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. 2003 Aug;132(4):2174-83.
doi: 10.1104/pp.103.023945.

Overproduction of cytokinins in petunia flowers transformed with P(SAG12)-IPT delays corolla senescence and decreases sensitivity to ethylene

Hsiang Chang  1 , Michelle L Jones, Gary M Banowetz, David G Clark
Affiliations

Overproduction of cytokinins in petunia flowers transformed with P(SAG12)-IPT delays corolla senescence and decreases sensitivity to ethylene

Hsiang Chang et al. Plant Physiol. 2003 Aug.

Abstract

Plant senescence is regulated by a coordinated genetic program mediated in part by changes in ethylene, abscisic acid (ABA), and cytokinin content. Transgenic plants with delayed senescence are useful for studying interactions between these signaling mechanisms. Expression of ipt, a cytokinin biosynthetic gene from Agrobacterium tumefaciens, under the control of the promoter from a senescence-associated gene (SAG12) has been one approach used to delay senescence. We transformed petunia (Petunia x hybrida cv V26) with P(SAG12)-IPT. Two independently transformed lines with extended flower longevity (I-1-7-22 and I-3-18-34) were used to study the effects of elevated cytokinin content on ethylene synthesis and sensitivity and ABA accumulation in petunia corollas. Floral senescence in these lines was delayed 6 to 10 d relative to wild-type (WT) flowers. Ipt transcripts increased in abundance after pollination and were accompanied by increased cytokinin accumulation. Endogenous ethylene production was induced by pollination in both WT and IPT corollas, but this increase was delayed in IPT flowers. Flowers from IPT plants were less sensitive to exogenous ethylene and required longer treatment times to induce endogenous ethylene production, corolla senescence, and up-regulation of the senescence-related Cys protease phcp1. Accumulation of ABA, another hormone regulating flower senescence, was significantly greater in WT corollas, confirming that floral senescence was delayed in IPT plants. These results extend our understanding of the hormone interactions that regulate flower senescence and provide a means of increasing flower longevity.

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Figures

Figure 1.
Figure 1.
Real-time PCR quantification of relative ipt mRNA levels in IPT22 and IPT34 corollas at 0, 12, 18, 24, 36, 48, and 72 hap. Ipt mRNA was quantified relative to an external RNA standard curve and normalized using actin as an endogenous control. All samples were amplified in triplicate. Data presented are the average of the three replications ± se.
Figure 2.
Figure 2.
Pollination-induced ethylene production and senescence in WT versus IPT22 and IPT34 flowers. A, Ethylene production from corollas at various times after pollination. Values presented are the average of three independent experiments ± se (n = 12). B, Visual appearance of WT, IPT22, and IPT34 flowers after pollination. C, Changes in the mRNA levels of the ethylene biosynthetic gene ACC oxidase (phaco1). Signal intensity was quantified, and all values were normalized for RNA loading differences using ribosomal RNA. The sample with the highest hybridization signal was set at 100%, and all other values are presented relative to that sample.
Figure 3.
Figure 3.
Detached flowers were treated with 2 μL L-1 ethylene for 0, 6, 9, 12, 18, 24, 36, and 48 h to compare the sensitivity of WT and IPT corollas to ethylene. Control flowers were treated with air (data not shown). A, Ethylene production from WT, IPT22 and IPT34 corollas after ethylene treatment. Experiments were conducted three times with similar results. Data presented are for one experiment and represent an average of four ethylene measurements ± se. B, Changes in the mRNA levels of the senescence-related Cys protease, phcp1,in corollas after treatment of flowers with ethylene. Signal intensity was quantified, and all values were normalized for RNA loading differences using ribosomal RNA. The sample with the highest hybridization signal was set at 100%, and all other values are presented relative to that sample.
Figure 4.
Figure 4.
Detached WT, IPT22, and IPT34 flowers were treated with 2 μL L-1 ethylene or air (control) for 12 h. After treatment, flowers were left on the laboratory bench and visual symptoms of senescence (i.e. corolla wilting) were assessed every 24 h. Zero represents the flowers immediately following the 12 h treatment.
Figure 5.
Figure 5.
Changes in total cytokinin content (A) and the relative quantity of ipt mRNAs in corollas (B) after treatment of WT, IPT22, and IPT34 flowers with 2 μL L-1 ethylene for 0 or 12 h. Values represent the average of three replicates ± se.
Figure 6.
Figure 6.
Quantification of endogenous ABA levels in WT, IPT22, and IPT34 corollas at 0, 12, 18, 24, 36, 48, and 72 hap. Values represent the average of three replicates ± se.
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