AU5496199A - Transgenic plants with altered senescence characteristics - Google Patents

Description

4 S F Ref: 357743D1

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

s Name and Address of Applicant: Actual Inventor(s): Address for Service: Invention Title: The following statement best method of performi Wisconsin Alumni Research Foundation 614 North Walnut Street P.O. Box 7365 Madison Wisconsin 53707-7365 UNITED STATES OF AMERICA Richard M Amasino and Susheng Gan Spruson Ferguson, Patent Attorneys Level 33 St Martins Tower, 31 Market Street Sydney, New South Wales, 2000, Australia Transgenic Plants with Altered Senescence Characteristics is a full description of this invention, including the ng it known to me/us:gQ O *SS ece ed C0n

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e"6 3 gg 1 'a\G 3o 5845 1 -1- TRANSGENIC PLANTS WITH ALTERED SENESCENCE

CHARACTERISTICS

Field Of The Invention In general, the present invention relates to the field of plant molecular biology. Specifically, the present invention relates to transgenic plants with inserted transgenes that are activated by developmentspecific promoters.

Background Leaf senescence is a phase of development during which cells undergo distinct metabolic and structural changes prior to cell death (Nood6n, Senescence and ing in Plants, D. Nood6n and A. C. Leopold, Ed.), pp. 391-439, Academic Press, San Diego, CA, 1988). It is an important phase in the plant life cycle that is thought to contribute to fitness by recycling nutrients to actively growing regions. The initiation of leaf senescence can be induced by a variety of external factors such as shading, mineral deficiency, drought and pathogen infection (Thomas, et al., Ann. Rev. Plant Physiol. 31:83-111, 1980) and by developmental processes such as seed development (Nooden, 1988, supra). In the absence of such factors, leaf senescence occurs in an age-dependent manner in many 30 species (Batt, et al., J. Exp. Bot. 26:569-579, 1975; Hensel, et al., Plant Cell 5:553-564, 1993; Jiang, et al., Plant Physiol. 101:105-112, 1993).

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Physiological and genetic studies indicate that senescence is a highly regulated process (Nooden, 1988, supra; Thomas, 1980, susra). The progression of a leaf through the senescence program is visibly marked by the loss of chlorophyll and consequent yellowing, a result of the disassembly of the chloroplast (Thomson, et al.

Plant Senescence: Its Biochemistry and Physioloy, pp.

20-30, 1987; Woolhouse, Can. J_ Bot. 62:2934-2942, 1984). Leaf senescence involves degradation of proteins, nucleic acids and membranes, and the subsequent transport of the nutrients resulting from this degradation to other regions of the plant, such as developing seeds, leaves, or storage organs (Nooden, 1988, supra; Woolhouse, 1984, sura).

Molecular studies indicate that changes in gene expression are associated with the senescence program.

The levels of mRNAs encoding proteins involved in photosynthesis decrease during senescence (Bate, et al., J_ EXE_ Bot. 42:801-811, 1991; Hensel, et al., Plant Cell 5:553-564, 1993; Jiang, et al., Plant Physiol. 101:105-112, 1993), while mRNA levels of genes encoding proteins thought to be involved in the senescence program increase (Graham, et al., Plant Cell S' 4:349-357, 1992, Hensel, et al., Plant Cell 5:553-564, 25 1993; Kamachi, et al., Plant Physiol. 93:1323-1329, 1992; Taylor, et al., Proc. Natl. Acad. Sci. USA 90:5118-5122, 1993). The activities of several enzymes that are likely to play a role in the breakdown and mobilization of nutrients have also been shown to increase during senescence (Blank, et al., Plant .P hysio 97:1409-1413, 1991; Debellis, et al., Plant Cell Physiol. 32:1227-1235, 1991; Friedrich, et al.

Plant Physiol. 65:1103-1107, 1980; Pistelli, et al., J.

Plant Physiol. 19:723-729, 1992).

35 Although the general changes that occur during senescence are known, many of the biochemical details of how nutrient remobilization occurs remain to be determined. Furthermore, little is understood of how the changes in gene expression that accompany senescence are regulated.

Promoters capable of promoting gene expression during the plant developmental stage of senescence are needed in the art of plant molecular biology.

As a first step towards obtaining this goal, we investigated macromolecular changes that occur during leaf senescence in Arabidopsis thaliana. The onset of leaf senescence in Arabidopsis is determined by leaf age (Hensel, et al., su§ra). This predictability of the senescence program in Arabidopsis facilitated an integrated study of changes in RNA, chlorophyll, protein, and gene expression associated with natural leaf senescence in the intact plant. We also used this system, as recited here, to isolate and characterize the temporal expression patterns of mRNAs that increase and decrease in abundance during leaf senescence.

These senescence-specific mRNAs allowed us, as described below, to isolate and characterize novel senescence-specific promoters.

Summary Of The Invention The present invention is a genetic construct comprising an SAG12 promoter sequence operably connected to a protein-coding DNA sequence not natively 25 connected to the promoter sequence. Preferably, the SAG12 promoter sequence is the SAG12-1 sequence. Most preferably, the SAG12 promoter is the first 602 bp of SEQ ID NO:2 and the protein-coding DNA sequence encodes isopentenyl transferase.

The present invention is also a cell or a plant ~containing the genetic construct.

It is an object of the present invention to provide a genetic construct with a promoter sequence enabling senescence-specific gene expression operably linked to a protein-coding sequence.

It is another object of the present invention to provide a senescence-specific promoter linked to a -4sequence encoding an enzyme that catalyzes the synthesis of a plant hormone, preferably cytokinin.

It is another object of the present invention to provide a senescence-specific promoter linked to an isopentenyl transferase sequence.

It is another object of the present invention to provide a transgenic plant that contains a transgene expressed only in senescing tissue.

It is a feature of the present invention that gene expression can be targeted specifically to senescing tissue, thus avoiding constitutive expression that could be damaging.

Other objects, advantages, and features of the present invention will become apparent after review of the specification, drawings, and claims.

Description Of The Drawings Fig. 1 is a schematic map of SAG12-1 promoter/GUS/MAS-ter construct in a binary vector.

Fig. 2 is a schematic map of SAG12-1 promoter/IPT/NOS-ter construct in a binary vector.

Fig. 3 is the nucleotide sequence of SAG12-1 promoter/IPT/NOS-ter construct. The and labels correspond to and in Figs. 1 and 2.

2 Description Of The Invention One aspect of the present invention is a genetic construct comprising a senescence-specific promoter operably linked to a foreign gene sequence that is not natively associated with the promoter. A useful .0 senescence-specific promoter, identified here as the SAG12 promoter, has been characterized. The availability of a senescence-specific promoter has also enabled the creation of transgenic plants with altered senescence morphology e.g. delayed senescence. This finding offers a mechanism to extend the growth of useful plants.

Isolation of a particular SAG12 promoter from Arabidopsis thaliana, SAG12-1, is described in detail below. Basically, a senescence-specific cDNA, here called "SAG12", was isolated along with the genomic clone corresponding to the SAG12 cDNA. The SAG12-1 promoter was isolated from this genomic material. The term "SAG" designates a senescence associated gene.

SEQ ID NO:1 and Fig. 3 contain a nucleotide sequence for one embodiment of the SAG12-1 promoter.

SEQ ID NO:2 describes a truncated version of this promoter. Both versions of the SAG12-1 promoter are sufficient to promote gene expression in a senescencespecific manner.

Also described below is a second senescencespecific promoter, isolated from Arabidopis in a similar manner. The second promoter is here designated "SAG13.,, The SAG13 promoter was also isolated from the ArabidoEss genome. SEQ ID NO:3 contains the nucleotide sequence for the SAG13 promoter, including 1782 base pairs upstream of the transcription start site.

By "senescence-specific promoter" it is meant to indicate that the SAG12-1 and SAG13 promoters are capable of preferentially promoting gene expression in a plant tissue in a developmentally regulated manner 25 such that expression of a 3' protein coding region occurs substantially only when the plant tissue is undergoing senescence.

Preferably, the SAG12 promoter includes 0 nucleotides sufficiently homologous to the first 602 bp of SEQ ID NO:2 so that the promoter is capable of expressing genes preferably in a senescing tissue Also, the senescence-specific promoter can consist of the nucleotide sequence of SEQ ID NO:2.

referably the SAG13 promoter includes a portion of the sequence set forth in SEQ ID NO:3 below. While this entire sequence is sufficient for senescencespecific promoter activity, it is also likely that a smaller sequence will also be sufficient. The bounds of such a smaller sequence can readily be determined by truncation of the sequence of SEQ ID NO:3 below, followed by empirical testing of such truncations for senescence specific promoter activity.

The Examples below describe the creation of senescence-specific cDNA clones from Arabidosis, the characterization of these clones, and the use of these cDNA clones to obtain a specific SAG12 senescencespecific promoter, SAG12-1 and a second promoter SAG13.

It is believed that there are other senescence-specific promoters with sufficient homology to SAG12-1 or SAG13 to be suitable for the present invention. One could easily use the techniques described below to obtain these homologous promoters.

Creation of an SAG12 Promoter In the Examples below, described is the isolation of the SAG12 promoter using the SAG12 cDNA clone. This cDNA clone was obtained from an RNA molecule that appears to be expressed only during senescence.

The SAG12 cDNA has been used to screen an SArabidopsis library to obtain the SAG12 gene. The gene was originally designated SAG12-1 in the belief that there were two SAG12 genes in Arabidopsis, although it is now believed that there is only one. The SAG12-1 25 promoter was obtained from the SAG12-1 genomic clone.

SEQ ID NO:1 and Fig. 3 disclose the sequence of 2073 bp of the SAG12-1 promoter. Further studies, also described below, showed that the SAG12-1 promoter could be truncated to 602 bp and still remain functional.

30 SEQ ID NO:2 describes the 602 bp linked to a untranslated region of the SAG12-1 gene.

To obtain a SAG12 promoter, one could follow several paths. Most easily, one could create an oligonucleotide probe from the sequences disclosed in SEQ ID NOs:l and 2 or Fig. 3 and probe an Arabidopsis genomic library to recover a copy of the SAG12 promoter.

It is envisioned that minor nucleotide additions, deletions, and mutations will not affect the function of the SAG12-1 promoter. Furthermore, it is possible, if not likely, that there may be variations in sequence of the SAG12 gene (or SAG13) and promoter among populations of Arabidopsis stocks because of normal allelic variations. Furthermore, it is likely and anticipated that homologous sequences can be recovered from other plants. Therefore, the sequence of a suitable SAG promoter might not be identical to that disclosed in SEQ ID NOs:l or 2. Detailed below is an assay by which one may determine whether a candidate genomic sequence is sufficiently homologous to the senescence-specific SAG12-1 promoter to be suitable for the present invention.

Additionally, it is envisioned that the 602 bp of SEQ ID NO:1 may be further truncated and still produce a suitable SAG12 promoter. One of ordinary skill in this technology can readily appreciate that 5' or 3' truncations, or internal deletions, from this 602 bp sequence can be made, and those truncations empirically tested for senescence-specific activity, to find such smaller truncations of the SAG12-1 promoter.

Preferably, a portion of the 5' untranslated 25 region of the SAG12-1 gene will be added to the promoter sequence. SEQ ID NOs:l and 2 disclose this sequence. In Fig. 3, the 5' untranslated region is the region between the +1 symbol and the "Nco I" symbol.

Creation of SAG13 Promoter A similar method was used to isolate and identify the SAG13 promoter set forth in the Examples below.

Variations in SAG13 sequence, due to allelic variations and the like, are expected as well. SAG12 and SAG13 are not notably homologous.

-8- Assay of a Candidate Promoter Once a candidate genomic sequence has been isolated, one may wish to determine whether or not this DNA sequence is a SAG12 or a SAG13 promoter. One could sequence the DNA sequence by techniques familiar to those skilled in the art of plant molecular biology and determine whether the sequence is identical to either SEQ ID NO:1, 2 or 3. If the candidate sequence is identical or homologous to a portion of the first 2073 bp of SEQ ID NO:1, the first 602 bp of SEQ ID NO:2, or the first 1782 bp of SEQ ID NO:3, then the sequence is a suitable SAG12 or SAG13 promoter.

If the sequence is not identical, however, and is closely homologous, i.e. more than 95% homologous, one may have isolated a copy of an allelic SAG12 or SAG13 promoter. One would wish to do a functional assay to determine whether or not this sequence was sufficiently homologous to the first 602 bp of SEQ ID NO:2, the first 2073 bp of SEQ ID NO: 1, or the first 1782 bp of SEQ ID NO:3 to be suitable for the present invention.

By "sufficiently homologous" it is meant that a candidate promoter is at least 95% homologous in nucleotide sequence and is substantially equivalent to the SAG12-1 promoter sequence in its ability to 25 preferentially promote gene expression in senescing plant tissue. An assay for determining whether a candidate sequence is suitable is described below.

To make this determination, one could follow the examples described below and attach the candidate promoter to a reporter protein coding sequence, such as the GUS sequence encoding the enzyme betaglucuronidase. The sequence of the GUS gene is described in U.S. patent 5,268,463. Transformation of a plant with an expression cassette including the GUS 35 sequence allows one to determine whether or not the GUS reporter sequence was expressed in only the senescing tissues, was constitutively expressed, or was not expressed at all. Only a result indicating that the reporter sequence is only expressed in senescing tissues and not other tissues would indicate a suitable promoter.

Alternatively, the candidate sequence could be attached to the isopentenyl transferase sequence and transformed into tobacco plants, as we have described below. Table 2 of the Examples discloses specific differences between plants transformed with the SAG12 promoter linked to an IPT gene and transgenic control plants containing a construct with the SAG12 promoter linked to the GUS reporter gene. A candidate promoter would have to perform equivalently to be suitable for the present invention.

Therefore, a candidate promoter must satisfy three criteria. First, it must be isolatable or hybridizable with an oligonucleotide probe created from the first 2073 bp or SEQ ID NO:1, the first 602 bp of SEQ ID NO:2, or th corresponding portion of SEQ ID NO:3.

Second, it must be sufficiently homologous to either the first 2073 bp of SEQ ID NO:1, the first 602 bp of SEQ ID NO:2, or the corresponding portion of SEQ ID NO:3 so as to promote senescence-specific expression of a reporter gene, such as GUS. Third, it must provide equivalent senescence-specific expression as the SAG12 25 or SAG13 promoter described in Table 2 of the Examples.

Creation of Genetic Construct Once one has obtained an SAG12 or SAG13 promoter, a genetic construct .must be created containing both that promoter and a protein-coding sequence. By 30 "genetic construct" it is meant to describe an operably connected promoter and gene sequence. Typically the promoter sequence is 5' or "upstream" of the gene sequence. The promoter will be able to promote transcriptional activity using the gene sequence as a template.

A suitable foreign gene sequence is capable of expressing an RNA molecule. This RNA molecule may or may not be translated into a mature protein.

A

foreign gene sequence" may alternatively be in the antisense orientation in order to express antisense mRNA. Preferably, the foreign gene sequence encodes a protein.

In one embodiment of the invention, the foreign gene sequence encodes an enzyme catalyzing biosynthesis of a plant hormone, preferably a cytokinin. Most preferably, the enzyme is IPT (isopentenyl transferase).

Standard molecular biological procedures may be used to link the cloned promoter to a protein-coding sequence, such as the IPT sequence. Several genes encoding IPT have been isolated, sequenced and published. The bacterial strains harboring these genes have been deposited with, and are available from, ATCC.

With published sequence information, PCR and other gene amplification and recovery techniques may be used to isolate IPT genes. Examples of IPT sequences (also referred to as tmr or tzs) are presented in: Crespi et al., EMBO J. 11:795-804 (1992); Goldberg et al., Nucleic Acids. Res. 12:4665-4677 (1984); Heide Kamp et al., Nucleic Acids Res., 11:6211-6223 (1983); Strabala o *et al., Mol. Gen. Genet. 216:388-394 (1989).

25 The genetic construct may be created using either plasmid or viral vectors or other methods known in the art of molecular biology to create a construct capable of being transformed into a plant cell. We describe the creation of a genetic construct suitable to be 30 transformed via the Agrobacterium system. However, there are other means of transformation of plants, and creation of transgenic plants, such as particle bombardment and electroporation, that require many different vector systems. The ability to construct and adopt such vectors to the transformation system to be used is well known to those of skill in the art.

-11- Modification of Plant Senescence The availability of effective plant senescencespecific promoters makes possible the creation of transgenic plants with altered senescence characteristics. Genetic constructs can be inserted into plants which become effective only upon plant cells entering senescence. Such senescence-specific expression permits the expression in plants of genes which might be disruptive of plant morphology or productivity if expressed at any other stage of plant development. For example, it now becomes possible to insert a gene encoding a cytokinin biosynthetic enzyme under the control of a senescence-specific promoter without having the tissues of the plant exposed to the excess cytokinin during pre-senescence growth. Then, at the onset of senescence, the senescence-specific promoter activates cytokinin production to alter the progression of senescence in the plant. It has been found, in particular, that the combination of a senescence-specific promoter and a cytokinin-producing gene sequence creates a transgenic plant that, in essence, has a delayed senescence. Such a plant will vegetatively grow longer, producing more flower, seed or fruit, than a corresponding non-transgenic plant.

25 It is anticipated that other coding regions affecting plant maturation and senescence may also be placed behind the senescence-specific promoter and transformed into plants to produce useful transgenic plants with altered senescence.

30 Examples :Materials and methods Plant materials Arabidopsis thaliana ecotype Landsberg erecta seed was sterilized in 2.5% sodium hypochlorite for 5 min and rinsed with five changes of sterile water. Sterile seed was imbibed at 4 0 C in 1 mM gibberellic acid A 3 for hours prior to sowing on a mixture of peat moss, -12vermiculite and perlite saturated with Arabidopsis nutrient solution as described in Somerville, et al., Methods in Chlroplast Molecular Biology, Elsevier Biomedical Press, New York, NY, pp.

129-137, 1982. Plants were grown at 230C and relative humidity under 120 ymol m-2 s- of continuous light from a mixture of cool-white fluorescent and incandescent bulbs and sub-irrigated as needed with water. Under these conditions the plants grew vegetatively for about 3 weeks forming 6-7 rosette leaves prior to bolting. Rosette leaves 5 and 6 were harvested at various times after full expansion. All tissues were frozen in liquid

N

2 immediately after harvest and stored at uantification of chlorophll and rein Forty-five cm of fresh leaves were soaked at 65 0

C

for 2 h in ethanol, and the amount of chlorophyll was determined spectrophotometrically (Wintermans, et al Biochem Biophvs Acta. 109:448-453, 1965). After ethanol incubation the same leaves were used for total protein extraction after they had been briefly dried under vacuum. The leaf residue from forty-five cm 2 of leaf material was ground in liquid

N

2 resuspended in S9 ml of 10 mM Na 2 Citrate, 1 mM EDTA, 1% SDS, pH 8 and 25 incubated at 70 0 C with stirring for 30 min. The soluble and insoluble components were separated by centrifugation. The pelletable fraction was solubilized in 10 ml 1 N NaOH overnight at 30 0

C.

30 Protein levels in the soluble and pelletable fractions were subsequently quantified according to Lowry, et A j Biol. Chem. 193:265-275, 1951 combining the modifications of Peterson, Anal. Biochem. 83:346-356 1977 and Larson, et al., Anal. Biochem. 155:243-248, 1986. Three replica samples from three independent 35 batches of Arabidopsis were analyzed.

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-13- RNA analysis Total RNA was extracted as described in Puissant, et al., BioTechniques 8:148-149, 1990 and quantitated spectrophotometrically (Sambrook, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY., 1989). For RNA gel blot analyses,

RNA

samples were electrophoretically fractionated on formaldehyde-agarose gels, transferred to polysulfone membranes (Gelman, Ann Arbor, MI), and hybridized to "P-labelled probes made by the random prime method (John, et al., J. Bacteriol. 170:790-795, 1988).

RNA

was loaded on a mass basis (5 pg of RNA per lane) and an area basis (a half leaf equivalent of RNA per lane).

The amount of probe hybridized to the RNA-was quantitated using a Betagen 9-particle scanner (IntelliGenetics, Inc., Mountain View, CA). RNA gel blots prepared from three independent batches of tissue were analyzed for each cDNA clone.

Construction and screening of cDNA libraries Poly RNA used for construction of cDNA libraries was isolated as described in Crowell, et al.

Proc. Natl. Acad. Sci. USA 87:8815-8819, 1990. RNA isolated from S2 and pooled S3 and S4 leaves was used Sto construct two cDNA libraries. First-strand cDNA was 25 synthesized using oligo (dT),,-Xba I as primer with SuperScript T RNase H- reverse transcriptase and secondstrand cDNA was synthesized using E. coli DNA Polymerase I, E. coli DNA ligase and RNAse H as recommended by the manufacturer (BRL, Gaithersberg, MD). Double-stranded cDNA was size-fractionated on a BioGel A 0.5m column (BioRad, Richmond, CA) to remove cDNAs less than 200 bp in length. EcoR I linkeradapters (Promega, Madison, WI) were ligated onto the cDNA then the 5' ends of the cDNA were then phosphorylated with polynucleotide kinase. The cDNA was size fractionated by agarose-gel electrophoresis and cDNAs >500 bp were electroeluted and ligated into -14pBluescript SKII(+) (Stratagene, La Jolla, CA) that had been cut with EcoR I and dephosphorylated. The ligation products were electroporated into E. coli strain DH5a. Both S2 and S3/4 cDNA libraries contained 1X10 s recombinant clones. For library screening, replica filters of the libraries were prepared as described (Sambrook, et al., 1989, supra) and hybridized to cDNA probes made by reverse transcription of poly RNA using deoxyadenosine 5- [Ce- 32

J]

triphosphate. For cross-hybridization analysis, probes corresponding to cDNA inserts were prepared using the random prime method and hybridized to dot blots of candidate plasmids (Sambrook, et al. 1989, supra) Leaf Senescence in Arabidopsis thaliana Proceeds throuch Defined Phenotypic and Biochemical Chanes We divided Arabidopsis thaliana rosette leaf senescence into five stages designated S1 through based on phenotypic appearance and measured the amount of RNA, protein, and chlorophyll present at each stage.

Leaves at the S1 stage of senescence show the first visible sign of senescence loss of chlorophyll at the tip of the leaf. As a leaf progresses through senescence, additional loss of chlorophyll occurs. In stage

S

2,

S

3,

S

4, and S5 leaves approximately 25%, *25 50%, 50-75%, and greater than 75% of the leaf area has become yellow. Our visual assessment of these stages corresponds to specific levels of chlorophyll loss.

Under our growth conditions, leaves reach stage Sl, S2, S3, S4, and S5 at 3, 5, 7, 9, and 10 days after full leaf expansion, respectively.

During senescence, the amount of RNA, protein, and chlorophyll present in a leaf declines. This decrease of RNA and protein has begun by the time chlorophyll loss is first noticeable (stage Sl), and continues as 35 the leaf progresses through the senescence program.

There is a highly reproducible correlation between the amount of chlorophyll loss and the decline in protein and RNA levels.

Isolation of Senescence-Associated Genes To identify mRNAs that increase in abundance in Arabidopsis leaves during senescence, we differentially screened a cDNA library constructed from mRNA from senescing leaves. Specifically, two cDNA libraries were constructed from template RNA isolated from S2 leaves and a mixture of S3 and S4 leaves. The S2 and S3/4 cDNA libraries were differentially screened with cDNA probes made by reverse transcribing poly RNA isolated from non-senescent (NS) leaves and poly

RNA

isolated from S2 or S3/4 leaves, respectively.

Differential screening of the S3/4 cDNA library identified mRNAs that increase in abundance during senescence. From this library, 23 cDNA clones that hybridized more strongly to the S3/4 cDNA probe than the NS cDNA probe were selected for further characterization. We refer to this class as senescence-associated genes (SAGs). Crosshybridization analyses indicated that this collection comprised six cDNA species. The longest cDNA of each family was used in subsequent analyses. The sizes of the mRNAs that correspond to the SAG cDNAs are presented below in Table 1.

Table 1. Approximate mRNA sizes in nucleotides of SAGs 25 Table 1. Approximate mRNA sizes in nucleotides of SAGs F.

30 SAG Size SAG Size 12 1360 15 4560 13 1340 16 1150 14 1140 17 800 Differential screening of the S2 cDNA library with NS and S2 cDNA probes revealed that the vast majority of the differentially expressed clones hybridized more strongly to the NS cDNA probe than to the S2 cDNA probe. Such cDNA clones correspond to mRNAs that -16decrease in abundance during senescence. During senescence the photosynthetic output of a leaf and the levels of transcripts encoding proteins required for photosynthesis declines (Hensel, et al., 1993, supra).

Therefore, cDNAs corresponding to transcripts encoding photosynthesis-associated proteins are likely to be in this group of clones that decrease in abundance during senescence. Six cDNAs that hybridized more strongly to the NS than the S2 cDNA probe were arbitrarily chosen for further study to provide a contrast to the SAG cDNAs. We designated these clones senescence-downregulated genes (SDGs) 1 through 6. We wish to emphasize that the SDGs 1-6 correspond to only a small fraction of the cDNAs in the library showing a sharp decline in abundance during senescence.

Gene Expression During Natural Leaf Senescence The steady-state mRNA levels corresponding to the isolated cDNA clones were investigated temporally throughout leaf senescence. This collection of cDNAs was isolated on the basis of differential expression on a mass basis. Specifically, replica filters of the libraries were screened with an equal mass (measured by dpm) of "P-labeled cDNA made by reverse transcription of poly RNA isolated from NS or senescing leaves.

Since the amount of total RNA present in a leaf decreases during senescence, it is possible that the levels of poly mRNA decline correspondingly. If the levels of poly mRNA decline during senescence, the differential cDNA screening may have revealed

SAG

clones corresponding to messages that remain constant during senescence when expression is examined on a per S• cell basis but increase in abundance when expression is examined as a function of RNA mass. For example, an S. SAG message that remains at a constant level on a per *35 cell basis would appear to increase in abundance on a mass basis if the levels of the majority of mRNAs were declining.

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wrvV -17- To address whether SAG mRNA levels increase during senescence, we examined the expression of these messages as a function of both mass and leaf area at each stage of senescence. The steady-state RNA levels corresponding to the SAG genes increase during senescence when examined on both a mass and area basis.

The increase based upon leaf area demonstrates that

SAG

mRNA levels per cell are increasing during senescence.

When examined on a mass basis, the levels of all SAG mRNAs are maximal at the later stages of senescence (S3 S5). However, when measured on a leaf area basis, certain SAG mRNAs 13 and 15) reproducibly exhibit maximal levels at earlier stages of senescence.

SAG12 exhibits one of the highest levels of induction and, within the limits of detection methods, appears to be expressed only during senescence. There is no detectable SAG12 signal in lanes of RNA from nonsenescent leaves even with long exposures of the autoradiograph or when measured by a 0 particle collector. The levels of SAG12 mRNA increase throughout the progression of senescence and reach maximal levels at the last stage of senescence examined.

The steady-state RNA levels corresponding to the 25 six downregulated genes decrease during senescence when Sexamined as a function of both RNA mass and leaf area.

As expected, the reduction is much greater when the expression is examined as a function of area than of Smass. As discussed above, the majority of mRNAs in the 30 leaf appear to follow this pattern, including mRNAs corresponding to nuclear-encoded genes involved in photosynthesis such as the chlorophyll a/b binding protein (CAB) and the small subunit of ribulose bisphosphate carboxylase/oxygenase (Rubisco) (Hensel, 35 et a1 .,1993, sugra). We also examined CAB mRNA levels during the stages of senescence that we have defined.

We found that CAB mRNA levels drop during leaf senescence at approximately the same rate as the SDGs.

-18- However, cross-hybridization analyses indicated that none of the 6 SDG clones were members of the CAB or Rubisco gene families.

Isolation of a Senescence-Specific Promoter We screened an Arabidopsis genomic library with the SAG12 cDNA forclones that contained the SAG12 promoter region of the SAG12. The library was provided by David Marks of the University of Minnesota.

We found that there is one copy of SAG12 in the Arabidopsis genome. Fig. 1 is a diagram of a construct containing 2073 bp of the SAG12-1 promoter and the untranslated region attached to the GUS reporter gene.

Fig 2 is a diagram of the nucleotide sequence of the SAG12-1 promoter linked to the SAG12-1 5' untranslated sequence, the isopentenyl transferase gene and the NOS termination sequence.

The SAG12-1 promoter fragment (from the EcoR V site at -2073 through an Nco I site artificially created at the SAG12-1 start codon by oligo mutagenesis) was cloned into pGEM5Zf(+) (Promega, Madison, WI) EcoR V-Nco I sites. This construct was named pSG499. A 2.6 kb Sal I-Sal I fragment containing 1.87 kb GUS and 0.8 kb MAS terminator was cloned into SpUC18 Sal I site. The MAS terminator is described in 25 Plant Mol. Biol. 15:373-381 (1990). This construct was named pSG468-2. The 2.2 kb SAG12-1 promoter from the Nco I site to the Pst I site in pSG499 was cloned into pSG468-2 at the Nco I-Pst I sites. This construct was named pSG506. The Pst I-Xba I fragment containing SAG12-1 promoter:GUS:MAS-ter was subsequently cloned into a binary vector at the Pst I-Xba I sites, resulting in the construct shown in Fig. 1.

A l kb Nco I-Xba I fragment containing 0.7 kb IPT and 0.3 kb NOS terminator sequences (Yi Li, et al., 35 Dev. Biol. 153:386-395, 1992) was cloned into pSG506 at the Nco I-Xba I sites to replace GUS:MAS-ter fragment.

This new construct was named pSG516. The Spe I-Spe I -19fragment containing SAG12-1 promoter:IPT:NOS-ter in pSG516 was then cloned into a binary vector at the Xba I site (both Spe I and Xba I have compatible cohesive restriction ends), resulting in the construct shown in Fig. 2.

We mapped the start site of transcription of SAG12-1 (indicated as +1 in Fig. 3) and fused a 2180 bp fragment containing 2073 bp upstream of this start site and the 107 bp SAG12-1 5' untranslated region (UTR) to two genes: the reporter gene beta-glucuronidase

(GUS)

and isopentenyl transferase (IPT), an enzyme catalyzing the rate-limiting step of cytokinin biosynthesis. The promoter fragment begins at point in Figs. 1, 2 and 3. SEQ ID NO:1 is the sequence of the SAG12-1 promoter, the IPT gene and the NOS-ter sequence.

These genes were introduced into the genome of both Arabidopsis thaliana (Arabidopsis) and Nicotiana tabacum (tobacco) by Agrobacterium-mediated transformation (Horsch, et al., Science 227:1229-1231, 1985; Valvekens, et al., Proc. Natl. Acad. Sci. USA 87:5536-5540, 1988). The resulting plants were fixed and assayed for expression of the GUS gene by colorimetric assay. Analysis of transgene expression demonstrated that the SAG12-1 genomic sequence fused to the reporter gene contains a senescence-specific promoter. In both Arabidopsis and tobacco, the GUS reporter gene was expressed in senescing leaves but was not detectable in leaves prior to senescence.

In transgenic tobacco we have done more extensive 30 analyses and found that the SAG12-1 promoter is also active in flower parts during senescence. This result is not surprising since floral organs are developmentally and evolutionarily related to leaves floral organs are thought of as modified 35 leaves).

We found that a 709 bp fragment (602 bp upstream of the start of transcription; point in Fig. 1) fused to the GUS gene results in a pattern of GUS expression in transgenic plants which is identical to that observed with the 2180 bp fragment. Thus, this smaller region contains all of the regulatory signals required for senescence-specific regulation. SEQ ID NO:2 is the 602 bp upstream from the start of transcription in the SAG12-1 gene and 107 bp of the untranslated region.

Use of the Senescence-Specific Promoter to Dela Senescence Cytokinins have been shown to be effective at blocking leaf senescence in both detached leaves and leaves undergoing natural senescence on the plant in many species including both monocots and dicots (for review see Nood6n, Senescence and Aqing in Plants,

PP

391-439, 1988 and Van Staden, et Senescence and Aqing in Plants, pp. 281-328, 1988). Moreover, the prevention of senescence by cytokinins results in the maintenance of a photosynthetically active leaf.

Several studies have demonstrated that cytokinin treatment stimulates photosynthesis and chloroplast and cytoplasmic protein synthesis while preventing chloroplast breakdown (Van Staden, et al., supra) While most studies on the effects of cytokinins on senescence have involved application of exogenous 25 cytokinins, there is evidence that endogenously produced cytokinins are a natural regulator of leaf :senescence. Nooden, et al. (Nooden, et al. Plant Phsiol. 93:33-39, 1990) have recently studied ':cytokinin fluxes in soybean leaves that are undergoing 30 natural senescence on the intact plant. During the later stages of seed development that trigger senescence in soybean, the flux of cytokinins from roots to leaves is drastically reduced. Moreover, removal of seed pods reverses senescence and restores 35 the flux of cytokinins to leaves. Further support is provided by transgenic plant studies. The isopentenyl transferase gene (IPT) from the T-DNA of the -21- Agrobacterium tumefaciens Ti plasmid catalyzes the rate-limiting step in the biosynthesis of cytokinins.

Transgenic plants that overexpress the IPT gene often exhibit some delay of leaf senescence (Li, et al., Dev.

Biol. 153:386-395, 1992; Ooms, et al., Plant Mol. Biol.

17:727-743, 1991; Smart, et al., The Plant Cell 3:647- 656, 1991). However, IPT expression in these transgenic plants was not leaf specific and therefore the transgenic plants displayed developmental abnormalities typical of general cytokinin overproduction such as stunted root growth and lack of apical dominance.

The goal was to target cytokinin production to senescing leaves at a level that will block senescence but does not interfere with other aspects of plant development.

Eight transgenic tobacco lines were created using the genetic construct illustrated in Fig. 2. All eight transgenic tobacco lines that expressed the SAG12-1/IPT fusion were perfectly normal phenotypically there were no alterations of branching, flower development, root growth, etc.) except that all of the leaves of the transgenic plants retained high levels of chlorophyll throughout flower and seed development.

25 Nontransformed control plants and plants transformed with a construct similar to the SAG12-1/IPT fusion, except that IPT sequences were replaced with the GUS gene, exhibited extensive senescence of lower leaves during flower and seed development. Thus, the goal of altering senescence was achieved without perturbing other aspects of plant development.

The transgenic plants had greatly enhanced yield of biomass and flower and seed production. As shown in Table 2 below, total biomass and flower number were greatly increased in the IPT transgenic plants as compared to transgenic controls that express GUS, although leaf number and flowering time were the same.

The seed yield per flower was the same in control and -22- IPT plants; therefore, the seed yield was almost doubled in the IPT transgenic plants. The IPT transgenics were still growing (the controls had stopped growing) when the experiment was terminated due to insect infestation and the actual increase in yield would probably have been greater if the experiment could have been continued. Thus, this system is of potential use to increase yield of both biomass and seed and enhance flower production in ornamental crops.

We have also put the SAG12-IPT construct shown in Fig. 2 into Arabidopsis and shown that it blocks leaf senescence in this species as well.

The SAG12-1/IPT construct was made with an IPT construct provided by Yi Li (Li, et al., Dev. Biol.

153:386-395, 1992). The useful feature of this IPT gene was the introduction of an Nco I site at the start of translation. The IPT gene was readily available from our previous work (See, for example, Akiyoshi, et al., Proc. Natl. Acad. Sci. USA 81:5994-5998), but we chose Li's construct to save a cloning step. This construct utilizes a "terminator" (a sequence that makes a proper 3' end on the mRNA) from the nopaline synthase gene (NOS) (Bevan, et al., Nucleic Acids ~Research 11:369-385, 1983).

25 Isolation of SAG13 Promoter In the mRNA library described above, 23 cDNA clones were identified associated with leaf senescence.

The identification of one, SAG12 is described above, and similar methods were used to identify SAG13 and its associated promoter.

The SAG13 clone contained a 1.24-Kb insert. This insert was used to make a probe to screen the Arabidopsis genomic library described above. Two unique genomic clones were found. there are two 35 copies of SAG13 in the Arabidopsis genome.) The two clones contained a 3.53 kb EcoRI-SalI fragment that contains the region upstream of the start site of -23transcription. These DNA fragments were subcloned into pBluescript II SK vector at the EcoRI and Sall sites and were subsequently sequenced. The fragment contained all the SAG13 cDNA sequence and an upstream promoter sequence. The sequence of the SAG13 upstream promoter sequence is set forth in SEQ ID NO:13 below.

The transcription start site is at nucleotide 1782 and the translation start site is at nucleotide 1957. The two sequences were identical except at position 1009 where one copy of the gene contains a G residue and the other copy an A residue.

w oo o e oo ooooo 0 C 0 0@ 0*

S

S

*eO

OS

"SS*

0* OS

S

0* 55 .555 5 0@ S Se 0 S. -4 5 @0 **5 S S. S OS S .5 5 Table 2. Comparison of some characteristics of SAG12-ipt transgenic and related plants Wisconsin 38 (Wild-type) SAG12-gus Plants SAG12-gus/ SAG12-ipt SAGl2-ipt Plants Plants Chlorophyll content (fg cm 2 leaf #7 39-day-old) 19.911 0.642 68-day-old) 1.239 0.719 Protein content (pg 39-day-old" 68-day-old' cm- 2 leaf #7) 52.47 1.75 16.00 5.29 21.627 1.893 1.797 1.575 52.27 1.01 19.60 10.65 22.117 1.944 16.905 1.551 71.33 7.04 54.40 3.49 25.638 1.877 18.527 2.855 Total flower Seed yield (g/plant) Biomaso: (g/plant) c Plant height (cm)d Leaf on main stem 178.3 28.1 176.2 51.1 318.6 44.2 20.436 4.182 107.51 14.41 176.25 14.27 33.3 0.5 21.142 3.683 101.64 10.97 172.54 6.70 33.0 0.9 30.240 4.037 151.80 20.40 178.38 10.54 33.1 1.0 71.60 3.86 49.60 5.88 327.5 46.3 31.154 4.100 150.79 20.15 180.15 7.91 33.5 1.4 The #7 leaves of all genotype plants were fully expanded but nonsenescent after 39 days of their emergence.

The #7 leaves of both wild-type and SAG12-gus plants were completely senesced after 68 days of emergence.

Dry weight of the above soil of the plant excluding seeds.

From the soil surface to the toppest floral stalk.

Sample Sizes: Wisconsin 38: 8 plants; SAG12-gus: 13 plants; SAG12-gus/SAG12-ipt: 8 plants; SAG12-ipt: 13 plants.

SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Amasino, Richard M Gan, Sushang (ii) TITLE OF INVENTION: Transgenic Plants with Altered Senescence Characteristics (iii) NUMBER OF SEQUENCES: 3 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Quarles Brady STREET: 1 South Pinckney Street CITY: Madison STATE: WI COUNTRY: US ZIP: 53703 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION: NAME: Seay, Nicholas J REGISTRATION NUMBER: 27,386 REFERENCE/DOCKET NUMBER: 960296.92808 (ix) TELECOMMUNICATION

INFORMATION:

TELEPHONE: 608-251-5000 35 TELEFAX: 608-251-9166 i. INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 3183 base pairs TYPE: nucleic acid 40 STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "SAG12-1 Promoter DNA" ooooo -26- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: GATATCTCTT TTTATATTCA AACAATAAGT TGAGATATGT TTGAGAAGAG

CTCGTGGAGC

AAAAAAGTAA

AAATGATTAG

TTTTCTCTCT

AATTAAAAAC

GAATAGTTGA

ATGAACTCAG

AAAAAGTGTC

GGATCTCTCA

GAGACGAGGA

CTTTGGTAGC

TCGTTATTAG

TGTGTTCATG

TAACAAAGTT

TTTACACCGC

GACAATTATA

TATGTCTTAC

20 AATCATTATA

TTATCACTTC

CTCTTGTCGT

TTTATATTGT

GGGAAACATC

ACTTAGCGTA

TTGTCGAATC

TGTGAATAAA

ACCGAGTCTG

AATCGTTGAT

TTATCATAGC

TTGGTGTTTT

AATATTTCCA

TTATGAATTA

TTGTTATACA

AGAATATGAC

AGAATATTTT

AGAAAGATTG

AAGTCGATTT

TTTGTACTTG

AGGTGATTGT

TTATAGATTT

ATTTTTCCCT

AGTTTATAAC

TTCTTCTTTG

TTTGTAAATA

AGCCAAATAT

CTAATGATTA

ATTCTAATGA

ATTGAACAGG

ATGAAGTTCA

ATTTTTCTTT

CAGAATCTTT

TTTTATATTA

TGTTAAAATT

TAATATAGCA

CTTAACATTA

AGTTTTATAT

GTTAGATCAA

AGAAATGAAA

ATGAAGCGTT

GGGCCATATT

GGTC.AAGTTA

ATTTGCCAGT

GTACCTTTGG

GATTTAATTT

TTTTTATAA

GTACAAGAAT

GTTTTTACAA

TGTAAGAAAA

TGCAGTTATT

GATTTGGATT

TTTCAATATT

CAGGGAAACT

AAACTTTTAG

CTTGTTGTGA

TTGATTTGAT

GAATTCAC

GAAACCCGAT

TAAAATTAGT

TGATTCTAAA

GAAGAACCCA

ACGAAACTTG

TACTCAATAT

ATGCTATTA

TTGTCCTACC

AGTTATATTT

ACAAAACAGA

AAAAACTTGG

TTAAGAAAAA

GTTGACTAGG

CATTTTTGCC

TCATATATTA

TTATTTAAAT

CTAACTATAT

TGTCAATTTT

TAAGTCCAAA

TCTTATATTA

TTCATAGAGA

CAAATCATAT

ATGACTATGA

TAAGCTTTTA

TATTTGATTA

TGTTATTTTT

TTCATCACGT

TTTGTTTTTT

TAACAATGTA

TTTTTTTAAT

ATGATCAATG

AATACCGATC

GGGTATCGAG

GGGCTTAAGC

GACACTCGTA

TACACAACTG

GTTGATATAG

GCGATTCCT

ACGCTTCGTA

TTTATTTATA

ACCATGTGAA

CACTATAATA

GAATTTAGTA

ATGCAATTTC

TCCCTAACTA

TTCAGATAGA

CGATTTATCT

TTTGATCAAA

ACTGCACGA

GTGAAAAGAC

GACAACTATT

AGACTGAGAC

TTCGATAAAA

GACACCCTTT

CGTTCAAATT

GAAAACAGTT

ATGTATATAT

ATGAAGTGTT

TTATAGGTTT

GTTTTGCAAA

TTAGTTGGTA

ACAACTCGTA

TTAAATCAGT

CACATCACAA

AAGTTTGGTA

TACTCCAGTT

GATCCAAGAA

AAATAATTCT

TTTTAGACGG

GTACGTATCC

CAGAGCTACA

TGAAATTGGT

ACAAAAGAAT

TTAGTTAATT

ATGGTTCTCT

AAAAGAAGAT

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560

S

S

~*TCCTTGTTTT TATGTGATTA GTGATTTTGA TGCATGAAAG GTACCTACGT ACTACAAGAA 1620 -27- ATGTAAAAGT ATTTTTTTCC AAATAATTTA 1680

AAATAAACAT

TACTCATGAT

TTTTAATTAA

TCGAAAATCT

ATCATCAATT

CCGAGCAAAG

TATCAAACAT

AAAAATGCTT

TTTTCACTAT

CATTTAACTT

AAGACGACGA

CGGGTCCAAT

AAAGGAACGA

AAGCAAGCTC

ATTCTTGAGG

GCAGATTTTC

GCGGCCAAGG

GAGTTGGTTT

TATCGATATG

20 CTTGACGCAA

GCGCGCCAAC

GGTCATCCGT

GGCAATAAAG

TTCTGTTGAA

GATGGGTTTT

TATGGCGCGC

TTC

GTACGTAACT

AGATTTTTTT

TTAAATAAGG

CTATAGTACA

ATAAATGTTT

TGAGTGAACA

CAACGATCAT

TGATTTGGAT

AAAACCCCAT

TCCTAAAACC

CCGCGATAGC

CGTGTCCTCA

CGCGTCTCTA

ATCATAGGCT

GAGGATCCAC

GTTGGCATAT

CCAGAGTTAA

ATCTTTGGAA

CCATGTTGTT

ATATGGAAGG

AGGAACAGAA

TCGGAATGTA

TTTCTTAAGA

TTACGTTAA6

TATGATTAGA

AAACTGGGAT

ACGTATCAGC

TTTTTGAAAT

AAATATATTT

CAAGTAGAGA

ACAAAACTAA

AGACTTGATT

TTAGTTATGT

CAATCACTTC

CTCAGTACCC

ATGGACCCTG

TCTTGCCCAG

ACTATCAACC

CCTTGATGAT

GATCGAGGAG

CTCGTTGCTC

TATTCGCCAC

GCAGATGTTG

TGAACCTCGG

TGCTAGCCAG

TAAGTTGATT

ATTCCCCCAA

TTAGGTTACG

TTGAATCCTG

CATGTAATAA

GTCCCGCAAT

AAATTATCGC

GTCAATTAAA

ATGCAAAACA

AAATAAATTT

TTAAACCCAC

TCAGGTTGAT

ATGAATGAAT

ATGTGAACAT

TTCTGAAGTA

CATCTAATTT

CAGACAGGGC

GGAAGCGGAC

CGGCCTCTGG

GTGTATAATC

AACTGCATGG

AAGTTACCCG

CACCCCGCTG

CTGAGGCCCA

AACCAGATCA

AATGGGATCG

GTTAACGCZAG

CCAGCCCTGA

TTGCCGGTCT

TTAACATGTA

TATACATTTA

GCGCGGTGTC

AATGCTTTCT

TCATCAACAC

TACTAGATAC

CACTAAAATT

GTAGGACTAA

GTAGTCATTA

TAGCAATTAC

ATCAAATTAA

TCGGTCCAAC

TTCCAGTCCT

GACCAACAGT

TGGAGGGTAT

ATGAGGCCAA

CGCGAAACAG

ACC1AAGAGAC

CAGGCCATTC

TTCTGAAAGA

CGGCAGATAT

CTCAGGAGTA

CCGCTTTCGA

GCTCGATCGT

TGCGATGATT

ATGCATGACG

ATACGCGATA

ATCTATGTTA

TAAATATTAA

ATATCCAACT

AAACTTCCTA

AACTAAAAAT

AATGGCTACG

CTTGTAAAAC

ATCAACCTTA

GAGCAAAAGT

TTGCACAGGA

TTCGCTTGAT

GGAAGAACTG

CAT CGCAGCC

CGGCGGGCTT

CTATTGGAGT

CTCATGAAA

TATTATTCAA

GATCGATGGA

GCTATTGCAG

TTTCATCCAT

CGGATTCGAA

TCAAACATTT

ATCATATAAT

TTATTTATGA

GAAAACAAAA

CTAGATCGAA

1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3 3120 3180 3183 0 0 o *.00 0 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 709 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc 1"SAG12-1 (truncated)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: AAGCTTTTA.A CTTGCACGAA TGGTTCTCTT GTGAATAAAC Promoter DNA

ATTTGATTAG

GCATGAAAGG

TGTAA.AAGTA

TCAATTAAAA

TGCAAAACAT

AATAAATTTT

TAAACCCACC

CAGGTTGATG

TGAATGAATG

TGTGAACATT

TCTGAAGTAA

TGAAAAGACA

TACCTACGTA

TTTTTTTCCA

ATGCTTTCTT

CATCAACACA

ACTAGATACA

ACTAAAATTA

TAGGACTAAA

TAGTCATTAC

AGCAATTACA

TCAAATTAAG

AAAGAAGATT

CTACAAGAAA

AATAATTTAT

AAATATTAAT

TATCCAACTT

AACTTCCTAA

ACTAAAAATC

ATGGCTACGT

TTGTAAAACA

TCAACCTTAT

AGCAAAAGTC

CCTTGTTTTT

AATAAACATG

ACTCATGATA

TTTAATTAAT

CGAAAATCTC

TCATCAATTA

CGAGCAAAGT

ATCAAACATC

AA.AATGCTTT

TTTCACTATA

ATTTAACTTT

AGAATCTTTG

ATGTGATTAG

TACGTAACTA

GATTTTTTTT

TAAATAAGGA

TATAGTACAC

TAAATGTTT.A

GAGTGAACAA

AACGATCATT

GATTTGGATC

AAACCCCATC

CCTAAAACC

AATTCAAACT

TGATTTTGAT

CGTATCAGCA

TTTTGAAATG

AATATATTTA

AAGTAGAGAA

CAAAACTAAT

GACTTGATTT

TAGTTATGTA

AATCACTTCA

TCAGTACCCT

120 180 240 300 360 420 480 540 600 660 709

-~I_~IIIIII~L-LIII~---~

-29- INFORMATION FOR SEQ ID NO:3: SEQUENCE

CHARACTERISTICS:

LENGTH: 1974 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "SAGl3 Promoter DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GAATTCTCAG TGTTCTCTTA AATCAAATCT CTCACACTAT a a a a a a.

a ATACATATCA

CAATTCCATT

CTATTTCGAT

CTTGTATTTA

TTTTTTCTTT

TTGAAGTTCA

CTTAGTTCAT

CAATTCTTTA

ACTCTATATA

TGTCATGTGG

GATCAAAGAT

AAAGAGTCTT

TGTCAATTCG

TTTGCAATAA

TTCCATGCAT

TTGATATTAT

AAAATCCAGT

TGACAGCATA

ATGTTACTGC CATGATTGAT CGCGGTTGGA

AATTATGATC

CACGTTTAGT

TGTTCTGTTG

ATTGTGGCTA

GTAAAATGCC

TCCATATTTT

TGCTGGGTTT

TACAAAATAT

AGAGTATTGT

ATAAAAAAAT

TAAGAAAGGT

AAGTCACATC

TTTAATTGAC

ATTGACAACA

ATGGTCATGC

GTAATATCAT TATGAAATGA GCAATCTTAG AAAAAAAAAA CAATGCATTT

AAAAAGAGAT

ATGGATATCT

ATAATGTTAA

ACAAATATAT

CTACACATAG

CATCTCTGGT

TCATGGTAAA

CTTACATTTG

TTTCTCTCTA

ATGAAGCTGG

GTTTTGATGG

AAACTCTTCA

TTTCTTATGG

ATCTACCTTA

AGTAGCTGAT

TGGAGTTATA

TTTTGAAAAT

ATATGTTTTG

ATGAAATGAA

AATAGAAGGT

AAAAAAAAAA

GGAAAGAATC

CCCAATCTAT

TACTCTGTTT

CAAAATAACT

AAACGTATTT

CATTGCTAGT

AATTCTCTTG

CAATAACATG

ATACATATAT

TACACCATAC

AATTAGTGTT

ATGTCACTTA

CTGCTTAATG

ATCCTATATA

ACGACGTTTA

TTGATTTTTG

GCATCTTCTA

TTTGTTTGTT

ATGTTTGTTG

ATATATTACA

AAAAAAGAAA

CGAGCTATCG

GAGTATATGA

CTCTCATACA

TAATTTGTGT

CAGAACCATT

ATCTTGTTTG

CACAGGTAAA

TAACTGGTGG

TCAGCCATAT

GATGTGTTAC

ATGCACTTGA

AAAGGATGGA

TCAAGAGAGC

TCTCCATCAA

TAAGTATAAC

TGGTTGTTAT

TTCATATTAG

GAATATATRT

TTTTTTTACT

TCAATTTTTT

AAATATTACC

AAGAAAAAGA

AATCCAAAGA

ACAAAATCAT TGAAAATGTT 120 ATCCTGATTT 180 ACTATTTTTT 240 ATCTACTTTG 300 AGTAAAAATT 360 AGATAGTAGA 420 TTATTTAAAT 480 GGTCATTCTA 540 TTATATATGG 600 CCCTCACTAA 660 TAATGACTAG 720 ATATTTAGAC 780 TAGATAATAA 840 TGAGTTTGAA 900 TTAACAAATA 960 ATATTCGAAA 1020 GGCCACACAA 1080 TTACTAACTT 1140 TAAAAGTAGA 1200 AACAAGATTA 1260 AGCATCTACT 1320 TCCTCCATCT GTTCTTGTAT CGTCTACCAG

TCTTAAAGAT

TCCAGATGTG

GTTTTTTGTC

ATTTACCAGG

TAAATTAGTT

TATGTGGTGC

ATACAAGACT

TATGTGTGGT

GTTCAAAACT

AGAAAAGAAT

GGTTGTTGGA

ATAGACAAAG

AATGGTTGTG

GGTTTAAGTG

TTAATTATAT

TCTATTAACT

TGTCAAAAGG

CGAGCAGTCA

ATCACAACTA

CGTCGTAGGT

GGGATCCTTT

GGCTGTGTGG

TATGCATAGA

GTTAAATTGA

AAATTT'rAAC

AAGGGGTGCT

AAA:AGTAGTA

TTATCATAAT

GTCTTGATCA

TGATATTTAA

AGATGGTGTT CCGGATCTCT GGCTATTATG GAGAACATTA CCTGTGAGAC

CGATGGCCAC

AATTCCCACA ACCGTTTGTG TACATGTAGA TCTAAAGTTT TACGCTCATG ACACGTAAAT TCATTATTAA TTCATAAAGA TTTTCGTACT ACGTCTACCC GTGGAATTTT GAATTGAGCG ATTCTATATA AGATCTGTGA CAAGGAATGG CAAAGGAAGG CGATCAATAT 1380 TTCGTTTATC 1440 TTAATTATTG 1500 CCTTAACACA 1560 TATGCTAATA 1620 GGTAGACCAA 1680 TTTCTTTACT 1740 CTCTCACGGA 1800 AGGTTTCAAA 1860 TCTTGGTTGA 1920 GGGC 1974 a a a a a a.

a a

Claims (29)

1. A genetic construct comprising an SAG12 or SAG13 promoter sequence operably connected to a protein-coding DNA sequence not natively connected to the promoter sequence.

2. The construct of claim 1 wherein the promoter sequence comprises the first 602 bp of SEQ ID NO:2.

3. The construct of claims 1 wherein the promoter sequence comprises the first 1782 pb of SEQ ID NO:3.

4. The construct of claim 1 wherein the protein- coding sequence encodes a plant hormone synthesizing enzyme.

5. The construct of claim 4 wherein the protein- 20 encoding sequence encodes an enzyme catalyzing the synthesis of the plant hormone cytokinin.

6. The construct of claim 5 wherein the protein- encoding sequence encodes isopentenyl transferase.

7. The construct of claim 1 additionally comprising the SAG12-1 5' untranslated region.

8. A genetic construct comprising a SAG12 promoter operably connected to a DNA sequence encoding an enzyme catalyzing the synthesis of cytokinin.

9. The construct of claim 8 wherein the DNA sequence codes for isopentenyl transferase.

A cell containing te construct of claim 2. -32-

11. A cell containing the construct of(claim 8.

12. A plant containing the construct of /claim 1.

13. A plant containing the construct of claim 8.

14. A genetic construct comprising a SAG13 promoter operably linked to a DNA sequence encoding an enzyme catalyzing the synthesis of cytokinin.

A transgenic plant with delayed senescence, the plant comprising in its genome, 5' to a genetic construction including a senescence associate promoter and a coding region for an enzyme catalyzing the synthesis of a cytokinin.

16. A transgenic plant as claimed in claim wherein the promoter is selected from the group consisting of the SAG12 promoter or a promoter sufficiently homologous to the SAG12 sequence to 5 preferentially express the enzyme in senescing tissue at a level similar to the SAG12 promoter when expressing the GUS gene in Arabidopsis.

17. A transgenic plant as claimed in claim wherein the promoter is SAG13. 10

18. A transgenic plant as claimed in claim wherein the enzyme is IPT. 33

19. A transgenic plant having delayed senescence characteristics comprising in its genome: a foreign genetic construction which comprises 5' to 3'; a senescence specific promoter; a protein coding region for an enzyme which, when expressed, will catalyse the production of a cytokinin in the cells of the plant; and a transcriptional termination sequence, wherein the foreign genetic construction is expressed in tissues entering senescence to delay the senescence of the plant tissues.

A transgenic plant according to claim 19, wherein the cytokinin is IPT. In

21. A transgenic plant according to claim 19 or claim 20, wherein the promoter is SAGI2-1.

22. A transgenic plant according to any one of claims 19 to 21, wherein the promoter is SAG13.

23. A senescence associated promoter sequence operably linked to a protein- 1i encoding DNA sequence not natively connected to the promoter sequence, wherein when the promoter is present in a Nicotiana tabacum plant and the protein-coding DNA sequence encodes an enzyme catalysing the synthesis of a cytokinin, the Nicotiana lahacum plant is unaltered in one or more of branching, flower development or root growth compared to the same Nicotiana labacum plant without the promoter operably 20 connected to the protein-coding DNA sequence encoding the enzyme catalyzing the synthesis of isopentenyl transferase.

24. A senescence associated promoter according to claim 23, wherein the enzyme is isopentenyl transferase.

25. A senescence associated promoter according to claim 23 or claim 24 which 25 forms part of a genetic construct.

26. A senescence associated promoter sequence operably linked to a protein- encoding DNA sequence not natively connected to the promoter sequence, wherein when the promoter is present in a Nicotiana labacum plant and the protein-coding DNA sequence encodes an enzyme catalysing the synthesis of a cytokinin, the Nicotiana o0 labacum plant is unaltered in one or more of branching, flower development or root growth compared to the same Nicotiana labacum plant without the promoter operably connected to the protein-coding DNA sequence encoding the enzyme catalyzing the synthesis of isopentenyl transferase, substantially as hereinbefore described with reference to any one of the Examples. [I:\DayLib\LIBZZ]06034#.doc:DKM N- 34

27. A transgenic plant comprising a senescence associated promoter sequence operably linked to a protein-encoding DNA sequence not natively connected to the promoter sequence, wherein when the promoter is present in a Nicotiana tabacum plant and the protein-coding DNA sequence encodes an enzyme catalysing the synthesis of a cytokinin, the Nicotiana tabacum plant is unaltered in one or more of branching, flower development or root growth compared to the same Nicotiana tabacum plant without the promoter operably connected to the protein-coding DNA sequence encoding the enzyme catalyzing the synthesis of isopentenyl transferase.

28. A transgenic plant according to claim 27, wherein the enzyme is isopentenyl I0 transferase.

29. A transgenic plant comprising a senescence associated promoter sequence operably linked to a protein-encoding DNA sequence not natively connected to the promoter sequence, wherein when the promoter is present in a Nicotiana tabacum plant and the protein-coding DNA sequence encodes an enzyme catalysing the synthesis of a cytokinin, the Nicotiana tabacum plant is unaltered in one or more of branching, flower development or root growth compared to the same Nicotiana tabacum plant without the promoter operably connected to the protein-coding DNA sequence encoding the enzyme catalyzing the synthesis of isopentenyl transferase, substantially as hereinbefore described with reference to any one of the Examples. Dated 15 October, 1999 Wisconsin Alumni Research Foundation Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON o DO* [I :\DayLib\LIBZZj06034#.doc:DKM