Molecular characterization and functional analysis of a Flowering locus T homolog gene from a Phalaenopsis orchid.

Warm day and cool night conditions significantly induce reproductive spike formation in Phalaenopsis plants; hence, determining the flowering mechanism regulating the reproductive transition is important. Flowering locus T (FT) plays important roles in flowering induction in several plants. To explore spike induction by warm days and cool nights in Phalaenopsis orchids, we isolated the FT (PhFT) from Phalaenopsis hybrid Fortune Saltzman. The cDNA of PhFT was 809-bp long and contained a 531-bp open reading frame encoding a putative protein of 176 amino acids, a 58-bp 5'-untranslated region (UTR), and a 220-bp 3'-UTR. The predicted molecular mass of PhFT was 19.80 kDa, with an isoelectric point of 8.68. The PhFT was predicted to possess the conserved functional regions of the phosphatidylethanolamine-binding protein superfamily. Nucleotide sequence data indicated that PhFT contained 3 introns and 4 exons. Sequence alignment and phylogenetic analyses of PhFT revealed high homology to the FT proteins of Cymbidium goeringii and Oncidium Gower Ramsey. Quantitative real-time polymerase chain reaction analysis indicated that PhFT mRNA was expressed in roots, apical leaves, mature leaves, and flowers. In flowers, PhFT was expressed more in developing floral buds than in mature flowers and was predominantly expressed in ovaries and petals. Ectopic expression of PhFT in Arabidopsis ft-1 mutants showed novel early-flowering phenotypes that lost their siliques. Our results indicated that the ectopic expression of PhFT could partially complement the late flowering defect in transgenic Arabidopsis ft-1 mutants. Our findings suggest that PhFT is a putative FT homolog in Phalaenopsis plants that regulates flowering transition.

Identification of warm day and cool night conditions induced flowering-related genes in a Phalaenopsis orchid hybrid by suppression subtractive hybridization.

The influence of warm day and cool night conditions on induction of spikes in Phalaenopsis orchids has been studied with respect to photosynthetic efficiency, metabolic cycles and physiology. However, molecular events involved in spike emergence induced by warm day and cool night conditions are not clearly understood. We examined gene expression induced by warm day and cool night conditions in the Phalaenopsis hybrid Fortune Saltzman through suppression subtractive hybridization, which allowed identification of flowering-related genes in warm day and cool night conditions in spikes and leaves at vegetative phase grown under warm daily temperatures. In total, 450 presumably regulated expressed sequence tags (ESTs) were identified and classified into functional categories, including metabolism, development, transcription factor, signal transduction, transportation, cell defense, and stress. Furthermore, database comparisons revealed a notable number of Phalaenopsis hybrid Fortune Saltzman ESTs that matched genes with unknown function. The expression profiles of 24 genes (from different functional categories) have been confirmed by quantitative real-time PCR in induced spikes and juvenile apical leaves. The results of the real-time PCR showed that, compared to the vegetative apical leaves, the transcripts of genes encoding flowering locus T, AP1, AP2, KNOX1, knotted1-like homeobox protein, R2R3-like MYB, adenosine kinase 2, S-adenosylmethionine synthetase, dihydroflavonol 4-reductase, and naringenin 3-dioxygenase accumulated significantly higher levels, and genes encoding FCA, retrotransposon protein Ty3 and C3HC4-type RING finger protein accumulated remarkably lower levels in spikes of early developmental stages. These results suggested that the genes of two expression changing trends may play positive and negative roles in the early floral transition of Phalaenopsis orchids. In conclusion, spikes induced by warm day and cool night conditions were complex in Phalaenopsis orchids; nevertheless, several molecular flowering pathway-related genes were found. The acquired data form the basis for a molecular understanding of spike induction by warm day and cool night conditions in Phalaenopsis orchids.