The bZIP transcription factor HY5 interacts with the promoter of the monoterpene synthase gene QH6 in modulating its rhythmic expression.

Fei Zhou,Lei Zhao,Tian-Hu Sun,Xi-Wu Pan,Shan Lu

doi:10.3389/fpls.2015.00304

Abstract

The Artemisia annua L. β-pinene synthase QH6 was previously determined to be circadian-regulated at the transcriptional level, showing a rhythmic fluctuation of steady-state transcript abundances. Here we isolated both the genomic sequence and upstream promoter region of QH6. Different regulatory elements, such as G-box (TGACACGTGGCA, −421 bp from the translation initiation site) which might have effects on rhythmic gene expression, were found. Using the yeast one-hybrid and electrophoretic mobility shift assay (EMSA), we confirmed that the bZIP transcription factor HY5 binds to this motif of QH6. Studies with promoter truncations before and after this motif suggested that this G-box was important for the diurnal fluctuation of the transgenic β-glucuronidase gene (GUS) transcript abundance in Arabidopsis thaliana. GUS gene driven by the promoter region immediately after G-box showed an arrhythmic expression in both light/dark (LD) and constant dark (DD) conditions, whereas the control with G-box retained its fluctuation in both LD and DD. We further transformed A. thaliana with the luciferase gene (LUC) driven by an 1400 bp fragment upstream QH6 with its G-box intact or mutated, respectively. The luciferase activity assay showed that a peak in the early morning disappeared in the mutant. Gene expression analysis also demonstrated that the rhythmic expression of LUC was abolished in the hy5-1 mutant.

Highlights

As sessile organisms, higher plants are capable of anticipating daily and annual requirements to coordinate biochemical and physiological activities
QH6 Sequence Analysis According to the alignment of the A. annua QH6 cDNA sequence (Lu et al, 2002) with A. thaliana ocimene/myrcene synthase (At4g16740) cDNA and genomic DNA sequences (Chen et al, 2003), approximate positions for introns and exons of QH6 were predicted (Supplementary Figure S1)
The A. annua β-pinene synthase gene QH6 is among a small number of terpene synthase genes reported to be regulated by the circadian clock at the transcriptional level

Summary

Introduction

Higher plants are capable of anticipating daily and annual requirements to coordinate biochemical and physiological activities This helps plants to optimize their growth and adaptation and may confer selective advantages (Green et al, 2002; Dodd et al, 2005). Kloppstech (1985) showed that mRNA levels of genes encoding light-harvesting chlorophyll a/b protein, Rubisco small subunit protein, and early light-inducible protein (ELIP) varied according to diurnal and circadian rhythms. This was probably the earliest report on plant circadian gene expression. It has been estimated that approximately one-third of Arabidopsis thaliana genes are regulated by the circadian clock at the transcriptional level (Michael and McClung, 2003; Edwards et al, 2006; Michael et al, 2008), for photosynthesis-related processes and for HY5 modulates monoterpene biosynthesis functions such as metabolic adaptation, hormone signaling and photomorphogenesis (Harmer et al, 2000; Covington and Harmer, 2007)

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