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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.
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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
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