Scientia Agricultura Sinica ›› 2023, Vol. 56 ›› Issue (7): 1377-1390.doi: 10.3864/j.issn.0578-1752.2023.07.014

• HORTICULTURE • Previous Articles     Next Articles

Identification of Salt-Tolerant Transcription Factors in the Roots of Pyrus betulaefolia by the Association Analysis of Genome-Wide DNA Methylation and Transcriptome

LI Hui1(), ZHANG YuFeng1,2, LI XiaoGang1, WANG ZhongHua1, LIN Jing1, CHANG YouHong1   

  1. 1 Institute of Pomology, Jiangsu Academy of Agricultural Sciences/Jiangsu Key Laboratory for Horticultural Crop Genetic Improvement, Nanjing 210014
    2 College of Biology and the Environment, Nanjing Forestry University, Nanjing 210037
  • Received:2022-05-05 Accepted:2023-02-01 Online:2023-04-01 Published:2023-04-03

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

Table 1

Primer sequences of qPCR and McrBC-qPCR in this study"

基因名称
Gene name
基因ID
Gene ID
正向引物
Forward primer (5′-3′)
反向引物
Reverse primer (5′-3′)
用途
Destination
PbDREB1 GWHGAAYT038287 CGACGAGGAGGTCATTTTGG TGTTCGGTTCTCTCATTTCGC qPCR
TCCCTAAGGACACTATGCGT AGAGCAAGATATTTCCATAAAAGAGCA McrBC-qPCR
PbERF1 GWHGAAYT040185 GAAATGCTTCTCTATGGAGTTCTTG TCTCTTATCTCCGCCGCAA qPCR
AGATCTAGTGATATTCCCTT TCGACA TCACAAATCCAAGCCAAAACCA McrBC-qPCR
PbERF2 GWHGAAYT045012 CTTCAGTACCCTGTACCCGTGT TTTACCGAAAAACCGTAGGAAC qPCR
TCTGAGTCGCCGAGATGGTA ATCGGGGATCATATTAGGGGT McrBC-qPCR
PbERF19 GWHGAAYT023975 GCAGCACAGCAAACAGAAAAC ACGCAAGCAATAATACGCAAC qPCR
ACCTCGTGTTTTTACTGTGGA TCAACCAAAAATAACAAGAAGCACA McrBC-qPCR
PbERF53 GWHGAAYT056118 TCTCAAGAAAGTCAAGAGCCCTG GTTGAACGGGTGGGGGAAT qPCR
GCTCAATGGCCTCCTAAACCT TCGCTCTGAGCAAGAATGAAGT McrBC-qPCR
PbGT3 GWHGAAYT011887 GAAGGAAAAGGGTCACCATCG CGCCGTGCTTCCCTTATTC qPCR
GCACCTTTAGATGTAAAAGTTCATGC TGGACTCCAAGCCTTTTAATTGC McrBC-qPCR
PbNAC29 GWHGAAYT016435 TAGCCTCCACTCCCTTACCTG TCGCACCATTCGGATACTTG qPCR
AGGCCATAAATTCGGACATTTGC AAGCACAAGCAAAAGCTCTCG McrBC-qPCR
PbNAC72 GWHGAAYT009070 GAGCCCTCTCGCAAAAATG CAGCGTCTTGAGCGAGTTTAT qPCR
AGGCTGGTATTTTGTCAAAAGATGG TCTAGATTGTAACATACTTTGGAGTGC McrBC-qPCR
PbSCL33 GWHGAAYT007070 GAAACTGTGGATTTGCGGAA CATCACCGTTAGGAGAAGCGT qPCR
GTCCCACTCTGTCCTTTGCT AGTTGAAGGACTATAACTGTACGT McrBC-qPCR
PbZAT10.1 GWHGAAYT041697 CGTTTGAAGAGGCGGAGGA GATGAGGCAGAGAGCGAGGT qPCR
TGCGGATTCGATACTTACAATCTCT GCCACATGAGATGTTTAACGCA McrBC-qPCR
PbZAT10.2 GWHGAAYT020116 GGACCCTCTCCTTTCCCATT CGGAGTTTGTTTCTTTAGTGTCTGT qPCR
GGATCAATACGACGTCGGCT ACTGTGCATTTAATTTTATACGCAAT McrBC-qPCR
PbZFP3 GWHGAAYT025897 TATTCGGGTTTTCGGTGACG GTACACTTGTCGCTACCCGTTG qPCR
AGCGTTGATAAACGTACTCATTTCT TTGACTTGGCCTTTTAGAATTAGG McrBC-qPCR

Table 2

Contents of sodium ions in different ecotypes of Pyrus betulaefolia after salt stress"

株系
Ecotype
组别
Group
全株钠含量
The sodium content
of whole plants (g·kg-1)
根系中钠占全株的比例
Ratio of root sodium to whole plant (%)
耐盐
Salt-tolerant
对照CK 7.45±0.64c 65.06a
NaCl 25.38±2.22b 47.09c
普通
Common
对照CK 7.05±0.53c 62.55b
NaCl 31.85±2.13a 35.79d

Fig. 1

Types and proportions of transcription factors detected in Pyrus betulaefolia root"

Fig. 2

The heatmaps of transcription factors expression among different roots of Pyrus betulaefolia ecotypes in response to salt stress DR: The root of salt-tolerant ecotype under normal conditions; DNR: The roots of salt-tolerant ecotype under 200 mmol·L-1 NaCl for 24 h; UR: The root of common ecotype under normal conditions; UNR: The roots of common ecotype under 200 mmol·L-1 NaCl for 24 h. The same as below"

Fig. 3

Distribution of methylation levels (MLs) in different regions of transcription factors in roots of Pyrus betulaefolia"

Fig. 4

The number of differentially methylated regions in transcription factors among different roots of Pyrus betulaefolia ecotypes in response to salt stress"

Fig. 5

The heatmaps of differentially methylated regions in transcription factors among different roots of Pyrus betulaefolia ecotypes in response to salt stress"

Table 3

Combined analysis of the number of differential transcription factors among different roots of Pyrus betulaefolia ecotypes in response to salt stress"

组别
Group
甲基化类型
Methylation type
甲基化水平上升 Hyper methylation 甲基化水平下降 Hypo methylation
基因区*
Gene region
启动子区**
Promoter region
基因区*
Gene region
启动子区**
Promoter region
耐盐株系对照根vs
耐盐株系盐胁迫根
DR vs DNR
mC 0 (0) 7 (7) 0 (0) 13 (13)
mCG 3 (3) 1 (1) 3 (3) 1 (1)
mCHG 1 (1) 2 (2) 5 (3) 6 (6)
mCHH 26 (25) 113 (104) 35 (32) 157 (150)
普通株系对照根vs
普通株系盐胁迫根
UR vs UNR
mC 7 (7) 16 (16) 2 (2) 13 (13)
mCG 2 (2) 1 (1) 2 (2) 0 (0)
mCHG 2 (2) 1 (1) 2 (2) 0 (0)
mCHH 47 (44) 144 (131) 20 (19) 96 (93)

Fig. 6

Expression analysis of DMR-associated transcription factors genes among different roots of Pyrus betulaefolia ecotypes in response to salt stress"

Fig. 7

DNA methylation levels of DMR-associated transcription factors among different roots of Pyrus betulaefolia ecotypes in response to salt stress and their differential methylation regions verification"

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