WO2000070061A2 - Promoteurs lies a la senescence - Google Patents

Promoteurs lies a la senescence Download PDF

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Publication number
WO2000070061A2
WO2000070061A2 PCT/GB2000/001849 GB0001849W WO0070061A2 WO 2000070061 A2 WO2000070061 A2 WO 2000070061A2 GB 0001849 W GB0001849 W GB 0001849W WO 0070061 A2 WO0070061 A2 WO 0070061A2
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Prior art keywords
promoter
plant material
senescence
dna construct
transcriptional initiation
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PCT/GB2000/001849
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WO2000070061A3 (fr
Inventor
Howard Thomas
Iain Simon Donnison
Paul Russell Henry Robson
Catherine Margaret Griffiths
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Institute Of Grassland & Environmental Research
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Priority to AU45971/00A priority Critical patent/AU4597100A/en
Publication of WO2000070061A2 publication Critical patent/WO2000070061A2/fr
Publication of WO2000070061A3 publication Critical patent/WO2000070061A3/fr

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    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
    • C12N9/6405—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals not being snakes
    • C12N9/641—Cysteine endopeptidases (3.4.22)
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/8223—Vegetative tissue-specific promoters
    • C12N15/8225—Leaf-specific, e.g. including petioles, stomata
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/823—Reproductive tissue-specific promoters
    • C12N15/8235—Fruit-specific
    • 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
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance

Definitions

  • the present invention relates to promoters associated with plant senescence and uses thereof .
  • a promoter may be defined as a region of DNA involved in binding of RNA polymerase to initiate transcription.
  • Plant senescence may be defined as the sequence of biochemical and physiological events comprising the final stage of plant development.
  • the changes taking place m a plant (that: is in the whole plant or in plant material) during senescence form a genetically programmed sequence, with close co-ordination at a cellular and tissue level .
  • Plant senescence may be caused by a variety of external factors or physical constraints (such as light flux, shading, day length, temperature, water/mineral relations, stress, pathogen attack etc.) or internal factors (such as light, space, nutrients, flowering/pollination, reproduction, and plant growth regulators (such as cytokinins, giberellins, auxins, ethylene, abscissic acid, jasmonates)) .
  • 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.
  • Plant senescence-related genes a senescence-related gene being a gene involved m the process of senescence
  • Housekeeping genes which control the primary metabolic activities of viable cells, for example, respiration, ribosomal RNA synthesis d protein synthesis.
  • Genes expressed early whose effects become apparent later. Examples include homeotic genes, which may influence all subsequent gene expression by defining which parts of the genome can be accessed, and genes which encode mRNAs or proteins that become active later in the life of a leaf, for example, vacuolar enzymes, and zymogens .
  • RNAs or proteins induced de novo or showing enhanced expression during senescence for example, enzymes of pigment breakdown. These genes may be classified as "consequential" .
  • Plant senescence-related genes provide the most direct evidence that senescence is controlled by gene expression. However, plant senescence-related genes that have been isolated so far all appear to be members of classes 5 and 6 above. Thus these genes seem to encode products with a role in the symptoms of plant senescence (classes 5 and 6) rather than the control of plant senescence initiation (class 4) .
  • PCT patent application O95/07993 describes a DNA construct adapted to modify tne expression of at least one gene comprising a DNA sequence corresponding to at least part of a plant senescence-related gene preceded by a transcriptional initiation region operative m plants.
  • Such aims of the present invention can be achieved by modifying the expression of at least one gene or coding region by use of a plant senescence enhanced promoter.
  • novel promoter or transcriptional initiation regions have been isolated and characterised from maize (including Zea mays) .
  • Such isolated promoters are useful m tne modification of gene expression during senescence of plant material .
  • the present invention accordingly provides a promoter or transcriptional initiation region of a plant genome that is capable of modifying gene expression during senescence of plant material, said promoter or transcriptional initiation region being obtainable from any of sequence ID Nos 1 to 6.
  • Sequence ID No 1 is a sequence from the promoter region upstream of the senescence-enhanced Seel gene isolated from Zea mays .
  • Sequence ID No 2 is a first version of a sequence from the promoter region upstream of the senescence-enhanced See2a gene isolated from maize.
  • Sequence ID No 3 is a second variation of a sequence from the promoter region upstream of the senescence-enhanced See2a gene isolated from maize.
  • Sequence ID No .4 is a sequence from the promoter region upstream of the senescence-enhanced See2b gene isolated from maize .
  • Sequence ID No. 5 is a sequence from the promoter region upstream of the senescence-enhanced See5 gene isolated from Zea mays .
  • Sequence ID No. 6 is a sequence from the promoter region upstream of the senescence-enhanced See ⁇ ' gene isolated from Zea mays .
  • the DNA and deduced protein sequence of ID No . 5 are homologous to receptor kinases induced by plant pathogens or fungal elicitors.
  • the DNA and deduced protein sequence of ID No. 6 are homologous to sugar transporters .
  • the present invention further provides a DNA construct adapted to modify the expression of a gene or coding region during senescence of plant material, said DNA construct being obtainable from any of sequence ID Nos 1 to 6.
  • a promoter or transcriptional initiation region of a plant genome obtainable from any of sequence ID Nos 1 to 6 , for use in modifying gene expression during senescence of plant material.
  • the present invention further provides a DNA construct obtainable from any of sequence ID Nos 1 to 6, for use in modifying gene expression during senescence of plant material .
  • a method for producing plant material having modified gene expression during senescence comprises transformation of the plant material with the promoter or transcriptional initiation region and/or the DNA construct according to the present invention and subsequent selection of plant material m which gene expression has been modified
  • Transformation of the plant material may be carried out using standard procedures. Exemplary procedures include, transformation using the Ti plas ⁇ d of Agrobacterium tumefaci ens or direct gene transfer using, for example, a biolistic gun. The method chosen will vary according to the plant to be transformed.
  • the modification of plant material gene expression may be achieved using known techniques. For example, it is known for the expression of either specific antisense RNA or partial sense RNA to be utilised to reduce tne expression of various target genes m plant material. These techniques, which involve the incorporation of a synthetic gene designed to express either antisense or partial sense R ⁇ A into the genome of the plant material, have been successfully used to down-regulate the expression of a range of individual genes.
  • genes to increase the expression of a target gene have also been developed.
  • additional genes designed to express R ⁇ A containing the complete coding region of the target gene may be incorporated into the genome of the plant material to "over-express" the gene product.
  • Various other methods to modify gene expression are known, for example, the use of alternative regulatory sequences.
  • the expression of genes, or each gene, or coding region may be either decreased or increased depending on the characteristics desired for the modified plant material .
  • “Antisense” or “partial sense” , or other techniques may be used to decrease gene expression.
  • gene expression may be increased, for example, by incorporation of additional genes.
  • the additional genes may be designed to give either the same, or different spatial and temporal patterns of expression m the plant .
  • Plant senescence may ne inhibited by inhibiting a gene which is normally activated during senescence Additionally or alternatively, senescence may D ⁇ inhibited by increasing the expression of a gene which is normally down-regulated during senescence .
  • senescence characteristics of plant material may be substantially delayed or slowed by transforming the plant material with a promoter or transcriptional initiation region and/or a DNA construct according to the present invention to preferably enhance expression of strong senescence antagonists.
  • the present invention further provides a promoter or transcriptional initiation region or a DNA construct according to the present invention, for use m substantially delaying senescence characteristics of plant material .
  • the promoter or transcriptional initiation region or DNA construct according to the present invention may be used for modifying expression of strong senescence antagonists.
  • Plant 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 .
  • plant 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.
  • a DNA construct according to the present 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 5' to 3 ' direction) is capable cf pairing with the corresponding base (G with C and A with U) in the mRNA sequence read in the 5 ' to 3' direction.
  • 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 senescence constructs may be used to inhibit gene expression or to over-express an enzyme.
  • the DNA construct and/or the promoter or transcriptional initiation region according to the invention may be inserted into any appropriate or suitable plant material to regulate the expression of one or more genes or coding regions.
  • Suitable plant material includes material of dicotyledonous or monocotyledonous plants.
  • expression of the target gene may be increased or decreased, either throughout or at particular stages in the life of the plant.
  • These promoters and DNA constructs may be particularly responsive to plant senescence related events and conditions .
  • 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.
  • 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 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 material gene. Constructs expressing sense RNA encode RNA with a base sequence which is homologous to part, or all, of the sequence of mRNA. In constructs which express the functional gene product, the whole of the coding region of the gene is linked to transcriptional control sequences capable of expression in plant material .
  • Recombinant DNA constructs according to the present invention may be made using standard techniques. For example, a suitable vector containing the desired base sequence for transcription is treated with restriction enzymes to cut the sequence out. The DNA strand obtained is cloned (if desired, in reverse orientation) into a second vector containing the desired promoter sequence (typically including at least 10% of any of sequence Nos ID1 , ID2 , ID3 , ID4 , ID5 and ID6) and the desired terminator sequence . Plant material senescence-related gene expression (and hence senescence characteristics) may be modified to a greater or lesser degree by controlling the extent of sense or antisense mRNA production in the plant cells. This may be done by a 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 promoter or transcriptional region and the DNA construct according to the present invention have many applications, the preferred applications are detailed below.
  • a method for substantially delaying senescence characteristics of plant material by transforming the plant material with a promoter or transcriptional initiation region and/or a DNA construct according to the present invention, to enhance expression of an IPT gene.
  • a further application of the present invention involves prevention of protease expression during plant material senescence .
  • proteases which display an enhanced level of expression during senescence, are also expressed at other stages of the plant material growth cycle and may have an important role to play m plant development .
  • antisense or sense protease sequences may be expressed from a senescence-enhanced promoter.
  • a method to substantially prevent protease expression during senescence of plant material comprises expression of antisense or sense protease sequences under the control of a promoter or transcriptional initiation region and/or a DNA construct according to the present invention.
  • a promoter or transcriptional initiation region or a DNA construct according to the present invention, for use m expression of antisense or sense protease sequences .
  • Another application of the present invention involves transgenic approaches to delay fruit ripening.
  • the use of a senescence-enhanced transgene would target the expression of the products of these transgenes to the specific period of plant development during which ripening occurs (that is during plant material senescence) and thereby reduce the potential for adverse effects during early stages of plant growth.
  • a method for substantially delaying fruit ripening comprises transforming plant material with a transgene capable of substantially delaying fruit ripening.
  • the transgene is preferably under the control of a promoter or transcriptional initiation region and/or a DNA construct according to the present invention.
  • a promoter or transcriptional initiation region for use in substantially delaying fruit ripening.
  • a number of crops, particularly those mechanically harvested require a period of extensive dry-down in order to facilitate harvesting.
  • the expression of abscisic acid or other plant products, during plant material senescence may facilitate the dry-down process, allowing crops to be harvested earlier and with greater efficiency than would otherwise be possible.
  • One example is the maturation of cotton which is currently achieved by spraying with environmentally recalcitrant ethylene derivatives. If these ethylene derivatives were expressed by a senescence-enhanced transgene, it may decrease environmental damage and increase crop value .
  • a method for substantially enhancing senescence characteristics of plant material comprises transforming the plant material with a promoter or transcriptional initiation region and/or a DNA construct according to the present invention, to preferably enhance expression of at least one plant growth regulator.
  • a promoter or transcriptional initiation region, or a DNA construct according to the present invention for use m substantially enhancing senescence characteristics of plant material.
  • the promoter or transcriptional initiation region or the DNA construct may be used for modifying expression of at least one plant growth regulator.
  • Modification of gene expression during senescence of plant material may also be useful for the harvesting process of plant material.
  • the production of silage involves both the breakdown of cell walls and of proteins.
  • the breakdown of cell walls increases nutritional value, whereas the breakdown of proteins decreases nutritional value.
  • Preferential inhibition of proteolysis in silage would lead to an increase in nutritional value while allowing the breakdown of cellulose to continue.
  • Post harvest chemical treatment of crops include spraying with anti-spoilage compounds.
  • Expressing protease inhibitors in senescing tissue may advantageously decrease spoilage without affecting plant growth and development and furthermore reduce the need for inefficient external application.
  • a method for substantially inhibiting proteolysis in silage comprises transforming the plant material with a promoter or transcriptional initiation region and/or a DNA construct according to the present invention, to enhance expression of at least one protease inhibitor during senescence of the plant material .
  • promoter or transcriptional initiation region or a DNA construct according to the present invention for use in modifying expression of protease inhibitors .
  • plant material may be transformed with at least one transgene, the transgene being under the control of a promoter or transcriptional initiation region and/or a DNA construct according to the present invention, such that a compound expressed by the transgene is expressed during senescence of the plant material .
  • a promoter or transcriptional initiation region for use in transforming the plant material with a transgene, the transgene being under the control of the promoter or transcriptional initiation region, or the DNA construct.
  • a further application of the present invention would be in the expression of pesticidal genes during plant senescence.
  • Some plants accumulate pesticidal products as they mature; however, for others a reduction in vigour associated with maturation results in susceptibility to insect infestation. Additionally, conventional insecticide application becomes less effective as crop canopies mature and become closed.
  • These problems in controlling economically important insect pests may be addressed by the transgenic expression of insecticidal compounds regulated by senescence-enhanced promoters.
  • Other crop pests including insects and fungus may also be targeted towards the end of the plant's growth cycle, thereby allowing resistance to be enhanced before harvesting and storage.
  • a method for enhancing expression of pesticidal genes during senescence of plant material comprises transforming the plant material with a promoter or transcriptional initiation region, and/or a DNA construct according to the present invention, to enhance expression of at least one pesticidal gene.
  • a preferred feature of the present invention includes transforming the plant material with a transgene capable of expressing a pesticidal compound.
  • the transgene is preferably under the control of a promoter or transcriptional initiation region and/or a DNA construct according to the present invention, such that the pesticidal compound may be produced during senescence of the plant material.
  • the term "pesticidal” relates to insecticidal and fungicidal genes/compounds .
  • a promoter or transcriptional initiation region or a DNA construct according to the present invention, for use in modifying expression of pesticidal genes.
  • the above method may also be applied to the temporal expression of chemical products.
  • Plants are used as a source of food and also as a direct source of many chemical products.
  • the accumulation of chemical products early in a crop's growth cycle may have a deleterious effect on overall yield.
  • the metabolic load required to express high levels of a chemical product in young plants may significantly divert resources away from essential processes such as growth, resulting in an overall decrease in product quantity.
  • the product may be toxic.
  • a method for the temporal expression of chemical product genes during senescence of plant material comprises transforming the plant material with a promoter or transcriptional initiation region, and/or a DNA construct according to the present invention, to enhance expression of at least one chemical product gene during senescence.
  • a promoter or transcriptional initiation region, or a DNA construct according to the present invention for use m the temporal expression of at least one chemical product gene.
  • a method to substantially modify stress responses m plant material which method comprises expression of See2 under stress conditions .
  • Another embodiment of the present invention includes a polypeptide obtainable from expression of a promoter or transcriptional initiation region and/or a DNA construct according to the present invention.
  • DNA molecule including a promoter or transcriptional initiation region, and/or a DNA construct according to the present invention.
  • a plant cell and/or a transgenic plant containing a promoter or transcriptional initiation region and/or a DNA construct according to the present invention.
  • some of the further advantages of being able to alter a gene or coding region during plant senescence include: a) Prolonged life of the whole, or part, of the plant due to delayed or slowed senescence. b) Increased yield due to delayed senescence of the leaf and resulting prolonged photosynthetic activity. c) Increased protein content of fruits and vegetables due to reduced rate of protein breakdown. d) Improved quality of leafy vegetables due to the reduced rate of senescence . e) Improved disease tolerance due to the presence of less senescing tissue. f) Improved tolerance of drought and other stresses. g) Improved storage life of harvested plant or plant material.
  • delaying senescence m gram can have various beneficial effects on crop phenotype .
  • the resulting increase m leaf area (increased duration of green leaf area) and rise m photosynthetic capacity during gram-fillmg results m increased yield.
  • Improved stalk integrity due to delayed senescence m stalk tissue) increases resistance to pathogenic organisms and allows improved harvesting, yield is again increased.
  • Delaying senescence early-maturmg silage, maize or sorghum will maintain leaf integrity and greenness throughout the continuing growing season to increase the crop's overall biomass.
  • Figure 1 shows sequence alignment of the predicted Seel polypeptide with other sequences of the same family ( this case, the oryzam y/ aleurain family) of cysteme protemases;
  • Figure 2 is a dendrogram illustrating the degree of sequence similarity between different cysteme protemases expressed m leaf senescence and/or germinating seeds;
  • Figure 3 shows Northern analysis using RNA from maize leaves and germinating seeds hybridised to cDNA probe Seel ;
  • Figure 4 shows sequence alignment of the predicted See2 polypeptides with other sequences of the legumam/hemoglobmase family of cysteme proteases;
  • Figure 5 is a dendrogram illustrating the degree of sequence similarity between different legumain-like cysteine proteases;
  • Figure 6 shows Northern analysis using RNA from maize leaves and germinating seeds hybridised to See2 cDNA probe
  • Figure 7 is an illustrative map showing positions of /the promoter sequences relative to the See genes.
  • Senescence-enhanced cDNAs have been isolated from a range of species including tomato, radish, cucumber, Arabidopsis , Brassica, Petunia, barley and maize.
  • the identity of the cloned sequences may be inferred from their similarity to known sequences in some cases, for example, cysteine proteinases have been found in Arabidopsis (SAG2 and SAG12) , Petunia (P21) , maize ( Seel and See2 , ) and tomato (SENU2 and SENU3) .
  • Seel The further characterisation of Seel , including sequence analysis, mRNA expression pattern (m natural senescence, germination, chilling, regreen g, dark-induced senescence and nutrient or water stress) , gene copy number and chromosome location oy RFLP mapping is detailed below.
  • the senescence-enhanced cDNA Seel was isolated by differential screening of a - ⁇ gtlO cDNA library from early senescing maize leaves 12-20 days after pollen shed (DAPS) with probes made from cDNA of 0 DAPS and 16 DAPS Complete DNA sequencing of Seel revealed an open reading frame capable of encoding a polypeptide of 360 ammo acids, with a molecular weight of 39kDa.
  • the first lane of Figure 1 shows the 360 ammo acid polypeptide sequence encoded by the maize Seel gene isolated.
  • Figure 1 further shows sequence alignment of the predicted Seel polypeptide with other sequences of the same family (the oryzam y/aleuram family) of cysteme proteinases.
  • the solid triangle (3) indicates the likely site of cleavage to generate the mature protein.
  • conserveed residues around the cysteme, histidme and asparagme triad (open circles (4)) of this family of cysteine proteinases are shown by the dotted lines (5) .
  • the proteins were aligned using the PILEUP program of the Wisconsin Genetics Computer Group software package and are displayed using PRETTYBOX .
  • Table 1 shows DNA and protein sequence homologies between cysteine proteinases expressed in leaf senescence and/or germinating seeds.
  • the figures in parentheses show the length of DNA sequence homology in base pairs or the length of protein sequence homology in amino acids. The relevant references are written in square brackets.
  • Figure 2 is a dendrogram compiled using the results from Table 1.
  • the dendrogram illustrates the degree of sequence similarity between different cysteine proteinases expressed in leaf senescence and/or germinating seeds.
  • Table 1 and Figure 2 she * .-; that the senescence-related tomato and Petunia sequences belong to the same family as Seel , and are thus more similar to Seel than other monocot cysteine proteinases, such as oryzain ⁇ and ⁇ . Senescence-related proteinases more similar to oryzains ⁇ and ⁇ do exist, as the position of the Arabidopsi s SAG12 sequence in Figure 2 illustrates.
  • the maize CCP1 proteinase which is upregulated during germination, is a member of yet another cysteine proteinase family.
  • CCP2 cysteine proteinase
  • Figure 3 shows Northern analysis using the extracted RNA. Each lane contained lO ⁇ g of total RNA which was hybridised at high stringency to the cDNA probe Seel .
  • Figure 3a shows Northern analysis of naturally- senescing leaves of mature LS (later senescence) plants.
  • Lanes 1 to 10 show RNA extracted at 5 -day intervals from the ear leaves of mature LS plants -5 to +40 days after pollen shed (DAPS) .
  • DAPS pollen shed
  • a peak in the level of the 1.4kb message can be seen at 35 DAPS (lane 9 of Figure 3a) , approximately 10 days after the start of visible senescence as measured by a major drop in relative chlorophyll content.
  • Lanes 11 to 14 of Figure 3b show RNA extracted from LS seedling leaf 6 (lane 11) , leaf 5 (lane 12) , leaf 4 (lane 13) and leaf 3 (lane 14) at a single date of harvesting, when leaf 3 was visibly yellowing while leaf 6 was still green.
  • Figure 3b shows that the level of Seel mRNA increases the older the seedling leaf. The level of Seel mRNA is highest in the older seedling leaf 3 (lane 14 of Figure 3b) .
  • Table 1 showed that Seel had a high degree of similarity to cysteine proteinases expressed in germinating seeds (maize CCP2 , rice oryzain ⁇ ) . Therefore, the pattern of expression of Seel was checked using RNA extracted from LS seedlings. Lanes 15 to 17 of Figure 3b show RNA extracted from LS maize seeds 1 day (lane 15) , 3 days (lane 16) and 5 days (lane 17) after germination. The Seel message level peaks at 3 days after germination (lane 16 of Figure 3b) .
  • the level of Seel mRNA was also investigated in mature maize plants of two additional genotypes, CT (chilling- tolerant ) and CS (chilling-sensitive; plants, which are respectively more and less chilling tolerant than LS .
  • CT chilling- tolerant
  • CS chilling-sensitive; plants, which are respectively more and less chilling tolerant than LS .
  • Lanes 18 to 19 of Figure 3c show RNA from the ear leaf of mature CT plants before (lane 18) and after (lane 19) 12 days of severe chilling.
  • Lanes 20 to 21 of Figure 3c show RNA from the ear leaf of mature CS plants before (lane 20) and after (lane 21) 12 days of severe chilling.
  • the pronounced greyish-white lesions and brown patches of chilling-sensitive plants such as CS appear to resemble visually the hypersensitive response to pathogen attack, more than natural senescence.
  • the hypersensitive response is a form of plant programmed cell death.
  • the data presented on Seel mRNA levels in the maize line CS provide evidence that the Seel gene, which is up-regulated in natural senescence, is down-regulated when more sudden cell death occurs.
  • the level of Seel mRNA was also studied in LS seedlings subjected to nutrient or water stress under high light conditions.
  • Six seeds per 15cm diameter pot were germinated, grown for 20 days at a temperature of 25°C (l ⁇ h day, PAR>900 ⁇ mol ⁇ r 2 s "1 ) , 22°C (night) in sand or soil and watered daily with full -strength Hoagland's solution, deionised water (nutrient stress) , tap water or no water for the last 8 days (water stress).
  • Leaves of the sand-grown seedlings given Hoagland's solution were broad and green while leaves of seedlings watered with deionised water were thinner, shorter and less green (Relative chlorophyll contents for leaf 3 were 22.8 and 17.6, respectively) .
  • leaf 3 was generally the oldest living leaf of a four- or five-leaf seedling
  • Lanes 29 to 32 of Figure 3d show RNA extracted from leaf 3 of the LS seedlings grown under the above growth conditions.
  • the growth media and daily watering solutions were sand/full-strength Hoagland ' s solution (lane 29), sand/deionised water (lane 30) , soil/tap water (lane 31) and soil/no water for last 8 days of experiment (lane 32) .
  • the level of Seel mRNA m leaf 3 of the seedlings given different treatments was much higher naturally-senescmg seedlings (sand/Hoagland ' s , lane 29; soil/tap water, lane 31, Figure 3d) than m stressed seedlings (sand/deionised water, lane 30; soil/drought, lane 32, Figure 3d) .
  • results of northern analysis may be summarised as follows. Leaves of naturally-senescmg seedlings or mature maize plants contain more Seel mRNA than younger leaves There is also a transient peak m Seel mRNA during seedling germination. Seedlings and mature plants of chilling-tolerant maize lines contain more Seel mRNA m their leaves during chilling treatments than is found m chillmg-sensitive maize. Regreenmg, detachment and dark- incubation, nutrient stress and drought all result in a decrease m Seel mRNA.
  • Both these proteases comprising the senescence-ennanced See2 m maize and the ⁇ penmg-upregulated Citvac m Citrus, have strong DNA and ammo-acid sequence homology to vacuolar processing enzymes expressed m maturing seeds of castor bean, soybean and Arabidopsi s thaliana and germinating seedlings of castorbean, soybean, pumpkin, vetch, j ackbean and soybean.
  • a partial See2 clone was isolated by differential screening of a ⁇ gtlO cDNA library made from early senescing maize leaves 12-20 days after pollen shed (DAPS) using 3 P labelled cDNA from 0 DAPS and 16 DAPS as probes. Although full length cDNAs of other genes were isolated from a maize senescent leaf cDNA library, all versions of See2 isolated appeared truncated on comparison to homologous sequences from other species. However, during the analysis of a senescence-enriched cDNA population, a 255 bp Dpnll fragment was isolated which, like See2, was homologous to the legumam family of proteases.
  • the senescence-enriched cDNA population was generated by a subtractive hybridisation using cDNA, after reverse transcription from mRNA, isolated from green and yellowing maize leaves.
  • the subtraction method was a modification of representational difference analysis (RDA) for cDNA analysis.
  • the subtracted clone (dd8) which was not homologous to the existing See2 clone, was similar to the 5 ' region of other legumam type genes which were homologous at the 3 ' end to the version of See2 originally isolated.
  • dd8 cDNA was labelled with 32 P and used to probe a maize genomic library ⁇ FixII (Stratagene) .
  • Primers were designed downstream of the putative TATA box for each version of See2 , and "full-length" cDNA copies of each sequence amplified by PCR using these and one other primer to the 3' end of the gene. These PCR fragments were cloned mto vector pGEM-T Easy (Promega) and sequenced in both directions as described above .
  • the See2a gene is predicted to encode a polypeptide of 408 amino-acids with a molecular mass of 45 kDa, whilst the predicted polypeptide of See2b is two amino-acids longer and has approximately the same mass.
  • Figure 4 shows sequence alignment of the two predicted See2 polypeptides with other sequences of the legumain/ hemoglobinase family of cysteine proteases. Black shading shows identical amino acids while grey shading indicates preferred amino acids and conservative substitutions.
  • Figure 5 is a dendrogram illustrating the degree of sequence similarity between different legumain-like cysteine proteases.
  • Both the DNA sequence and the predicted amino acid sequence of See2a and See2b show a strong homology to the legumain/haemoglobinase group of cysteine proteases.
  • This group of proteases include vacuolar processing enzymes expressed in maturing seeds of castor bean, soybean, and Arabidopsi s thaliana, germinating seedlings of castorbean, soybean, pumpkin and vetch; ripening fruit of Citrus and hemoglobinase in human and human parasites.
  • rice (C24770) and apple (At000252) partial DNA sequences in the EST data libraries which show strong homology, albeit over their limited lengths.
  • the two See2 DNA sequences and deduced amino acid sequences are most similar to Citvac a legumain isolated from the flavedo (outer coloured part of the fruit peel) of Ci trus fruits.
  • the DNA sequences of Citvac and See2a and See2b are 69% and 70% identical, respectively.
  • the ammo acid sequences of Citvac and See2a and See2b are 84% and 85% similar, and 72% and 73% identical, respectively.
  • See2 was cloned from a differential screen and a cDNA subtraction as a leaf senescence up-regulated message, and this has been subsequently confirmed by northern analysis. See2 is therefore the only legumam-like sequence to be implicated at this developmental stage, however a similarity between leaf senescence and seed germination has already been reported for Seel . The necessity m both these stages of development to mobilise resources and m particular protein is thought to explain this. Moreover legumams have also been isolated from ripening Citrus and developing apple fruits and this is another example of the links between leaf senescence and fruit ripening.
  • the most notable feature of the two See2 genes is that the predicted ammo-acid sequence is truncated by at least 55 amino-acids compared to all known 5' sequences for legumam-like sequences including from human and human parasites, and 66 ammo acids from all 5 ' sequences for plant legumams . 5 ' untranslated regions have also been isolated for the two See genes so that this truncation is not due to incomplete sequence data. Moreover, the 30 bases immediately upstream of the putative translation start site m maize is homologous to the comparable, but translated, region other legumams. This homology stops at the equivalent of the 1st mtron/ 2nd exon junction of Arabidopsis genomic clones.
  • the See2 predicted amino acid sequences of maize are not only shorter than other legumains but also lack the signal sequence which, in plants, targets the protease to the vacuole, and a conserved active site not only shared in all legumains but many other cysteine proteases.
  • One other putative active site is conserved in See2s and all other legumains, and one of two potential glycosylation sites described in Citvac is also conserved in both See2s .
  • the protein sequences are remarkably well conserved. In other words, it is highly likely that See2 has a similar functional role to legumains.
  • the antibody to See2 detects a band of approximately 34kDa (data not shown) which is mucn smaller than the predicted size of 45Kda cased on the sequence of the two cDNAs .
  • the predicted precursor proteins of other legumains are even larger and when Citvac cDN . v.as transcribed in vitro using a rabbit reticulocyte translation system, a specific polypeptide of 53 kDa was observed.
  • the size compares more favourably with the western data for legumam-like sequences of vetch (38Kda) , soybean (39 kDa) , and castor bean (37 kDa) , which suggests that the protein detectable m maize and these other species has already been processed.
  • Figure 6 shows Northern analysis using RNA from maize leaves and germinating seeds. Each lane contained lO ⁇ g of total RNA which was hybridised at high stringency to the See2 cDNA probe.
  • Lanes 1 to 10 of Figure 6a show RNA extracted at 5 -day intervals from the ear leaves of mature RNA extracted at 5-day intervals from the ear leaves of mature LS plants -5 to +40 days after pollen shed (DAPS) .
  • Figure 6a shows that See2 message increases with leaf age and peaks at 5-10 DAPS (lanes 3 and 4) and more significantly at 30-40 DAPS (lanes 8 to 10) .
  • Visible senescence in other words de-greening
  • a reduction in chlorophyll content started at approximately 25 DAPS. Relative chlorophyll content in senescing leaves was approximately half that of a mature leaf by 30-35 DAPS.
  • Lane 14 of Figure 6b shows RNA extracted from the roots of young seedlings grown in hoaglands solution. It can be seen from Figure 6b, lane 14, that See2 was also detected in these roots.
  • CS and CT seedlings were transferred to a temperature regime of 10°C day/5°C night (14 h day, PAR > 750 ⁇ ol m 2 s - • ) for 13 days.
  • Lanes 15 and 16 of Figure 6c show RNA from the ear leaf of mature CT plants before (lane 15) and after 13 days (lane 16) of severe chilling.
  • Lanes 17 and 18 of Figure 6c show RNA from the ear leaf of mature CS plants before (lane 17) and after (lane 18) 13 days of severe chilling.
  • Figure 6c shows that the concentration of See2 mRNA was found to increase after severe chilling m both mature CS and CT plants.
  • the concentration of See2 mRNA was found to increase m seedlings of a moderately tolerant genotype and in mature plants of chilling sensitive and chilling tolerant genotypes.
  • the concentration of mRNA of the maize cysteine protease Seel was found to increase m LS and CT genotypes following a chilling treatment whilst it decreased m a CS genotype.
  • Leaf 8 of the detopped plant had a relative chlorophyll content of 37%.
  • Leaf 8 was harvested and the total RNA was extracted.
  • RNA from leaf 8 shows RNA from leaf 8 (re-greening) .
  • the concentration of See2 mRNA in the re-greened leaf 8 (lane 22) was slightly lower than that of leaf 7 (lane 21) , even though the greener leaf was older. Under the experimental conditions used, at least one maize leaf senesced every week.
  • Seel and See2 have different roles. For example after dark detached senescence, chilling treatments, and re-greening experiments, the reduction in See2 expression, if any, is much less than for Seel .
  • an endopeptidase such as Seel is susceptible to regulation dependent on signals from the rest of the plant, whilst See2 is more autonomous.
  • the growth media and daily watering solutions were sand/full strength Hoagland's solutions (lane 25) , sand/de-ionised water (lane 26), soil/tap water (lane 27) and soil/no water for last 8 days of experiment (lane 28) .
  • the concentration of See2 was greatest in the leaves of non-stressed plants undergoing normal senescence (lanes 25 and 27) , and comparatively less in nutrient starved (lane 26) or droughted
  • Figure 7 is an illustrative map showing positions of the promoter sequences relative to the See genes.
  • See2a two potential promoter sequences (ID2 and ID3) have been identified and are indicated.
  • a full-length Seel and partial-length See2 clone were isolated by a differential screen of a ⁇ gtlO cDNA library made from early senescing maize leaves 12-20 days after pollen shed (DAPS) using 32 P labelled cDNA from 0 DAPS and 16 DAPS as probes.
  • DAPS pollen shed
  • full length cDNAs of other genes were isolated from a maize senescent leaf cDNA library, all versions of See2 isolated appeared truncated on comparison to homologous sequences from other species.
  • a 255 bp Dpnll fragment was isolated which, like See2, was homologous to the legumain family of proteases.
  • the senescence-enriched cDNA population was generated by a subtractive hybridisation using cDNA from green and yellowing maize leaves.
  • the subtracted clone (dd8) which was not homologous to the existing See2 clone, was similar to the 5' region of other legumain type genes which were homologous at the 3' end to the version of See2 originally isolated. Seel and dd8 cDNAs were labelled with 32 P and used to probe a maize genomic library in ⁇ FixII (Stratagene) .

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Abstract

La présente invention concerne de nouveaux promoteurs liés à la sénescence ou des régions d'amorçage transcriptionnel isolés et caractérisés à partir du maïs (y compris le Zea mays). Le promoteur ou la région d'amorçage transcriptionnel isolé est capable de modifier l'expression de gène durant la sénescence d'une matière végétale. L'invention concerne également un promoteur ou une région d'amorçage transcriptionnel d'un génome végétal et une construction d'ADN utilisable pour modifier l'expression de gène durant la période de sénescence d'une matière végétale. L'invention concerne en outre un procédé de production d'une matière végétale présentant une expression de gène modifiée durant la sénescence.
PCT/GB2000/001849 1999-05-15 2000-05-15 Promoteurs lies a la senescence WO2000070061A2 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002020772A1 (fr) * 2000-09-06 2002-03-14 Agriculture Victoria Services Pty Ltd Manipulation de senescence de plantes au moyen d'un promoteur de gene myb et de genes de biosynthese de la cytokinine
WO2008128293A1 (fr) * 2007-04-24 2008-10-30 Agriculture Victoria Services Pty Ltd Manipulation de sénescence dans les plantes à l'aide de promoteurs modifiés
EP2088200A1 (fr) 2000-11-17 2009-08-12 Genencor International, Inc. Manipulation du contenu d'acide phénolique et digestibilité des parois de cellules végétales par l'expression ciblée de gènes codant les enzymes dégradantes des parois des cellules
WO2012114117A1 (fr) * 2011-02-25 2012-08-30 Consejo Nacional De Investigaciones Cientificas Y Tecnicas Protéase de plante
US8399739B2 (en) 2000-09-06 2013-03-19 Agriculture Victoria Services Pty Manipulation of plant senescence using modified promoters

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Publication number Priority date Publication date Assignee Title
WO1995007993A1 (fr) * 1993-09-13 1995-03-23 Zeneca Limited Regulation de la senescence
WO1996029858A1 (fr) * 1995-03-29 1996-10-03 Wisconsin Alumni Research Foundation Plantes transgeniques aux caracteristiques de senescence modifiees

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WO1995007993A1 (fr) * 1993-09-13 1995-03-23 Zeneca Limited Regulation de la senescence
WO1996029858A1 (fr) * 1995-03-29 1996-10-03 Wisconsin Alumni Research Foundation Plantes transgeniques aux caracteristiques de senescence modifiees

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BUTT ADRIAN ET AL: "Differential expression of a senescence-enhanced metallothionein gene in Arabidopsis in response to isolates of Peronospora parasitica and Pseudomonas syringae." PLANT JOURNAL, vol. 16, no. 2, October 1998 (1998-10), pages 209-221, XP002147532 ISSN: 0960-7412 *
CHUNG BYOUNG-CHULL ET AL: "The promoter activity of sen1, a senescence-associated gene of Arabidopsis, is repressed by sugars." JOURNAL OF PLANT PHYSIOLOGY, vol. 151, no. 3, 1997, pages 339-345, XP000946111 ISSN: 0176-1617 *
GRIFFITHS C M ET AL: "Protease genes in maize leaf senescence." JOURNAL OF EXPERIMENTAL BOTANY, vol. 49, no. SUPPL., May 1998 (1998-05), page 46 XP000946131 The Society for Experimental Biology Annual Meeting;London, England, UK; March 22-27, 1998 ISSN: 0022-0957 *
GRIFFITHS CATHERINE M ET AL: "Sequencing, expression pattern and RFLP mapping of a senescence-enhanced cDNA from Zea mays with high homology to oryzain gamma and aleurain." PLANT MOLECULAR BIOLOGY, vol. 34, no. 5, July 1997 (1997-07), pages 815-821, XP002147531 ISSN: 0167-4412 *
SMART CATHERINE M ET AL: "The timing of maize leaf senescence and characterisation of senescence-related cDNAs." PHYSIOLOGIA PLANTARUM, vol. 93, no. 4, 1995, pages 673-682, XP000946128 ISSN: 0031-9317 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002020772A1 (fr) * 2000-09-06 2002-03-14 Agriculture Victoria Services Pty Ltd Manipulation de senescence de plantes au moyen d'un promoteur de gene myb et de genes de biosynthese de la cytokinine
US7227055B2 (en) 2000-09-06 2007-06-05 Agriculture Victoria Services Pty Manipulation of plant senescence
US8399739B2 (en) 2000-09-06 2013-03-19 Agriculture Victoria Services Pty Manipulation of plant senescence using modified promoters
US8829277B2 (en) 2000-09-06 2014-09-09 Agriculture Victoria Services Pty Ltd. Manipulation of plant senescence using modified promoters
EP2088200A1 (fr) 2000-11-17 2009-08-12 Genencor International, Inc. Manipulation du contenu d'acide phénolique et digestibilité des parois de cellules végétales par l'expression ciblée de gènes codant les enzymes dégradantes des parois des cellules
WO2008128293A1 (fr) * 2007-04-24 2008-10-30 Agriculture Victoria Services Pty Ltd Manipulation de sénescence dans les plantes à l'aide de promoteurs modifiés
WO2012114117A1 (fr) * 2011-02-25 2012-08-30 Consejo Nacional De Investigaciones Cientificas Y Tecnicas Protéase de plante

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