Molecular Cloning and Characterization of Senescence

Plant Physiol. (1989) 90, 690-696

Received for publication November 28, 1988 and in revised form February 8, 1989

0032-0889/89/90/0690/07/$01 .00/0

Molecular Cloning and Characterization of SenescenceRelated Genes from Carnation Flower Petals1 Kay A. Lawton, Bin Huang, Peter B. Goldsbrough, and William R. Woodson* Department of Horticulture, Purdue University, West Lafayette, Indiana 47907 ABSTRACT

senescence in carnation. Treatment of flowers with inhibitors of ethylene biosynthesis (4) or action (4, 22, 23) prevents typical petal senescence. Furthermore, exposure of presenescent flowers to ethylene hastens the onset of petal senescence. The senescence of flower petals is associated with a series of physiological and biochemical changes. These include an increase in hydrolytic enzymes (1, 9), degradation of macromolecules (1, 14), increased respiratory activity (17), and a loss of cellular compartmentalization (8, 15, 20). Many of these processes are highly regulated and are the result of active metabolism (1, 2, 20). Analysis of total proteins isolated from carnation petals during the development of senescence revealed both increases and decreases in the levels of several polypeptides (25). In vitro translation of mRNAs isolated from carnation petals at various stages of development has shown that senescence is accompanied by changes in mRNA populations (25). Taken together, these results indicate that the development of petal senescence is associated with regulated changes in gene expression. There is growing evidence that responses of plants to ethylene can include changes in gene expression (3, 6, 11, 12, 19, 26). In climacteric fruits such as avocado and tomato, ethylene has been shown to modulate the expression of several ripening-specific genes (5, 12). In addition, ethylene has been reported to increase the steady state levels of several mRNAs for enzymes involved in plant defense responses including chitinase (3), 4-coumarate:CoA ligase (6), and L-phenylalanine ammonia-lyase (6). We recently reported (26) that exposure of presenescent carnation petals to ethylene leads to premature petal senescence and induces changes in gene expression. Furthermore, most of the ethylene-induced mRNAs are similar to those which accumulate during natural senescence (25), based on the mol wt of their in vitro translation products. We are interested in understanding how senescence in plants is controlled and have chosen to study the regulation of carnation petal senescence. In this organ, senescence is rapid and predictable and is regulated by the phytohormone ethylene. Analysis of petal senescence should serve as a model for senescence of other plant tissues and is an interesting system for studying the action of a specific hormone at the molecular level. Here we report the identification of several cDNA clones which have been used to examine the expression of senescence-related mRNAs during flower petal senescence and in response to ethylene. MATERIALS AND METHODS

The senescence of carnation (Dianthus caryophyllus L.) flower petals is associated with increased production of ethylene which plays an important role in regulating this developmental event. Three senescence-related cDNA clones were isolated from a cDNA library prepared from mRNA isolated from senescing petals. These cDNAs are representative of two classes of mRNAs which increase in abundance in senescing petal tissue. The mRNA for one class is present at low levels during the early stages of development and begins to accumulate in mature petals prior to the increase in ethylene production. The accumulation of this mRNA is reduced, but not eliminated, in petals treated with aminooxyacetic acid, an inhibitor of ethylene biosynthesis, or silver thiosulfate, an ethylene action inhibitor. In contrast, expression of the second class of mRNAs appears to be highly regulated by ethylene. These mRNAs are not detectable prior to the rise in ethylene production and increase in abundance in parallel with the ethylene climacteric. Furthermore, expression of these mRNAs is significantly inhibited by both aminooxyacetic acid and silver thiosulfate. Expression of these mRNAs in vegetative and floral organs was limited to floral tissue, and predominantly to senescing petals.

Senescence represents the final stage in the development of a whole plant, organ, tissue, or cell. Flower petals are often the plant organ with the shortest life span, and as such provide a useful tissue for studying the mechanisms underlying control of senescence. It is clear that petal senescence is a highly controlled developmental event, which plays an important role in the overall reproductive development of the plant (2). In many species, petals function in the attraction of insects for pollination. Once pollination has occurred, and the function of the petals is complete, the metabolic resources of the petals are rapidly mobilized to the developing ovary leading to petal senescence. In the flowers of carnation (Dianthus caryophyllus L.), pollination induces senescence by signaling the petals to produce the phytohormone ethylene (18). In the absence of pollination, petal senescence still occurs and is associated with a climacteric-like increase in ethylene production (2, 16). It is clear that the increase in ethylene production plays a critical role in the coordination and regulation of petal ' Supported in part by a grant from the Indiana Corporation for Science and Technology and an Incentive Grant from the Purdue University Agricultural Experiment Station. K. A. L. was supported by a Davis Ross Fellowship from Purdue University. Publication No. 1 1,530 of the Purdue University Agricultural Experiment Station.

Plant Material

Carnation (Dianthus caryophyllus L. cv 'White Sim') flowers were harvested at anthesis from plants grown under green690

EXPRESSION OF SENESCENCE-RELATED GENES

house conditions and held in the laboratory as described (25). Vegetative and reproductive tissues were collected, immediately frozen in liquid nitrogen, and stored at -70°C until used for RNA isolation. Chemicals

L-[35S]Methionine at 1200 Ci/mmol and a[32P]dCTP at >3000 Ci/mmol were obtained from New England Nuclear. Restriction enzymes were purchased from Promega Biotec and Bethesda Research Laboratories. Nick translation kit was obtained from Amersham. Chemicals used in cDNA synthesis were purchased from Bethesda Research Laboratories. XgtlO and Gigapack phage packaging extracts were purchased from Stratagene. All other chemicals and reagents were from Sigma Chemical Company. RNA Extraction and Poly(A ) Isolation RNA was extracted from frozen tissue and poly(A+) RNA isolated as described elsewhere (25, 26).

Construction and Screening of a cDNA Library Double-stranded cDNA was synthesized from poly(A+) RNA isolated from senescing carnation petals (6 d after harvest) essentially as described by Gubler and Hoffman (7). Following methylation, ligation of EcoRI linkers and digestion with EcoRI, cDNA molecules larger than 0.5 kb2 were ligated to EcoRI digested XgtlO (10). The recombinant DNA

molecules were packaged in vitro and used to infect Escherichia coli strain C600 hfl+. Approximately 5 x 105 recombinant phage were present in this cDNA library. The library was screened by differential hybridization (10) using 32p_ labeled single-stranded cDNA, prepared from preclimacteric (day of harvest) and climacteric (6 d after harvest) petal poly(A+) RNAs. Briefly, a portion of the library was plated at a density of 500 pfu/9 cm x 9 cm plate. Duplicate nitrocellulose lifts were taken from each plate, denatured, neutralized, and baked for 2 h at 80°C under vacuum (10). Each replicate lift was prehybridized for 4 h at 68C in a solution containing 6 x SSC (1 x SSC is 0.15 M NaCl2, 15 mM Na-citrate, pH 7.0), 0.1I% SDS, 5 x Denhardt's mixture (1 x Denhardt's is 0.02% PVP, 0.02% Ficoll, 0.02% BSA), and 100 lg/mL denatured salmon sperm DNA. Hybridization was carried out for 18 h at 68°C in identical buffer containing 5 x 105 cpm/ mL 32P-labeled cDNA. Filters were washed at 68°C for 4 h in 4 changes of 3 x SSC, 0.1% SDS (w/v), dried, and exposed to Kodak XAR-5 film with an intensifying screen at -70°C for 18 h. A number of recombinant phage that hybridized more intensely with climacteric cDNA were picked and plaque purified through several rounds of screening. After purification, the cDNA inserts of these phage were isolated and subcloned into the EcoRI site of pUC1 8. Phage and plasmid DNAs were isolated and purified as described by Maniatis et al. (13). 2 Abbreviations: kb, kilobase pair; AOA, aminooxyacetic acid; NBD, 2,5-norbornadiene; STS, silverthiosulfate; pfu, plaque-forming unit.

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Northem and Slot Blot Hybridizations

RNA was electrophoretically separated on 1.2% agarose gels containing 2.2 M formaldehyde and transferred to nitrocellulose. The nitrocellulose filter was prehybridized 4 h at 42°C in a solution containing 50% (v/v) formamide, 5 x Denhardt's, 0.1I% SDS, 6 x SSPE (1 x SSPE is 0.15 M mM NaCl, 10 mm NaH2PO4, 1 mM EDTA, pH 7.4), and 100 jg/ mL denatured salmon sperm DNA. Hybridization was carried out in identical buffer solution plus 5 x 105 cpm/mL denatured 32P-labeled DNA probe for 18 h at 42°C. Plasmid DNA was labeled by nick translation to a specific activity of 2 x 108 cpm/sg DNA. The filters were washed in 3 x SSC, 0.1% SDS (w/v) for 4 h at 42°C, exposed to Kodak XAR-5 film with an intensifying screen at -70°C for 18 to 36 h. Total RNA was slot blotted according to manufacturers instructions using a Schleicher and Schuell Minifold II Slot Blot apparatus. Prehybridization and hybridization were carried out as described above for RNA-blot analysis. Hybrid-Select Translation mRNA corresponding to the cDNA clones was selected according to Maniatis et al. (13). Briefly, 10 ,ug of linearized plasmid DNAs from selected clones were denatured in 0.5 M NaOH, neutralized with the addition of hybrid select translation buffer (1 M NaCl, 0.3 M Na-citrate, 0.5 M Tris [pH 8]), and spotted onto 1 cm2 nitrocellulose discs. The discs were air-dried and baked at 80°C under vacuum. Prehybridization was performed for 2 h at 42C in 0.6 M NaCl, 20 mm Pipes, 2 mm EDTA, 0.1% SDS (w/v), and 100 ,ug/mL tRNA. The hybridization solution was the same as prehybridization except for the addition of 10 ,g poly(A+) RNA from d 6 petals. Total hybridization volume was 100 ,uL. The filters were hybridized for 18 h at 42C. Following hybridization, filters were washed 10 times in 1 mL of buffer containing 10 mM Tris (pH 8.0), 150 mm NaCl, 1 mM EDTA, and 0.5% (w/v) SDS at 65°C, followed by two final washes in the same buffer without SDS. Bound mRNA was released into sterile H20 by heating to IOOC for 2 min. The hybrid-selected mRNA was precipitated overnight at 20°C by the addition of 5 ,sg carrier tRNA, 1/20 volume of 4 M Na-acetate, and 2 volumes ethanol. Hybrid-selected mRNA was translated in vitro using rabbit reticulocyte lysate, separated by SDS-PAGE, and visualized by fluorography as previously described (25).

RESULTS Ethylene Production and Carnation Petal Senescence Ethylene production by carnation flowers and visible morphological changes exhibited by the petals during senescence are shown in Figure 1. The increase in ethylene represents the 'climacteric' phase of development and has been shown to be associated with increased respiratory activity in carnation petals (2, 17). In addition, carnation petals exhibit specific morphological changes which can be associated with ethylene production. For the first 4 d after harvest, ethylene production remained low (