WO2004108931A1

WO2004108931A1 - Cytokinin receptor ahk3 involved in senescence regulation of plant and use thereof

Info

Publication number: WO2004108931A1
Application number: PCT/KR2004/001398
Authority: WO
Inventors: Hong-Gil Nam; Hyo-Jung Kim; Hye-Ryun Woo; In-Chul Lee
Original assignee: Genomine Inc.; Postech Foundation
Priority date: 2003-06-11
Filing date: 2004-06-11
Publication date: 2004-12-16
Also published as: KR100997612B1; KR20040106810A

Abstract

The present application relates to Cytokinin receptor AHK3 which is involoved in regulating senescence of a plant and its use. More detailedly, a method for delaying senescence of a plant by using Cytokinin receptor AHK3 and its variants. By overexpressing AHK3 and its variants from this invention in a plant, plant senescence could be possibly delayed. In addition, productivity and storage efficiency can also be improved.

Description

CYTOKININ RECEPTOR AHK3 INVOLVED IN SENESCENCE REGULATION OF PLANT AND USE THEREOF

TECHNICAL FIELD

The present invention relates to a protein involved in the regulation of plant senescence and the use thereof, and more particularly to a method of delaying plant senescence using such a protein.

BACKGROUND ART

Plant senescence is the final stage of plant development, and senescence initiation can be an abrupt turning point in the plant development stage. As the senescence of plants progresses, the plants show a gradual reduction in their synthetic ability and lose their cellular homeostasis with the successive degradation of intracellular structures and macromolecules, and ultimately reach death (Matile et al, Elservier, 413-440, 1992; Nooden et al., Senescence and Aging in Plant, Academic press 1988; Thiman et al., CRC press 85-115, 1980; Thomas et al., Annu. Rev. Plant Physiol. 123:193-219, 1993). This plant senescence is a series of continued biochemical and physiological events, and genetically intended so that it progresses at the level of cells, tissues and organs in a very elaborate and active manner. Plant senescence is a cell degeneration process as well as a process required for transferring nutrients from growing organs to reproductive organs in the winter season, and it is thought to be a genotype that is actively acquired so as to adapt to circumstances during the plant development process. Plant senescence is of high biological importance as well as high industrial importance due to the possibility of improvement in the production or post-harvest storage of crops. Accordingly, genetic, molecular biological, physiological and biochemical studies are being actively conducted in an attempt to elucidate the phenomenon of plant senescence and delay plant senescence.

Recently, success in delaying the senescence of plants was achieved by specifically regulating the synthesis of cytokinin, a plant growth hormone, in a certain senescence stage, through the linking of an IPT gene to the promoter of a senescence-specific SAG12 gene. In the case of tobacco plants whose senescence was delayed by this method, a more than 50% increase in production could be achieved without regulating flowering time or inducing other abnormalities (Gan et al., Science 22:1986-1988, 1995). Furthermore, other studies have been conducted in order to delay plant senescence by manipulating degradation-associated genes which have activity involved in a biochemical change occurring in a senescence process or are involved in signal transduction pathways. A typical example thereof includes the Flavr savr™ tomato whose softening is prevented by inhibiting the expression of a polygalacturonase gene involved in cell wall degradation using antisense RNA, thus improving its shipping and pre- or post-harvest storage properties (Giovannoni et al., Plant Cell l(l):53-63, 1989). Moreover, a study result was reported that indicates that the senescence caused by plant hormones is delayed when their expression is inhibited using antisense phospholipase D involved in lipid degradation (Fan et al., Plant Cell 9(12):2183-2196, 1997). Also, another study result was reported that the senescence of leaves in transgenic tobaccos introduced with knl (knotted 1), a maize homeobox gene linked to an SAG 12 promoter, is delayed (Ori et al., Plant Cell 11:917-927, 1999). In addition, it was reported that the photoreceptor phytochrome influences the senescence of plants. For example, in transgenic tobaccos where an oat PhyA gene had been overexpressed, a delayed- senescence phenotype, such as a delayed reduction in the amount of chlorophyll and total intracellular protein, was observed (Cherry et ah, Plant Physiology 96:775-785, 1991). Furthermore, extended longevity in the leaves of a potato plant where the PhyB gene of Arabidopsis thaliana had been overexpressed was observed, in which case the starting time of chlorophyll reduction in the leaves of the transgenic potato plant was the same as that of normal potato plants, but the elapsed time to complete the degradation of chlorophyll was about 3-4 weeks longer in the transgenic potato plants than that in the normal potato plants (Thiele et al, Plant physiology 120:73- 81, 1999).

However, since plant senescence is a series of continued biochemical and physiological events in which many genes are involved, studies on genes involved in the regulation of plant senescence, functions thereof and methods of regulating the longevity of plants using such genes, are still insufficient.

Meanwhile, cytokinin is an important plant hormone involved in shoot meristem, leaf formation, cell division, chloroplast biogenesis and senescence (Hwang et al, Nature 413(6854):383-389, 2001). Cytokinin signal transduction is achieved by a two-component system or two-component signalling circuit consisting of histidine protein kinase sensing the signal input and a response regulator mediating the signal output (Hwang et al, Nature 413(6854)383-389, 2001; Hwang et al, Plant Physiol. 129(2):500-515, 2002). The cytokinin signal transduction is initiated by the various activities of histidine kinase in cell membrane. This histidine kinase is a cytokinin receptor that recognizes a signal by binding with cytokinin, and the signal is continuously transduced by phosphorylation from the histidine residue of the histidine kinase into the aspartate residue of the response regulators. In this case, a variety of histidine phosphotransmitters serve as signalling shuttles between cytoplasm and nuclei, which depends on cytokinin. By the transduction mechanism as described above, the expression of a series of genes regulating intracellular metabolisms is regulated. Various histidine kinases, such as AHK2, AHK3, and AHK4, have now been known as cytokinin receptors (Yamada et al, Plane Cell Physiol. 42(9): 1017-1023, 2001), but their concrete functions are not yet established.

DISCLOSURE OF THE INVENTION

Previously, the present inventors found various genes involved in the regulation of leaf longevity, and methods of regulating plant longevity using the same (Korean Patent Registration No. 10-350213, Korean Patent Application No. 2001-50748, and Korean Patent Application No. 2001-50774). In addition to this, during our continued studies to find new genes involved in the regulation of plant senescence, the present inventors have found that AHK3, a species of a cytokinin receptor, is involved in the regulation of plant senescence, and the mutation of this receptor delays senescence in plants, thereby completing the present invention.

Accordingly, an object of the present invention is to provide the new use of cytokinin receptor AHK3 and its mutant. Another object of the present invention is to provide a promoter regulating the gene expression of cytokinin receptor AHK3, as well as the use thereof.

To accomplish the above object, in one aspect, the present invention provides a method for delaying the senescence of plants, which comprises introducing into plants a polynucleotide selected from the group consisting of (a) an isolated polynucleotide coding for cytokinin receptor AHK3 or (b) a polynucleotide having at least 70% homology with the isolated polynucleotide (a), and overexpressing the introduced polynucleotide.

In another aspect, the present invention provides a mutant of cytokinin receptor AHK3, which has a substitution of serine for proline at amino acid position 243 in the amino acid sequence of cytokine receptor AHK3 set forth in SEQ ID NO: 10, as well as an isolated polynucleotide encoding the same. Also, the present invention provides a recombinant vector containing the above polynucleotide, as well as cells transformed with the above recombinant vector.

In another aspect, the present invention provides a method for delaying the senescence of plants, which comprises generating a point mutation of cytosine (C) to thymine (T) at base position 727 in the base sequence of an AHK3 gene in plants, the base sequence of the AHK3 gene being set forth in SEQ ID NO: 9.

In still another aspect, the present invention provides a method of investigating a plant senescence-associated substance using any one selected from the group consisting of: (a) an isolated polypeptide set forth SEQ ID NO: 8 or 10; (b) an isolated polynucleotide encoding the polypeptide (a); (c) fragments of the polypeptide (a) or the polynucleotide (b); and (d) derivatives of the polypeptide (a) or the polynucleotide (b).

In still another aspect, the present invention provides the promoter of an AHK3 gene, the promoter having a base sequence set forth in SEQ ID NO: 13. In yet another aspect, the present invention provides a method for inducing the expression of a target gene at a certain development stage of a plant, the method comprising introducing into the plant a recombinant expression vector containing the target gene linked downstream of the AHK3 gene promoter.

As used herein, the term "ore!2" refers to a gain-of-function mutant showing a delayed senescence phenotype by a point mutation occurring on the base sequence of the AHK3 gene, in which the point mutation is a substitution of thymine (T) for cytosine (C) at base position 727 in an AHK3 gene's base sequence set forth in SEQ ID NO: 9.

As used herein, the term "ORE12 gene" refers to a point mutated AHK3 gene resulting from the mutation of orel2. The ORE12 gene according to the present invention has a base sequence set forth in SEQ ID NO: 7.

As used herein, the term "ahk3-l" or "ahk3-2" refers to a knockout mutant resulting from the inactivation of the AHK3 gene by T-DNA insertion.

As used herein, the term "S40-1" or "S75-1" refers to an overexpression mutant of the AHK3 gene, which was genetically manipulated such that the AHK3 gene is overexpressed under the regulation of a CaMV 35S promoter.

In addition, as used herein, the term "orel2ox-13", "orel2ox-14" or "ore!2ox-18" refers to an overexpression mutant of the ORE12 gene, which was genetically manipulated such that the ORE 12 gene is overexpressed under the regulation of a CaMV 35S promoter.

Hereinafter, the present invention will be described in detail. It was first found in the present invention that AHK3, a type of a cytokinin receptor involved in cytokinin signal transduction, regulates the senescence of plant leaves. Furthermore, it was confirmed by various experiments that a gain-of- function mutation of the AHK3 gene shows increased leaf longevity as compared to that of a wild type, and the expression of A-type ARR genes that are cytokinin early response genes is induced at a remarkably higher level than that of a wild type. Such results provide direct evidence indicating that AHK3 is a cytokinin receptor that regulates leaf senescence in Arabidopsis thaliana.

Accordingly, the present invention provides a method for delaying the senescence of plants using the cytokinin receptor AHK3. This method comprises introducing into plants a polynucleotide encoding the cytokinin receptor AHK3, and overexpressing the introduced polynucleotide.

The polynucleotide used in the present invention includes DNA or RNA. Any polynucleotide can be used without limitations if it has a base sequence encoding the cytokinin receptor AHK3 known in the art. Preferably, it may be an AHK3 -encoding polynucleotide having an amino acid sequence set forth in SEQ ID NO: 10. The polynucleotide most preferably has a base sequence set forth in SEQ ID NO: 9. In addition, the polynucleotide used in the present invention include polynucleotides encoding a functional equivalent of AHK3. The functional equivalent refers to a mutant having essentially the same physiological activity as that of a wild-type AHK3 protein, in which the mutant has an amino acid sequence resulting from the substitution of all or parts of wild-type protein amino acids or the deletion or addition of parts of the amino acids. Thus, polynucleotides having at least 70%, preferably at least 80%, and more preferably at least 90% sequence homology with either a polynucleotide encoding the AHK3 protein or a polynucleotide having a base sequence complementary thereto, may all be used in the present invention. Most preferably, a polynucleotide that encodes a mutant-type cytokinin receptor AHK3 where proline at amino acid position 243 in the amino acid sequence of cytokinin receptor AHK3, set forth in SEQ ID NO: 10, had been substituted with serine, may be used. The polynucleotide encoding the mutant-type cytokinin receptor AHK3 preferably contains a base sequence set forth in SEQ ID NO: 7.

The method as described above comprises linking the polynucletide to a promoter capable of regulating the expression of the polynucleotide, introducing the linked polynucleotide into plants, and overexpressing the introduced polynucleotide. As used herein, the term "overexpressing" means that the gene is expressed at a higher level than that in wild-type plants. As the promoter, a constitutive promoter that induces the expression of a target gene constitutively in all time zones, may be used. Examples thereof include a CaMV 35S promoter (Odell et al, Nature 313:810-812, 1985), an Rsyn7 promoter (US Patent Application No. 08/991,601), a rice actin promoter (McElroy et al, Plant Cell 2:163-171, 1990), an ubiquitin promoter (Christensen et al, Plant Mol. Biol. 12:619-632, 1989) and an ALS promoter (US Patent Application No. 08/409,297). In addition, promoters disclosed in US Patent Nos. 5,608,149, 5,608,144, 5,604,121, 5,569,597, 5,466,785, 5,399,680, 5,268,463, and 5,608,142 may all be used. Preferably, the CaMV 35S promoter is used. Furthermore, the introduction of the polynucleotide into plants according to the present invention can be performed by any plant transformation method known in the art. For example, agrobacterium-mediated transformation, electroporation, microparticle bombardment, and polyethylene glycol-mediated uptake may be used. The agrobacterium-mediated transformation is preferably used. In a preferred embodiment of the present invention, each of an ORE 12 gene having a base sequence set forth in SEQ ID NO: 7 and an AHK3 gene having a base sequence set forth in SEQ ID NO: 9 was linked to the CaMV 35S promoter, and introduced into Arabidopsis thaliana wild-type by the agrobacterium-mediated transformation method. The delayed-senescence phenotype of the resulting two transgenic plants was examined and the results confirmed that both the two transgenic plants show a delayed-senescence phenotype (see FIGS. 7 and 8).

Furthermore, the present invention provides a method for delaying the senescence of plants by mutating an AHK3 gene in the plants. Concretely, it provides a method of delaying the senescence of plants, which comprises generating a point mutation of cytosine to thymine at base position 727 of the base sequence of the AHK3 gene in the plants, the AHK3 gene's base sequence being set forth in SEQ ID NO: 9. The point mutation can be induced either by treatment with a mutagen, such as EMS (ethyl methane sulfonate), nitrous acid or UV, or by site-directed mutagenesis (N. Swamy et al, Biochemistry 39:12162- 12171 , 2000). In a preferred embodiment of the present invention, Arabidopsis thaliana wild-type plants were treated with EMS to induce mutation, and it was confirmed that a plant (orell) where cytosine at base position 727 in the AHK3 gene's base sequence set forth in SEQ ID NO: 9 had been point-mutated to thymine by the EMS treatment shows a delayed-senescence phenotype (see FIGS. 1 and 2).

Plants to which the method for delaying plant senescence according to the present invention is applicable include dicotyledonous plants, such as lettuce,

Chinese cabbage, potato and radishes, and monocotyledonous plants, such as rice, barley and bananas. Concrete examples include: food crops, such as rice, wheat, barley, corn, bean, potato, red bean, oat and American millet; vegetable crops, such as Arabidopsis, Chinese cabbage, radish, red pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, Welsh onion, onion and carrot; industrial crops, such as ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, green perilla, peanut and rape; fruit trees, such as apple tree, pear tree, jujube tree, peach, chinensis Planch, grape, mandrain orange, persimmon, plum, apricot and banana; flowers, such as rose, Gladiolus, Gerbera, carnation, mum, lily and tulip; and forage crops, such as Ryegrass, red clover, Orchardgrass, Alfalfa, Tallfescue and Perennial ryegrass. Particularly, if the inventive method for delaying plant senescence is applied to edible vegetables or fruits showing a rapid deterioration in quality by senescence resulting from their thin skin, such as tomatoes, and plants whose leaves are major goods, the inventive method will effectively improve the pre- or post-harvest storage of the plants.

Furthermore, the present invention provides mutant-type cytokinin receptor

AHK3 that delays the senescence of plant leaves, as well as a polynuclotide encoding the same. The mutant-type cytokinin receptor AHK3 which is provided according to the present invention is a mutant where proline at amino acid position 243 in the amino acid sequence of cytokinin receptor AHK3 set forth in SEQ ID NO: 10 had been substituted with serine. Concretely, the mutant-type cytokinin receptor has an amino acid sequence set forth in SEQ ID NO: 8. Also, the polynucleotide encoding the inventive mutant-type cytokinin receptor AHK3 is a mutant where cytosine at base position 727 in an AHK3 gene's base sequence set forth in SEQ ID NO: 9 had been substituted with thymine. Preferably, this polynucleotide has a base sequence set forth in SEQ ID NO: 7. The polynucleotide encoding the inventive mutant-type cytokinin receptor AHK3 may be operably linked to an expression control sequence. As used herein, the term "operably linked" means that one nucleic acid fragment binds to a second nucleic acid fragment so that its function or expression is influenced by the second nucleic acid fragment. Also, the term "expression control sequence" refers to a DNA sequence that controls the expression of an operably linked nucleic acid sequence in certain host cells. Such a control sequence comprises a promoter for initiating transcription, an optional operator sequence for controlling transcription, a sequence coding for a suitable mRNA ribosome binding site, and a sequence controlling termination of transcription or translation.

The inventive polynucleotide can be inserted into a suitable expression vector. As used herein, the term "expression vector" refers to plasmids, viruses or other mediators known in the art, into which the inventive polynucleotide can be inserted. Vectors suitable to introduce the inventive polynucleotide into plant cells include but are not limited to a Ti plasmid, a root-inducing (Ri) plasmid and a plant virus vector. Preferably, a pNB96 vector can be used. The recombinant vector containing the inventive polynucleotide can be introduced into cells by any method known in the art. The cells may be eukaryotes, such as yeasts and plant cells, or prokaryotes, such as E. coli cells. Preferred examples of the cells include E.coli or Agrobacteήum sp.. The known methods for introducing the expression vector into host cells, which can be used in the present invention, include but are not limited to Agrobacterium-mediated transformation, particle gun bombardment, silicon carbide whiskers, sonication, electroporation and PEG(polyethyleneglycol)- mediated uptake. Thus, the present invention provides host cells transformed with the recombinant vector. Examples of such host cells include eukaryotes, such as yeasts and plant cells, or prokaryotes, such as E. coli cells.

In one embodiment of the present invention, in order to identify new genes regulating the leaf senescence of plants, leaf longevity during the natural senescence process and leaf longevity during a senescence process induced by dark treatment were compared to each other by leaf yellowing, and mutants showing a delayed- senescence phenotype were selected. First, seeds treated with EMS (ethyl-methyl sulfonic acid), a mutagen, were sowed, and from grown individual plants, an individual plant having a slow yellowing rate when observing visually was selected (see FIG. 1). The delayed-senescence mutant selected as such was designated "orel2". Then, the phenotype of orel2 was analyzed by the measurement of leaf chlorophyll content and photosynthetic activity, and the measurement result confirmed that orel2 shows a delayed-senescence phenotype as compared to that of a wild-type plant (see FIG. 2).

It is known that the expression of photosynthesis-associated genes, such as a chlorophyll a/b binding protein and a chloroplast ribosomal protein SI 7, increases during leaf growth and then reduces at a senescence stage, but the expression of various senescence-associated genes, such as SAG12, SEN4 and SEN5, increases as senescence progresses (Nam et al, Curr. Opin. Biotech. 8:200-207, 1997). Thus, in another embodiment of the present invention, in order to confirm whether delayed senescence occurring in ore 12 occurs not only at a physiological level but also at a molecular level, the expression patterns of a photosynthetic gene (cab) and a senescence-associated gene (SAG12) were examined. The results showed that, in a wild-type plant, the expression of the cab gene was reduced in an age dependent manner, but in orel2, it was maintained at a high level until 24 days after fourth rosette leaf emergence (DAE) (see FIG. 3). At the same time, the expression of the SAG12 gene was increased in the wild-type plant whereas it was not detected in orel 2. This indicates that the delayed-senescence phenotype of orel2 appears not only at a physiological level and but also at a molecular level.

Generally, although leaf senescence is thought to be predetermined in genes, it is known that the initiation and progression of senescence can be changed either by treatment with plant hormones, such as ABA (abscicsic acid), MeJA (methyl jasmonate) and ethylene, or by dark treatment (Hensel et al, Plant Cell 5:553, 1993; Weaver et al, Plant Mol. Biol. 37(3):455-69, 1998; Zeevaart et al, Annu. Rev. Plant. Physiol. Plant Mol. Biol. 39:439-473, 1988; He et al, Plant Physiol. 128(3):876-884, 2002; Grbic et al, Plant J. 8:595-602, 1995). Thus, in another embodiment of the present invention, a change in leaf longevity in orel 2 by treatment with this plant hormone or dark treatment was analyzed by the measurement of leaf chlorophyll content and photosynthetic activity. The results showed that in a wild-type plant, the leaf chlorophyll content and photosynthetic activity were greatly reduced by treatment with the plant hormone or dark treatment, promoting senescence, but in ore!2, the effect of the plant hormone or dark treatment was greatly reduced, delaying senescence even upon treatment with senescence-promoting hormones (data not shown).

In another embodiment of the present invention, in order to find a gene that induces delayed senescence in orel2, genetic mapping was performed and the corresponding gene was identified. The results confirmed that an ORE12 gene is located 6.18±0.92 centimorgans (cM) from the M235 locus on chromosome 1 of Arabidopsis thaliana, particularly located at BAC F17L21 (see FIG. 4). The base sequence of the region containing the ORE12 gene was analyzed and the result confirmed that ORF is present in this region. The ORF sequence was analyzed in NCBI BLAST and the results confirmed that a single base pair of an AHK3 gene (Atlg27320) encoding for histidine kinase 3 has a substitution of thymine (T) for cytosine (C) at nucleotide position 727 adjacent to the translation-initiation site. And a substitution of serine for proline at amino acid position 243 in the amino acid sequence of the AHK3 gene results from this point mutation. Then, in order to confirm whether the point-mutated AHK3 gene coincides with an ORE12 gene, the AHK3 gene was introduced into orel 2 and subjected to a complementation test, and the test results confirmed that the AHK3 gene is sufficient to complement the delayed leaf senescence phenotype of orel 2. This suggests that the ORE12 gene is a gene resulting from the point mutation of the AHK3 gene known as a cytokinin receptor in Arabidopsis thaliana.

A mechanism that the mutant-type cytokinin receptor AHK3 according to the present invention has a delaying effect on plant senescence may be inferred as follows. In a cytokinin signal transduction system in which cytokinin binds to an AHK3 receptor so that cytokinin signal transduction is made to delay senescence, it is believed that, even in the absence of cytokinin, the inventive mutant-type cytokinin receptor AHK3 enters either a condition where it binds to cytokinin or a condition where it receives a signal from cytokinin, so that it induces continuous signal transduction. Alternatively, the affinity of the mutant-type cytokinin receptor to cytokinin may also be increased to induce continuous signal transduction. Furthermore, the delaying effect on plant senescence by the overexpression of the AHK3 receptor can be explained on the assumption that the amount of AHK3 to which endogenous cytokinin can bind in plants is limited. Namely, in normal conditions, cytokinin signal transduction cannot be continuously made due to the limited amount of AHK3, but when the AHK3 gene is overexpressed, continuous cytokinin signal transduction occurs since the amount of AHK3 that can bind to endogenous cytokinin becomes larger.

In addition, the present invention provides a method for identifying a plant senescence-associated substance using cytokinin receptor AHK3 or a mutant thereof, or a polynucleotide encoding the same, or fragments or derivatives thereof. Concretely, the present invention provides a method for identifying a plant senescence-associated substance using any one selected from the group consisting of: (a) an isolated polypeptide set forth in SEQ ID NO: 8 or 10; (b) an isolated polynucleotide encoding the polypeptide (a); (c) fragments of the polypeptide (a) or the polynucleotide (b); and (d) derivatives of the polypeptide (a) or the polynucleotide (b). The senescence-associated substance may be genes, proteins or chemicals. More concretely, a gene having high sequence homology with the base sequence of an AHK3 gene encoding the cytokinin receptor AHK3 or a mutant-type gene thereof can be identified by sequence comparison, or a similar gene to the AHK3 gene can be identified by hybridizing a part of the AHK3 gene as a probe with cDNA prepared using a RNA or mRNA template extracted from plants treated with a senescence inducer (promoter). Furthermore, either substances binding to the AHK3 gene or a mutant gene thereof, or chemicals inhibiting or activating the expression thereof, can be directly identified. In addition, a senescence-associated protein can also be identified by analyzing the binding pattern of the protein to the AHK3 receptor or a mutant thereof, and a chemical that inhibits or activates the activity of the inventive AHK3 receptor or a mutant thereof can be identified. Such identifications can be performed by generally known methods, such as DNA chip analysis, protein chip analysis, polymerase chain reaction (PCR), Northern blot analysis, Southern blot analysis, Western blot analysis, enzyme-linked immunosorbent assay (ELISA), 2-D gel analysis, yeast two-hybrid systems, and in vitro binding assay.

Furthermore, the present invention provides the promoter of an AHK3 gene encoding the cytokinin receptor AHK3 involved in senescence regulation. Concretely, the promoter has a base sequence set forth in SEQ ID NO: 13, and induces the expression of a target gene specifically at a plant development stage. It induces the expression of a target gene, mainly at a senescence stage. Namely, the inventive promoter induces little or no expression of a target gene in developing organs while it induces the target gene expression at a very high level in mature leaves. Moreover, it induces the target gene expression at a much higher level in shoot organs than that in plant roots. In a preferred embodiment of the present invention, the promoter of the AHK3 gene was linked to a GUS reporter gene and introduced into Arabidopsis thaliana wild-type plants. The transformed plants were subjected to GUS staining, and the result showed that the expression of the GUS gene was limited to the mature parts of the plants (e.g., aged parts of completely grown leaves or roots), and detected at a higher level in shoots than that in roots (see FIG. 6). This suggests that the inventive promoter can be used as a component of an expression vector to induce the expression of a target gene at a certain development stage of plants, and the use of the expression vector can induce the expression of a target gene at a certain development stage of plants. Particularly, genes encoding proteins delaying the senescence of a plant, such as IPT genes, PhyA genes or PhyB genes, are linked to the inventive promoter and introduced into the plant, so that the expression of the genes at a senescence stage can be induced to delay the senescence of the plants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the comparison of delayed-senescence phenotypes between Arabidopsis thaliana wild-type (Col) and delayed-senescence mutant orel 2.

A: a photograph showing the extent of leaf senescence according to days after fourth rosette leaf emergence (hereinafter, referred to as "DAE").

B: a photograph showing the appearance of whole plants observed at 28 DAE. FIG. 2 is a graphic diagram showing a change in chlorophyll content (A) and a change in photosynthetic activity (B) with DAE in Arabidopsis thaliana wild- type (Col) and delayed-senescence mutant orel2. The error bar represents the standard deviation (SD; n=60).

FIG. 3 is the result of Northern blot analysis, which shows the expression patterns of a photosynthetic gene (cab) and a senescence-associated gene (SAG12) in Arabidopsis thaliana wild-type (Col) and delayed-senescence mutant ore 12 with DAE.

FIG. 4 is a gene map showing the location of an ORE12 gene on the Arabidopsis thaliana genome. bar with slant lines: a 7.2-kb DNA fragment used in a complementation test. FIG. 5 is a photograph showing the intracellular location of an AHK3-GFP fusion protein.

A: a transmission electron microscopic photograph.

B: a confocal microscopic photograph. FIG. 6 is a photograph showing the results of GUS staining of Arabidopsis thaliana introduced with a GUS reporter gene linked to the promoter of an AHK3 gene.

A: an individual plant kept for 7 days under light conditions.

Box in A: the roots of an individual plant kept for 7 days under light conditions.

B: an individual plant kept for 10 days under light conditions.

C: an individual plant kept for 15 days under light conditions.

FIG. 7 shows the results of RT-PCR performed to determine the expression level of an AHK3 gene in overexpression mutants of the AHK3 gene (A), and changes in the photosynthetic activity of such mutants with days after dark treatment (DAT) (B). col: Arabidopsis thaliana wild-type. orel 2: a point mutant of an AHK3 gene.

S40-1 and S75-1 overexpression mutants of AHK3 gene. FIG. 8 shows changes in the photosynthetic activity of ORE 12 gene overexpression mutants with DAT. orel2ox-13, -14, and -18: overexpression mutants of ORE12 gene.

FIG. 9 shows the results of RT-PCR performed to determine the expression level of type-A-response regulator genes (ARR3-7, ARR9 and ARR15) in Arabidopsis thaliana wild-type (col) and orel 2. Actinδ: a positive control.

Best Mode for Carrying Out the Invention

The present invention will hereinafter be described in further detail by examples. It will however be obvious to a person skilled in the art that the present invention is not limited to or by the examples.

Example 1: Screening of delayed-senescence mutant M2 seeds (obtained from ABRC seed stock center) treated with EMS were sowed and grown in a greenhouse at a controlled temperature of about 23 °C. The extent of leaf yellowing caused by a reduction in chlorophyll resulting from age- dependent plant senescence was observed with the naked eye, and one individual plant showing a slower yellowing rate than that of a wild type was selected. The selected mutant was designated "orel2". As shown in FIG. 1, the mutant orel 2 when observed with the naked eye showed delayed-senescence symptoms in an age dependent manner in planta leaf senescence. However, its whole plant appearance showed no great difference from that of the wild type.

Example 2: Examination of delayed-senescence phenotype of orel '2

Chlorophyll content and photosynthetic activity that are typical senescence- associated markers were measured to examine the leaf longevity of orel 2 obtained in

Example 1 (Fan et al, Plant Cell 9:2183-2196, 1997; Oh et al, Plant J. 12:527-535,

1997). For this purpose, delayed-senescence mutant orel 2 and a wild-type individual to be used as a control were grown in an environmentally controlled growth room (Korea Instrument Inc.) at 22 °C with a 16-hour light 8-hour dark cycle. In each test, third and fourth rosette leaves were used. 2-1: Measurement of chlorophyll content

Leaves were detached from each plant at 4-day intervals from 12 days to 40 days after fourth rosette lead emergence (DAE). Then, individual sample leaves were boiled in 95% ethanol at 80 °C to extract chlorophyll from the leaves. Thereafter, the absorbance of the extract at 648nm and 664nm was measured to examine chlorophyll content. The chlorophyll content was expressed as chlorophyll concentration per fresh weight of leaves (Vermon et al, Anal. Chem. 32: 1142-1150, 1960). The results showed that, in the wild type, chlorophyll content was reduced by about 70% at 24 DAE, but in orel 2 at the same DAE, leaf yellowing had just started (see A of FIG. 2).

2-2: Measurement of photosynthetic activity

Photosynthetic activity was measured according to the method described in Oh et al, Plant Mol. Biol. 30:939, 1996. For this purpose, leaves at each DAE were subjected to dark treatment for 15 minutes and then measured for their chlorophyll fluorescence using a plant efficiency analyzer. Photosynthetic activity was expressed as the photochemical efficiency of photosystem II deduced from the characteristic of chlorophyll fluorescence. The photochemical efficiency was calculated as the ratio of maximum variable fluorescence (Fv) to maximum value of fluorescence (Fm). The higher the calculated value, the better the photosynthetic activity. The results showed that a reduction in photosynthetic activity was delayed in ore 12 as compared to that in a wild-type plant (see B of FIG. 2).

The above results suggest that, in ore 12, biochemical changes caused by senescence, which are expressed as reductions in chlorophyll content and photosynthetic activity, are progressed at a slower rate than that in the wild-type plant, so that ore 12 shows a delayed leaf senescence phenotype.

Example 3: Examination of expression patterns of photosynthetic gene and senescence-associated gene in orel 2

Leaves at 16, 20, 24, 28 and 32 DAE were detached from orel 2. Also, leaves at 16, 20 and 24 DAE were detached from a wild type as a control. Total RNA was extracted from each sample leaf using Tri-reagent (Sigma), and each lane was loaded with 10 μg RNA. Then, Northern blot analysis was performed using senescence-associated gene SAG12 having a base sequence set forth in SEQ ID NO: 1 and photosynthetic gene Cab (chlorophyll ab binding protein) having a base sequence set forth in SEQ ID NO: 2, as probes (Woo et al, Plant Cell 13:1779-1790, 2001).

As shown in FIG. 3, the analysis results showed that in the wild-type plant, the expression of photosynthetic gene Cab was decreased in an age-dependent manner, whereas in orel2, it was maintained at high levels until 24 DAE. Meanwhile, at 24 DAE, the expression of senescence-associated gene SAG12 was increased in the wild-type plant, whereas it was not detected in orel 2. These facts suggest that ore 12 delays senescence initiation not only at a physiological level but also a molecular level to extend leaf longevity. Furthermore, such test results coincide with previous study results indicating that anabolic activity, such as photosynthesis, and self-maintenance gene activity, are increased in leaf growth and then decreased at a senescence stage (Nam H. G. Curr. Opin. Biotech. 8:200-207, 1997). Example 4: Examination of senescence phenotype of ore 12 by treatment with senescence inducer

Whether the leaf longevity of orel2 is changed by dark treatment and treatment with various plant hormones known to induce (or promote) senescence was examined by the measurement of changes in chlorophyll content and photosynthetic activity.

4-1 : Dark treatment

72 individual leaves at 12 DAE were detached from Arabidopsis thaliana wild-type (col) and orel 2, and floated on 2 ml of 3 mM 2-[N-morpholino]- ethanesulfonic acid (pH 5.8)(hereinafter, referred to as "MES buffer"). Then, the resulting leaves were placed in a light-tight box at 22 °C while photosynthetic activity and chlorophyll content were measured for 6 individual leaves every day in the same manner as in Example 2. The measurement results showed that after 6 days of dark treatment, the photosynthetic activity of the wild-type plant was decreased to 30%, whereas orel 2 was maintained at a photosynthetic activity of 90% (data not shown). Furthermore, similarly to the case of photosynthetic activity, a reduction in chlorophyll content was much shower in orel 2. Namely, after 6 days of dark treatment, the wild-type plant showed a chlorophyll content of 10%, whereas ore 12 showed a chlorophyll content of 80% that is eight times the chlorophyll content of the wild-type plant (data not shown).

4-2: Treatment with plant hormones

36 individual leaves at 12 DAE were floated on 2 ml of MES buffer containing 100 μM ABA or 100 μM MeJA. Control leaves were floated on MES buffer containing no ABA or MeJA. Such treatments with plant hormones were performed for 5 days at 22 °C under continuous lighting. Then, photosynthetic activity and chlorophyll content were measured in the same manner as in Example 2.

The measurement results showed that the photosynthetic activities of the wild-type plant were reduced to 30% and 40% after treatment with ABA and MeJA, respectively, whereas the photosynthetic activities of orel 2 were all maintained at

80%. Also, similarly to the case of photosynthetic activity, a reduction in chlorophyll content was much slower in orel2 (data not shown). Such results suggest that orel 2 has low sensitivity to plant senescence-promoting hormones so that it can inhibit the progression of senescence caused by such hormones, thus extending the longevity of plants.

Example 5: Genetic segregation analysis of phenotype of orel 2 mutant

Delayed-senescence mutant orel2 gene according to the present invention was crossed to Arabidopsis thaliana wild-type (col) to obtain FI and F2 progenies. On these progenies, the genetic segregation analysis of senescence phenotypes was performed. In the analysis results, all the FI plants showed normal senescence symptoms, but about 1/3 of the F2 plants showed a delayed-senescence phenotype. This F2 segregation indicates that the orel2 mutant is a monogenic recessive nuclear mutation. The results are given in Table 1 below. Table 1. Phenotype segregation of progeny crossed wild-type with orel 2

Example 6: Cloning and sequencing of ORE 12 gene based on genetic mapping

To obtain accurate genetic information on ORE12, gene mapping was performed using cleaved amplified polymorphic sequence (CAPS) markers. First, orel 2 was crossed to Landsverg erecta (Ler) to obtain F2 progenies. Among the F2 progenies, 919 plants showing a delayed-senescence phenotype were selected and used in experiments. The CAPS markers were constructed using Arabidopsis thaliana genome sequence data (http://www.arabidopsis.org). As shown in FIG. 4, the results of genetic mapping showed that an ORE12 located 6.18±0.92 centimorgans (cM) from the M235 locus on chromosome 1, particularly located at BAC F17L21.

Thereafter, to clone the ORE 12 gene, the present inventors generated two

CAPS markers (designated F17L21-3A and F17L21-4A genes) at locations where one recombinant chromosome per 919 individual plants can be obtained from the

ORE12 gene. Among the CAPS markers, F17L21-3A is a 1.2-kb product amplified by PCR using oligonucleotide primers having base sequences set forth in SEQ ID NO: 3 and SEQ ID NO: 4, respectively, and has two Dde I cutting sites derived from Col and two Dde I cutting sites derived from Ler. Also, another CAPS marker, F17L21-4A, is a 1.4-kb product amplified by PCR using oligonucleotide primers having base sequences set forth in SEQ ID NO: 5 and SEQ ID NO: 6, respectively, and has one EcoRI cutting site derived from Col and two EcoRI cutting sites derived from Ler. Genetic mapping was performed using the two CAPS markers, and the result showed that an open reading frame (ORF) was located at a region expected to have the ORE12 gene. The base sequence of this region was analyzed and compared to that of the wild-type plant. The sequence comparison results showed that point mutation occurred at

ORF in orel2. The result of search in NCBI BLAST revealed that the ORF is the base sequence of an AHK3 gene (Genbank accession No. AB046870) encoding the histidine kinase 3 of Arabidopsis thaliana. Concretely, the point mutation in orel 2 occurred at base position 727 in the base sequence of the AHK3 gene. Namely, cytosine at base position 727 was substituted with thymine. Also, due to the point mutation of the base sequence, proline at amino acid position 243 in the amino acid sequence of a protein encoded by this gene was substituted with serine. The base sequence of the point-mutated AHK3 gene (i.e., ORE12 gene) is set forth in SEQ ID NO: 7, and the amino acid sequence of the protein encoded by this gene is set forth in SEQ ID NO: 8. Furthermore, the base sequence of the AHK3 gene disclosed in Genbank accession No. AB046870 is set forth in SEQ ID NO: 9, and the amino acid sequence of an AHK3 receptor (i.e., histidine kinase 3) encoded by the AHK3 gene is set forth in SEQ ID NO: 10.

Example 7: Complementation test on ORE12 gene To confirm that the point-mutated AHK3 gene corresponds to the ORE12 gene, the present inventors introduced an AHK3 gene-containing DNA fragment into orel 2. For this purpose, a 7.2-kb DNA fragment containing the AHK3 gene was first amplified by PCR using oligonucleotide primers having base sequences set forth in SEQ ID NO: 11 and SEQ ID NO: 12, respectively. In the PCR amplification, template DNA was denatured by heating at 94 °C for 2 minutes, and then subjected to 30 PCR cycles, each cycle consisting of 30 seconds at 94 °C, and 8 minutes at 68 °C, followed by a final reaction of 10 minutes at 72 °C. The amplified PCR product was corifirmed by 1% agarose gel electrophoresis, and then separated from gel and inserted into a GEM T easy vector (Promega, USA). Next, the AHK3 gene was separated from the vector and subcloned into plant transformation vector pCAMBIA1300 (MRC, USA). The recombinant vector containing the AHK3 gene was introduced into orel 2 and subjected to a complementation test. Antibiotic resistance and phenotype in the T2 generation of the transformed individual plants (orel2/AHK3) were observed. As shown in Table 2 below, the observation results confirmed that the DNA fragment containing the AHK3 gene could complement orel2. This suggests that the ORE12 gene is a point mutant of the AHK3 gene known as a cytokinin receptor in Arabidopsis thaliana.

X value: calculated by the equation of X =Σ(observed value-expected value)²/expected value, for a phenotype ratio of 3:1 or 15:1 (Hyg^r:Hyg^s or wild type : delayed senescence phenotype) which is expected to be expressed by progenies. Hyg^r: hygromycin resistance

Hyg^s: hygromycin sensitivity

Example 8: Examination of expression characteristic of AHK3 gene

8-1: Examination of intracellular expression location

Previously, it was found by hydrophobicity analysis that three putative transmembrane segments are located in the N-terminal parts of the AHK3 gene, suggesting that the AHK3 receptor is located in a plasma membrane (Ueguchi et al, Plant Cell Physiol. 42(2) :231-235, 2001). Thus, the present inventors examined the intracellular location of the AHK receptor using an AHK3-GFP (green fluorescence protein) fusion protein. PCR was performed in the same manner as in Example 7 to amplify the AHK3 gene. The amplified gene was inserted into a plasmid containing 35SC4PPK- sGFP (Wan-ling Chiu et al, Current Biology 6:325-330, 1996). The plasmid expressing an AHK3-GFP fusion protein was purified using a CsCl-EtBr maxiprep. By a method using polyethylene glycol, 4 x 10⁴ protoplasts made from the leaf tissue of Arabidopsis thaliana were transfected with 40 μg of the plasmid. The expression of the fusion plasmid was observed at 23 °C at 15 hours after transformation, and GFP fluorescence was observed under a Nikon TE200 fluorescent microscopy and a Leica TCSNT confocal microscopy. As shown in FIG. 5, the observation results showed that the expression of the AHK3-GFP fusion protein was detected mainly in a plasma membrane. This suggests that AHK3 can transduce a cytokinin signal across the plasma membrane of Arabidopsis thaliana.

8-2: Examination of expression patterns in plants

To examine gene expression patterns, a 2-kb promoter region of SEQ ID NO: 13, which is located upstream of the AHK3 gene to control the expression of the gene, was amplified by PCR. In the PCR amplification, oligonucleotides having base sequences set forth in SEQ ID NO: 14 and SEQ ID NO: 15 were used as primers. Also, template DNA was denatured by heating at 94 °C for 2 minutes and then subjected to 35 PCR cycles, each cycle consisting of 40 seconds at 94 °C, 1 minute at 57°C and 1 minute and 30 seconds at 72 °C, followed by a final reaction of 10 minutes at 72 °C. Thereafter, the amplified PCR product was inserted into the Smal restriction enzyme site of binary vector pCAMBIA1303 (MRC, USA) containing a GUS gene. The resulting vector was introduced into Arabidopsis thaliana wild-type (col), and T3 homoline plants were selected. Then, the transformed T3 homoline plants were grown for 7 days, 10 days and 15 days under white light and subjected to GUS staining to examine the expression of a GUS gene at various development stages of the plants. As shown in A of FIG. 6, the results showed that the GUS gene was expressed strongly in the cotyledon and hypocotyl of 7-day old seedlings, whereas it was expressed very weakly in emerging primary leaves and vascular bundles. Also, as the plants grew, the expression of the GUS gene was limited to the mature parts of the plants, such as fully expanded leaves and older parts of roots. Furthermore, the expression of the GUS gene in roots was relatively weaker than that in the aerial parts of the plants (see B and C of FIG. 6). Particularly, the GUS gene was expressed at high levels in shoots.

It was reported that AHK4, known already as a cytokinin receptor, was expressed predominantly in roots (Ueguchi et al, Plant Cell Physiol. 42(2) :231-235, 2001), and the phenotype of a loss-of-function mutant of AHK4 was observed mainly in roots other than shoots (Ueguchi et al, Plant Cell Physiol. 42(7): 751-755, 2001). When considering these facts, the test results in this Example suggest that AHK3 functions as a main cytokinin receptor in the shoots of Arabidopsis thaliana, particularly in mature parts, to maintain the antisenescence action-associated intracellular level of cytokinin.

Example 9: Examination of senescence phenotype in knockout mutant of AHK3 gene

In order to examine the role of AHK3 as a cytokinin receptor in the leaf senescence of plants, the antibiotic resistance of SIGnAL Arabidopsis knockout pools (nttp://signal.salk.edu/cgi-bin/tdnaexpress was examined by PCR and T-DNA insertion, and homolines where the AHK3 gene had been knock out were selected. To examine whether the AHK3 gene was inactivated or not, PCRs were performed with primers having base sequences set forth in SEQ ID NO: 16 and SEQ ID NO: 17 and with primers having base sequences set forth in SEQ ID NO: 18 and SEQ ED NO: 19, respectively. Two individual plants where the AHK3 gene had been knocked out by T-DNA insertion were selected, and they were named "ahk3-l " and "ahk3-2 ", respectively. The selected ahk3-l and ahk3-2 were subjected to dark treatment for 4-7 days to induce senescence. Then, whether a delayed-senescence phenotype is shown was observed visually. The results showed that the delayed- senescence phenotype of leaves did not appear (data not shown). This suggests that a single amino acid change in the extracytoplasmic domain of AHK3 is a gain-of- function mutation that causes a delayed-senescence phenotype.

Example 10: Examination of senescence phenotype of overexpression mutant of AHK3 gene

In order to further confirm that the single amino acid change in AHK3 is the gain-of-function mutation, the overexpression of an AHK3 gene was induced with a CaMV 35S promoter that induces the constitutive expression of genes. First, the full-length DNA of the AHK3 gene was amplified by RT-PCR with oligonucleotide primers having base sequences set forth in SEQ ID NO: 11 and SEQ ID NO: 12. In the PCR amplification, a total RNA template isolated from Arabidopsis thaliana was subjected to reaction for 30 minutes at 50 °C to synthesize primary cDNA. Then, the cDNA was denatured by heating at 94 °C for 2 minutes, and subjected to 30 PCR cycles, each cycle consisting of 30 seconds at 94 °C, 30 seconds at 52 °C, and 5 minutes at 72 °C, followed by a final reaction of 10 minutes at 72 °C. The amplified PCR product was inserted into a pNB96 vector (distributed from Laboratory of Plant Molecular Genetics, Pohang University of Science and Technology, and the resulting vector was introduced into an Agrobacterium tumefaciens AGLl strain (Lazo et al, Biotechnology 9:963-967,1991)(ATCC BAA- 101). Thereafter, Arabidopsis thaliana wild-type (col) was transformed with the transformed Agrobacterium strain using the floral dip method (Clough et al, Plant J. 16(6) :735-743, 1998). Following this, two T3 homoline plants showing antibiotic resistance were selected, and named "S40-1" and "S75-1", respectively. Total RNAs were extracted from the selected T3 homolines, and subjected to

RT-PCR in the same manner as described above. As shown in A of FIG. 7, the results of RT-PCR confirmed that the expression level of the AHK3 gene was remarkably higher than that of the wild type. Also, the selected T3 homolines were cultured under dark conditions for 5-6 days, and then, fourth rosette leaves at 12 DAE were detached. Next, the detached leaves were measured for their photosynthetic activities according to the method described in Example 2-1 to examine delayed-senescence patterns. As shown in B of FIG. 7, the results showed that delayed-senescence patterns of the transformed plants are weaker than that of the delayed-senescence phenotype of orel 2 but delayed more than that of the wild-type plant. Also, similarly to orel 2, an adult morphological phenotype caused by continuous cytokinin signal transduction did not appear in the transformed plants (data not shown). This suggests that ORE12 (i.e., point mutated AHK3) is a cytokinin receptor that is more specifically associated to the delayed senescence of Arabidopsis thaliana, as compared to normal AHK3. Example 11: Examination of senescence phenotype of overexpression mutant of ORE 12 gene (point mutated AHK3 gene)

In order to examine whether a delayed-senescence phenotype appears even if a point mutated AHK3 gene (i.e., an ORE12 gene of SEQ ID NO: 7) is overexpressed in Arabidopsis thaliana wild-type, the following test was performed.

Template DNA was extracted from orel 2, and subjected to PCR in the same manner as in Example 10, using oligonucleotide primers having base sequences set forth in SEQ ID NO: 11 and SEQ ID NO: 12. In the PCR reaction, the template DNA was denatured by heating at 94 °C for 2 minutes, and then subjected to 30 PCR cycles, each cycle consisting of 30 seconds at 94 °C, 30 seconds at 52 °C, and 5 minutes at 72 °C, followed by a final reaction of 10 minute at 72 °C. Thereafter, Arabidopsis thaliana wild-type plants were transformed in the same manner as in Example 10, and from the transformed plants, three T2 line individuals were selected. The selected transgenic plants were named "ore!2ox-13 ", ^uorel2ox-14" and "orel2ox-18". Next, the delayed-senescence phenotype of the ORE12 gene- overespressed plants was observed in the same manner as in Example 10. As shown in FIG. 8, the results showed that a delayed-senescence phenotype appeared as in orel2.

Example 12: Examination of expression patterns of cytokinin-responsive genes in ore 12

In order to confirm that a delayed leaf senescence phenotype appearing in orel 2 is caused by continuous cytokinin signal transduction, the present inventors examined the expression pattern of type-A-response regulator genes, which is very specifically induced by cytokinin (Brandstatter et al, Plant Cell 10:1009- 1020, 1998; Taniguchi et al, FEBS Lett. 429:259-262, 1998; Imamura et al, Plant Cell Physiol. 40:733-742, 1999). Total RNAs were extracted from the third and fourth rosette leaves of wild-type plants and ore 12 at 16 DAE using a Tri-Reagent kit (molecular research center, USA). 1 μg of each RNA template was subjected to reaction at 65 °C for 5 minutes, at 42 °C for 6 minutes and at 85 °C for 5 minutes, using a first strand cDNA synthesis kit (Roche, Germany), to synthesize primary cDNA. Thereafter, the synthesized cDNA template was subjected to PCR with primers specific to various ARR genes as set forth in Table 3 below. In the PCR reaction, the template DNA was denatured by heating at 94 °C for 2 minutes, and then subjected to 35 PCR cycles, each cycle consisting of 40 seconds for 94 °C, 1 minute at 52 °C, and 1 minute and 30 seconds at 72 °C, followed by a final reaction of 10 minutes at 72 °C. Next, the PCR products were analyzed by 1% agarose gel electrophoresis.

As shown in FIG. 9, the analysis results showed that the expression levels of various type-A ARR genes in orel2 were remarkably higher than that in the wild- type plant. This suggests that a delayed-senescence phenotype appearing in orel2 is caused by continuous cytokinin signal transduction resulting from the phosphorylation of a two-component system.

Table 3. Type-A ARRs

INDUSTRIAL APPLICABILITY

As described above, it was found in the present invention that AHK3 known already as a cytokinin receptor is involved in the regulation of plant senescence, and a mutation of the AHK gene delays senescence in plants. The introduction and overexpression of the inventive AHK3 gene or a mutant gene thereof in plants can delay the senescence of the plants, resulting in an increase in the production of the plants as well as an improvement in the pre- or post-harvest storage of the plants. Furthermore, the inventive AHK3 gene, a mutant gene thereof, or a protein expressed therefrom, will be useful in studies on mechanisms of plant senescence, and the identification of plant senescence-associated substances, etc.

Claims

WHAT IS CLAIMED IS:

1. A method for delaying the senescence of plants, which comprises: introducing into the plants a polynucleotide selected from the group consisting of (a) an isolated polynucleotide coding for cytokinin receptor AHK3, and (b) a polynucleotide having at least 70% homology with the polynucleotide (a); and overexpressing the introduced polynucleotide.

2. The method of Claim 1, wherein the polynucleotide (a) has a base sequence set forth in SEQ ID NO: 9.

3. The method of Claim 1, wherein the polynucleotide (b) has a base sequence set forth in SEQ ID NO: 7.

4. The method of Claim 1, wherein the plants are dicotyledonous plants or monocotyledonous plants.

5. An isolated polynucleotide encoding a mutant of cytokinin receptor AHK3, in which the mutant has a substitution of serine for proline at amino acid position 243 in the amino acid sequence of the cytokinin receptor AHK3, the amino acid sequence being set forth in SEQ ID NO: 10.

6. The polynucleotide of Claim 5, which has a base sequence set forth in SEQ ID NO: 7.

7. A recombinant vector containing the polynucleotide of Claim 5.

8. Cells transformed with the recombinant vector of Claim 7.

9. A method for delaying the senescence of plants, the method comprising generating a point mutation of cytosine (C) to thymine (T) at base position 727 in the base sequence of an AHK3 gene in the plants, the base sequence of the AHK3 gene being set forth in SEQ ID NO: 9.

10. The method of Claim 9, wherein the point mutation is generated by mutagen treatment or site-directed mutagenesis.

11. The method of Claim 9, wherein the plants are dicotyledonous plants or monocotyledonous plants.

12. A method for identification a plant senescence-associated substance using any one selected from the group consisting of:

(a) an isolated polypeptide set forth in SEQ ID NO: 8 or 10;

(b) an isolated polynucleotide encoding the polypeptide (a); (c) fragments of the polypeptide (a) or the polynucleotide (b); and

(d) derivatives of the polypeptide (a) or the polynucleotide (b).

13. The method of Claim 12, which is performed by DNA chip analysis, protein chip analysis, polymerase chain reaction (PCR), Northern blot analysis, Southern blot analysis, Western blot analysis, enzyme-linked immunosorbent assay (ELIS A), 2-D gel analysis, yeast two-hybrid systems, or in vitro binding assay.

14. The promoter of an AHK3 gene, in which the promoter has a base sequence set forth in SEQ ID NO: 13.

15. The promoter of Claim 14, which induce the expression of a target protein specifically in a plant development stage.

16. A recombinant expression vector comprising a target gene linked downstream of the promoter of Claim 14.

17. A method for inducing the expression of a target gene in a certain development stage of plants, the method comprising introducing the recombinant expression vector of Claim 16 into the plants.