EP1045631A1 - Genes associes a la senescence des plantes - Google Patents

Genes associes a la senescence des plantes

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Publication number
EP1045631A1
EP1045631A1 EP98962909A EP98962909A EP1045631A1 EP 1045631 A1 EP1045631 A1 EP 1045631A1 EP 98962909 A EP98962909 A EP 98962909A EP 98962909 A EP98962909 A EP 98962909A EP 1045631 A1 EP1045631 A1 EP 1045631A1
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Prior art keywords
gene
plant
sark
nucleotide sequence
promoter
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English (en)
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Shimon Gepstein
Taleb Hajuoje
Amalia Rosner
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Vitality Biotechnologies Inc
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Vitality Biotechnologies Inc
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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
    • 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
    • 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
    • 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
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • C12N15/8266—Abscission; Dehiscence; Senescence

Definitions

  • the present invention relates to isolated genes which are expressed early m the process of plant senescence. These genes are referred to as senescence-associated genes or sag genes.
  • the present invention is directed to a class of sag genes which encode protein kinase.
  • the present invention is directed to a senescence-associated receptor-like protein kinase gene, or sark gene, which was isolated from bean and is expressed early m the plant senescence process.
  • the present invention is directed to another class of sag- gene which encode S-adenosyl methionine (SAM) synthase designated sam .
  • a sag gene promoter is used to drive expression of a gene product that inhibits or accelerates the senescence process.
  • the invention further relates to isolation of the promoter from a sag gene, such as the sark or sam gene promoter, and operably linking this promoter to a foreign gene.
  • a sag gene promoter is used to drive expression of a desired product, such as a pharmaceutical, during the process of plant maturation.
  • a sag gene promoter is used to drive expression of a gene which confers resistance, or enhances resistance to, a pathogen or pest during senescence when the plant is particularly susceptible to pathogen infection or pest infestation.
  • a first sag gene promoter is used to drive expression of a gene product that inhibits the senescence process
  • a second sagr gene promoter is used to drive expression of the foreign gene, such as gene encoding a pharmaceutical or disease resistance product, at later stages of plant maturation.
  • the invention further relates to induction of sark gene expression m a detached plant part.
  • the promoter of a sark gene is operably linked to a foreign gene to drive expression m a detached plant part.
  • Senescence refers to an active developmental process which is genetically controlled by the plant . Plants and their parts develop continuously and the latter part of this developmental process, which includes maturity and ultimately the loss of organization and function, is termed senescence.
  • plant parts e.g. leaves
  • older parts such as older leaves, senesce and die.
  • senescence of a part of the plant such as the top of an overwintering perennial, while the rest of the plant remains alive.
  • certain cell types such as xylem vessel and tracheids, may undergo senescence and die while the plant as a whole _s growing vigorously.
  • Patterns of senescence differ with respect to process and reversibility.
  • some types of senescence are closely related to developmental events m the whole plant.
  • Senescence m monocarpic plants for example, is closely related to flowering and growth of fruits. If flower or fruits are removed from a monocarpic plant, senescence may be postponed. Many monocarpic crop plants, including legumes and cereals, undergo abrupt chlorosis and death following fruit production, even under optimal growing conditions.
  • the rapid senescence of a detached flower or leaf can be reversed by application of plant hormone, such as cytokinin or rooting the leaf. The senescence of older leaves on bean plants can be reversed if the top of the plant is removed.
  • senescence process is carefully regulated. Decreases m DNA, RNA, and proteins occur during the senescence process.
  • the export of a substantial portion of plant nutrients from tissues undergoing senescence to the growing shoot is also associated with the process of senescence.
  • senescmg cells undergo a reduction m their structure as the membranous subcellular compartments are disrupted. Morphological changes such as chlorosis of cotyledons and older leaves, or withering and shedding of flower petals following pollination, are aspects of senescence.
  • chloroplasts are the first organelles to deteriorate during onset of leaf senescence. Thylakoid protein components and stomal enzymes disappear m an ordered sequence.
  • the metabolism of senescing tissues requires the de novo synthesis of various hydrolytic enzymes such as proteases, nucleases, lipases and chlorophyll -degrading enzymes.
  • hydrolytic enzymes such as proteases, nucleases, lipases and chlorophyll -degrading enzymes.
  • the levels of the ma ority of leaf mRNAs significantly decline during senescence while the abundance of certain other transcripts increases. Watanable and Imaseki, Plant Cell Physiol . 22 : 489-497 (1982) .
  • senescence down-regulated genes include genes which encode proteins involved m photosynthesis.
  • senescence-associated gene a gene in this category is referred to as a senescence-associated gene or sag gene.
  • sag genes About 30 different sag genes have been isolated and identified in several plant species including Arabidopsis, tomato, maize, barley, asparagus, Brassica napus, and carnation. These genes are expressed in different plant tissues. The functions of the proteins encoded by these genes generally have been deduced on the basis of their sequence homology to known genes .
  • sag genes encode degradative enzymes such as proteases, ribonucleases and lipases. See Hensel et al . (1993), supra ; Oh et al . , Plant Mol . Biol . 3_p_: 739-754 (1996); Taylor et al . , Proc . Natl Acad . Sci . USA SH): 5118-5122 (1993); Ryu and
  • SAM synthase comprises the first step in the ethylene biosynthetic pathway.
  • SAM S-adenosyl methionine
  • the product of SAM synthase or SAM has additional roles in the plant cell, i.e. in the synthesis of polyamines and in the methylation of DNA. Therefore, the gene encoding SAM synthase, unlike the genes encoding ACC synthase or ACC oxidase, was not expected to be preferentially expressed during senescence.
  • Yet another class of sag genes encode products having secondary functions in senescence. These genes code for enzymes involved in the conversion or remobilization of breakdown products. One of these enzymes is glutamine synthetase (GS) which catalyzes the conversion of ammonium to glutamine and is responsible for nitrogen recycling from senescing tissues. Watanable et al . , Plant Mol . Biol . . 26: 1807-1817 (1994). Transgenic plants expressing antisense of genes coding for enzymes involved in the ethylene biosynthetic pathway, such as ACC synthase and ACC oxidase, synthesize ethylene at very low levels.
  • the antisense mutants have been shown to exhibit delayed leaf senescence as well as delayed fruit ripening in tomato. Oeller et al . , Science 254 : 437-439 (1991); Hamilton et al . , Nature 346: 284-287 (1990); John et al . , Plant Molecular Biology 30 (2) : 297-306 (1996) . The total life-span of these mutants increased by only 30% over the wild-type. Accordingly, ethylene appears to modulate the rate of senescence rather than completely control the process. Senescence was also delayed by transforming plants with a gene encoding isopentenyl transferase (IPT) , a key enzyme m cytokinm biosynthesis. Overexpression of IPT causes an overproduction of cytokimns, leading to delayed leaf senescence. Smart et al . , Plant Cell 3 (7) . :
  • IPT isopentenyl transferase
  • Some of these difficulties include: (1) gene mactivation; (2) recombination as a result of pairing along homologous regions within the nucleotide sequence of the promoter leading to cross-over events and loss of the intervening region prior, or subsequent to, integration; and (3) competition among different copies of the same promoter region for binding of promoter-specific transcription factors or other regulatory DNA-binding proteins.
  • expression control sequences such as transcription control sequences which can be operably linked to a foreign gene to drive expression during plant maturation.
  • senescence- associated regulatory genes are needed for coordinated regulatory control of multiple senescence phenomena, for example, tissue senescence, organ senescence, hypersensitivity response, plant death, and programmed cell death (PCD) .
  • PCD programmed cell death
  • senescence-associated regulatory genes which are expressed in detached plant parts are highly desirable.
  • sag gene promoters are not tissue-specific. Tissue-preferred sag gene promoters would be useful to direct production of desired gene products in specific senescing plant tissues. Promoters exhibiting closely controlled temporal expression, different from the expression of known sag genes, are also needed to expand the repertoire of gene expression regulatory sequences.
  • an object of the present invention to provide an isolated gene which is expressed during the process of plant senescence. It is another object of the present invention to provide an isolated gene which is expressed early in the plant senescence process .
  • Yet another object of the present invention is to provide an isolated gene which encodes a protein that regulates the plant senescence process.
  • Another object of the present invention to provide a gene which is preferentially expressed during the senescence process in a detached plant part such as a detached leaf or flower.
  • Yet another object of the present invention is to provide a gene promoter which is preferentially expressed during the process of plant senescence. This promoter is operably linked to a foreign gene to direct expression of the foreign gene product during plant maturation.
  • a ligand, or ligand analog to a protein receptor m the signal transduction pathway for plant senescence .
  • a sark gene which encodes a receptor-like senne/threonine protein kinase.
  • an isolated DNA molecule which is (a) a nucleotide sequence comprising SEQ ID NO: 1; (b) a nucleotide sequence that has substantial sequence similarity with SEQ ID NO: 1 which encodes a senescence-associated receptor-like senne/threonine protein kinase ( sark) ; or (c) a functional fragment of (a) or (b) , wherein the DNA molecule encodes a SARK. Also provided is an expression vector and transformed host comprising a DNA molecule encoding SARK.
  • an isolated DNA molecule comprising a nucleotide sequence selected from the group consisting of (a) a nucleotide sequence comprising SEQ ID NO: 5; (b) a nucleotide sequence that has substantial sequence similarity with SEQ ID NO: 5 and has the transcriptional activity of a sark gene promoter;
  • a nucleotide sequence comprising SEQ ID NO: 8;
  • an expression vector and transformed host comprising an isolated DNA molecule which has the transcriptional activity of a sark gene promoter.
  • a method of producing a foreign protein m a transformed host plant or plant cell comprising the steps of (a) constructing an expression vector comprised of a promoter operably linked to a foreign gene, wherein said promoter comprises a nucleotide sequence selected from the group consisting of (1) a nucleotide sequence comprising SEQ ID NO: 5;
  • nucleotide sequence that has substantial sequence similarity with SEQ ID NO: 5 and has the transcriptional activity of a sark gene promoter; (m) a nucleotide sequence comprising
  • SEQ ID NO: 8 (iv) a nucleotide sequence that has substantial sequence similarity with SEQ ID NO: 8 and has the transcriptional activity of a sark gene promoter; and (v) a functional fragment of d), (ii) , (in) or ( v) wherein sa d nucleotide sequence has the transcriptional activity of a sark gene promoter; and (b) transforming a nost
  • a method of inhibiting plant senescence comprising the steps of (a) constructing an expression vector comprised of a promoter, wherein said promoter comprises a nucleotide sequence selected from the group consisting of (l) a nucleotide sequence comprising SEQ ID NO: 5; (ii) a nucleotide sequence that has substantial sequence similarity with SEQ ID NO: 5 and has the transcriptional activity of a sark gene promoter (m) a nucleotide sequence comprising SEQ ID NO: 8; (iv) a nucleotide sequence that has substantial sequence similarity with SEQ ID NO: 8 and has the transcriptional activity of a sark gene promoter; and (v) a functional fragment of (l) , (n) , (in) or
  • nucleotide sequence has the transcriptional activity of a sark gene promoter; (b) operably linking said promoter to a foreign gene which is an antisense gene of a senescence-associated gene, a sark antisense gene, a S-adenosyl methionine synthase antisense gene, an ACC synthase antisense gene, an ACC oxidase antisense gene or gene encoding sopentenyl transferase, a gene encoding ribozyme or a external guide sequence gene; and (c) transforming a host.
  • a foreign gene which is an antisense gene of a senescence-associated gene, a sark antisense gene, a S-adenosyl methionine synthase antisense gene, an ACC synthase antisense gene, an ACC oxidase antisense gene or gene encoding sopentenyl transferase, a
  • a method of increasing plant resistance to pathogen infection or pest infestation comprising the steps of (a) constructing an expression vector comprised of a promoter, wherein said promoter comprises a nucleotide sequence selected from the group consisting of (1) a nucleotide sequence comprising SEQ ID NO: 5; (11) a nucleotide sequence that has substantial sequence similarity with SEQ ID NO: 5 and has the transcriptional activity of a sark gene promoter (m) a nucleotide sequence comprising SEQ ID NO: 8; (iv) a nucleotide sequence that has substantial sequence similarity with SEQ ID NO: 8 and has the transcriptional activity of a sark gene promoter; and (v) a functional fragment of (I) , (ii) , (m) or (iv) wherein the nucleotide sequence has the transcriptional activity of a sark gene promoter; (b) operably linking the promoter to a disease resistance gene;
  • the disease resistance gene may be an antisense gene, a coat protein gene, a ribozyme gene, a protease inhibitor gene, a Bacillus thurmgi ensis toxin gene, or a chitmase gene, among others.
  • a method of preferentially producing a foreign protein m the detached part of transformed plant comprising the steps of (a) constructing an expression vector comprised of a promoter, wherein said promoter comprises a nucleotide sequence selected from the group consisting of (I) a nucleotide sequence comprising SEQ ID NO: 5; (n) a nucleotide sequence that has substantial sequence similarity with SEQ ID NO: 5 and has the transcriptional activity of a sark gene promoter (in) a nucleotide sequence comprising SEQ ID NO: 8; (iv) a nucleotide sequence that has substantial sequence similarity with SEQ ID NO: 8 and has the transcriptional activity of a sark gene promoter; and (v) a functional fragment of (I) , (ii) , (m) or (iv) wherein the nucleotide sequence has the transcriptional activity of a sark gene promoter; (b) transforming said
  • an isolated DNA molecule comprising the nucleotide sequence of SEQ ID NO: 6.
  • an isolated peptide comprising the ammo acid sequence of SEQ ID NO . 4.
  • Figure 1 presents the nucleotide sequence [SEQ ID NO: 1] of a senescence-associated receptor-like protein kinase structural gene (sark) isolated from bean and its corresponding ammo acid sequence [SEQ ID NO: 2).
  • the mRNA 5' untranslated sequence is included. Also included is the downstream untranslated region.
  • the TAA stop codon is represented by a star (*) .
  • a leucme rich ammo acid region typical of domains involved ligand binding is shown m bold.
  • a domain expected by its hydrophobicity to be membrane-traversing is highlighted.
  • a sequence which is expected to be involved in export of the protein to the cellular membrane is double underlined.
  • Figure 2 presents the nucleotide sequence [SEQ ID NO: 3] , and its corresponding ammo acid sequence [SEQ ID NO: 4] of a region from the sark gene selected for expression in Escherichia coli .
  • Figure 3 presents the DNA sequence [SEQ ID NO: 5] upstream of the sark gene's transcribed sequence (the promoter) .
  • the underlined sequence overlaps the nucleotide sequence of Figure 1, i.e. the underlined sequence represents the 5 ' -end of the mRNA.
  • Figure 4 presents the partial nucleotide sequence of a gene [SEQ ID NO: 6] encoding S-adenosyl methionine (SAM) and its corresponding am o acid sequence [SEQ ID NO: 7] .
  • SAM S-adenosyl methionine
  • Figure 5 represents the nucleotide sequence [SEQ ID No : 8] upstream of the structural gene. It incorporates but extends further upstream of the sark gene than the sequence shown in
  • Figure 3 The sequence m italics is the nucleotide sequence presented also Figure 3.
  • the underlined sequence overlaps the nucleotide sequence of Figure 1, i.e. the underlined sequence represents the 5 ' -end of the mRNA.
  • Figure 6 presents the senescence of detached potato leaf discs incubated for 9 days m the dark.
  • WT are leaves from nontransgenic plants.
  • the numbers on the y-axis represent % of leaves observed.
  • SARK are leaves from transgenic potato plants containing the sark gene expressed constitutively from the 35S promoter.
  • the number 1 designates leaves that are green, i.e. exhibit the least senescence.
  • the numbers 2, 3, 4 and 5 designate leaves which exhibit progressively more senescence as measured by extant visable chlorosis.
  • Senescence m plants, plant parts, or organs is a genetically controlled process leading to morphological and biochemical changes associated with aging and death. Transcription of DNA into mRNA is generally reduced although expression of certain genes increases during senescence. Chlorophyll and protein degradation occurs during senescence. Increased transcription of genes encoding proteins responsible for conversion and mobilization of the breakdown products occurs during plant senescence. A senescence-associated down-regulated gene is referred to as a sdg. A senescence-associated gene which exhibits increased transcription during senescence is designated sacf.
  • a class of sag genes is the senescence-associated receptorlike protein kinase gene or sark which encodes a receptor-like protein kinase which is preferentially expressed early m the process of plant senescence.
  • a sark gene is expressed prior to apparent changes m plant morphology or biochemistry generally associated with senescence.
  • a sark gene product has a senescence regulatory function.
  • a structural gene is a DNA sequence that is first transcribed into messenger RNA (mRNA) and then translated into a sequence of ammo acids characteristic of a specific polypeptide.
  • a promoter is a DNA sequence that directs the transcription of a structural gene. Typically, a promoter is located m the 5i region of a gene, proximal to the transcriptional start site of a structural gene. If a promoter is an inducible promoter, then the rate of transcription increases m response to an inducing agent.
  • a promoter may be regulated m a tissue-preferred manner such that it is predominantly active m transcribing the associated coding region m a specific tissue type(s) such as leaves, roots or me ⁇ stem.
  • the promoter is a constitutive promoter. If transcription from the promoter is predominant only at certain stages of plant development, then the promoter is a temporal promoter or a developmentally-regulated promoter. If the promoter controls transcription of the sag gene, then the promoter is a senescence associated promoter or sag promoter.
  • An isolated DNA molecule is a fragment of DNA that is not integrated m the genomic DNA of an organism.
  • a cloned sark gene is an illustration of an isolated DNA molecule.
  • Another example of an isolated DNA molecule is a chemically- synthesized DNA molecule that is not integrated m the genomic DNA of an organism.
  • cDNA Complementary DNA
  • cDNA is a single-stranded DNA molecule that is formed from an mRNA template by the enzyme reverse transc ⁇ ptase .
  • a primer complementary to portions of mRNA is employed for the initiation of reverse transcription.
  • cDNA refers to a double-stranded DNA molecule consisting of such a single-stranded DNA molecule and its complementary DNA strand.
  • RNA polymerase II catalyzes the transcription of structural genes to produce mRNA.
  • a DNA molecule can be designed to contain an RNA polymerase II template which the RNA transcript has a sequence that is complementary to at least a significant section (at least 10 nucleotides) of a specific mRNA. This particular RNA transcript is termed an antisense RNA and a
  • Antisense RNA molecules are capable of hybridizing in vivo to mRNA molecules, resulting m an inhibition of gene expression.
  • a ribozyme is an RNA molecule that contains a catalytic center. The term includes RNA enzymes, selfsplicing RNAs, and self-cleaving RNAs.
  • a DNA sequence that encodes a ribozyme is termed a ribozyme gene.
  • An external guide seguence is an RNA molecule that directs the endogenous ribozyme, RNase P, to a particular species of mtracellular mRNA, resulting m the cleavage of the mRNA by RNase P .
  • a DNA sequence that encodes an external guide sequence is termed an external guide sequence gene.
  • gene expression refers to the biosynthesis of a gene product.
  • expression involves transcription of the structural gene into mRNA and the translation of mRNA into one or more polypeptides.
  • a cloning vector is a DNA molecule, such as a plasmid, cosmid, or bacte ⁇ ophage, that has the capability of replicating autonomously m a host cell .
  • Cloning vectors typically contain one or a small number of restriction endonuclease recognition sites at which foreign DNA sequences can be inserted in a determ able fashion without loss of an essential biological function of the vector, as well as a marker gene that is suitable for use m the identification and selection of cells transformed with the cloning vector. Marker genes typically include genes that provide tetracyclme resistance or ampicillm resistance.
  • An expression vector is a DNA molecule comprising a gene that is expressed m a host cell. Typically, gene expression is placed under the control of certain regulatory elements, including constitutive or mducible promoters, tissue-specific regulatory elements, and enhancers. Such a gene is said to be "operably linked to" regulatory elements.
  • a recombinant host may be any prokaryotic or eukaryotic cell that contains either a cloning vector or expression vector. This term also includes those prokaryocic or eukaryotic cells that have been genetically engineered to contain the cloned gene(s) in the chromosome or genome of the host cell.
  • a foreign gene or a transgene refers m the present description to a DNA sequence that is operably linked to at least one heterologous regulatory element.
  • any gene other than a sag and sark gene is considered to be a foreign gene if the expression of that gene is controlled by the sag and sark gene promoter, respectively.
  • a foreign gene includes an antisense gene.
  • a transformed host may be any prokaryotic or eukaryotic cell that contains either a foreign gene, cloning vector or expression vector. This term includes those prokaryotic or eukaryotic cells that have been genetically engineered to contain the cloned gene(s) m the chromosome or genome of the host cell or transiently express the cloned gene.
  • a transgenic plant is a plant having one or more plant cells that contain an expression vector or a stably integrated foreign gene .
  • a nucleotide sequence has substantial sequence similarity to the coding sequence of a sag, sark or sam gene if the former sequence shares a similarity of at least 70%, preferably at least
  • nucleotide sequence of a plant sag, sark or sam gene encodes a protein which functions as a senescence-associated protein, protein kinase, or SAM synthase, respectively.
  • nucleotide sequence has substantial sequence similarity to the promoter sequence associated with a sag, sark or sam gene if the former sequence shares a similarity of at least 60%, preferably 70%, more preferably at least 80%, most preferably at least 90% with the nucleotide sequence of a plant sag, sark or sam gene promoter and has the same transcriptional activity as the sag, sark or sam gene promoter, respectively.
  • the nucleotide sequence of the promoters is compared over the region responsible for control of transcriptional activity. Sequence similarity determinations can be performed, for example, using the FASTA program (Genetics Computer Group; Madison, WI ) .
  • sequence similarity determinations can be performed using BLAST (Basic Local Alignment Search Tool) of the Experimental GENIFO ® BLAST Network Service. See Altschul et al . , J. Mol . Biol . 215 :403 (1990);
  • a convenient visual marker for senescence onset is chlorosis of a first leaf. Plants grown under greenhouse conditions exhibit chlorosis of the first leaf at about 45 days following seed germination. The time of senescence onset is dependent on many factors including environmental conditions and genotype of the plant, but chlorosis of the first leaf provides a convenient visual marker under any standard set of conditions.
  • the isolated cDNAs of the present mvention were obtained by differential display.
  • the two mRNA pools used m the differential display process were extracted from (1) fully expanded young bean leaves harvested from plants that been grown for not more than 15 days post-germination and (2) primary leaves displaying initial chlorosis harvested 45 days post -germination.
  • Two cDNA clones were isolated by differential display which are preferentially expressed m leaves during senescence, as confirmed by Northern blot analysis. Each cDNA clone was amplified, labeled, and used as a hybridization probe against mRNA extracted from young leaves and senescence- stage leaves.
  • the isolated sag genes were inserted into plasmid pUC55 and subjected to nucleotide sequence analysis.
  • Nucleotide sequence analysis of one isolated cDNA clone obtained through differential display revealed that it encodes a protein which resembles the C-termmus a protein kinase.
  • the cloned cDNA was used as a hybridization probe to obtain an isolated nucleotide sequence which is a senescence-associated receptor-like protein kinase (sark) gene.
  • the nucleotide sequence of the sark gene and its corresponding ammo acid sequence are shown m Figure 1.
  • the coding sequence of the sark gene is found between nucleotides 152 and 2863 of Figure 1. Accordingly, the sark gene encodes a protein having 904 ammo acids .
  • the sark gene is expressed early m the senescence process.
  • Another early event m plant senescence is a decrease in the level of chlorophyll protein LHC2.
  • the sark gene is expressed prior to a detectable decrease m LHC2. Additionally, the sark gene is expressed prior to chlorosis of the first leaf of bean plants grown under greenhouse conditions.
  • Northern analysis using the sark gene as a hybridization probe revealed that the sark gene is expressed m a detached bean leaf tissue, m the dark, 1 day after removal of the leaf tissue. Further analysis revealed that sark gene expression is developmentally controlled. The time of expression of the sark gene was constant relative to the age of an individual leaf. Accordingly, the youngest leaves near the top of the plant express the sark gene later m the overall life span of the plant than older leaves near the bottom of the plant. The combined observations that the sark gene is expressed early in the senescence process and encodes a receptor-like protein kinase led to the expectation that sark is a senescence regulator, responsible for control of other senescence phenomena.
  • Nucleotide sequence analysis revealed that another cDNA clone obtained through differential display corresponds to a sam gene.
  • Figure 4 shows the partial nucleotide sequence of the isolated sam gene and the corresponding ammo acid sequence.
  • SAM synthase catalyses the first step m plant ethylene biosynthesis.
  • Northern analysis using the cDNA clone corresponding to the same gene as a hybridization probe revealed that transcription of the sam gene occurs m young leaves, then decreases leaves at about 20 days post-germination and then increases as the leaves mature. Expression of the sam gene continues to increase as the leaves mature.
  • Isolation of sag genes relies on identification of genes expressed preferentially during senescence. Approaches are well known to the skilled artisan for identification of mRNA expressed differentially m certain cell types at specific stages in the plant development process or during infection by a parasite or a virus. Those studies generally employ subtractive hybridization to reveal the differentially expressed mRNA(s).
  • the subtractive hybridization method generally employs preferential amplification of novel cDNA. "Tester” and “driver” cDNA pools are created.
  • the tester DNA may have short DNA “tails” or “adapters” attached by ligation to allow for amplification via polymerase chain reaction (PCR) primers complementary to these tails.
  • PCR polymerase chain reaction
  • the tester and driver cDNA pools are mixed, heated and allowed to reanneal .
  • the remaining single- stranded cDNA is enriched for the unique sequences.
  • the remaining single-stranded cDNA is PCR amplified as above, reannealed to driver cDNA, and the process is repeated, to allow further enhancement of the unique cDNA.
  • a variation of the method employs restriction enzyme sites withm the tails allowing addition of new adapter molecules to the unique cDNA to enhance amplification of only unique cDNA m subsequent rounds.
  • the adapters are attached to the driver cDNA and biotmylated. This allows use of streptavidm or avidm to effectively subtract the background cDNA.
  • the locking mechanism involves extending the poly dT primer m the 3 ' direction, by either one nucleotide (A, C or G) or by two nucleotides (also A, C or G for the nucleotide proximal to the poly dT stretch and yet one more of the four possible nucleotides for the 3' most nucleotide of the primer) .
  • the differential display method of Liang further employs a decanucleotide of arbitrary sequence as a primer for PCR, internal to the mRNA, m conjunction with a lock-dockmg oligo at the 3 ' -end of mRNAs.
  • a decanucleotide of arbitrary sequence as a primer for PCR, internal to the mRNA, m conjunction with a lock-dockmg oligo at the 3 ' -end of mRNAs.
  • sark genes encode protein kmases enables yet another approaches to identify these genes.
  • plant protein kmases have regions of high homology.
  • the internal decanucleotide sequence can reflect the conserved regions of protein kmases.
  • nucleic acid probes based on the highly conserved regions can identify other protein kinase genes, preferably by screening of cDNA libraries.
  • the temporal expression of any identified sark gene can be deduced from hybridization of the cDNA to mRNA isolated at different time points during plant development to identify isolated nucleotide sequences having the transcriptional activity of a sark gene promoter.
  • Promoters associated with a sag or sark gene are isolated by identifying genes having substantial sequence similarity with the coding sequence of a plant sag or sark gene. Regions upstream of the sag or sark coding sequences are isolated by chromosomal walking techniques; i.e., sequencing of a coding sequence and continuously employing new primers pased on the newly revealed sequence. Alternatively, a specific primer designed on the basis of the known sequence and a random primer m the appropriate orientation are used m a PCR reaction to clone a DNA fragment upstream of the known sequence. The nucleotide sequence of the isolated DNA is determined.
  • the promoter associated with the sag or sark gene is isolated by conventional methods.
  • the coding sequence of a bean sark gene is provided m Figure 1 and the upstream regulatory region of this gene is snown m Figure 3.
  • a portion of the coding sequence of a bean sag gene, the sam gene, is shown m
  • the entire sag or sark gene coding region, or fragments thereof are labeled, for example radiolabeled, by conventional methods and used to detect related nucleotides sequences in plant genomic libraries by means of DNA hybridization. See Yang, supra and Sambrook (1989), supra .
  • the probe can be a single and relatively short oligonucleotide of defined sequence, pools of short oligonucleotides whose sequences are highly degenerate or pools of long oligonucleotides of lesser degeneracy.
  • a plant genomic DNA library can be prepared by means well- known m the art. See, for example, Slightom et al . "Construction of ⁇ Clone Banks," Glick (1993), pages 121-46.
  • Genomic DNA can be isolated from plant tissue, for example, by lysmg plant tissue with the detergent Sarkosyl , digesting the lysate with protemase K, clearing insoluble debris from the lysate by cent ⁇ fugation, precipitating nucleic acid from the lysate using isopropanol, and purifying resuspended DNA on a cesium chloride density gradient. Ausubel et al . (eds.), CURRENT
  • DNA fragments that are suitable for the production of a genomic library can be obtained by the random shearing of genomic DNA or by digestion of genomic DNA with restriction endonucleases . See, for example, Ausubel et al . , supra, at pages
  • Genomic DNA fragments can be inserted into a vector, such as a bacteriophage or cosmid vector, m accordance with conventional techniques, such as the use of restriction enzyme digestion to provide appropriate termini, the use of alkaline phosphatase treatment to avoid undesirable joining of DNA molecules, and ligation with appropriate ligases. Techniques for such manipulation are disclosed by Slightom et al . , supra, and are well-known m the art. Also see Ausubel et al . , supra, at pages
  • a library containing genomic clones is screened with DNA hybridization probes based on the nucleotides sequence of the sag, sark or sam gene coding sequence by standard techniques.
  • Genomic clones can be analyzed using a variety of techniques such as restriction analysis, Southern analysis, primer extension analysis, and DNA sequence analysis. Primer extension analysis or SI nuclease protection analysis, for example, can be used to localize the putative start site of transcription of the cloned gene. Ausubel et al . , supra, at pages 4.8.1-4.8.5; Walmsley et al . , "Quantitative and Qualitative Analysis of Exogenous Gene
  • the general approach of such functional analysis involves subclonmg fragments of the putative promoter into an expression vector which contains a reporter gene, introducing the expression vector into various plant tissues, and assaying the tissue to detect the transient expression of the reporter gene.
  • Methods for generating fragments of a genomic clone are well-known.
  • enzymatic digestion is used to form nested deletions of genomic DNA fragments. See, for example, Ausubel et al . , supra, at pages 7.2.1-7.2.20; An et al . , supra .
  • the vector contains:
  • prokaryotic DNA elements coding for a bacterial replication origin and an antibiotic resistance marker to provide for the growth and selection of the expression vector m the bacterial host; (2) DNA elements that control the processing of transcripts, such as a transcription termmation/polyadenylation sequence; (3) convenient cloning s ⁇ te(s) for introducing the putative promoter into the vector to control transcription of a reporter gene; and (4) a reporter gene that is operably linked to the DNA elements that control transcription initiation.
  • Useful reporter genes include ⁇ -glucuronidase, ⁇ -galactosidase, chloramphenicol acetyl transferase, green florescent protein (GFP) , luciferase, and the like.
  • the reporter gene is either the ⁇ -glucuromdase (GUS) gene or the luciferase gene.
  • GUS ⁇ -glucuromdase
  • the reporter gene is either the ⁇ -glucuromdase (GUS) gene or the luciferase gene.
  • Methods of introducing vectors into plant tissue include the direct infection or co- cultivation of plant tissue with Agrobacte ⁇ um tumefaciens .
  • Agrobacterium vector systems and methods for Agrobacterium- mediated gene transfer are provided by Gruber et al . (1993), supra , and Miki et al . (1993), supra .
  • Methods of introducing vectors into plant tissue also include direct gene transfer methods such as microprojectile-mediated delivery, DNA injection, electroporation, and the like. Id .
  • Variants, or functional fragments, of the sag, sark or sam gene promoter can be produced by deleting, adding and/or substituting nucleotides. Such variants or functional fragments can be obtained, for example, by oligonucleotide-directed mutagenesis, linker-scanning mutagenesis, mutagenesis using the polymerase chain reaction, and the like.
  • agronomically important, foreign genes can be operably linked to a sag, sark or sam gene promoter and expressed m transformed plants during plant maturation.
  • a sag, sark or sam gene promoter is advantageously utilized to overexpress an operably linked gene because gene expression does not occur until later stages m the development of the plant thereby limiting the demand on plant metabolic resources that could reduce plant vigor during early stages of development.
  • a first sag gene promoter is used to drive expression of a gene product that inhibits the senescence process and a second sag gene promoter is used to drive expression of the foreign gene, such as gene encoding a pharmaceutical or disease resistance product, at later stages of plant maturation.
  • a gene can be selectively expressed m detached plant parts, for example a stem of cut flowers and leaves, or m fruit .
  • genes encoding enzymes involved m cytokmm biosynthesis such as isopentyl transferase, are operably linked to a sag, sark or sam gene promoter and expressed m transformed plants during plant senescence. Production of increased cytokmm during senescence inhibits the senescence process. Cytokmms play a role m leaf senescence.
  • the sag or sark gene promoters are particularly suited for expression of a gene encoding isopentenyl transferase, the enzyme that catalyzes the rate- limiting step m cytokmm biosynthesis.
  • an antisense gene of an ethylene biosynthetic gene such as ACC synthase, ACC oxidase or SAM synthase is operably linked to a sag, sark or sam gene promoter and expressed m transformed plants during plant senescence to inhibit the senescence process.
  • An antisense gene of a plant senescence- associated gene such as a sag, sark or sam gene, is operably linked to a sag, sark or sam gene promoter and expressed m transformed plants during plant senescence to inhibit the senescence process.
  • other alternative strategies for control of expression include use of genes encoding ribozyme or external guide sequences and used to control expression of senescence-associated genes.
  • Plant or pest disease resistance genes or genes which enhance resistance to plant pathogens or pests, are operably linked to a sag, sark or sam gene promoter and expressed in transformed plants during plant senescence. Plants are particularly susceptible to infection by certain plant pathogens and infestation by certain insect pests during senescence. Certain plant pathogenic fungi such as Botrytis sp. or
  • insects toxins include an msect-specifIC hormone or pheromone such as an ecdysteroid and juvenile hormone, a variant thereof, a mimetic based thereon, or an antagonist or agonist thereof. See, for example, the disclosure by Hammock et al . ,
  • Chitmase genes are useful for inhibiting insect pests. Chitmase also can be used for combating fungal pathogens. Additional antifungal genes include genes encoding ⁇ -1,3- glucanase, which degrades a major polysaccharide of fungal cell walls, and ribosome inactivating protein, which inactivates fungal ⁇ bosomes. Full-length cDNAs of glucanase and ⁇ bosome- mactivatmg protein are disclosed Leah et al . , J. Biol . Chem.
  • polypeptides include the bactericidal native and recombinant cecropms, insect attacm, frog magaimn, cereal thionms, T4 and hen egg white lysozyme, horseshoe crab tachyplesm I, Erwinia oligogalacturonide lyase .
  • plant disease resistance genes are available for use. See, for example, Bent, The Plant Cell 8 . 1151 (1996).
  • Preferred antibacterial and antifungal genes include DNA molecules that encode natural and synthetic lytic peptides and plant defensms.
  • Lytic peptides are broad-spectrum antibiotic peptides that are active against Gram-negative and Gram-positive bacteria, fungi and protozoa. These peptides can be classified into many categories based upon their structure (e . g. , linear vs. cyclic), their size (20-45 ammo acids) and their source (e.g., insect, amphibian, plant) .
  • numerous defense-related peptides have the common features of being highly basic and being capable of forming amphipathic structures. These unifying features suggest that most peptides appear to act by a direct lysis of the pathogenic cell membrane. Their basic structure facilitates their interaction with the cell membrane, and their amphipathic nature allow them to be incorporated into the membrane ultimately disrupting its structure.
  • Magamms 1 and 2 have 23 ammo acid residues length, contain no cysteme, and form an amphipathic ⁇ -helix.
  • PGL a is a small peptide processed from a larger precursor and is both cationic and amphipathic m nature. It has the somewhat unusual feature of containing a COOH-termmal amide group rather than the expected carboxyl group.
  • magamm 2 but not magamm 1
  • PGL a can interact synergistically with one another to exert enhanced levels of anti-microbial activity.
  • Insects have also been demonstrated to possess a variety of defense-related peptides. Cecrop s from moths and flies are slightly larger than the frog-derived peptides (31-39 residues) , are basic due to the presence of multiple arginme and lysme residues, and therefore interact strongly with the negatively charged lipid bilayer. Boman, Cell 65 : 205 (1991) . Studies of these peptides have shown that they form an N-termmal ⁇ -helical region connected by a hinge region to a C-termmal cy-helical domain . In addition to the naturally-occurring peptides, a wide array of synthetic analogs representing deletion, substitution and variable chain length derivatives have been generated for structure/activity relationship studies.
  • transgenic plants express a viral protein.
  • the accumulation of viral coat or replicase proteins m transformed plant cells provides resistance to viral infection and/or disease development by the virus from which the coat protein gene was derived, as well as by related viruses. See Beachy et al . , Ann. fiev. Phytopathol . 28 : 451 (1990); Beachy, "Virus Resistance
  • RNA for example, antisense RNA has been used to confer resistance to cucumber mosaic virus, as disclosed by Rezaian et al . , Plant Molec . Biol . 11 : 463 (1988). Moreover, Day et al . ,
  • a transgenic plant expresses pokeweed antiviral protein (PAP) , a ribosome-inhibiting protein found m the cell walls of Phytolacca ameri cana . Lodge et al . , Proc . Na t ' l Acad .
  • PAP pokeweed antiviral protein
  • genes have been shown to create a more compact habit and earlier flowering m transgenic plants. These include the rol genes (A, B, and C) from Agrobacterium rhizogenes (U.S.
  • Patent No. 5,648,598 phytochrome genes such as phyA (McCormac et al . , Planta 185 : 162-170 (1991)), developmental genes such as lfy (Wegel and Nilsson, Nature 377 : 495-496 (1995)), and the
  • MADS-box containing family of genes such as apetala (Mandel and Yanofski, Nature 377 : 522-524 (1995)), and OsMADSl (Chung et. al . , Plant Mol . Biol . 2_6: 657-665, (1994)).
  • genes have been shown to create modified color expression m transgenic plants. These include the crtO gene involved synthesis of the bright red pigment called astaxanthm, the lycopene cyclase gene involved m synthesis of the orange pigment ⁇ -carotene, the ⁇ -carotene hydroxylase gene involved m synthesis of the golden pigment zeaxanthm, as well as the genes m the flavonoid biosynthesis pathway which are involved m the synthesis of various anthocyananm pigments which can be red, blue, pale yellow, as well as a wide range of intermediates and pastels.
  • crtO gene involved synthesis of the bright red pigment called astaxanthm
  • the lycopene cyclase gene involved m synthesis of the orange pigment ⁇ -carotene
  • the ⁇ -carotene hydroxylase gene involved m synthesis of the golden pigment zeaxanthm
  • the genes m the flavonoid biosynthesis pathway which are involved m the synthesis
  • genes which affect plant fragrance. These genes include, but are not limited to, the lmalool synthase gene which causes the synthesis of aromatic lmalool and the limonene synthase gene which causes synthesis of the fragrant limonene (Alonsa et al . , J. Biol . Chem . 267 : 7582-7587 (1992).
  • Vi treoscibba hemoglobin gene ( "vhb gene")
  • vhb gene Vi treoscibba hemoglobin gene
  • Synthesis of agronomic genes of interest can be effected by the polymerase chain reaction. See, for example, Ausubel et al .
  • the expression may comprise a selectable or screenable marker.
  • Many of the commonly used positive selectable marker genes for plant transformation were isolated from bacteria and code for enzymes that metabolically detoxify a selective chemical agent which may be an antibiotic or a herbicide.
  • Other positive selective marker genes encode an altered target which is insensitive to the inhibitor.
  • neomycm phosphotransferase II (nptll) gene, isolated from Tn5 , which when placed under the control of plant regulatory signals confers resistance to kanamycm. Fraley et al . , Proc . Na t ' l Acad . Sci . U. S . A . 8_0: 4803 (1983) .
  • Another commonly used selectable marker is the hygromycm phosphotransferase gene which confers resistance to the antibiotic hygromycm. Vanden Elzen et al . , Plant Mol . Biol . 5 . :
  • Additional positive selectable marker genes of bacterial origin that confer resistance to antibiotics include gentamicm acetyl transferase, streptomycin phosphotransferase, ammoglycos ⁇ de-3 ' -adenyl transferase and the bleomycm resistance determinant.
  • genes for plant transformation are not of bacterial origin. These genes include mouse dihydrofolate reductase, plant 5-enolpyruvylsh ⁇ k ⁇ mate-3- phosphate synthase and plant acetolactate synthase. Eichholz et al . , Soma ti c Cell Mol . Genet . 13.: 67 (1987); Shah et al . , Science
  • European Patent application No. 0 333 033 and U.S. Patent No. 4,975,374 disclose nucleotide sequences of glutamine synthetase genes which confer resistance to herbicides such as L- phosphmothricm.
  • the nucleotide sequence of a phosphmoth ⁇ cm- acetyl-transferase gene is provided m European application No. 0 242 246.
  • De Greef et al . , Bio/Technology 1 : 61 (1989) describe the production of transgenic plants that express chimeric jbar gene coding for phosphmothricm-acetyl-transferase activity.
  • marker genes for plant transformation requires screening of presumptively transformed plant cells rather than direct genetic selection of transformed cells for resistance to a toxic substance such as an antibiotic. These genes are particularly useful to quantify or visualize the spatial pattern of expression of a gene m specific tissues and are frequently referred to as reporter genes because they can be fused to a gene or gene regulatory sequence for the investigation of gene expression.
  • a sag gene including a sark or sam gene of the instant invention, is inactivated in a transgenic plant by expression of a gene construct that inhibits expression of the senescence gene thereby retarding the senescence process.
  • expression of a sark gene is targeted for inhibition.
  • Strategies that allow suppression of a specific gene are known and include antisense, ribozymes and external sequence guide genes.
  • the expression of an anti -senescence DNA construct is operably linked to a plant compatible developmentally regulated promoter such as that isolated from a sag or sark gene, or an mducible promoter.
  • This temporal expression pattern provides an opportunity to transcribe agronomic genes operably linked to the sark promoter prior to onset of chlorophyll degradation but before plant senescence is fully expressed.
  • Expression of heterologous proteins plants might be toxic to the plant, cause a significant decrease m available plant metabolic resources leading to poor plant vigor, and/or prevent normal plant development if produced at earlier developmental stages. Inhibition of plant growth can be advantageously avoided by producing the protein encoded by the agronomic gene during later stages of plant development.
  • a sag promoter such as a sark gene promoter
  • a cytokmm biosynthetic gene such as isopentyl transferase.
  • the sag gene promoter is operably linked to an antisense gene of a sag, sark, sam or ethylene biosynthetic gene.
  • a sark gene promoter can be used to express agronomic genes in the detached plant part including color genes, fragrance genes, or ethylene biosynthesis genes important for fruit ripening.
  • nucleic acid sequences are commonly identified as protein kinase receptor genes if conserved membrane targeting, transmembrane domains and kinase domains are present .
  • certain ammo acids are invariant or nearly invariant m the protein kmases.
  • plant protein kmases have also been identified which maintain structure similarity to animal kmases.
  • the C-terminal domains of plant and animal protein kmases comprise eleven well conserved domains. See Hanks et al . , Science 241 : 42-52 (1988); Chang et al . , Plant Cell 4 1263-71 (1992); Zhou et al . , Cell 83 . : 925-35
  • Bean plants Phaseolus vulgaris cv. bulgarian were grown in a temperature-regulated greenhouse at 25°C. Primary leaves (at the bottom) were harvested 15 days (young) and 45 days (senescing) post-germination. Leaves were macerated and total mRNA was extracted as described by Puissant, C. and Houdeb e, L.M., Bio techniques ]3_ : 148-149 (1990).
  • bean DNA was digested with EcoRV and recircularized by ligation. Inverse PCR as described above was performed with the following PCR primers: 5 ' - CTCATTCAGAGACAACGAGCA -3 ' , and 5 ' - GTGGAGGTGTTTGGTATAAGG -3 ' . An approximately 2.5 kb DNA fragment was isolated. The DNA fragment was isolated and cloned after PCR reactions with the following primers:
  • Bean seeds Phaseolus vulgaris cv. bulgarian were germinated and grown for 40 days in temperature controlled greenhouse. A total of 10 plants were evaluated. The 40 day-old plants show initial yellowing of the 2 primary leaves (at the bottom) . Bean plants produce a pair of primary leaves which are the first to develop and are the oldest leaves.
  • the expression of the sark gene was highest in the oldest leaf (1) . Expression of the sark gene in the second oldest leaf
  • Bean seeds (Phoseolus vulgaris cv. bulgarian) were germinated and grown in a temperature controlled greenhouse at 25°C.
  • Leaf discs were removed with a 10 mm cork borer from fully expanded leaves of 15 day-old bean plants. The 15 day-old bean plants exhibited no visual evidence of senescence such as chlorosis of the oldest leaf. The leaf discs were incubated in distilled water in the dark.
  • Total RNA and protein was extracted at time zero and at 24 hr intervals for a total of 6 consecutive days. Total RNA was extracted as described above. Total protein was extracted according to Ben-David supra .
  • Chlorophyll levels were measured and no decrease was detectable after 24 hours. Chlorophyll levels decreased following approximately 48-72 hrs incubation of the leaf discs in distilled water.
  • isopentyl transf erase is therefore operably linked to a sag gene promoter, such as sark or sam gene promoter.
  • a sag gene promoter such as sark or sam gene promoter.
  • the cytokinin biosynthetic gene(s) is operably linked to an inducible promoter. Expression of the cytokinin biosynthetic gene leads to increased in planta concentrations of cytokinin and inhibits senescence.

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Abstract

L'invention concerne des gènes isolés qui sont exprimés pendant le vieillissement des plantes. En particulier, l'invention traite d'un gène qui code une protéine kinase de type récepteur qui est, de préférence, exprimé dans les feuilles des plantes au début du processus de sénescence des plantes. En outre, l'invention a aussi pour objet un gène codant la synthase du S-adénosyl méthionine qui est exprimé pendant le processus de sénescence des plantes. Enfin, l'invention traite aussi de promoteurs de gènes exprimés pendant le vieillissement de la plante qui sont liés, de manière opérationnelle, à un gène étranger pour assurer l'expression spécifique au développement du gène étranger dans une plante transformée.
EP98962909A 1997-12-08 1998-12-08 Genes associes a la senescence des plantes Withdrawn EP1045631A1 (fr)

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EP1130104A1 (fr) * 2000-02-16 2001-09-05 Stichting Dienst Landbouwkundig Onderzoek Réduction de la dégradation des produits végétales in planta
KR100604195B1 (ko) * 2004-09-02 2006-07-25 고려대학교 산학협력단 식물 노화에 특이적으로 발현되는 유전자 및 그 유전자의프로모터
MX2007011612A (es) * 2005-03-21 2007-10-18 Univ California Plantas resistentes a la sequia.

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