Pollination of Orchid Flowers: Quantitative and Domain-Specific Analysis of Ethylene Biosynthetic and Hormone-Induced Gene Expression

Abstract

Pollination initiates a suite of postpollination developmental events in orchid flowers. Ethylene plays a key role in coordinating these developmental changes. We examined the temporal-spatial patterns of expression of genes encoding 1-aminocyclopropane 1-carboxylate acid (ACC) synthase and ACC oxidase following pollination and associated hormonal treatments using quantitative procedures. We present the first in situ hybridization analysis of ACC synthase and an earlier time course of ACC oxidase gene expression. In immature ovaries, critical morphological changes involving cell differentiation and growth were affected by pollination and various hormone and inhibitor treatments. Patterns of ACC synthase and ACC oxidase mRNA accumulation were determined by RNA blot hybridization analysis. Gene expression analysis using real-time quantitative polymerase chain reaction indicated that the unpollinated ovary had very low levels of ACC oxidase expression at 6 h and that pollination caused near-maximal levels in this organ by 12 h. Quantitative mRNA analysis showed that auxin could mimic pollination by inducing a similar pattern of organ-specific expression, but earlier and to higher levels. Ethylene had the most dramatic effect by inducing gene activity to its highest levels at all time points and in all floral organs. The early induction in situ hybridization analyses contribute new findings that ACC synthase and ACC oxidase transcripts were localized sequentially in epidermal, phloem, and parenchyma cells of the stigma and that by 8–12 h postpollination, saturation had occurred throughout the stigma. Finally, phylogenetic analysis of orchid ACC synthase proteins leads to the conclusion that at least a subgroup of the angiosperm ACC synthase gene family is induced by auxin but may not be posttranscriptionally regulated, indicating regulatory sequence but not functional divergence of orchid genes with other seed plant ACC synthase proteins.