关键词: 杜梨, 盐胁迫, 转录因子, DNA甲基化, 转录组

Abstract:

【Objective】 Here, two ecotypes of P. betulaefolia from Huaguo Mountain, Lianyungang (the salt-tolerant ecotype, D) and Purple Mountain, Nanjing (the common ecotype, U) were collected for this research. The purpose of this study was to analyze the role of transcription factor genes in the roots of two ecotypes of P. betulaefolia differing in terms of salt stress. Transcription factors involving in the regulation of the salt tolerance of different P. betulaefolia ecotypes were identified on the grounds of differential expression under salt stress and the relationship between the methylation status and the relative expression level of relevant tolerance genes after exposure to salt stress was investigated. 【Method】 The 90-day-old P. betulaefolia seedlings were grown hydroponically in Hoagland’s nutrient solution supplemented with 200 mmol∙L^-1 NaCl, with seedlings grown in Hoagland’s nutrient solution as the control. The sodium ion content in the tissues was determined by flame graphite furnace atomic absorption spectrometry. Whole-genome DNA methylation analysis and transcriptome sequencing were performed on three replicates for the following four root samples: ecotype D and ecotype U, each grown in the presence or absence of salt stress. Bioinformatics analysis of transcription factor gene expression under salt stress at the levels of transcriptional regulation and epigenetic methylation were carried out using transcriptome sequencing data and whole-genome DNA methylation results, respectively. Then, McrBC-PCR and real-time fluorescence quantitative PCR (qPCR) were used to confirm the levels of methylation and transcription of differential transcription factor genes. 【Result】 After exogenous NaCl treatment for 24 h, the concentration of sodium ions in P. betulaefolia roots increased significantly, with the increase in sodium ion concentration in the salt-tolerant ecotype being significantly less than that in the common ecotype. In the whole seedling, the final salt concentration of tolerant ecotype was only 73.1% of that of the common ecotype. Whereas, in the roots, the sodium content of the salt-tolerant ecotype was 1.1 times of that in the common ecotype. These results indicated that the salt-tolerant ecotype could store more sodium ions in roots and limit their upward transport after salt stress. A total of 2 682 transcription factor (TF) genes from 69 gene families were detected in roots. Among them, 243 TF genes displayed differential expression in response to salt stress, including 37 AP2/ERF, 19 bHLH, 7 bZIP, 10 HD-Zip, 30 MYB, 18 NAC, 8 WRKY, and 23 ZFP family genes. The global methylation level of transcription factor genes in the genome of the salt-tolerant rootstock ecotype decreased, whereas the overall methylation level of these genes in the common ecotype increased after exposure to 200 mmol∙L^-1 NaCl. The differentially methylated regions in both ecotypes were mainly in the position of gene promoters, with the type of differentially methylated sequences being mostly mCHH, constituting more than 93% of the sum of all three types of methylated sequences. The expression levels of twenty-three transcription factor genes, which belonged to the AP2/ERF, bHLH, DREB, GRAS, GT factor, HB Zip, MYB, NAC, Trihelix, and zinc-finger ZFP gene families, were upregulated, and their methylation levels were downregulated in both two ecotypes in response to salt stress. These genes may be involved in the regulation of sodium uptake and accumulation in roots under salt stress. The expression patterns and promoter methylation of representative candidate genes identified by bioinformatics analysis were confirmed by qPCR and McrBC-qPCR.【Conclusion】 The differentially expressed genes in roots of P. betulaefolia under salt stress included 243 transcription factor genes in both ecotypes. The methylation changes in DNA sequences in eight transcription factor genes (PbERF2, PbGT3, PbZAT10.1, PbSCL33, PbDREB1, PbZAT10.2, PbERF53, and PbNAC72) were correlated with their transcriptional activity. Our results provided preliminary experimental evidence for supporting a relationship between promoter DNA methylation and expression of TF genes in P. betulaefolia in response to salt stress as part of the molecular role of TFs involved in the regulation of salt tolerance among different P. betulaefolia ecotypes, which would increase our understanding of the role of epigenetics in the response of woody trees to abiotic stress.

Key words: Pyrus betulaefolia, salt stress, transcription factors, DNA methylation, transcriptome