Molecular cloning and characterization of violaxanthin de-epoxidase (VDE) in Zingiber officinale

Abstract

Ginger ( Zingiber officinale Rosc.), an important horticultural crop in tropical Southeast Asia, is prone to photoinhibition under intense sunlight and grows well at low light intensity. Violaxanthin de-epoxidase (VDE) as the key enzyme of xanthophyll cycle plays an important role in protecting photosynthesis apparatus from the damage of excessive light. In this study, a full length (2000 bp) cDNA encoding violaxanthin de-epoxidase ( GVDE) (GenBank accession no. AY876286) was cloned from ginger using RT-PCR and 5′, 3′ rapid amplification of cDNA ends (RACE). The expression patterns of GVDE in response to light were characterized. GVDE has a 1431 bp open reading frame and the predicted polypeptide contains 476 amino acids with the molecular mass of 53.7 kDa. Northern blot analysis showed that the GVDE was mainly expressed in leaves. GVDE mRNA level increased as the illumination time prolonged under high light. For determining the GVDE function, its antisense sequence was inserted into tobacco plants via EHA105. PCR-Southern blot analysis confirmed the integration of antisense GVDE in the tobacco genome. Chlorophyll fluorescence measurements showed that, transgenic plants had lower values of non-photochemical quenching (NPQ) and the maximum efficiency of PSII photochemistry (Fv/Fm) compared with the untransformed controls under high light. The size of xanthophyll cycle pigment pool (V + A + Z) and the ratio of (A + Z)/(V + A + Z) were lower in T-VDE tobacco plants than in control, indicating that GVDE was suppressed in antisense T-VDE tobacco. These results showed that VDE plays a major role in alleviating photoinhibition.