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WO1995007993A1 - Regulation de la senescence - Google Patents

Regulation de la senescence Download PDF

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Publication number
WO1995007993A1
WO1995007993A1 PCT/GB1994/001990 GB9401990W WO9507993A1 WO 1995007993 A1 WO1995007993 A1 WO 1995007993A1 GB 9401990 W GB9401990 W GB 9401990W WO 9507993 A1 WO9507993 A1 WO 9507993A1
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WIPO (PCT)
Prior art keywords
senescence
sequence
dna
clone
dna construct
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PCT/GB1994/001990
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English (en)
Inventor
Catherine Margaret Smart
Howard Thomas
Sally Elizabeth Hosken
Wolfgang Walter Schuch
Caroline Rachel Drake
Donald Grierson
Aldo Onorio FARRELL
Isaac John
John Andrews GREAVES
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Zeneca Limited
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Priority to AU76194/94A priority Critical patent/AU696417B2/en
Priority to EP94926310A priority patent/EP0719341A1/fr
Publication of WO1995007993A1 publication Critical patent/WO1995007993A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—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
    • C12N15/8243—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
    • C12N15/8249—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 ethylene biosynthesis, senescence or fruit development, e.g. modified tomato ripening, cut flower shelf-life

Definitions

  • This application relates to novel DNA constructs, plant cells containing such constructs and plants derived therefrom. In particular, it relates to the modification of the senescence process in plants.
  • Senescence is a controlled series of biochemical and physiological events comprising the final stage of development. The changes taking place in senescence form a genetically programmed sequence, with close co-ordination at the cell and tissue level. Cells remain viable and show tight metabolic regulation until the end of senescence. Senescence may be caused by a variety of external factors (including light, temperature, water/minerals, pathogens) or internal factors (including space, light, nutrients, flowering/pollination, growth substances) .
  • a genetic switch is triggered which modifies gene expression at the transcriptional and/or post-transcriptional level and induces a change in cell/tissue function resulting in senescence.
  • the photosynthetic apparatus is dismantled and leaf function changes from carbon assimilation to nitrogen/phosphorus mobilisation.
  • Senescence involves pigment degradation, proteolysis and nucleic acid breakdown leading to nitrogen redistribution and phosphorus remobilisation to other plant parts. It also involves the respiration of lipids and carbohydrates.
  • leaf expansion and senescence are quantified as leaf area index and leaf area duration, and these factors are known to be major determinants of yield in many crops (Thomas, 1992 in Crop Photosynthesis: Spatial and Temporal Determinants, Baker and Thomas (eds) , Elsevier, pages 11-41) .
  • the importance of delayed senescence in increasing the yield of determinate crops has been confirmed by studies on several species (for example, Tollenaar and Daynard, 1978, Plant Sci, 78:199-206; Thomas, 1987 in Developmental Mutants in Higher Plants, Thomas and Grierson (eds) , Cambridge Univ Press, pages 245-265) .
  • the modification of plant gene expression has been achieved by several methods.
  • the molecular biologist can choose from a range of known methods to decrease or increase gene expression or to alter the spatial or temporal expression of a particular gene.
  • the expression of either specific antisense RNA or partial sense RNA has been utilised to reduce the expression of various target genes in plants (as reviewed by Bird and Ray, 1991, Biotechnology and Genetic Engineering Reviews 9:207-227).
  • These techniques involve the incorporation into the genome of the plant of a synthetic gene designed to express either antisense or sense RNA. They have been successfully used to down-regulate the expression of a range of individual genes, for example those involved in the development and ripening of fruit (Gray et al, 1992, Plant Molecular Biology, 19:69-87).
  • RNA containing the complete coding region of the target gene may be incorporated into the genome of the plant to "over-express" the gene product.
  • Various other methods to modify gene expression are known; for example, the use of alternative regulatory sequences.
  • Senescence-related genes having a function in foliar senescence may be classified according to their patterns of expression during leaf development (Smart, 1994, New Phytol, 126:419-448; Thomas, 1994, Reviews in Clinical Gerontology, 4:5-20) . Six broad categories may be recognised:
  • a senescence-related gene is a gene which has a role in senescence.
  • the senescence-related gene may be activated during senescence, or may be down-regulated during senescence, or may show an unchanged level of expression during senescence.
  • Senescence may be inhibited by inhibiting a gene which is normally activated during senescence. Additionally or alternatively, senescence may be inhibited by increasing the expression of a gene which is normally down-regulated during senescence.
  • Senescence may also be delayed or slowed by transforming a plant with a construct in which a promoter from a senescence-activated gene drives expression of a strong senescence antagonist.
  • Senescence may be accelerated by inhibiting a gene which is normally down-regulated during senescence. Additionally or alternatively, senescence may be accelerated by increasing the expression of a gene which is normally activated during senescence.
  • senescence may be inhibited or accelerated by respectively inhibiting or increasing the expression of a gene which normally shows an unchanged level of expression during senescence.
  • the method of the present invention can be applied to any plant, including tomato, lettuce, broccoli, cabbage, carrot, beet, melon, banana, strawberry, wheat, maize rice, canola, rape, sunflower, soybean. Selected plants will show modified senescence phenotypes, which may include one or more of the following characteristics :
  • onset and rate of senescence can be adapted to the specific environment or other requirements by producing crops with particular characteristics or by inducing those characteristics when needed.
  • delaying senescence in grain maize, sorghum, wheat or barley can have various beneficial effects on crop phenotype.
  • the resulting increase in leaf area (increased duration of green leaf area) and rise in photosynthetic capacity during grain-filling results in increased yield.
  • Improved stalk integrity due to delayed senescence in stalk tissue
  • yield is again increased.
  • Delaying senescence in early-maturing silage maize or sorghum will maintain leaf integrity and greenness throughout the continuing growing season to increase the crop's overall biomass.
  • the generation of improved crop varieties is not limited by the naturally-available genotypes: it is possible to generate a different range of allelic forms (for example, those having greatly inhibited or greatly accelerated senescence) . Genetic modification of single genes will probably result in a dominant "modified senescence" phenotype, which can be more easily incorporated into traditional breeding programmes.
  • stay-green plant lines could be produced by inhibiting the chlorophyll degradation pathway or by preventing lipid degradation.
  • stay-green silage maize could be produced with improved quality and palatability and with post-harvest stability.
  • the putative pathway of chlorophyll breakdown in senescence involves various enzymes including chlorophyllase, magnesium dechelatase, dioxygenase, proteases and ferredoxin (Matile, 1992) .
  • the putative pathway of galactolipid breakdown in senescence involves gluconeogenesis and hence the enzyme pyruvate,. orthophosphate dikinase which converts pyruvate to PEP (Matile, 1992) .
  • low proteolysis plant lines could be produced by down-regulating physiological protein mobilisation or by preventing autolytic degradation.
  • low proteolysis maize lines would have high silage quality and allow improved nitrogen intake and retention by the animal, with a consequent reduction in slurry nitrogen.
  • Genetic modification of single senescence-related genes may also be used in combination with a "gene switch" allowing control of the senescence process according to circumstances. If the relevant senescence-related genes could be switched on or off at will, the timing and progress of senescence and the phenotype of the crop could be directly controlled. For example, induction of senescence (“switching on” senescence) can allow early harvest of the crop. Modifying the expression of senescence-related genes using a gene switch may also be used to improve the quality of the crop. For example, induction of senescence in late-maturing silage maize or sorghum can result in better quality silage and better harvest timing. Silage consists of about 50% grain and 50% leaf/stem material.
  • a method for producing plants having modified senescence characteristics which comprises transformation of plants with a DNA construct adapted to modify the expression of at least one senescence-related gene and subsequent selection of plants in which the senescence process is either inhibited or accelerated.
  • the expression of the or each senescence-related gene may be either reduced or increased depending on the characteristics desired for the modified plant. "Antisense” or “partial sense” or other techniques may be used to reduce gene expression or expression may be increased, for example, by incorporation of additional senescence-related genes.
  • the additional genes may be designed to give either the same or different spatial and temporal patterns of expression in the plant.
  • the invention further provides a DNA construct adapted to modify the expression of at least one senescence-related gene comprising a DNA sequence corresponding to at least part of a senescence-related gene preceded by a transcriptional initiation region operative in plants so that the construct can generate RNA in plant cells.
  • a DNA construct according to the invention may be an "antisense” construct generating "antisense” RNA or a “sense”, construct (encoding at least part of the functional gene product) generating “sense” RNA.
  • "Antisense RNA” is an RNA sequence which is complementary to a sequence of bases in the corresponding mRNA: complementary in the sense that each base (or the majority of bases) in the antisense sequence (read in the 3' to 5' sense) is capable of pairing with the corresponding base (G with C, A with U) in the mRNA sequence read in the 5' to 3' sense.
  • Such antisense RNA may be produced in the cell by transformation with an appropriate DNA construct arranged to generate a transcript with at least part of its sequence complementary to at least part of the coding strand of the relevant gene (or of a DNA sequence showing substantial homology therewith) .
  • Sense RNA is an RNA sequence which is substantially homologous to at least part of the corresponding mRNA sequence.
  • Such sense RNA may be produced in the cell by transformation with an appropriate DNA construct arranged in the normal orientation so as to generate a transcript with a sequence identical to at least part of the coding strand of the relevant gene (or of a DNA sequence showing substantial homology therewith) .
  • Suitable sense constructs may be used to inhibit gene expression (as described in International Patent Publication O91/08299) or to over-express the enzyme.
  • the constructs of the invention may be inserted into any plant to regulate the expression of one or more senescence-related genes.
  • the constructs may be transformed into any dicotyledonous or monocotyledonous plant.
  • expression of the senescence-related gene may be increased or reduced, either throughout or at particular stages in the life of the plant.
  • gene expression is enhanced only by full-length sense constructs which express RNA homologous to the substantially complete coding region of the gene.
  • Constructs containing an incomplete DNA sequence shorter than that corresponding to the complete gene generally inhibit the expression of the gene, whether they are arranged to express sense or antisense RNA.
  • Full-length antisense constructs also inhibit gene expression.
  • the transcriptional initiation region may be derived from any plant-operative promoter.
  • the transcriptional initiation region may be positioned for transcription of a DNA sequence encoding RNA which is complementary to a substantial run of bases in a senescence-related mRNA (making the DNA construct a full or partial antisense construct) .
  • DNA constructs according to the invention may comprise a DNA sequence at least 10 bases (preferably at least 20 bases) in length for transcription into sense or antisense RNA. There is no theoretical upper limit to the base sequence - it may be as long as the relevant mRNA produced by the cell - but for convenience it will generally be found suitable to use sequences between 100 and 1000 bases in length. The preparation of such constructs is described in more detail below.
  • any senescence-related gene may be used in the DNA constructs as suitable genes may be isolated from any plant species.
  • suitable genes may be isolated from any plant species.
  • the cDNA of these genes has been cloned and characterised, and may be used to modify the senescence process of plants, including foliar senescence, fruit senescence and senescence of other plant parts.
  • the genes in question are encoded in the following clones: pSENUl, pSENU2, pSENU3, pSENU4, pSENU5, pSEND31, pSEND32, pSEND33, pSEND34, pSEND35, pSENE71 (all isolated from tomato: the pSENU clones correspond to genes which are up-regulated during senescence; the pSEND clones correspond to genes which are down-regulated during senescence; the pSENE clone corresponds to a gene which exhibits even expression during senescence) ;
  • SEE1, SEE2, SEE3, SEE4, SEE5, SEE6, SEE7, SEE8, SEE9, SEC1, SEC2, SED1, SED2 all isolated from maize; the SEE clones correspond to genes whose expression is enhanced during senescence; the SED clones correspond to genes whose expression is diminished during senescence; the SEC clones correspond to genes whose expression is constant during senescence) .
  • the DNA sequence in a construct according to the invention may be derived from cDNA, genomic DNA or synthesised ab initio.
  • SEE4 NCIMB 40583 cDNA clones similar to pSENUl-5 or pSEND31-35 or pSENE71 may be obtained from the mRNA of senescent tomatoes leaves.
  • cDNA clones similar to SEE1-4 may be obtained from the mRNA of senescent maize leaves . In this way may be obtained sequences coding for the whole, or substantially the whole, of the mRNA produced by the cDNA clones.
  • An alternative source of DNA for the base sequence for transcription is a suitable genomic sequence encoding a senescence-related mRNA.
  • This gene may differ from the cDNA in that introns may be present. The introns are not transcribed into mRNA (or, if so transcribed, are subsequently cut out) .
  • Oligonucleotide probes or the cDNA clone may be used to isolate the actual senescence-related gene(s) by screening genomic DNA libraries. Such genomic clones may include control sequences operating in the plant genome.
  • promoter sequences which may be used to drive expression of the senescence-related protein or any other protein. These promoters may be particularly responsive to senescence-related events and conditions. Senescence-related promoters may be used to drive expression of any target gene.
  • a further way of obtaining a suitable DNA base sequence for transcription is to synthesise it ab initio from the appropriate bases, for example using the sequences in SEQ ID NOs 1 to 39
  • the proposed full sequence of SENU2 by comparison with clone C14 is shown as SEQ ID NO 5 as a guide.
  • Recombinant DNA constructs according to the present invention may be made using standard techniques.
  • the DNA sequence for transcription may be obtained by treating a vector containing said sequence with restriction enzymes to cut out the appropriate segment.
  • the DNA sequence for transcription may also be generated by annealing and ligating synthetic oligonucleotides or by using synthetic oligonucleotides in a polymerase chain reaction (PCR) to give suitable restriction sites at each end.
  • PCR polymerase chain reaction
  • the DNA sequence is then cloned into a vector containing upstream promoter and downstream terminator sequences. If antisense DNA is required, the cloning is carried out so that the cut DNA sequence is inverted with respect to its orientation in the strand from which it was cut.
  • RNA in a construct expressing antisense RNA the strand that was formerly the template strand becomes the coding strand, and vice versa.
  • the construct will thus encode RNA in a base sequence which is complementary to some or all of the sequence of the senescence-related mRNA.
  • the two RNA strands are complementary not only in their base sequence but also in their orientations (5' to 3') .
  • RNA In a construct expressing sense RNA, the template and coding strands retain the assignments and orientations of the original plant gene. Constructs expressing sense RNA encode RNA with a base sequence which is homologous to part or all of the sequence of the mRNA. In constructs which express the functional senescence-related gene product, the whole of the coding region of the gene is linked to transcriptional control sequences capable of expression in plants.
  • constructs according to the present invention may be made as follows.
  • a suitable vector containing the desired base sequence for transcription such as pSEND35
  • restriction enzymes to cut the sequence out.
  • the DNA strand so obtained is cloned (if desired, in reverse orientation) into a second vector containing the desired promoter sequence (for example cauliflower mosaic virus 35S RNA promoter or the promoter of the pSENUl gene or other genes which are switched on at the onset of senescence) and the desired terminator sequence (for example the 3' of the Agrobacterium tumefaciens nopaline synthase gene, the nos 3' end) .
  • the desired promoter sequence for example cauliflower mosaic virus 35S RNA promoter or the promoter of the pSENUl gene or other genes which are switched on at the onset of senescence
  • the desired terminator sequence for example the 3' of the Agrobacterium tumefaciens nopaline synthase gene, the nos 3' end
  • the transcriptional initiation region (or promoter) operative in plants may be a constitutive promoter (such as the 35S cauliflower mosaic virus promoter) or an inducible or developmentally regulated promoter (such as fruit-specific promoters), as circumstances require.
  • a constitutive promoter will tend to affect the senescence process in all parts of the plant, while use of a tissue specific promoter allows more selective control of gene expression and affected functions as the antisense or sense RNA is only produced in the organ in which its action is required.
  • fruit development and/or ripening-specific promoters that could be used include the ripening-enhanced polygalacturonase promoter (International Patent Publication Number WO92/08798), the E8 promoter (Diekman S_ Fischer, 1988, EMBO, 7:3315-3320) and the fruit specific 2A11 promoter (Pear et al, 1989, Plant Molecular Biology, 13:639-651). Inducible promoters may also be used.
  • the transcriptional initiation region in the DNA construct comprises a "gene switch" .
  • Such chemically-inducible promoter sequences may be used in "gene switches” to regulate transcription of an associated DNA sequence (or “target gene”) in plants or plant tissue.
  • the gene switch may be a positive switch, where the inducible promoter directly controls the target gene. In the presence of the chemical inducer, the target gene is switched on and the encoded protein is expressed.
  • the inducible GST 11-27 promoter can be operatively linked to one or more target genes to give a chemically switchable construct: expression of the target gene(s) is controlled by application of an effective exogenous inducer.
  • the gene switch construct may be inserted into a plant by transformation.
  • the inducible GST 11-27 promoter is functional in both monocotyledons and dicotyledons, and in a variety of tissues including roots, leaves, stems and reproductive tissues.
  • Effective inducers for use with the GST 11-27 promoter include N,N-diallyl-2, 2-dichloroacetamide (common name: dichloramid) ; benzyl-2-chloro-4- (trifluoromethyl) -5-thiazole-carboxylate (common name: flurazole) ; naphthalene-1, 8-dicarboxylic anhydride; 2-dichloromethyl-2-methyl-l,3-dioxolane and several others as described in International patent application publication numbers WO90/08826 and WO93/01294. The contents of the said applications are incorporated herein by reference.
  • the gene switch may be a negative switch, where the inducible promoter indirectly controls the target gene via a repressor/operator system. In the presence of the chemical inducer, the target gene is switched off and the encoded protein is not expressed.
  • the switch comprises a chemically-inducible promoter (A) driving expression of a repressor gene encoding a repressor protein, and a promoter (B) containing an operator sequence and driving expression of a target gene.
  • A chemically-inducible promoter
  • B promoter
  • the operator region may be introduced into promoter(B) by biotechnological techniques) . If present, the repressor protein binds to the operator sequence, preventing expression of the target gene.
  • promoter (A) In the absence of inducer, promoter (A) is not active and the repressor protein is not expressed: hence the target gene is expressed. In the presence of .the chemical inducer, the repressor protein prevents expression of the target gene.
  • Promoter (A) may be GST 11-27 or any other chemically-inducible promoter sequence.
  • the repressor gene/operator sequences may be taken from the E coli lac operon.
  • Senescence-related gene expression may be modified to a greater or lesser extent by controlling the degree of sense or antisense mRNA production in the plant cells. This may be done by suitable choice of promoter sequences, or by selecting the number of copies or the site of integration of the DNA sequences that are introduced into the plant genome.
  • the DNA construct may include more than one DNA sequence encoding a senescence-related gene or more than one recombinant construct may be transformed into each plant cell.
  • a first plant may be individually transformed with a senescence-related DNA construct and then crossed with a second plant which has been individually transformed with a construct adapted to modify the expression of another gene.
  • single plants may be either consecutively or co-transformed with senescence-related DNA constructs and with appropriate constructs for modification of the other gene(s) .
  • An alternative example is plant transformation with a senescence-related DNA construct which itself contains an additional gene for modification of the activity of the other gene(s).
  • the senescence-related DNA constructs may contain sequences of DNA for regulation of the expression of the other gene(s) located adjacent to the senescence-related sequences. These additional sequences may be in either sense or antisense orientation as described in International patent application publication number W093/23551 (single construct having distinct DNA regions homologous to different target genes) . By using such methods, the benefits of modifying the activity of the senescence-related gene may be combined with the benefits of modifying the activity of other genes.
  • the senescence characteristics of plants may be modified by transformation with a DNA construct according to the invention.
  • the invention further provides plant cells containing constructs of the invention; plants derived therefrom showing modified senescence characteristics; and seeds of such plants.
  • a DNA construct of the invention is transformed into a target plant cell.
  • the target plant cell may be part of a whole plant or may be an isolated cell or part of a tissue which may be regenerated into a whole plant.
  • the target plant cell may be selected from any monoc ⁇ tyledonous or dicotyledonous plant species. Suitable plants include any fruit-bearing plant (such as tomatoes, mangoes, peaches, apples, pears, strawberries, bananas and melons) and other important crops such as maize, rice, wheat, barley, sorghum, sugar beet, canola, rape, soybean.
  • the DNA sequence used in the transformation construct may be derived from the same plant species, or may be derived from any other plant species (as there will be sufficient sequence similarity to allow modification of related enzyme gene expression) .
  • Constructs according to the invention may be used to transform any plant using any suitable transformation technique to make plants according to the invention.
  • Both monocotyledonous and dicotyledonous plant cells may be transformed in various ways known to the art. In many cases such plant cells (particularly when they are cells of dicotyledonous plants) may be cultured to regenerate whole plants which subsequently reproduce to give successive generations of genetically modified plants.
  • Any suitable method of plant transformation may be used.
  • dicotyledonous plants such as tomato and melon may be transformed by Agrobacterium Ti plasmid technology, such as described by Bevan (1984, Nucleic Acid Research, 12:8711-8721) or Fillatti et al (Biotechnology, July 1987, 5:726-730) . Such transformed plants may be reproduced sexually, or by cell or tissue culture.
  • Figure 1 is a diagram illustrating the construction of a pSENUl sense construct .
  • Figure 2 is a diagram illustrating the construction of a pSENUl antisense construct .
  • Figure 3 is a diagram illustrating the construction of a pSENU5 sense construct.
  • Figure 4 is a diagram illustrating the construction of a pSENU5 antisense construct.
  • a tomato leaf senescence cDNA library was constructed using polyA RNA from the onset and mid stages of senescence in the ration 4:1.
  • Stratagene lambda Uni-ZAP XR cDNA library was g generated with 1x10 pfu, average insert size
  • pSENUl pSENUl (also known as clone 4S1) is a cDNA of approximately l.OkB, encoding a mRNA of approximately 1.4 kB.
  • the mRNA encoded by pSENUl is expressed during the onset of senescence in tomato leaves.
  • pSENUl encodes a protein of unknown function.
  • the pSENUl DNA sequence does not show any significant homology to sequences in publicly-available sequence databases.
  • the sequence of pSENUl is shown as SEQ ID NO 1.
  • the clone was deposited at The National Collections of Industrial and Marine Bacteria (Scotland) on 13 July 1993 under the accession number NCIMB 40571.
  • PSENU2 pSENU2 (also known as clone 8S1) is a cDNA of approximately 1.2kB, encoding a mRNA of approximately 1.4 kB. The mRNA encoded by pSENU2 is expressed during the onset of senescence in tomato leaves.
  • the pSENU2 sequence exhibits 100% homology with C14 cDNA induced in unripe tomato fruit in response to low temperature.
  • DNA sequence analysis indicated that C14 mRNA encodes a polypeptide with a region that is homologous to the plant thiol proteases actinidin and papain and to animal thiol protease cathepsin H (Schaffer and Fischer, 1988, Plant Physiol, 87:431-436) .
  • a similar thiol protease gene is also expressed in pea ovaries during senescence (Granell et al, 1992, Plant J, 2:907-915) .
  • This kind of protease is also expressed during leaf senescence. This protease may play an important role in the degradation of peptides at the onset and during foliar senescence.
  • Partial sequences of pSENU2 are shown as SEQ ID NO 2 (5' end) to SEQ ID NO 4 (3' end) .
  • the proposed full sequence of SENU2 by comparison with clone C14 is shown as SEQ ID NO 5.
  • the clone pSENU2 was deposited at The National Collections of Industrial and Marine Bacteria (Scotland) on 13 July 1993 under .the accession number NCIMB 40572.
  • PSENU3 pSENU3 (also known as clone 77S3) is a cDNA of 1.1982kB, encoding a mRNA of approximately 1.4 kB. The mRNA encoded by pSENU3 is expressed during the onset of senescence in tomato leaves.
  • the pSENU3 sequence exhibits 70% homology with oryzain gamma, a cysteine proteinase expressed in rice seeds and induced by gibberellin, GA3 (Watanabe et al, 1991, J Biol Chem,
  • pSENU3 The complete sequence of pSENU3 is shown as SEQ ID NO 6.
  • the clone was deposited at The National Collections of Industrial and Marine Bacteria (Scotland) on 13 July 1993 under the accession number NCIMB 40573.
  • pSENU4 pSENU4 (also known as clone 73S7) is a cDNA of 0.525kB, encoding a mRNA of approximately 0.7kB.
  • the clone is double-stranded from base 179 to base 323; the rest is single-stranded.
  • the mRNA encoded by pSENU4 is expressed during the onset of senescence in tomato leaves .
  • the predicted amino acid sequence encoded by pSENU4 matches that originally determined by Lucas et al (1985, EMBO J, 4:2745-2749) for the tomato extracellular pathogenesis related protein P14.
  • the P14 protein contains two isomers, P4 and P6 (van Kan et al, 1992, Plant Mol Biol, 20:513-527) .
  • P6 is 15.5kD and is serologically related to the PR1 protein family of tobacco, but has not been assigned a function.
  • the pSENU4 sequence is homologous to the cDNA encoding P6 (clone LEPRP6) which has been isolated from Cladosporiu fulvum infected tomato, but pSENU4 lacks 160bp at the 5'end of the P6 cDNA.
  • An identical cDNA (clone LEP1P14A) has also been isolated from ethylene treated tissue (Vera P and Tonero P, unpublised data) which encodes a protein named PI (P14a) .
  • pSENU4 The complete sequence of pSENU4 is shown as SEQ ID NO 7.
  • the clone was deposited at The National Collections of Industrial and Marine Bacteria (Scotland) on 13 July 1993 under the accession number NCIMB 40574.
  • PSENU5 pSENU5 (also known as clone 72S3) is a cDNA of 0.847kB, encoding a mRNA of approximately 2.0kB. The mRNA encoded by pSENU5 is expressed during the onset of senescence in tomato leaves.
  • pSENU5 encodes a protein of unknown function.
  • the pSENU5 DNA sequence does not show any significant homology to sequences in publicly-available sequence databases.
  • the complete sequence of pSENU5 is shown as SEQ ID NO 8. The clone was deposited at The National Collections of Industrial and Marine Bacteria (Scotland) on 13 July 1993 under the accession number NCIMB 40575.
  • PSEND31 pSEND31 (also known as clone 1M4) is a cDNA of approximately 0.9kB, encoding a mRNA of approximately l.OkB.
  • the mRNA encoded by pSEND31 is expressed in green leaves of tomatoes plants but at the onset of senescence its expression is switched off.
  • the pSEND31 sequence exhibits 100% homology to the tomato cDNA clone TAS14 which is inducible by salt stress and ABA in tomato seedlings (Godoy et al, 1990, Plant Mol Biol, 15:695-705) . Southern analysis suggests that there is one gene per haploid genome. We now show this gene is specifically reduced during tomato leaf senescence.
  • Partial sequences of pSEND31 are shown as SEQ ID NO 9 (5' end) to SEQ ID NO 10 (towards the 3' end) .
  • the proposed full sequence of SEND31 by comparison with clone TAS14 is shown as SEQ ID NO 11.
  • the clone pSEND31 was deposited at The National Collections of Industrial and Marine Bacteria (Scotland) on 13 July 1993 under the accession number NCIMB 40576.
  • PSEND32 pSEND32 (also known as clone 8M2 ) is a cDNA of 0 . 8kB , encoding a mRNA of approximately 0 . 6kB .
  • the mRNA encoded by pSEND32 is expressed in green leaves of tomatoes plants but at the onset of senescence its expression is switched off.
  • pSEND32 encodes a protein of unknown function.
  • the pSEND32 DNA sequence does not show any significant homology to sequences in publicly-available sequence databases.
  • the sequence of pSEND32 is shown as SEQ ID NO 12.
  • the clone was deposited at The National Collections of Industrial and Marine Bacteria (Scotland) on 13 July 1993 under the accession number NCIMB 40577.
  • PSEND33 pSEND33 (also known as clone 59S3) is a cDNA of approximately 0.8kB, encoding a mRNA of approximately 0.6kB.
  • the mRNA encoded by pSEND33 is expressed in green leaves of tomatoes plants but at the onset of senescence its expression is switched off.
  • the pSEND33 sequence exhibits 60% homology to ferredoxin-1 of pea and spinach.
  • the amino acid sequence of plant ferredoxins is highly conserved. Plant leaves contain at least two distinct forms of chloroplast-type ferredoxins. Ferredoxin-1 appears more closely related to other angiosperm ferredoxins. Ferredoxin-1 is encoded by a single gene in pea (Elliott et al, 1989, Plant Cell, 1:681-690) .
  • pSEND33 may encode ferredoxin. We have now shown that expression of this gene is specifically reduced during tomato leaf senescence. The down-regulation of ferredoxin during senescence may be counteracted by overexpression of the pSEND33 gene. This may lead to prolonged photosynthesis and increased yield of plants.
  • the sequence of pSEND33 is shown as SEQ ID NO 13.
  • the clone was deposited at The National Collections of Industrial and Marine Bacteria (Scotland) on 13 July 1993 under the accession number NCIMB 40578.
  • PSEND34 pSEND34 (also known as clone 91S3) is a cDNA of 0.558kB, encoding a mRNA of approximately 0.6kB. The mRNA encoded by pSEND34 is expressed in green leaves of tomatoes plants but at the onset of senescence its expression is switched off.
  • the pSEND34 sequence exhibits 90% homology to the potato photosystem II lOkD polypeptide at both the nucleotide and the predicted amino acid level (Eckes et al, 1986, Mol Gen Genet, 205:14-22) . Homology with the same protein from spinach and Arabidopsis is about 80% (Lautner et al, 1988, J Biol Chem, 263:10077-10081; Gil-Gomez et al, 1991, Plant Mol Biol, 17:517-522) . We have now shown that this gene is specifically reduced during tomato leaf senescence.
  • PSEND35 pSEND35 (also known as clone 72S6) is a cDNA of approximately 0.7kB. The mRNA encoded by pSEND35 is expressed in green leaves of tomatoes plants but at the onset of senescence its expression is switched off.
  • pSEND35 encodes a protein of unknown function.
  • the pSEND35 DNA sequence does not show any significant homology to sequences in publicly-available sequence databases.
  • the sequence of pSEND35 is shown as SEQ ID NO 15.
  • the clone was deposited at The National Collections of Industrial and Marine Bacteria (Scotland) on 13 July 1993 under the accession number NCIMB 40580.
  • pSENE71 (also known as clone 75S3) is a cDNA of 0.6kB. The mRNA encoded by pSENE71 is expressed in green leaves of tomatoes plants and during senescence.
  • pSENE71 encodes a protein of unknown function.
  • the pSENE71 DNA sequence does not show any significant homology to sequences in publicly-available sequence databases.
  • the 5' sequence of pSENE71 is shown as SEQ ID NO 16.
  • the clone was deposited at The National Collections of Industrial and Marine Bacteria (Scotland) on 13 July 1993 under the accession number NCIMB 40581.
  • Leaf senescence after pollen shed was studied in two maize lines. Relative chlorophyll content, photosystem II efficiency (determined by analysis of chlorophyll fluorescence) and photosynthetic C0 _schreibt- fixation (measured by infrared gas analyser) declined during senescence. Statistical analysis of the fitted curves revealed that yellowing in the first line was significantly delayed compared with the second line, but the decline in photosynthesis occurred simultaneously in the two lines. Western blotting detected a transition point during senescence when pronounced quantitative and qualitative changes occurred in a number of leaf proteins. This point, coinciding with the onset of visible senescence, was delayed in the first line.
  • a cDNA library was constructed from the poly(A) RNA of leaves judged to be in the early stages of senescence and differential screening was employed to isolate senescence- related clones, which were investigated further by northern analysis. Partial sequencing, followed by comparison with all known sequences in the GenEMBL database, indicated that a number of cDNAs were related to genes of known identity, including oryzain, pyruvate, orthophosphate dikinase and ferredoxin I, while others showed similarity to cDNAs of unknown function, or did not exhibit any significant homology.
  • Non-destructive measurements of chlorophyll fluorescence and the level of greenness were made on ear leaves from two maize genotypes (X and Y) . Ear leaves were harvested at intervals from 5 days before pollen shed until 35 days after pollen shed. The data indicated a significant decrease in the chlorophyll fluorescence parameter and greenness between 20 and 25 days after pollen shed
  • RNA samples were then analyzed for protein and RNA changes.
  • Two cDNA libraries were therefore made: one from RNA extracted from leaves 10-20 days after pollen shed (initiation/early senescence) and the other from RNA extracted from leaves 25 days after pollen shed (mid senescence) .
  • Total RNA was extracted from leaves at the appropriate stages, polyA RNA was purified and cDNA made using a Pharmacia cDNA Synthesis Kit .
  • Two cDNA libraries (early and mid senescence) were constructed in lambda gtlO (average insert size 1.3 kB) .
  • Senescence-related cDNA clones were identified by differential screening, using RNA from leaves at pollen shed and RNA from the same stages as were selected to make the library to provide the necessary probes.
  • the cDNA SEE1 shows homology to genes for two thiol proteases, oryzain gamma from rice and aleurain from barley.
  • the cDNA SEE2 shows some homology to a castor bean vacuolar processing enzyme.
  • the cDNA SEE3 is identical to part of the maize pyruvate, orthophosphate dikinase mRNA while the cDNA SEE4 shows homology to maize and Silene ferredoxin mRNAs.
  • Proteases and ferredoxin are thought to play a part in chlorophyll breakdown during senescence, while pyruvate, orthophosphate dikinase has a role in gluconeogenesis, which has been suggested to occur during galactolipid breakdown in senescence.
  • the maize cDNAs represent senescence-related genes and may be used to modify the senescence process.
  • SEE1 is a senescence-enhanced cDNA clone of approximately 1.7kB, encoding a mRNA of approximately 1.2kB.
  • the mRNA encoded by SEE1 increases in abundance during maize leaf senescence.
  • SEE1 shows homology to genes for two thiol proteases: oryzain gamma from rice (GenEMBL ID code OSOZC; 77.6% identity in a 304bp overlap) and aleurain from barley (GenEMBL ID code HVLEU; 77.9% identity in a 222bp overlap) .
  • a more detailed comparison of the SEE1 sequence with the rice oryzain gamma DNA sequence shows a 78.9% identity over 1417 base pairs, with 80% homology over 607 base pairs at the 5' end and 74% homology over 665 base pairs at the 3' end.
  • the clone SEE1 may thus encode a protease. This is supported by the finding that a IkB DNA fragment is amplified by PCR when one of the primers used is derived from a region which is conserved in a range of thiol proteases.
  • SEE1 The sequence of SEE1 is shown as SEQ ID NO 17. It is 1442 base pairs in length with a single long open reading frame between bases 78 to 1160.
  • the predicted amino acid sequence suggests that the encoded protein is 360 amino acids long with a molecular weight of 39 kDa.
  • the predominantly hydrophilic protein sequence suggests that the protein is soluble.
  • the SEE1 sequence shows 85.6% identity over 355 amino acids to the thiol protease aleurain precursor and 83.7% identity over 363 amino acids to the oryzain gamma precursor.
  • SEE2 is a senescence-enhanced cDNA clone of approximately 1.3kB, encoding a mRNA of approximately 1.8kB.
  • the mRNA encoded by SEE2 increases in abundance during maize leaf senescence.
  • SEE2 shows homology to a castor bean vacuolar processing enzyme (Hara-Nishimura et al, 1993) : 67% homology over 374 base pairs at the 5' end (and at the protein level, 75% homology over 130 amino acids at the 5' end) but no homology over 431 base pairs at the 3' end.
  • SEE2 Partial sequences of SEE2 are shown as SEQ ID NO 18 (isolated using a T7 primer) and SEQ ID NO 19 (isolated using a T3 primer) .
  • the clone SEE2 was deposited at The National Collections of Industrial and Marine Bacteria (Scotland) on 13 July 1993 under the accession number NCIMB 40584.
  • SEE3 is a senescence-enhanced cDNA clone of approximately 2.5kB, encoding a mRNA of approximately 3.4kB.
  • the mRNA encoded by SEE3 increases in abundance during maize leaf senescence.
  • the SEE3 sequence exhibits 100% identity to the maize pyruvate, orthophosphate dikinase mRNA (GenEMBL ID code ZMPOD) in a 141bp overlap.
  • a more detailed comparison to the dikinase shows a 91% homology over 132 base pairs at the 5' end and an 89% homology over 262 base pairs at the 3' end.
  • SEE3 also exhibits 97.7% identity to the maize pyruvate, orthophosphate dikinase gene, exons 2-19 (GenEMBL ID code ZMPPDK2) in a 128 bp overlap.
  • SEE3 has an internal EcoRI site, and partial sequences of SEE3 are shown as SEQ ID NOs 20 to 23 (SEQ ID NO 20 being the most 5' and SEQ ID NO 23 being the 3' end) .
  • the clone SEE3 was deposited at The National Collections of Industrial and Marine Bacteria (Scotland) on 13 July 1993 under the accession number NCIMB 40570.
  • SEE4 is a senescence-enhanced cDNA clone of approximately 0.9kB, encoding a mRNA of approximately 1.2kB.
  • the mRNA encoded by SEE4 increases in abundance during maize leaf senescence.
  • the SEE4 sequence exhibits homology to the following ferredoxin mRNAs: maize ferredoxin I isoprotein mRNA, pFDl (GenEMBL ID code ZMFD1; 80.6% identity in a 170bp overlap) ; maize ferredoxin I isoprotein mRNA, pFDl' (GenEMBL ID code ZMFD1P; 80.6% identity in a 170bp overlap); maize ferredoxin isoprotein mRNA, pFD5 (GenEMBL ID code ZMFD5; 81.8% identity in a 159bp overlap) ; maize ferredoxin III isoprotein mRNA (GenEMBL ID code ZMFD3; 67.9% identity in a 140bp overlap) ; Silene pratensis mRNA for ferredoxin precursor (GenEMBL ID code SPFER1; 67.5% identity in a 120bp overlap) .
  • SEE4 The 5' sequence of SEE4 is shown as SEQ ID NO 24 and the 3' sequence as SEQ ID NO 25.
  • the clone was deposited at The National Collections of Industrial and Marine Bacteria (Scotland) on 13 July 1993 under the accession number NCIMB 40583.
  • SEE5 (pl6.1) shows homology to a maize catalase (Abler and Scandalios, unpublished) : 87% over 79 base pairs at the 3' end.
  • the 3' sequence of SEE5 is shown as SEQ ID NO 26.
  • SEE6 shows homology to a maize cab-1 (Sullivan et al, 1989) : 97% over 270 base pairs at the 5' end and 98% over 349 base pairs at the 3'end.
  • the 5' sequence of SEE6 is shown as SEQ ID NO 27 and the 3' sequence as SEQ ID NO 28.
  • SEE7 shows homology to a maize GRP (Didierjean et al, 1992) : 61% over 198 base pairs at the 5' end but no homology over 450 base pairs at the 3' end.
  • the 5' sequence of SEE7 is shown as SEQ ID NO 28 and the 3' sequence as SEQ ID NO 30.
  • SEE8 (pl6.6) shows no homology to known sequences over 388 base pairs at the 5' end.
  • the partial sequence of SEE8 is shown as SEQ ID NO 31 (isolated with the T3 primer) .
  • SEE9 (pl6.11) shows no homology to known sequences over 209 base pairs at the 5' end and over 390 base pairs at the 3' end. Partial sequences of SEE9 are is shown as SEQ ID NO 32
  • SEC1 (pl6.7) shows homology to an Arabidopsis thaliana PSl type III cab (Wang, Zhang and Goodman, unpublished) : 79% homology over 136 base pairs at the 5' end.
  • the partial sequence of SEC1 is shown as SEQ ID NO 34 (isolated with the T3 primer) .
  • SEC2 (pl6.9) shows no homology to known sequences over 151 base pairs at the 5' end and over 385 base pairs at the 3' end.
  • the 5' sequence of SEC2 is shown as SEQ ID NO 35 and the 3' sequence as SEQ ID NO 36.
  • SED1 (pl6.13) shows homology to an Arabidopsis thaliana AP52 ATP sulfurylase (Leustek, unpublished) : 62% homology over 282 base pairs at the 5' end but no homology over 444 base pairs at the 3' end.
  • the 5' sequence of SED1 is shown as SEQ ID NO 37 and the 3' sequence as SEQ ID NO 38.
  • SED2 (p20.3) shows homology to an Arabidopsis thaliana cDNA 20D6T7 (Newman, unpublished) : 68% over 131 base pairs at the 3' end (and at the protein level, 85% over 26 amino acids at the 3' end) .
  • the partial sequence of SED2 is shown as SEQ ID NO 39 (isolated with the T7 primer) .
  • a vector is constructed using sequences corresponding to a restriction fragment obtained from a senescence-related cDNA clone described in Example 1 or 2 and is cloned into the vectors GA643 (An et al, 1988, Plant Molecular Biology Manual A3: 1-19) or pDH51 (Pietrzak et al, 1986, Nucleic Acids Research, 14:5875-5869) which has previously been cut with a compatible restriction enzyme (s) .
  • GA643 Al, 1988, Plant Molecular Biology Manual A3: 1-19
  • pDH51 Pieris factor-related clone
  • s a compatible restriction enzyme
  • EXAMPLE 4 Construction of antisense RNA vectors with the polygalacturonase promoter.
  • pJR3 is a Binl9 based vector, which permits the expression of the antisense RNA under the control of the tomato polygalacturonase promoter.
  • This vector includes approximately 5 kb of promoter sequence and 1.8 kb of 3' sequence from the PG promoter separated by a multiple cloning site.
  • the fragment of senescence-related cDNA described in Example 3 is also cloned into the vectors described in Example 3 in the sense orientation.
  • the vectors with the sense orientation of senescence-related sequence are identified by DNA sequence analysis.
  • EXAMPLE 6 Construction of sense RNA vectors with the polygalacturonase promoter.
  • the fragment of senescence-related cDNA that was described in Example 3 is also cloned into the vector pJR3 in the sense orientation.
  • the vectors with the sense orientation of senescence-related sequence are identified by DNA sequence analysis.
  • EXAMPLE 7 Construction of an over-expression vector using the CaMV35S promoter.
  • Vectors are transferred to A ⁇ robacterium tumefaciens LBA4404 (a micro-organism widely available to plant biotechnologists) and are used to transform tomato plants.
  • Transformation of tomato cotyledons follows standard protocols (e.g. Bird et al Plant Molecular Biology 11, 651-662, 1988) . Transformed plants are identified by their ability to grow on media containing the antibiotic kanamycin. Plants are regenerated and grown to maturity.
  • Plants are analysed for modifications to their senescence characteristics.
  • senescence-related sequence encoded by SEEl is incorporated into DNA constructs in the sense orientation and under the control of (a) the ubiquitin promoter and (b) the GST II promoter. Similar constructs are made using the SEE2 sequence. These constructs are used to transform maize.
  • TCATGGCCAT ATGCCACATG AATCAACCAA CTTCTCAAGT AGCACAAGCA TGGTCCATAG 300 TGATGGTGGT TATGGTAGTG GAATGAACCA ATCATCCCAC GCCCATATGT CGTCCATGGC 360
  • CTACTGCTAT CTTAATTTAA ATTATCTATG TATCTGCTTT ATCATTGACA AATGATGAAT 660
  • CTTCCATCTG TACTTGGTTT CAACATTAAT TAAAAAAAAG GACTATCTTC TGTACCTTTC 960
  • CTCCTTCGCT CGCTTTGCTA TCAGGCATCG GAAAAGGTAT GACTCCGTTG AAGAGATCAA 240
  • TACTCCCTAC TGGCTCATAA AGAACTCATG GGGAGCAGAT TGGGGTGAGG ATGGATACTT 1020
  • AAAGTGTAGA CATTATACTC AAGTAGTCTG GCGCAACTCA GTCCGACTAG GTTGTGGTCG 240
  • AAAAAAGAAT TTTTGATCAA AGATGGCACA ATACGGCAAT CAAGACCAAA TGCGCAAGAC 60
  • AAAAAAGAAT TTTTGATCAA AGATGGCACA ATACGGCAAT CAAGACCAAA TGCGCAAGAC 60
  • GAAGATAATG GAGAAGATGC CTGGACAACA TGAAGGTGAG TATGGACAAA CAACAGGTGA 360
  • ATACAGTCCC ATCTACAACA CGGATGAATG GTCTCCAAGT GGTGATGTCT ATGTTGGAGG 300
  • GGT 423 (2) INFORMATION FOR SEQ ID NO: 19:
  • CAACTGCAAA GCAGCAAGCT CTACTCTTCT TCTGTACTGA ACGTGTGACT AGATAACAAT 60
  • CAAGTCCTCC TTCGCGGGCT CCCGGCTCCC TTCGGCCACG CGCACCACCA CCCCGTCGTC 120

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Abstract

On peut transformer des plantes avec un assemblage d'ADN adapté pour modifier l'expression d'au moins un gène relatif à la sénescence. On choisit ensuite des plantes ayant des caractéristiques de sénescence modifiées. Sont ici décrits les gènes spécifiques de plantes relatifs à la sénescence, leur incorporation dans des assemblages d'ADN ainsi que leur utilisation pour inhiber ou accélérer la sénescence des plantes.
PCT/GB1994/001990 1993-09-13 1994-09-13 Regulation de la senescence WO1995007993A1 (fr)

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EP0804066A4 (fr) * 1995-03-29 1999-01-27 Wisconsin Alumni Res Found Plantes transgeniques aux caracteristiques de senescence modifiees
WO1996037613A1 (fr) * 1995-05-22 1996-11-28 Germinal Holdings Limited Gene controlant la senescence chez les plantes
WO1997041242A1 (fr) * 1996-04-18 1997-11-06 Geron Corporation Element transcriptionnel sensible a la senescence
WO1998011228A3 (fr) * 1996-09-10 1998-04-23 Zeneca Ltd Modulation genetique du murissement de fruits
US7910326B2 (en) 1996-09-11 2011-03-22 Arborgen, Inc. Materials and methods for the modification of plant lignin content
US5952486A (en) * 1996-09-11 1999-09-14 Genesis Research & Development Corporation Limited Materials and methods for the modification of plant lignin content
WO1998011205A3 (fr) * 1996-09-11 1998-08-20 Genesis Res & Dev Corp Ltd Materiaux et procedes permettant de modifier le contenu de plantes en lignine
US6204434B1 (en) 1996-09-11 2001-03-20 Genesis Research & Development Corporation Limited Materials and methods for the modification of plant lignin content
US7087426B2 (en) 1996-09-11 2006-08-08 Agrigenesis Biosciences Ltd. Materials and methods for the modification of plant lignin content
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AU696417B2 (en) 1998-09-10
GB9318927D0 (en) 1993-10-27
AU7619494A (en) 1995-04-03
EP0719341A1 (fr) 1996-07-03
CA2172842A1 (fr) 1995-03-23

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