Enhanced expression of EsWAX1 improves drought tolerance with increased accumulation of cuticular wax and ascorbic acid in transgenic Arabidopsis

Drought can activate several stress responses in plants, such as stomatal closure, accumulation of cuticular wax and ascorbic acid (AsA), which have been correlated with improvement of drought tolerance. In this study, a novel MYB gene, designed as EsWAX1, was isolated and characterized from Eutrema salsugineum. EsWAX1 contained a full-length open reading frame (ORF) of 1068 bp, which encoding 355 amino acids. Transcript levels of EsWAX1 were quickly inducible by drought stress and ABA treatment, indicating that EsWAX1 may act as a positive regulator in response to drought stress. Ectopic expression of EsWAX1 increased accumulation of cuticular wax via modulating the expression of several wax-related genes, such as CER1, KCS2 and KCR1. Scanning electron microscopy further revealed higher densities of wax crystalline structures on the adaxial surfaces of leaves in transgenic Arabidopsis plants. In addition, the expression of several AsA biosynthetic genes (VTC1, GLDH and MIOX4) was significantly up-regulated in EsWAX1-overexpressing lines and these transgenic plants have approximately 23-27% more total AsA content than WT plants. However, the high-level expression of EsWAX1 severely disrupted plant normal growth and development. To reduce negative effects of EsWAX1 over-expression on plant growth, we generated transgenic Arabidopsis plants expressing EsWAX1 driven by the stress-inducible RD29A promoter. Our data indicated the RD29A::EsWAX1 transgenic plants had greater tolerance to drought stress than wild-type plants. Taken together, the EsWAX1 gene is a potential regulator that may be utilized to improve plant drought tolerance by genetic manipulation.