Regulatory Mechanism Of Grapevine Ethylene Response Factor ERF057 And ERF080 In Cold Resistance

Grapevine (Vitis vinifera L.) is one of important fruit crops in our country. The north producing areas always sustain cold and drought stresses in winter, while the cold resistance of the main cultivars is poor. So these cultivars need be protected by covering with soil, which greatly increases the cost of production and the risk of cultivation. It is reported that ethylene is a gaseous plant hormone that plays important roles in biotic and abiotic stress responses in plants. ERF (ethylene response factor) transcription factors, located in the downstream of ethylene signal pathway, regulate stress related genes and improve the plants stress tolerance. Here micropropagated V. amurensis and V. vinifera cv. Muscat Hamburg plantlets were used in this study. We investigated the relationship between cold stress and ethylene biosynthesis, and the effect of exogenous 1-aminocyclopropane-l-carboxylicacid (ACC, ethylene precursor) and aminoethoxyvinylglycine (AVG, ethylene biosynthesis inhibitor) application on the cold tolerance of grapevine. Two ERF transcription factors, ERF057 and ERF080, were cloned from V. amurensis and V. vinifera cv. Muscat Hamburg. The expression patterns of ERF057 and ERF080 were detected in two grapevine cultivars after different abiotic stress treatments. The function of VaERF057 was analyzed by ectopic expression in Arabidopsis. RNA-Seq was performed with VaERF057-overexpressing Arabidopsis transgenic lines to determine its putative downstream genes. The main research results are as follows:1. The ethylene production, ACC content and ACO activity under cold stress in V. amurensis and V. vinifera cv. Muscat Hamburg were meausred. The results showed that cold stress increased the ACC content and ACO activity, which increased ethylene biosynthesis rate in grapevine. Relative electric conductivity of grapevine leaves treated with ACC and AVG were measured. The semi-lethal temperature (LT50) was calculated to evaluate the cold tolerance of grapevine. The results showed that the application of exogenous ACC increased the cold tolerance of grapevine. By contrast, the application of the ethylene biosynthesis inhibitor AVG reduced the cold tolerance of grapevine. These findings suggested that ethylene positively affected cold stress responses in grapevine.2. According to the reference genomic sequence, two ERF transcription factors, ERF057 and ERF080, were cloned in V. amurensis and V. vinifera cv. Muscat Hamburg. Sequence analysis showed that ERF057 and ERF080 contained a conserved AP2/ERF domain composed by 59 amino acids and belonged to group VII and group IX ERF transcription factor, respectively. They might have the same biological function with their homologues and be involved in biotic and abiotic stress responses.3. The expression patterns of ERF057 and ERF080 after different treatment were analyzed by qRT-PCR. The results showed that the expression of ERF057 and ERF080 upregulated under either cold or ACC treatment in V. amurensis and V. vinifera cv. Muscat Hamburg. However, the effects of cold stress on the expression of ERF057 and ERF080 were totally inhibited when the plants were subjected with AVG under cold condition. Because cold stress promotes the ethylene production in grapevine, so the upregulated expression of ERF057 and ERF080 might be ethylene-dependent.4. VaERF057 and VaERF080 were introduced into Arabidopsis using Agrobacterium-mediated transformation. The results showed that overexpressing VaERF057 and VaERF080 significantly enhanced the cold tolerance of transgenic Arabidopsis. The transgenic Arabidopsis had lower MDA content than wild type and empty vector, while had higher SOD, POD, and CAT activities. So VaERF057 and VaERF080 might decrease the membrane lipid peroxidation and increase the ability of wiping off ROS, and then enhanced the cold tolerance of transgenic Arabidopsis.5. VaERF057 was demonstrated binding activity to GCC-box and DRE motifs by using Y1H system. The results of transcriptome sequencing (RNA-Seq) showed that 89 genes significantly upregulated, and 51 downregulated. Among the 140 significant differentially expressed genes, there were some key genes involved in cold stress signal pathyway, and some downstream genes of ethylene signal pathyway were also included. Promotor analysis showed that 24 genes contain GCC-box and 54 genes contain at least one DRE core motif upstream of their 1 kb promoter region. These results suggested that VaERF057 may regulate the expression of target genes by binding to GCC-box and DRE motifs in their promoter regions and participate in the cold stress response in transgenic Arabidopsis.