Characterization of Chromatin Accessibility and Gene Expression upon Cold Stress Reveals that the RAV1 Transcription Factor Functions in Cold Response inVitis Amurensis

Abstract

Cold tolerance is regulated by a variety of transcription factors (TFs) and their target genes. Except for the well-characterized C-repeat binding factors (CBFs)-dependent transcriptional cascade, the mechanisms of cold tolerance mediated by other transcriptional regulatory networks are still largely unknown. Here we used the assay for transposase-accessible chromatin with sequencing (ATAC-seq) and RNA-seq to identify cold responsive TFs in Vitis amurensis, a grape species with high cold hardiness. A number of 9 TFs, including CBF4, RAV1 and ERF104, were identified after cold treatment. Weighted gene co-expression network analysis (WGCNA) and gene ontology (GO) analysis revealed that these TFs may regulate cold response through different pathways. As a prime candidate TF, overexpression of VaRAV1 in grape cells improved its cold tolerance. The transgenic cells exhibited low electrolyte leakage and malondialdehyde (MDA) content and high peroxidase (POD) activity. Moreover, the TF gene TCP8 and a gene involving in homogalacturonan biosynthesis were found to be regulated by VaRAV1, suggesting that the contribution of VaRAV1 to cold tolerance may be achieved by enhancing stability of cell membrane and regulating the expression of target genes involved in plant cell wall composition. Our work provides novel insights into plant response to cold stress and demonstrates the utility of ATAC-seq and RNA-seq for rapid identification of TFs in response to cold stress in grapevine. The VaRAV1 may play an important role in adaption to cold stress.

Overexpression of grape ABA receptor gene VaPYL4 enhances tolerance to multiple abiotic stresses in Arabidopsis

“…In an attempt to identify cold-responsive PYL genes in grapevine, we analyzed gene expression of the 9 members of PYL family (Fig. 1 a) in V. amurensis based on our previous transcriptome data [ 33 ]. Among the 9 PYL genes, VaPYL1 , VaPYL4 , VaPYL5 and VaPYL13 were strongly induced by cold, whereas the expression of VaPYL3 was significantly decreased in response to cold stress (Fig.…”

“…4 c). Low temperature exotherms (LTEs) assay is usually used to evaluate cold tolerance of plant tissues or calli/cells [ 33 , 35 , 36 ]. Thus, the cold tolerance of KO-1 and KO-2, as well as the three OE lines, was evaluated by measuring LTEs using a differential thermal analysis system according to Sun et al [ 36 ].…”

“…The derived in vitro plants of V. amurensis and V. vinifera were cultured in an artificial climate chamber with a 16-h light/8-h dark photoperiod at 25 ± 1℃. Two-month-old V. amurensis plants were used for cold treatment, which was conducted as previously described with some modifications [ 33 ]. The grapevine plants with uniform growth status were divided into two groups: one group of plants were transferred to the incubator set to 4℃ for cold treatment and the other kept at 25℃ were used as the control.…”

“…The fluorescence was detected 3 days after infiltration using Leica TCS SP8 confocal laser scanning microscopy. The histone H2B-mCherry was used as an indicator of nucleus as previously described [ 33 ].…”

“…The reactions were carried out as described by Ren et al [ 56 ]. Grape Actin 1 and GAPDH [ 33 ] and Arabidopsis Actin 2/8 were used as internal controls. Gene expression relative to internal controls was determined using 2 −∆∆CT method [ 57 ].…”