The grapevine basic helix-loop-helix (bHLH) transcription factor positively modulates CBF-pathway and confers tolerance to cold-stress in Arabidopsis

Abstract

Basic helix-loop-helix (bHLH)-type transcription factors play diverse roles in plant physiological response and stress-adaptive regulation network. Here, we identified one grapevine bHLH transcription factor from a cold-tolerant accession 'Heilongjiang seedling' of Chinese wild Vitis amurensis (VabHLH1) as a transcriptional activator involved in cold stress. We also compared with its counterpart from a cold-sensitive Vitis vinifera cv. Cabernet Sauvignon (VvbHLH1). These two putative proteins are characterized by the presence of the identically conserved regions of 54 amino acid residues of bHLH signature domain, and shared 99.1% amino acid identity, whereas several stress-related cis-regulatory elements located in both promoter regions differed in types and positions. Expressions of two bHLHs in grapevine leaves were induced by cold stress, but evidently differ between two grapevine genotypes upon cold exposure. Two grapevine bHLH proteins were exclusively localized to the nucleus and exhibited strong transcriptional activation activities in yeast cells. Overexpression of either VabHLH1 or VvbHLH1 transcription factor did not affect the growth and development of transgenic Arabidopsis plants, but enhanced tolerance to cold stress. The improved tolerance in VabHLH1- or VvbHLH1-overexpressing Arabidopsis plants is associated with multiple physiological and biochemical changes that occurred during the time-course cold stress. These most common changes include the evaluated levels of proline, decreased amounts of malondialdehyde and reduced membrane injury as reflected by electrolyte leakage. VabHLH1 and VvbHLH1 displayed overlapping, but not identical, roles in activating the corresponding CBF cold signaling pathway, especially in regulating the expression of CBF3 and RD29A. Our findings demonstrated that two grapevine bHLHs act as positive regulators of the cold stress response, modulating the level of COR gene expression, which in turn confer tolerance to cold stress.