R2R3-MYB genes coordinate conical cell development and cuticular wax biosynthesis in Phalaenopsis aphrodite

Petals of the monocot Phalaenopsis aphrodite (Orchidaceae) possess conical epidermal cells on their adaxial surfaces, and a large amount of cuticular wax is deposited on them to serve as a primary barrier against biotic and abiotic stresses. It has been widely reported that subgroup 9A members of the R2R3-MYB gene family, MIXTA and MIXTA-like in eudicots, act to regulate the differentiation of conical epidermal cells. However, the molecular pathways underlying conical epidermal cell development and cuticular wax biosynthesis in monocot petals remain unclear. Here, we characterized two subgroup 9A R2R3-MYB genes, PaMYB9A1 and PaMYB9A2 (PaMYB9A1/2), from P. aphrodite through the transient overexpression of their coding sequences and corresponding chimeric repressors in developing petals. We showed that PaMYB9A1/2 function to coordinate conical epidermal cell development and cuticular wax biosynthesis. In addition, we identified putative targets of PaMYB9A1/2 through comparative transcriptome analyses, revealing that PaMYB9A1/2 act to regulate the expression of cell wall-associated and wax biosynthetic genes. Furthermore, a chemical composition analysis of cuticular wax showed that even-chain n-alkanes and odd-chain primary alcohols are the main chemical constituents of cuticular wax deposited on petals, which is inconsistent with the well-known biosynthetic pathways of cuticular wax, implying a distinct biosynthetic pathway occurring in P. aphrodite flowers. These results reveal that the function of subgroup 9A R2R3-MYB family genes in regulating the differentiation of epidermal cells is largely conserved in monocots and dicots. Furthermore, both PaMYB9A1/2 have evolved additional functions controlling the biosynthesis of cuticular wax.

Molecular Characterization and Expression Profile of PaCOL1, a CONSTANS-like Gene in Phalaenopsis Orchid

CONSTANS (CO) and CONSTANS-like (COL) genes play important roles in coalescing signals from photoperiod and temperature pathways. However, the mechanism of CO and COLs involved in regulating the developmental stage transition and photoperiod/temperature senescing remains unclear. In this study, we identified a COL ortholog gene from the Taiwan native orchid Phalaenopsis aphrodite. The Phalaenopsis aphrodite CONSTANS-like 1 (PaCOL1) belongs to the B-box protein family and functions in the nucleus and cytosol. Expression profile analysis of Phalaenopsis aphrodite revealed that PaCOL1 was significantly expressed in leaves, but its accumulation was repressed during environmental temperature shifts. We found a differential profile for PaCOL1 accumulation, with peak accumulation at late afternoon and at the middle of the night. Arabidopsis with PaCOL1 overexpression showed earlier flowering under short-day (SD) conditions (8 h/23 °C light and 16 h/23 °C dark) but similar flowering time under long-day (LD) conditions (16 h/23 °C light and 8 h/23 °C dark). Transcriptome sequencing revealed several genes upregulated in PaCOL1-overexpressing Arabidopsis plants that were previously involved in flowering regulation of the photoperiod pathway. Yeast two-hybrid (Y2H) analysis and bimolecular fluorescence complementation (BiFC) analysis revealed that PaCOL1 could interact with a crucial clock-associated regulator, AtCCA1, and a flowering repressor, AtFLC. Furthermore, expressing PaCOL1 in cca1.lhy partially reversed the mutant flowering time under photoperiod treatment, which confirms the role of PaCOL1 function in the rhythmic associated factors for modulating flowering.

Spike Activator 1, Encoding a bHLH, Mediates Axillary Bud Development and Spike Initiation in Phalaenopsis aphrodite

Double-spikes Phalaenopsis orchids have greater market value than those with single-spike. In this study, a gene designated as Spike Activator 1 (SPK1), which encodes a basic helix-loop-helix (bHLH) transcription factor, was isolated and characterized from Phalaenopsis aphrodite (moth orchid). SPK1 was highly expressed in the meristematic tissues. In the axillary bud, SPK1 was highly upregulated by a moderately low temperature of 20 °C but downregulated by a spike inhibition temperature of 30 °C. SPK1 protein is localized in the nucleus. Another bHLH, bHLH35, which is also highly expressed in young tissues in the same way as SPK1 was also identified. In contrast to SPK1, bHLH35 transcripts are downregulated at 20 °C but upregulated at 30 °C. Bimolecular florescence complementation assay and yeast two-hybrid assays indicated that SPK1 interacts with bHLH35 and forms a heterodimer. Virus-induced gene silencing (VIGS) showed that 7 out of 15 vector control plants produced double spikes but that only 1 out of 15 VIGS-spk1 plants produced double spikes. RT-qPCR results indicated that VIGS-spk1 downregulated gene expression levels of SPK1, FT, CYCB, and EXPA8. Overall, we propose that SPK1 plays an essential role in early axillary bud development and spike initiation of P. aphrodite.