Towards Understanding the Onset of Petal Senescence: Analysis of Ethylene Production in the Long-Lasting Carnation cv. White Candle

In senescing carnation (Dianthus caryophyllus L.) flowers, ethylene is produced from the gynoecium, and acts as a diffusible signal received by petals to induce the expression of 1-aminocyclopropane-1-carboxylate (ACC) synthase (DC-ACS1) and ACC oxidase (DC-ACO1) genes in the petals. This results in autocatalytic ethylene production in the petals. We investigated ethylene production in cut flowers of cv. White Candle, which produce ethylene only in trace amounts and have a long vase life. The low ethylene production in these flowers was due to low ethylene production in the gynoecium, accompanied by low accumulation of ACC synthase transcripts. These findings further support the importance of ethylene production from the gynoecium in the senescence of carnation flowers. Cv. White Candle flowers had water relations that were different from those in cv. Light Pink Barbara, a cultivar that showed the normal climacteric rise of ethylene production during senescence. We discuss a factor that possibly induces ethylene production in the carnation gynoecium. INTRODUCTION Senescence in carnation flowers culminates in autocatalytic ethylene production and petal wilting. The ethylene production in the petals begins by the expression of ACC synthase (ACS) (DC-ACS1) and ACC oxidase (ACO) (DC-ACO1) genes upon perception of ethylene that comes from the gynoecium or is applied exogenously (Jones and Woodson, 1997; ten Have and Woltering, 1997; Shibuya et al., 2000). The ethylene production in the gynoecium is induced by compatible pollination or, in the case of nonpollinated flowers, it starts by itself several days after full opening of the flower. Several papers deal with pollination-induced ethylene production in the carnation gynoecium (Nichols, 1977; Jones and Woodson, 1997). However, many carnation cultivars do not have anthers, and thus cannot be pollinated by their own pollen. They nonetheless show an increase in ethylene production in gynoecium, which must be induced by factors other than pollination. The mechanism of gynoecium ethylene production without pollination has thus far received little attention. It has been established, however, that the increase in ethylene production in unpollinated flowers starts in the ovary (part of the gynoecium), whereas in pollination-induced senescence it starts in the style of the gynoecium. We are interested in factors that induce ethylene production in the gynoecium of unpollinated carnation flowers. In the present study, we investigated ethylene production in flowers of a cv. White Candle (WC), which has long lasting flowers, in comparison with that of the conventional cv. Light Pink Barbara (LPB). The work was undertaken to obtain further evidence of the role of ethylene production from the gynoecium in carnation flower senescence. Furthermore we explored a factor that induces ethylene production in the gynoecium of unpollinated senescing carnation flowers. MATERIALS AND METHODS We used carnation flowers (Dianthus caryophyllus L.) of the cvs. White Candle (WC) and Light Pink Barbara (LPB). Flowers were harvested at the full opening stage (day Proc. VIII IS Postharvest Phys. Ornamentals Eds. N. Marissen et al. Acta Hort. 669, ISHS 2005 176 0). Stem length was trimmed to 3 cm. The stem ends were placed in 20 ml distilled water in 50-ml glass vials (one flower per vial). The flowers were left under white fluorescent light (15 μmol m s) at 23 °C, and water was replaced daily. Flowers were observed daily to record senescence symptoms, and flower ethylene production was measured once each day. For ethylene treatment, the vials with flowers on day 0 were placed in a glass chamber with ethylene at 2 μl L for 0 to 18 h under the conditions described above. Flowers were taken at 6-h intervals from the glass chamber, and held in open air for 1 h to allow the accumulated ethylene to diffuse out of the flower tissue. Ethylene production from the whole flower was measured by enclosing the flowers in 350-ml glass containers (one flower per container) for 1 h at 23 °C. A 1-ml gas sample was taken from the container, and analyzed for ethylene with a gas chromatograph. Then the flowers were separated into gynoecium (ovary plus styles), petals, and the remaining parts (sepals plus stem). The gynoecium and petals were subjected to measurement of ethylene production by enclosing them for 1 h in glass containers of appropriate sizes. After the assay for ethylene production, the petals and gynoecium were weighed, immediately frozen in liquid N2 and stored at – 80 °C until isolation of RNA. Total RNA from petals and gynoecium was isolated using the SDS–phenol method (Palmiter, 1974). The presence or absence of transcripts of an ACO gene (DCACO1) and ACS genes (DC-ACS1, DC-ACS2, DC-ACS3) in total RNA fractions was determined by amplification by RT-PCR. RT-PCR was performed according to standard procedures with necessary optimization. The PCR amplicates were separated on a 2.0% agarose gel and visualized by ethidium bromide staining. To examine the identities of the operation of PCR and the amount of template RNA, we amplified the fragment of actin (DC-ACT1, Waki et al., 2001). The upstream and downstream primers for RT-PCR, the sizes of amplified cDNA fragments and their positions in the original cDNAs will be reported elsewhere. Water uptake and transpiration of ‘WC’ and ‘LPB’ flowers were determined by daily measurement of flower fresh weight and the weight of the water that had been taken up from the incubation solutions. RESULTS AND DISCUSSION Ethylene Production in Whole Flowers, Gynoecium and Petals of Unpollinated ‘WC’ and ‘LPB’ Flowers Fig. 1 compares ethylene production from whole flowers, petals and gynoecia in ‘WC’ and ‘LPB’ flowers. Ethylene production of whole ‘WC’ flowers was below the detection limit, throughout the 19 d study period. When petals and gynoecia were detached from the whole flowers, their ethylene production was also mostly below detection limit, throughout the 19 d period. By contrast, whole ‘LPB’ flowers produced ethylene in a significant amount. The production rate was maximal (2.70 nmol g h) on day 5. Isolated gynoecia and petals produced ethylene with a time course similar to that of whole flowers. We determined whether the absence of ethylene production in the ‘WC’ flowers was a result of malfunction of ethylene biosynthesis in the gynoecia and/or the petals. We found that treatment of ‘WC’ flowers with exogenous ethylene caused both an increase in ethylene production (Fig. 2), and accumulation of transcripts for DC-ACS1 and DC-ACO1 genes in the gynoecia and petals (data not shown). This suggests that the machinery for induction of ethylene biosynthesis genes was fully intact, and that the resulting enzymes were fully functional. Changes in the Levels of ACO and ACS Transcripts in the Gynoecium and Petals of ‘WC’ and ‘LPB’ Flowers during Natural Senescence Since three genes for ACS (DC-ACS1, DC-ACS2, DC-ACS3) and one gene for ACO (DC-ACO1) have been identified in carnation (Wang and Woodson, 1991; Park et al., 1992; Henskens et al., 1994; Jones and Woodson, 1999), we determined the presence or absence of transcripts of these genes in the gynoecium and petals of unpollinated ‘WC’ and ‘LPB’ flowers (Fig. 3). In the gynoecium of the ‘LPB’ flowers, the DC-ACS3 transcript was