Molecular cloning and characterization of the light-regulation and circadian-rhythm of the VDE gene promoter from Zingiber officinale

Abstract

Ginger (Zingiber officinale Rosc.) is prone to photoinhibition under intense sunlight. Excessive light can be dissipated by the xanthophyll cycle, where violaxanthin de-epoxidase (VDE) plays a critical role in protecting the photosynthesis apparatus from the damage of excessive light. We isolated *2.0 kb of ginger VDE (GVDE) gene promoter, which contained the circadian box, I-box, G-box and GT-1 motif. Histochemical staining of Arabidopsis indicated the GVDE promoter was active in almost all organs, especially green tissues. b-glucuronidase (GUS) activity driven by GVDE promoter was repressed rather than activated by high light. GUS activity was altered by hormones, growth regulators and abiotic stresses, which increased with 2,4-dichlorophenoxyacetic acid and decreased with abscisic acid, salicylic acid, zeatin, salt (sodium chloride) and polyethylene glycol. Interestingly, GUS activities with gibberellin or indole-3-acetic acid increased in the short-term (24 h) and decreased in the long-term (48 and 72 h). Analysis of 5 0 flank deletion found two crucial functional regions residing in -679 to -833 and -63 to -210. Northern blotting analysis found transcription to be regulated by the endogenous circadian clock. Finally, we found a region necessary for regulating the circadian rhythm and another for the basic promoter activity. Key message A novel promoter, named GVDE promoter, was first isolated and analyzed in this study. We have determined one region crucial for promoter activity and another responsible for keeping circadian rhythms.

Characterization of cucumber violaxanthin de-epoxidase gene promoter in Arabidopsis

“…Defining the strong green tissue-specific promoter fragment and the functional regulative regions of the CsVDE promoter A previous study showed the GVDE promoter drives a weaker expression of GUS than that driven by the 35S promoter in transgenic plants leaves (18). In this study, the CsVDE promoter showed a strong activity in green tissues in transgenic Arabidopsis.…”

“…This indicated that green tissue-specificity seen in Arabidopsis is governed by sequences located in this most 3' fragment. Studies on the Ginger VDE promoter determined that the regulatory elements involved in green tissue-specific induction were present in a fragment from À386 to À295 respective to the ATG translation initiation site (18). Thus it is obvious that the necessary cis-elements for green tissue-specificity and constitutive GUS expression in CsVDE promoter are much nearer to the ATG translation start codon (from À194 to À1) than in GVDE promoter (from À386 to À295).…”

“…Both of these two promoter fragments contain a TATA box and a CAAT box as the necessary transcript start cis-elements. However the light response elements in these two promoter fragments are different, i.e., a GA-motif and Sp1 motif in the À194/-1 CsVDE promoter fragment, and two I-boxes and two GT-1 boxes in the À386/À295 GVDE promoter fragment (18). A similar GUS expression pattern of these two fragments indicated these cis-elements may act in the same light response regulation, and may contribute to the green-tissue specific expression.…”

“…PEG, Mannitol and NaCl are often used to also noted a similar reduction in the expression of a reporter gene driven by GVDE promoter due to treatment with PEG, NaCl and ABA. This is likely due to ABA functioning as a cellular signaling molecule, mediating the response of the gene to drought and salt stresses (18).…”

“…VDE is located in the thylakoid lumen as a soluble protein, but becomes attached to the thylakoid membrane under acidification of the thylakoid lumen which occurs upon exposure to excess light (13,14). Although much physiological research has been addressed to xanthophyll cycles, and several VDE genes from different plant species have been isolated and studied (15e17), the characterization of the VDE promoter has only been studied in ginger (18). That study on the ginger violaxanthin de-epoxidase (GVDE) promoter mainly focused on the response to high light stress and circadian clock regulation.…”