Results Directory UMM :Data Elmu:jurnal:P:PlantScience:PlantScience_Elsevier:Vol158.Issue1-2.Sept2000:

Fig. 2. Changes in the levels of transcripts for ACC oxidase, ACC synthase and CPase in petals of carnation flowers treated with DPSS. Carnation flowers at day 0 were treated with 0.1 mM DPSS for 24 h, then left for sampling of the petals at given time. Two micrograms of mRNAs isolated from petals was separated on an agarose gel and hybridized with 32 P-labeled DC-ACO 1 , DC-ACS 1 or CPase probes. For reference, the mRNAs isolated from petals of flowers, left to senesce naturally for 5 days, were treated similarly. 3 . 2 . Two distinct responses to exogenous ethylene of the petals from the transgenic carnation To investigate whether the petals of the trans- genic line with the sense ACC oxidase transgene respond to ethylene, we treated the petals ob- tained from the transgenic and control lines with 10 ml l − 1 ethylene for 18 h at the full opening stage day 0, and compared their senescence be- havior. The petals of both lines exhibited in- rolling symptoms after the ethylene treatment Fig. 1A. On the other hand, ethylene production from the petals after the ethylene treatment was quite different between the two lines Fig. 1B. Petals of the transgenic line produced ethylene at 0.5 9 0.1 nmol h − 1 g − 1 , whereas those of the control line did so at 10.3 9 1.4 nmol h − 1 g − 1 . Ethylene production was negligible in the petals that had not been treated with ethylene in both lines. Fig. 1C shows the levels of the transcripts for ACC oxidase, ACC synthase and CPase in the petals of the transgenic and control lines after the treatment with or without ethylene. A large amount of transcripts for both ACC oxidase and ACC synthase accumulated in the petals of the control line after ethylene treatment, whereas did only a little in the petals of the transgenic line. The large difference in the levels of the transcripts between the control and the transgenic lines corre- sponded with the difference in ethylene produc- tion between the two lines. Also, the difference in the transcript levels between the two lines after ethylene treatment was probably a reflection of the difference found in the flowers of the two lines, which underwent natural senescence as men- tioned above. On the other hand, the transcript for CPase accumulated at almost the same level in the petals of both the transgenic and control lines. The present finding that neither ACC synthase nor ACC oxidase transcripts accumulated in the petals after the ethylene treatment were different from those reported by Savin et al. [29] who showed a substantial accumulation of these tran- scripts in the petals of a carnation plant trans- formed with an antisense ACC oxidase transgene after ethylene treatment. However, their results might be caused by the use of a high concentra- tion of ethylene, 150 ml l − 1 , which might cause an excess accumulation of transcripts. tions. Hybridization signals were detected with an image analyzer FLA2000, Fuji Photo Film.

3. Results

3 . 1 . Characteristics of senescence in flowers of a transgenic carnation harboring a sense ACC oxidase transgene We generated a transgenic carnation harboring a sense ACC oxidase transgene. Details for gener- ation and characterization of the transgenic car- nation will appear elsewhere. Here we describe briefly its characteristics to help understand the transformant as an experimental material. DNA gel blot analysis revealed that the transformant harbored multiple copies of a sense ACC oxidase transgene. The transgenic plant grew and flowered normally like the non-transformed control plant. Cut flowers of the transgenic carnation produced only a trace amount of ethylene during the senes- cence period, and had a vase-life about twofold longer than that of the control flowers. The sup- pressed ethylene production was accompanied by little or no accumulation of transcripts for ACC oxidase and ACC synthase in petals. These results suggested that the sense ACC oxidase transgene caused cosuppression of the expression of ACC oxidase gene, which was accompanied by the blockage of the expression of the ACC synthase gene resulting in the absence of autocatalytic in- duction of ethylene production. 3 . 3 . Difference in the expression of genes for ACC synthase, ACC oxidase and CPase in carnation petals treated with DPSS DPSS is an antisenescence preservative for cut carnation flowers [30]. DPSS prolongs the vase-life of carnation flowers by preventing ethylene pro- duction, but does not affect the ethylene-induced senescence in the flowers. Application of DPSS inhibits the increase in activities of ACC synthase and ACC oxidase, which occurred in non-treated control flowers during natural senescence [30,31]. DPSS does not inhibit in vitro activities of either ACC synthase or ACC oxidase obtained from senescing carnation petals [32]. Recently it was revealed that the inhibitory action of DPSS was specific to the ethylene production in carnation flowers undergoing natural senescence [31]. How- ever, the mechanism of DPSS action is not fully elucidated yet. As shown in Fig. 2, the administration of DPSS to carnation flowers at day 0 induced the accumu- lation of transcripts for ACC synthase DC- ACS 1 and ACC oxidase DC-ACO 1 from the next day on, but did not cause a significant change in the level of transcript for CPase. The increased level of transcripts for ACC synthase and ACC oxidase lasted until day 9. On the other hand, transcripts for all three genes were accumulated on petals, which underwent natural senescence for 5 days. The mechanism of the action of DPSS on the up-regulation of ACC synthase and ACC ox- idase genes and no increase in the activities of the enzymes in carnation petals is now under investi- gation. These findings clearly indicated that carna- tion petals can regulate the expression of genes for ACC synthase and ACC oxidase independent of that for CPase.

4. Discussion