Chang-Hsien Yang

Functional analysis of FT and TFL1 orthologs from orchid (Oncidium Gower Ramsey) that regulate the vegetative to reproductive transition.

The FLOWERING LOCUS T (FT) and TERMINAL FLOWER 1 (TFL1) genes play crucial roles in regulating the vegetative to reproductive phase transition. Orthologs of FT/TFL1 (OnFT and OnTFL1) were isolated and characterized from Oncidium Gower Ramsey. OnFT mRNA was detected in axillary buds, leaves, pseudobulb and flowers. In flowers, OnFT was expressed more in young flower buds than in mature flowers and was predominantly expressed in sepals and petals. The expression of OnFT was regulated by photoperiod, with the highest expression from the 8th to 12th hour of the light period and the lowest expression at dawn. In contrast, the expression of OnTFL1 was only detected in axillary bud and pseudobulb, and was not influenced by light. Ectopic expression of OnFT in transgenic Arabidopsis plants showed novel phenotypes by flowering early and losing inflorescence indeterminacy. In addition, ectopic expression of OnFT was able to partially complement the late flowering defect in transgenic Arabidopsis ft-1 mutants. In transgenic tfl1-11 mutant plants, 35S::OnTFL1 delayed flowering and rescued the phenotype of terminal flowers. Furthermore, substitution of the key single amino acid His85 by Tyr was able to convert the OnTFL1 function to OnFT by promoting flowering in 35S::OnTFL1-H85Y transgenic Arabidopsis plants. Further analysis indicated that the expression of APETALA1 (AP1) was significantly up-regulated in 35S::OnFT and 35S::OnTFL1-H85Y plants, and was down-regulated in 35S::OnTFL1 transgenic Arabidopsis plants. Our data indicated that OnFT and OnTFL1 are putative phosphatidylethanolamine-binding protein genes in orchids that regulate flower transition similar to their orthologs in Arabidopsis.

Four Orchid (Oncidium Gower Ramsey) AP1/AGL9-like MADS Box Genes Show Novel Expression Patterns and Cause Different Effects on Floral Transition and Formation in Arabidopsis thaliana

Four AP1/AGL9 functional MADS box genes were characterized from the orchid (Oncidium Gower Ramsey). OMADS6 is a SEP3 ortholog, OMADS11 is a SEP1/2 ortholog, OMADS7 is an AGL6-like gene and OMADS10 is a putative paleoAP1 ortholog. The identity of these four genes was further supported by the presence of conserved motifs in the C-terminal regions of the proteins. OMADS6 showed an expression pattern different from SEP3 orthologs, with expression in the sepal, petal, lip and carpel, and was barely detected in the stamen. The expression pattern for OMADS11 was similar to OMADS6 and different from SEP1/2 orthologs since its expression was undetectable in the stamen. The expression pattern for OMADS7 was nearly identical to OMADS6. The similarities in the expression patterns of the SEP/AGL6-like genes OMADS6, 11 and 7 indicated that their transcriptional regulation is highly evolutionarily conserved in the orchid. Unlike OMADS6/11/7, OMADS10 was only expressed in vegetative leaves and in the lip and carpel of mature flowers, which distinguishes it from most genes in the SQUA subfamily. Ectopic expression of OMADS6, 11 or 7 caused extremely early flowering, whereas 35S::OMADS10 only caused moderately early flowering in transgenic Arabidopsis plants. In addition, flower organ conversions, such as carpelloid sepals and staminoid petals, were observed in 35S::OMADS6 and carpelloid sepals were produced in 35S::OMADS7, while flower organ conversions were not observed in 35S::OMADS11 or 35S::OMADS10 transgenic flowers. This result reveals possible functional diversification of the orchid AP1/AGL9 genes OMADS6, 11, 7 and 10 in regulating flower transition and formation.

Functional Analysis of Three Lily (Lilium longiflorum) APETALA1-like MADS Box Genes in Regulating Floral Transition and Formation

Three cDNAs showing a high degree of homology to the SQUA subfamily of MADS box genes were isolated and characterized from the lily (Lilium longiflorum). Lily MADS Box Gene 5 (LMADS5) showed high sequence identity to oil palm (Elaeis guineensis) SQUAMOSA3 (EgSQUA3). LMADS6 is closely related to LMADS5 whereas LMADS7 is more related to DOMADS2, an orchid (Dendrobium) gene in the SQUA subfamily. The expression pattern for these three genes was similar and their RNAs were detected in vegetative stem and inflorescence meristem. LMADS5 and 6 were highly expressed in vegetative leaves and carpel, whereas LMADS7 expression was absent. Ectopic expression of LMADS5, 6 or 7 in transgenic Arabidopsis plants showed novel phenotypes by flowering early and producing terminal flowers. Homeotic conversions of sepals to carpelloid structures and of petal to stamen-like structures were also observed in 35S::LMADS5, 6 or 7 flowers. Ectopic expression of LMADS6 or LMADS7 was able to complement the ap1 flower defect in transgenic Arabidopsis ap1 mutant plants. These results strongly indicated that the function of these three lily genes was involved in flower formation as well as in floral induction. Furthermore, the ability of lily LMADS6 and 7 to complement the Arabidopsis ap1 mutant provided further evidence to show that the conserved motifs (paleoAP1 or euAP1) in the C-terminus of the SQUA/AP1 subfamily of MADS box genes is not strictly necessary for their function.

Characterization of Oncidium ‘Gower Ramsey’ Transcriptomes using 454 GS-FLX Pyrosequencing and Their Application to the Identification of Genes Associated with Flowering Time

Oncidium Gower Ramsey is a valuable and successful commercial orchid for the floriculture industry in Taiwan. However, no genome reference for entire sequences of the transcribed genes currently exists for Oncidium orchids, to facilitate the development of molecular biological studies and the breeding of these orchids. In this study, we generated Oncidium cDNA libraries for six different organs: leaves, pseudobulbs, young inflorescences, inflorescences, flower buds and mature flowers. We utilized 454-pyrosequencing technology to perform high-throughput deep sequencing of the Oncidium transcriptome, yielding >0.9 million reads with an average length of 328 bp, for a total of 301 million bases. De novo assembly of the sequences yielded 50,908 contig sequences with an average length of 493 bp from 796,463 reads and 120,219 singletons. The assembled sequences were annotated using BLAST, and a total of 12,757 and 13,931 unigene transcripts from the Arabidopsis and rice genomes were matched by TBLASTX, respectively. A Gene Ontology (GO) analysis of the annotated Oncidium contigs revealed that the majority of sequenced genes were associated with unknown molecular function, cellular process and intracellular components. Furthermore, a complete flowering-associated expressed sequence that included most of the genes in the photoperiod pathway and the 15 CONSTANS-LIKE (COL) homologs with the conserved CCT domain was obtained in this collection. These data revealed that the Oncidium expressed sequence tag (EST) database generated in this study has sufficient coverage to be used as a tool to investigate the flowering pathway and various other biological pathways in orchids. An OncidiumOrchidGenomeBase (OOGB) website has been constructed and is publicly available online (http://predictor.nchu.edu.tw/oogb/).

C/D Class MADS Box Genes from Two Monocots, Orchid (Oncidium Gower Ramsey) and Lily (Lilium longiflorum), Exhibit Different Effects on Floral Transition and Formation in Arabidopsis thaliana

We have characterized three C/D class MADS box genes from an orchid (Oncidium Gower Ramsey) and a lily (Lilium longiflorum). OMADS4 of orchid and LMADS10 of lily are C class gene orthologs, whereas OMADS2 of orchid is a putative D class gene ortholog. The identity of these three genes is further supported by the presence of conserved motifs in the C-terminal regions of the proteins. The mRNA for these three genes can be detected in flowers and is absent in vegetative leaves. In flowers, OMADS4 and LMADS10 show similar expression patterns, being specifically expressed in the stamens and carpels. The expression of OMADS2 is restricted to the stigmatic cavity and ovary of the carpel. The similarities of the expression patterns of OMADS4/LMADS10 and OMADS2 to those of C and D class genes, respectively, indicate that their transcriptional regulation is highly evolutionarily conserved in these monocot species. Yeast two-hybrid analysis indicates that both OMADS2 and OMADS4 form homodimers and heterodimers with each other. Similar interactions are observed for LMADS2 and LMADS10. Ectopic expression of LMADS10 causes extremely early flowering, terminal flower formation and conversion of the sepals into carpel-like structures, similar to ectopic expression of the lily D class gene LMADS2. In contrast, 35S::OMADS2 and 35S::OMADS4 cause only early or moderately early flowering in transgenic Arabidopsis plants without floral organ conversion. This result indicates that C/D class genes from the lily have stronger effects than those from the orchid in transgenic Arabidopsis, revealing possible functional diversification of C/D class genes from the two monocots in regulating floral transition and formation.

Functional analysis reveals the possible role of the C-terminal sequences and PI motif in the function of lily (Lilium longiflorum) PISTILLATA (PI) orthologues

Two lily (Lilium longiflorum) PISTILLATA (PI) genes, Lily MADS Box Gene 8 and 9 (LMADS8/9), were characterized. LMADS9 lacked 29 C-terminal amino acids including the PI motif that was present in LMADS8. Both LMADS8/9 mRNAs were prevalent in the first and second whorl tepals during all stages of development and were expressed in the stamen only in young flower buds. LMADS8/9 could both form homodimers, but the ability of LMADS8 homodimers to bind to CArG1 was relatively stronger than that of LMADS9 homodimers. 35S:LMADS8 completely, and 35S:LMADS9 only partially, rescued the second whorl petal formation and partially converted the first whorl sepal into a petal-like structure in Arabidopsis pi-1 mutants. Ectopic expression of LMADS8-C (with deletion of the 29 amino acids of the C-terminal sequence) or LMADS8-PI (with only the PI motif deleted) only partially rescued petal formation in pi mutants, which was similar to what was observed in 35S:LMADS9/pi plants. In contrast, 35:LMADS9+L8C (with the addition of the 29 amino acids of the LMADS8 C-terminal sequence) or 35S:LMADS9+L8PI (with the addition of the LMADS8 PI motif) demonstrated an increased ability to rescue petal formation in pi mutants, which was similar to what was observed in 35S:LMADS8/pi plants. Furthermore, ectopic expression of LMADS8-M (with the MADS domain truncated) generated more severe dominant negative phenotypes than those seen in 35S:LMADS9-M flowers. These results revealed that the 29 amino acids including the PI motif in the C-terminal region of the lily PI orthologue are valuable for its function in regulating perianth organ formation.

Overexpression of the Lily p70s6k Gene in Arabidopsis Affects Elongation of Flower Organs and Indicates TOR-Dependent Regulation of AP3, PI and SUP Translation

The p70 ribosomal S6 kinase (p70(s6k)) signaling pathway plays a key role in regulating the cell cycle via translational regulation of specific 5TOP mRNAs. However, the function of this signaling pathway is still poorly understood in plants. Ectopic expression of the lily putative p70(s6k) gene, LS6K1, resulted in up-regulation of NAP (NAC-LIKE, ACTIVATED BY AP3/PI) and PISTILLATA (PI) expression, and significantly inhibited cell expansion for petals and stamens, resulting in the male sterility phenotype in transgenic Arabidopsis. Sequence analysis revealed that the genes involved in petal and stamen development, such as APETALA3 (AP3), PI and SUPERMAN (SUP), probably encode 5TOP mRNAs. Green fluorescent protein (GFP), fused to oligopyrimidine tract sequences that were identified in the 5-untranslated region (UTR) of AP3, PI and SUP, was translationally regulated in human cells in response to mitogen stimulation and inhibition by the macrolide antibiotic rapamycin. Furthermore, 35S::LS6K1 significantly up-regulated beta-glucuronidase (GUS) activity in the flower buds of transgenic plants carrying the GUS transgene fused to the AP3 promoter and the 5 UTR. These results have identified a novel role for the p70(s6k) gene in regulating cell division and the expansion of petals and stamens by translational regulation of the 5TOP mRNAs once ectopically expressed in Arabidopsis.