Violaxanthin de-epoxidase: Properties of C-terminal deletions on activity, NPQ and pH-dependent lipid binding in tobacco

Abstract

Violaxanthin de-epoxidase (VDE) is localized in the thylakoid lumen and catalyzes the de-epoxidation of violaxanthin to form antheraxanthin and zeaxanthin. VDE is predicted to be a lipocalin protein with a central barrel structure flanked by a cysteine-rich N-terminal domain and a glutamate-rich C-terminal domain. The full-length Arabidopsis VDE cDNA under either the single 35S cauliflower mosaic virus (CaMV) promoter or the double 35S CaMV promoter was used to transformed tobacco plants. Overexpression of VDE under control of the double 35S CaMV promoter increased VDE specific activity in the thylakoid lumen by 18-fold, relative to wild-type. These plants also demonstrated an increase in the initial rate of non-photochemical quenching (NPQ) consistent with increased rates of de-epoxidation. The glutamate-rich C-terminal region of VDE was subjected to analysis using C-terminal deletion mutants to understand the importance of the C-terminal domain in binding to the thylakoid membrane. Transformation of tobacco with two deletion mutants demonstrated that 71 C-terminal amino acids could be removed without affecting activity. In-vitro lipid-micelle binding experiments using these mutants identified a region of 12 amino acids that is potentially part of the membrane-binding domain. These transformed tobacco plants are the first reported example of plants with an increased level of violaxanthin de-epoxidase activity.