997) Plant J. 12(2): 255-265; Kawamata et al (1997) Plant Cell Physiol. 38(7): 792-803; Hansen et al (1997) Mol. Gen. Genet. 254(3): 337-343; Russell et al. (1997) Transgenic Res. 6(2): 157-168; Rinehart et al (1996) Plant Physiol. 112(3): 1331-1341; Van Camp et al (1996) Plant Physiol. 112(2): 525-535; Canevascni et al (1996) Plant Physiol. 112(2): 513-524; Yamamoto et al (1994) Plant Cell Physiol. 35(5): 773-778; Lam (1994) Results Probl. Cell Differ. 20: 181-196; Orozco et al (1993) Plant Mol. Biol. 23(6): 1129-1138; Mutsuoka et al (1993) Proc. Natl. Acad. Sci. USA 90(20): 9586-9590; and Guevara-Garcia et al (1993) Plant J. 4(3): 495-50.

"Operably linked" means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter. DNA operably linked to a promoter is "under transcriptional initiation regulation" of the promoter.

In a preferred embodiment the promoter is an inducible promoter or a developmentally regulated promoter. Preferably said developmentally regulated promoter is a germination/post germinative promoter. Promoters of this type are known in the art, for example, the promoters of malate synthase and isocitrate lyase are strong promoters which have the requisite expression pattern.

Particular vectors are nucleic acid constructs which operate as plant vectors. Specific procedures and vectors previously used with wide success upon plants are described by Guerineau and Mullineaux (1993) (Plant transformation and expression vectors. In: Plant Molecular Biology Labfax (Croy RRD ed) Oxford, BIOS Scientific Publishers, pp 121-148). Suitable vectors may include plant viral-derived vectors (see e.g. EP- A- 194809).

Vectors may also include selectable genetic marker such as those that confer selectable phenotypes such as resistance to herbicides (e.g. phosphinotricin, chlorsulfuron, methotrexate imidazolinones and glyphosate) and antibiotics (kanamycin, hygromycin, gentamycin, spectinomycin).

According to a further aspect of the invention there is provided a plant comprising a cell according to the invention.

In a preferred embodiment of the invention there is provided a plant selected from the group consisting of: corn (Zea mays), canola (Brassica napus, Brassica rapa ssp.), flax (Linum usitatissimum), alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cerate), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower (Helianthus annus), wheat (Tritium aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum), sweet potato (Iopmoea batatus), cassava (Manihot esculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple (Anana comosus), citris tree (Citrus spp.) cocoa (Theobroma cacao), tea (Camellia senensis), banana (Musa spp.), avacado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifer indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia inter grif olid), almond (Prunus amygdalus), sugar beets (Beta vulgaris), oats, barley, vegetables.

Preferably, plants of the present invention are crop plants (for example, cereals and pulses, maize, wheat, potatoes, tapioca, rice, sorghum, millet, cassava, barley, pea), and other root, tuber or seed crops. Important seed crops are oil-seed rape, sugar beet, maize, sunflower, soybean,sorghum, and flax (linseed).

Horticultural plants to which the present invention may be applied may include lettuce, endive, and vegetable brassicas including cabbage, broccoli, and cauliflower. The present invention may be applied in tobacco, cucurbits, carrot, strawberry, sunflower, tomato, pepper.

Particularly preferred species are those of ornamental plants.

Grain plants that provide seeds of interest include oil-seed plants and leguminous plants. Seeds of interest include grain seeds, such as corn, wheat, barley, rice, sorghum, rye, etc. Oil-seed plants include cotton, soybean, safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants include beans and peas. Beans include guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava been, lentils, chickpea, etc.

According to a yet further aspect of the invention there is provided a seed comprising a cell according to the invention.

According to a further aspect of the invention there is provided a method to regulate phosphoenolpyruvate carboxykinase activity in a plant comprising the steps of: i) transfecting a plant cell with an expression cassette or vector according to the invention; ii) regenerating said cell into a plant; iii) monitoring phosphoenolpyruvate carboxykinase activity of said plant. According to a further aspect of the invention there is provided a screening method for the identification of an agent with the ability to inhibit plant growth and/or viability comprising the steps of: i) providing a polypeptide encoded by the nucleic acid selected from the following group; a) a nucleic acid represented by Fig 6 or 7; b) nucleic acids which hybridise to the sequences of (i) above and which have PEPCK activity; and c) nucleic acid sequences which are degenerate as a result of the genetic code to the sequences defined in (a) and (b) above; ii) providing at least one candidate agent; iii) forming a preparation which is a combination of (i) and (ii); iv) determining the interaction of the polypeptide and said candidate agent; and v) testing the effect of the agent on the growth and/or viability of plants.

In a preferred method of the invention said agent has herbicidal activity.

In a preferred method of the invention said polypeptide is encoded by the nucleic acid sequence represented by Fig 6 or 7.

In a further preferred method of the invention said polypeptide is represented by the amino acid sequence in Figure 8, or active fragment thereof. Alternatively said amino acid sequence is modified by addition, deletion or substitution of at least one amino acid residue.

According to a further aspect of the invention there is provided an agent identified by the screening method according to the invention.

In a preferred embodiment of the invention said agent is a herbicide. According to a further aspect of the invention there is provided a method for inhibiting the growth of undesired vegetation comprising applying an agent identified by the methods according to the invention.

According to a further aspect of the invention there is provided a method to regulate the phosphoenolpyruvate carboxykinase activity of a plant comprising the steps of: i) providing a cell according to the invention; ii) regenerating said cell into a plant; and iii) monitoring phosphoenolpyruvate carboxykinase acitivity in said plant.

In a preferred method of the invention a seed from a plant according to the invention has modulated germination and/or post-germinative growth.

In a preferred method of the invention said modulation is an inhibition of germination or post-germinative growth.

In an alternative method of the invention said modulation is the enhancement of germination or post-germinative growth.

An embodiment of the invention will now be described by example only and with reference to the following figures:

Figure 1 shows RT-PCR on RNA from 2 days old 35S PCK1 seedlings grown on 20 mM sucrose. PCR was carried out on undiluted cDNA (1), 1/10 dilution (2), 1/100 dilution (3) and 1/1000 dilutions of cDNA. ACT ACTIN, (E) Endogenous (T) transgene PEPCK transcript;

Figure 2 presents PEPCK activity in 2 day old ColO and 35 S PCK1 antisense seedlings grown on 20 mM sucrose. Values are the mean ± SE of 3 measurements made on three separate batches. Figure 3 A presents seedling establishment in high (160) and low (10 uM.m.sec) light intensities. Sucrose was not included in the media, values are the mean ± SE of measurements on three separate batches of 30 seedlings. (B) Hypocotyl length of antisense and wild type etiolated seedlings. Seedlings were grown in the dark on medium without sucrose for 5 days. Values are the mean ± SE of measurements on three separate batches of 30 seedlings;

Figure 4 represents relationship between seedling establishment and PEPCK activity in 2 day old 35S PCK1 seedlings grown without exogenous sucrose;

Figure 5A represents incorporation of ¹⁴C acetate in ColO and 35S PCK1 antisense 2 day old seedlings. Incorporation into sugars (O) and CO₂ (•). All values are relative to a wild type of 100% and are the mean ±SE of measurements made on 3 replicates. 5(B) Sucrose levels in 2 day old seedlings;

Figure 6 is the genomic sequence of Arabidopsis thaliana PEPCK1;

Figure 7 is the cDNA sequence of Arabidopsis thaliana PEPCK1; and

Figure 8 is the protein sequence of PEPCK1.

Materials and Methods

Antisense construct

The cDNA EST H36251 was identified in the Genbank DNA sequence database as a

PEPCK (PCK1) and the clone was subsequently obtained from the Arabidopsis

Biological Resource Center, DNA Stock Center. The cDNA clone was provided as an

E. coli bacterial colony containing the cloning vector λ-Ziplox (GIBCO BRL) into which the cDNA clone was directionally inserted 5' to 3'. The sequence was confirmed by partial sequencing of the 3' and 5¹ ends. The 1239bp PEPCK fragment was excised from λ-Ziplox by restriction digest of the plasmid with Xbal and Smal and ligated into pJO530 (a pBIN19 derivative, (21), which had been previously linearized by restriction digestion with Xbal and Smαl. Plant transformation using the binary vector system was conducted using Agrobacterium tumefaciens strain G3V101 containing a vir⁺ Ti-plasmid lacking the T-DNA region. Insertion of the PEPCK cDNA fragment in pJO530 in the antisense orientation was confirmed by restriction digest and PCR analysis.

Plant material and growth conditions

Arabidopsis thaliana, ecotype Columbia (Co/0) plants were used for transformation.

Plants were grown in growth chambers at 20 °C under a constant illumination of 70 μEM/m². Plants were transformed by vacuum infiltration (22). Tl seed was grown on hygromycin plates and resistant seedlings selected to soil. T2 and T3 progeny were screened for homozygous lines, by segregation on hygromycin. After initial characterisation, five lines with T-DNA insertions at 1-2 loci based on segregation analysis, and representing a range of PEPCK activities, were selected for further analysis. Seeds were germinated in continuous light on 0.8% (w/v) agar plates containing half strength Murashige and Skoog media (23) (plus 20 mM sucrose where indicated) at 20°C following three days imbibition at 4°C in the dark. For experiments with etiolated seedlings, plates were transferred back to the dark at 20 °C after 30 min exposure to white light.

RT-PCR Analysis

The cDNA was synthesised from 5μg of DNAse treated RNA and 0.5μg oligo dT (Invitrogen) were heated 95 °C for 2 min, chilled on ice then 5x 1^st strand buffer, 2μl 2.5 mM dNTPs, 2 μl 0.1M DTT, 0.5 μl RNAsin (Promega) and 100U Superscript reverse transcriptase (Gibco) and the reaction incubated at 42 °C for 90 mins. PCR was performed using serial dilutions of cDNA, the reactions were heated to 95 °C for 2 min followed by 40 cycles of 95 °C, 15 sec, 60 °C, 30 sec and 72 °C 1 min, then a single 72 °C for 10 min. Primers used were, for constitutive expression, primers to the actin (ACT2) gene were used (Genbank U41998) ACT2S CTT ACA ATT TCC CGC TCT GC and ACT2S GTT GGG ATG AAC CAG AAG GA. The endogenous PCK1 transcript was amplified using PCK1-72 (GAAGATAACGACCGGAGCAG) & PCK1-573 (GGGAGCACGACCAGTCTTAG). Endogenous PCK1 and Transgene transcripts were amplified using primers PCKl-1001

(AGGGTCTTTTCAGTGTGATGC) and PCK-1709

(CCATAACTGCCACCAGACCA). The PCK2 gene was amplified using PCK2-540 (CTGCATTTTCTCAGCCAACA) and PCK2-1208

(CCGGTATTTGATCGGAGATG).

Enzyme and sugar Assays

PEPCK assays were performed on plant tissue extracts according to the method of (24). Soluble sugars were measured according to the method of (25).

Metabolism of ¹⁴ C acetate

The metabolism of acetate was as described in Eastmond et al., 2000. Amounts incorporated into neutral and CO fractions were as described in (26).

Example 1

Analysis during early post-germinative growth

One hundred and ninety six Tl transformants were identified after selected on hygromycin, of these, sixty percent showed a significant reduction in the level of seedling establishment in the T2 generation (data not shown). After initial characterisation, five lines with T-DNA insertions at 1-2 loci, based on segregation analysis in the T2 and T3 generations, and representing a range of PEPCK activities, were selected for further analysis (lines 16, 64, 122,192 and 195). RT-PCR analysis indicated that transcript levels of PCK1 were decreased in all five lines, with line 16 showing the lowest level of PCK1 expression (Fig. 1). Transcript levels of PCK2 were much lower, less than 1% that of PCK1, and were not reduced in the antisense lines. Transcript levels of FBPase were also unaltered (results not shown).

The pattern of transcript expression was mirrored by the decrease in PEPCK activity; line 16 exhibited the lowest level of activity, <11% of untransformed ColO (Fig 2A). Hypocotyl lengths in 5 day old antisense seedlings grown without exogenous sucrose, were also reduced, with line 16 68% that of wild- type (Fig 3B). The ability of the 35S PCK1 antisense seedlings to establish without an exogenous supply of sucrose was further compromised by the reduction in light intensity (Fig 3A). Antisense seedlings that had failed to establish could be rescued by transferring to plates containing 20 mM sucrose. To test the relationship between PEPCK activity and seedling establishment, measurements were made on an additional 18 lines to provide a more complete range of PEPCK activity. There is a statistically significant correlation between PEPCK activity and seedling establishment (r = 0.750, PO.01) (Fig. 4). To investigate if the reduction in seedling establishment was due to a reduced supply of sucrose, we measured the soluble sugar levels in 2 day old seedlings grown without an exogenous supply of sucrose. We found a significant reduction in the levels of soluble sugars in the four lines with the lowest PEPCK enzyme activities. To examine if storage lipid catabolism was compromised in the antisense lines fatty acid levels were analysed. Seedlings from zero to 5 days after imbibition were grown without exogenous sucrose. Fatty acid analysis for all five lines revealed no significant alterations in the rate of storage lipid catabolism from that of untransformed Col0 seedlings (results not shown).

As lipid catabolism appeared unaltered, but soluble sugar levels were reduced, we investigated whether more of the storage carbon was being respired in the antisense lines than in untransformed ColO by feeding 2 day old seedlings ¹⁴ C acetate. To test the relationship between PEPCK activity and ¹⁴ C incorporation into CO₂ and sugars, measurements were made on an additional 18 antisense lines to provide a more complete range of PEPCK activities (Fig 5A). The slope of the regression line for incorporation into CO₂ decreased with increasing levels of PEPCK activity whilst the slope of the regression line for accumulation into sugars increased with increasing PEPCK activity. Although, the r values were not significant (r = -0.420 and r = 0.370 respectively), similar trends were seen in two separate experiments.

Example 2

Seedling Establishment

PCK1, the predominantly expressed PEPCK gene during Arabidopsis seedling germination plays an essential role in seedling establishment. The effects of decreased PEPCK activity on seedling establishment can be partially compensated for by increasing the amount of light available for photosynthesis. This growth restriction can also be overcome by supplying exogenous sugars, however, even without a supply of exogenous sugars, storage lipid is still catabolised during early post-germinative growth. There is most probably an increase in the respiration of acetyl CoA units released from β-oxidation and a corresponding decrease in the production of sugars in the antisense lines with decreasing PEPCK activity. These results indicate that when the capacity for the conversion of storage lipid to sucrose is reduced, lipid is used as a source of carbon for respiration. This has been reported for a mutant of the glyoxylate cycle gene, isocitrate lyase (27). In the id mutant, the glyoxylate cycle is not essential for seedling establishment under optimal conditions. Arabidopsis seeds contain, in addition to lipid, reserves of soluble carbohydrate and protein. This soluble carbohydrate is used to fuel germination, with the lipid reserves being catabolised later during early post-germinative growth (28). Under optimal conditions a percentage of seeds contain enough soluble carbohydrate and protein to attain photosynthetic competency before these additional reserves are depleted. Furthermore, increasing the amount of light available for photosynthesis can increase this percentage. However, under reduced light, photosynthetic competency is not reached before these additional reserves are depleted. Under these conditions, seedling growth of both the id mutant and PEPCK antisense lines is arrested unless an exogenous source of sucrose is provided. Whilst lipid can be respired, without an anaplerotic supply of carbon skeletons for the TCA cycle, seedling growth cannot continue. The id mutant (27) established that the glyoxylate cycle was not essential for lipid catabolism or seedling establishment in optimal conditions. Here, our results indicate that gluconeogenesis is also not essential for lipid catabolism or seedling establishment in optimal conditions. However, as reported for the id mutant, the ability to produce sugars from lipid, prior to attaining photosynthetic competency is important for seedling survival in the sub-optimal conditions that can be encountered in nature, for example prolonged darkness. Unlike id and the PEPCK antisense lines, the β-oxidation mutant pedl, which lacks 3-ketoacyl-CoA thiolase activity (29) is unable to catabolise storage lipid reserves even in the presence of an exogenous supply of sucrose. Without a supply of exogenous sucrose, post-germinative growth does not occur. Thus whilst both the glyoxylate cycle and gluconeogenesis are not essential for seedling establishment under optimal conditions, the β-oxidation cycle is essential under all conditions.

In conclusion, the complete catabolism of lipid to sucrose is not essential for seedling establishment under optimal conditions, however, a reduction in PEPCK levels does compromise seedling establishment under the sub-optimal conditions of low light. Seedling establishment can be rescued by the supply of exogenous sucrose or by increased light intensity.

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Claims

1. Use of phosphoenolpyruvate carboxykinase as a herbicide target.

2 Use according to Claim 1 wherein phosphoenolpyruvate carboxykinase is encoded by a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of: i) a nucleic acid sequence, or part thereof, represented by the sequence in Figure 6 or 7 ii) a nucleic acid sequence, or part thereof, which hybridizes to the sequence in Figure 6 or 7 and encodes at least part of a phosphoenolpyruvate carboxykinase polypeptide; and iii) a nucleic acid sequence which is degenerate as a result of the genetic code to the sequences in (i) and (ii).

3. Use according to Claim 2 wherein said nucleic acid molecule hybridises under stringent hybridisation conditions to the sequence in Figures 6 or 7.

4. Use according to Claim 1 wherein phosphoenolpyruvate carboxykinase is represented by the amino acid sequence in Figure 8.

5. Use according to Claim 4 wherein said amino acid sequence is modified by addition, deletion or substitution of at least one amino acid residue.

6. A screening method for the identification an agent with the ability to inhibit plant growth and/or viability comprising the steps of: i) providing a polypeptide encoded by a nucleic acid molecule selected from the following group; a) a nucleic acid molecule represented by Fig 6 or 7; b) nucleic acids which hybridise to the sequences of (a) above and which have phosphoenolpyruvate carboxykinase activity; and c) nucleic acid sequences which are degenerate as a result of the genetic code to the sequences defined in (a) and (b) above; ii) providing at least one candidate agent; iii) forming a preparation which is a combination of (i) and (ii); iv) determining the interaction of said polypeptide and said candidate agent; and optionally v) testing the effect of the agent on the growth and or viability of plants.

7. A method according to Claim 6 wherein said agent has herbicidal activity.

8. A method according to Claim 6 or 7 wherein said polypeptide is encoded the nucleic acid sequence represented by Fig 6 or 7.

9. A method according to any of Claims 6-8 wherein said polypeptide is represented by the amino acid sequence in Figure 8, or active fragment thereof.

10. A method according to any of Claims 6-8 wherein said polypeptide is modified by addition, deletion or substitution of at least one amino acid residue.

11 An agent identified by the screening method according to any of Claims 6-10.

12. An agent according to Claim 32 wherein said agent is a herbicide.

13. A method for inhibiting the growth of undesired vegetation comprising applying an agent according to Claim 12 to said vegetation.

14. A transgenic plant cell wherein the genome of said cell is modified such that the activity of phosphoenolpyruvate carboxykinase is modulated when compared to a non-transgenic reference cell of the same species characterised in that phosphoenolpyruvate carboxykinase expression is limited to germination and/or post- germinative growth.

15. A cell according to Claim 14 wherein phosphoenolpyruvate carboxykinase is down-regulated.

16. A cell according to Claim 14 wherein phosphoenolpyruvate carboxykinase is up-regulated.

17. A cell according to any of Claims 14-16 wherein phosphoenolpyruvate carboxykinase is encoded by a nucleic acid molecule selected from the group consisting of: i) a nucleic acid sequence, or part thereof, represented by the sequence in Figure 6 or 7; ii) a nucleic acid sequence, or part thereof, which hybridizes to the sequence in Figure 6 or 7 and encodes at least part of a phosphoenolpyruvate carboxykinase polypeptide; and iii) a nucleic acid sequence which is degenerate as a result of the genetic code to the sequences in (i) and (ii).

18. A method to regulate the phosphoenolpyruvate carboxykinase activity of a plant comprising the steps of: i) providing a cell according to any of Claims 14-17; ii) regenerating said cell into a plant; and iii) monitoring phosphoenolpyruvate carboxykinase acitivity in said plant.

19. A method according to Claim 18 wherein a seed from said plant has modulated germination and/or post-germinative growth.

20. A method according to Claim 19 wherein said modulation is an inhibition of germination or post-germinative growth.

21. A method according to Claim 19 wherein said modulation is the enhancement of germination or post-germinative growth.