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SENESCENCE-ASSOCIATED GENE EXPRESSION IN NARCISSUS ‘DUTCH MASTER’ Donald A. Hunter and Michael S. Reid Department of Environmental Horticulture, University of California, Davis, USA E-mail: [email protected] Keywords: Daffodil, remobilization, tepals, flower senescence, suppression-subtraction PCR, programmed cell death 1. Introduction Flower senescence is a highly controlled developmental event that culminates in the death of the floral organs. Most research on flower senescence has focused on the perianth, since it typically determines the commercial life of the flower. The mechanism by which the perianth dies differs between species: in some flowers, wilting is the primary symptom of senescence whereas in others, the perianth is shed prior to, or at the time of wilting. Daffodils (Narcissus) are widely grown commercial flowers that are prized for their beauty in spring. The flowers are short-lived and are reported to senesce independently of ethylene action (Woltering and van Doorn, 1988). In addition to the petals and sepals (tepals), daffodil flowers are unique in possessing an additional petaloid structure, the corona. We have used the flowers of the ‘Dutch Master’ cultivar as an experimental model to examine molecular changes associated with the onset of nonclimacteric flower senescence, and report here our preliminary results. 2. Materials and methods Plant material Bulbs of ‘Dutch Master’ daffodils were obtained from the Oregon Bulb Company (Oregonbulb.com) and were precooled in pots at 7°C for a total time of 15 weeks. They were then transferred to the greenhouse until just prior to bud break, when they were moved to an interior controlled environment at 20±2°C, relative humidity of ca. 45% and a 12 h photoperiod (15 µmol.m-2.sec-1 PAR from Cool White fluorescent lamps). Molecular procedures Total RNA was isolated from tepals and coronas by the method of Hunter and Reid (2000). Poly A+ RNA was isolated from total RNA with the polyATtract mRNA isolation system (Promega). The subtracted cDNA library was constructed by means of the PCR-select kit (Clontech). Subtracted cDNA sequences were ligated into the pGEM T-Easy vector (Promega) and cloned into E. coli. Isolated plasmids were sequenced at the sequencing facility on the U.C. Davis campus and their sequences were compared with sequences in the GenBank database with the BLASTx tool (Altschul et al., 1990). 3. Results and discussion We selected 94 of the subtracted PCR sequences for further analysis. Among the 94 sequences, we identified a large number as homologous with genes reported in the GenBank database to be associated with senescence or remobilization in plants (Table 1). Of particular interest is the identification of the enzymes of the glyoxylate cycle, presumably associated with remobilization of lipids. The homologies with genes known to be involved in regulation of growth and development are also interesting, as they may provide leads to exploration of the control of senescence in non-climacteric flowers. Some genes, interestingly, showed homology to sequences that in other systems Proc. 4th. Int. Conf. On Postharvest Eds. R. Ben-Arie & S. Philosoph-Hadas Acta Hort. 553, ISHS 2001 341 are associated with ethylene. For instance, E8 has been shown to be ethylene-inducible in tomato fruit and is thought to have a role in controlling the ripening-associated ethylene production of the fruit (Kneissl et al., 1995). We also identified a DNA transcription factor containing an ethylene response element binding motif. It would be interesting to determine the relevance of these sequences to the apparently ethylene-independent senescence of the daffodil flower. The presence of sequences with high homology to protein-degrading enzymes may indicate a significant role for proteases in the senescence of daffodil flowers, presumably in degradation of structural proteins, and perhaps in the control of senescence itself. Similarly, the S1-type endonuclease, an enzyme thought to cleave both RNA and single stranded DNA, may be responsible for the senescence-associated reduction in RNA we have observed in daffodil flowers. 4. Conclusion Sequence homology comparisons suggest that the PCR-select protocol is an effective tool for isolating sequences that encode enzymes associated with senescence of the daffodil flower. We have identified gene transcripts encoding proteins presumably involved in hydrolytic activity, remobilisation, signal transduction, and transcriptional regulation of the senescing flower. In future studies we will confirm their association with senescence using northern analysis and study the role of the different enzymes in the senescence cascade in daffodils and other flowers. References Altschul, S.F., Gish, W., Millar, W., Myers, E.W. and Lipman, D.J., 1990. Basic local alignment search tool. Journal of Molecular Biology, 215:403-410. Hunter, D.A. and Reid, M.S., 2000. A simple and rapid method for isolating high quality RNA from petals. Acta Horticulturae. In press. Kneissl, ML. and Deikman, J., 1995. The tomato E8 influences ethylene biosynthesis in fruit but not in flowers. Plant Physiology, 112:537-547. Woltering, E.J. and van Doorn, W.G. 1988. Role of ethylene in senescence of petals: Morphological and taxonomical relationships. J. Experimental Botany, 39:1605-1616. 342 Tables 1. Probable identity and homology with known senescence- or mobilization-associated sequences of cDNAs isolated by subtractive PCR from incipiently senescent daffodil tepals. Percentage identity is based on deduced amino acid sequences. Enzyme or protein Isolated from Enzymes associated with ripening and senescence Cysteine proteinase Senescing daylily tepals Vacuolar processing Ripening citrus fruit, ethylene inducible S1–type nuclease Tepal senescence in daylily Regulatory protein E8 Regulates tomato ethylene production Glyoxysomal enzymes Enoyl CoA hydratase Mangrove (Avicennia marina) Malate dehydrogenase Dock (Medicago sativa) Acyl CoA synthetase Arabidopsis thaliana Acyl CoA oxidase Moth orchid (Phalaenopsis) Proteins associated with control of growth and development Eukaryotic initiation factor Tobacco (Nicotiana tabacum) Heat shock protein Morning glory (Pharbitis nil) Auxin efflux carrier protein Arabidopsis thaliana Ankyrin-like protein Arabidopsis thaliana Receptor-like kinase Cabbage (Brassica oleracea) DNA binding factor (EREBP) Arabidopsis thaliana Identity (%) 76 65 87 51 72 89 55 89 92 83 64 63 57 48 343