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  • C12N15/8247—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified lipid metabolism, e.g. seed oil composition
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  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
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  • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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  • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
  • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
  • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

• the present invention alleviates the aforesaid problems by providing plants which specifically respond to elevated carbon dioxide concentrations by increasing the number of stomata on their leaf surfaces. In the absence of other limiting factors this would be expected to increase carbon dioxide uptake for photosynthesis and present a greater effective surface area for water loss resulting in increased transpiration and thereby counteracting calcium deficiency. Data from carbon isotope discrimination studies in wheat, barley, rice and Phaseolus vulgaris indicate that genotypes with lower stomatal resistance are higher yielding.
• genes in plants is controlled by a number of regulatory components, including nucleic acid and protein elements. Where the plant gene exists as double stranded DNA, the primary steps of expression involve the production of a messenger RNA by a polymerase enzyme. The initiation of this part of the expression process is controlled by a region commonly referred to as the "promoter".
• the promoter lies upstream (5') of the protein encoding region and may be constitutive or tissue-specific, developmentally-regulated and/or inducible.
• the core promoter region contains the characteristic CAAT and TATA boxes plus surrounding sequences, and represents a transcription initiation sequence which defines the transcription start point for the structural gene.
• the precise length of the core promoter region is indefinite but it is usually easily recognisable. Such a region is normally present, with some variation, in all promoters.
• the base sequences lying between the various well-characterised “boxes” appear to be of lesser importance.
• Also provided is a method of producing plants with an increased number of stomata relative to control like plants comprising the steps of: (i) transforming plant material with a polynucleotide comprising the sequence depicted as SEQ LD No 1 or 2 or SEQ LD No 8. (ii) selecting the thus transformed material;
• the polynucleotides for use in this method may be under expression control of a plant operable promoter and may further comprise a transc ⁇ ption termination region which is downstream of the protein encoding region of the said polynucleotide.
• the following promoters may be used: CaMV35S; FMV35S; NOS, OCS and E9. More preferably the promoter may be a stomatal guard cell specific promoter. Even more preferably the promoter may be comprised by the polynucleotide sequence depicted as SEQ ID No 2 or SEQ ID No 8. Also provided is an isolated polynucleotide comprising the sequence depicted as SEQ ID No 2 or SEQ ID No 8..
• the polynucleotide may comprise the sequence depicted in SEQ LD No. 2 or SEQ LD No 8.
• the protein encoding region comprised by the polynucleotide may be bounded by a plant operable promoter and terminator.
• promoters and terminators which are per se not germane to the invention, are well known to the skilled man and include, for example, the CaMV35S, FMV35S, NOS, OCS and E9 (derived from the small subunit of RUBISCO) promoters and terminators. It is particularly preferred, however, that the protein encoding region of the polynucleotide according to the invention is under expression control of a stomatal guard cell specific promoter.
• the protein capable of providing for herbicide resistance may be selected from the group consisting of glyphosate oxido-reductase (GOX), 5-enol-pyravyl-3-phosphoshikimate synthetase (EPSPS), phosphinothricin acetyl transferase (PAT), hydroxyphenyl pyruvate dioxygenase (HPPD), glutathione S transferase (GST), cytochrome P450, Acetyl-COA carboxylase (ACCase), Acetolactate synthase (ALS), protoporphyrinogen oxidase (PROTOX), dihydropteroate synthase, polyamine transport proteins, superoxide dismutase (SOD), bromoxynil nitrilase, phytoene desaturase (PDS), the product of the tfdA gene obtainable from Alcaligenes eutrophus, and known mutagenised or otherwise modified variants of the said proteins.
• the polynucleotide with which the plant material may be transformed may comprise 5' of the protein encoding regions which encode: (i) a peptide which is capable of targeting the translation products of the regions to plastids such as chloroplasts, mitochondria, other organelles or plant cell walls; and/or (ii) non-translated translational enhancing sequences.
• the polynucleotide may be codon-optimised, or otherwise altered to enhance at least transcription once it is incorporated into plant material.
• Transformation techniques are well known and include particle mediated biohstic transformation, Agrobacterium-mediated transformation, protoplast transformation
• the skilled man may, however, prefer to transform plant material with a polynucleotide comprising a sequence which is complementary to one which when incubated at a temperature of between 60 and 65°C in 0.3 strength citrate buffered saline containing 0.1% SDS followed by rinsing at the same temperature with 0.3 strength citrate buffered saline containing 0.1% SDS still hybridises with the sequence depicted in SEQ ID No. 1.
• the plants may be selected on the basis of resistance to an antibiotic, which resistance is produced by an antibiotic resistance conferring gene which has been co-introduced into the plant material together with genes capable of increasing the number of stomata.
• an antibiotic which resistance is produced by an antibiotic resistance conferring gene which has been co-introduced into the plant material together with genes capable of increasing the number of stomata.
• Stomatal index which in essence is the number of stomata in a particular area of a leave (for example) when expressed as a percentage of the total number of cells contained within that area. Stomatal index may be defined as follows:
• the invention still further includes plants which result from the method disclosed above, the progeny of such plants, the seed of such plants and progeny, and parts of such plants and progeny. Particularly prefeoed parts are fruits, flowers and seeds.
• the invention still further includes the use of the polynucleotide or vector of the invention in a method for the production of plants which have an increased number of stomata relative to non transformed control plants.
• the invention will be further apparent from the following description, taken together with the associated Figures and Sequence Listings.
• Figure 4 depicts the pFL13 scematic drawing.
• pMOG553 promoterless GUS on T-DNA.
• pMOG553 is depicted schematically m Figure 2.
• Genomic DNA was isolated from Tag 590 plant material (rosette leaves). The copy No. of the T-DNA insert was determined by restriction enzyme analysis of the said genomic DNA using EcoR V, Msc I with the 5' part of the GUS gene being used as a probe. This restriction regime yielded a single band on suitable electrophoresis. An inverse PCR was performed on the gDNA of Tag590 using the following GUS gene primers (according to the method described by Barthels, see p. 2131, Fig. 7 of that document).
• primer 7 5'-GTA.ATG.CTC.TAC.ACC.ACG.CCG-3' (SEQ LD No. 5).
• primerS 5'-CTT.TCC.CAC.CAA.CGC.TGA.TC-3' (SEQ LD No. 6).
• the following primers were used to in the production of a PCR fragment which was generated on genomic DNA from original TAG590 using pfu polymerase (Proofreading capacity). This fragment was then cloned into the pGEMT making the construct labelled as pFL44.
• GUS 9 S'-CAG.AAA.CTT.ACG.TAC.ACT.TTT.C-S' (SEQ ID 7)
• pFL44 does not contain any recognizable promoter sequences, it has been shown that it gives specific expression when transformed to plants indicating that it comprises some tissue specific regulatory elements.
• the PCR fragment (pFL44) has been completely sequenced on both strands. It appears that the GUS tag has inserted within a gene with extensive similarity but clearly non- identity to an Arabidopsis gene that has been previously identified by transposon tagging experiments and is designated as FAE I.
• the sequence depicted in SEQ LD No. 2 contains at least two exons and does have putative translation initiation and termination points but appears to encode a peptide significantly shorter than FAE I (110 and 12 amino acids missing at the N- and C- terminal ends respectively). Therefore it is possible that there are additional coding regions that are not within this cloned fragment.
• the GUS tag has inserted 3' to the second exon (180bp 3' to the putative translation stop codon) this is probably close to the end of the transcribed sequence or alternatively maybe within an intron. It is unlikely that another gene could be within this distance from the Fae-like coding sequences.
• the cooesponding region of the genomic clone (pFL30) has been sequenced using the same Internal primers. This longer clone is sequenced with a view (i) to identifying further exons, if they exist; and (ii) to characterise the potential gene promoter regions which may lie upstream of the identified coding sequence.
• FAE I is thought to encode a fatty acid elongase necessary for the production of very long chain fatty acids.
• a transposon tagged FAE I mutant fails to accumulate fatty acids longer than C18 (i.e. 20:0, 20: 1 and 22: 1) in its seed.
• FAE I is thought to encode a seed specific ketoacyl synthase which catalyses the condensation reaction with malonyl CoA.
• a region of protein having approximately 50 amino acid in FAE I has been identified which shares some sequence similarity to regions within other plant malonyl CoA condensing enzymes (e.g. CHS and STS).
• CHS and STS e.g. CHS and STS.
• At the DNA level there is some similarity to 4 Arabidopsis ESTS, T6700, T44939, T44368 and ATTS 1282. These homologies lie entirely within the proposed coding regions of the SEQ ID No. 2 sequence.
• Tag 590 gene encodes a putative fatty acid elongase which is expressed specifically in developing guard cells. This enzyme plays a role in determining stomatal density in response to altered carbon dioxide concentration (described below).
• pMOG1017 was used for the Arabidopsis C24 transformation following which 40 transgenic lines were generated and their leaves were histochemically tested for GUS expression. 14 Lines show clearly specific expression of the GUS gene in the stomata only see photo's 1 or 2. The remaining 26 lines did not show clear expression and were thought to be low or non expressors.
• Controlled environment chambers were used to grow plants under ambient (350- 450ppm) and elevated (ambient +650ppm) carbon dioxide. Under these conditions flower and leaf appearance, plant development and flowering time were all recorded. Wild type C24 were used as untransformed controls and 35S constitutive GUS expressors were used as GUS positive controls. Plants were transfeoed to the chambers at 16 or 35 days after exposure to lighting (dal). In the case of the 16 day age group, plants were grown on to 35 days whilst the older plants were grown to seed set before termination of the experiments. Initiation of flowering was recorded for individual plants up to 67 days. On harvest all plants were photographed.
• Xantopren dental impression material (Dental Links Products) was used to take impressions of the abaxial epidermis (Weyers and Johansen 1985) from 2 to 3 leaves per plant, choice of leaf being based on comparative size rather than leaf number.
• Optically clear acetone based varnish was used to make positives from the Xantopren impressions.
• Stomatal and epidermal cell counts per unit area (9.2 ). were taken from three different parts of each positive under light microscopy at x200 magnification.
• Guard cells are known to exhibit two distinct responses to elevated carbon dioxide (CO?).
• CO? elevated carbon dioxide
• the other is a developmental response and is manifested in certain species by a reduction in the number of stomata in plants grown under elevated CO?.
• the results described below come from experiments in which the phenotype of the Tag 590 plants and C24 controls were compared under elevated and ambient CO?.
• the objective of this experiment was to use more carefully controlled growth conditions to investigate the TAG 590 phenotype more accurately.
• an ambient level of CO? was maintained for the duration of the experiment.
• the CO? level was kept at 650 ppm above ambient. This was a higher concentration that used in Experiment 1 but was chosen as it more accurately reflects the CO 2 regimes employed by commercial growers.
• the seeds were germinated in a growth room, potted on and at 45 days old were transfeoed to growth cabinets. They were then grown for a further 22 days and at the end of the experiment flowering was recorded as was stomatal number. It is important to note that stomatal number was only recorded in leaves which had grown during the experimental treatment.
• the delayed flowering of the tagged plants in elevated CO? was seen again in this experiment. All the tagged lines flowered later than the controls in both elevated and ambient treatments (delayed by approximately 7 days).
• the results of the stomatal number determination are summarised in Table 2 where it is apparent that stomatal number was reduced in control plants grown in elevated CO? but that in both tagged lines it was increased thus confirming the results of the previous experiment which indicated that stomatal numbers increased in response to elevated CO 2 .
• the delay in flowering observed in the tagged mutant lines may result from a secondary effect of the gene disruption. That is, the altered stomatal density in these plants causes changes in the rate of carbon assimilation which in turn affects the initiation of the floral meristem.

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• 1998
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