To ensure the fine control of metabolic processes associated to the physiological functions, plants have developed an endogenous system for a precise measurement of photoperiod, represented by circadian rhythms, synchronized with the prevailing environmental conditions. The endogenous system is the regulatory mechanism of gene expression. For example, the temporal regulation of plant scent emission is correlated with the expression pattern of biosynthetic genes in the metabolic pathway of scent production [26–28]. The scent emission is closely tied with the oscillation of expression pattern of metabolic genes. In the scent biology, several model plants, such as rose, snapdragon, tobacco, and petunia have been comprehensively studied for the oscillatory emission patterns. In this regard, Petunia hybrida has been perhaps the most complete model in scent emission [29]. The analysis of gene expression within the floral volatiles benzenoid/ phenylpropanoid (FVBP) pathway provided key insights into the mechanism of scent regulation. Thus far, most studies of the regulatory mechanism of oscillatory emission of floral volatiles are focused on FVBP pathway. FVBP pathway is composed of a series of enzymes. For example, the oscillation of methyl benzoate compound is closely correlated with the expression patterns of the biosynthetic enzymes- BSMT1 and BSMT2 (Salicylate/benzoate carboxyl methyltransferase 1 and 2). PAL (phenylalanine lyase) mRNA expression pattern also oscillates in a similar pattern to that of benzoic acid synthesis in diurnal conditions [30]. Recently, two R2R3-type MYB transcription factors, ODORANT1 (ODO1) and EMISSION BENZENOIDS II (EOBII) were identified as regulatory components of floral scent metabolism. Both are involved in FVBP synthesis [31]. Up-regulation and down-regulation of their transcription increases and decreases the transcripts of many key enzymes in the FVBP pathway and subjective to affect scent production. Most recently, it revealed the identification of putative binding sites of clock gene, i.e. LHY (LATE ELONGATED HYPOCOTYL) in the promoter of ODO1 of P. hybrida [32]. When PhLHY was over-expressed, the floral emission of hybrida was almost lost, and many genes involved in FVBP pathway, including ODO1, EPSPS (enolpyruvylshikimate 3-phosphate synthase), CM1 (chorismate mutase 1), ADT (arogenate dehydratase), and PAL, were down-regulated [15]. However, when PhLHY was mutated, the peak of floral scent emission and gene expression levels in FVBP pathway were move up from dusk to afternoon. As its homologs in other plants, PhLHY peaks around dusk. Further investigation reported it can bind to other genes in the FVBP pathway and controls the expression phase of these genes. These researches delivered the information that temporal expression of scent appears to be primarily regulated through manipulation of the timing of transcriptional regulators in the biosynthetic pathway [16]. In recent years, MYB transcription factors were reported to regulate the production of terpenoids [7].
Our current study firstly found that the floral scent emitted from Onc. Sharry Baby closely related with the expression of terpene synthase. Its relative less expression level influences the scent quantity. The circadian expression is controlled by the promoter structure. Analysis on promoter structure of OgTPS further disclosed a putative CBS (CCA1 binding site), -AGATTTTT-, located at -422〜-430bp of TPS promoter (Fig. 4). The cis-acting element is a morning-specific element, so-called CBS, instead of the evening element (EE), -AAATATCT-. Therefore, volatiles are emitted by diurnal pattern. The EMSA assay identified that OgCCA1 transcriptional factor is able to bind on CBS sequence of TPS promoter. It strongly confirmed the affinity between CCA1 transcription factor and CBS motif (Fig. 6). Similar to the emission pattern of floral scent, TPS and CCA1 are expressed at peak of 6:00〜10:00 AM in the morning, demonstrating the circadian clock function in scent emission. Interestingly, circadian oscillation of OgTPS and OgCCA1 expression showed distinct patterns in light- dependent manners. In the previous reports, the analysis of promoter sequences of target genes of CCA1 showed that there were three other elements, -GCCACGTGTC-, -CAGATATT- and –GTGGGGCC-, were found repeatedly and controlled by CCA1 beside CBS element, suggested that CCA1 might not affect target genes directly, but also indirectly by combining proteins which had interactions with these elements [9]. However, in promoter of Onc. Sharry Baby, the CBS element was the only cis element that is capable to interact with CCA1. Certainly, CBS is the only cis-acting element in TPS promoter that can interact with CCA1. We infer that CCA1 was the major transcription factor to control the circadian rhythm of TPS in Sharry Baby. When Onc. SB orchid plants were kept in continuous light (LL), robust oscillation of OgTPS and OGCCA1 abolished in the first day (Fig. 3). In continuous dark, robust oscillation ceased in the second day (Fig. 3). The results indicated that the proper light/dark cycle is the critical condition for robust circadian oscillation of OgTPS and OgCCA1 in Onc. SB. Evidence suggested that both light and internal clock interact in the rhythmic expression of clock genes [33]. Interestingly, under constant light (LL) conditions, the expression level of TPS recovered at 6:00, day3 (Fig. 3c), suggesting that another factors might exist to affect the gene expression of TPS in plant. In constant dark conditions, there were also several peaks of TPS gene expression.