Genome-wide identification and low temperature expression analysis of the XTH gene family in Chongyi wild mandarin (Citrus reticulata)

【Objective】Chongyi wild mandarin (Citrus reticulata) was discovered in 1977 in the primeval forests of Niedu Township, Chongyi County, Ganzhou City, Jiangxi Province, China. Remarkably, it is more cold-tolerant than Poncirus trifoliata, a widely used cold-hardy citrus rootstock. Xyloglucan endotransglucosylase/hydrolase (XTH) can catalyze the breakage and reconnection of the xyloglucan-cellulose cross-linking network, functioning as an indirect loosening agent controlling cell wall remodeling. It catalyzes the cutting and reconnection of xyloglucan, thereby achieving dynamic reconstruction of the cell wall and regulating cell wall relaxation and reinforcement. However, current knowledge on XTH genes in Chongyi wild mandarin remains extremely limited. In this study, we systematically identified the CrXTH gene family members, analyzed their sequence characteristics, and examined their expression patterns in response to low temperature treatment.【Methods】Based on the whole-genome data of Chongyi wild mandarin and the hidden Markov models (HMMs) for XTH conserved domains (PF00722 and PF06955), CrXTH gene family members were identified using BLAST, HMMER3.0, and SMART. TBtools was employed to extract genomic features of CrXTH family members, including ami-no acid length, chromosomal distribution, gene IDs, and collinearity. Protein molecular weight (MW) and isoelectric point (pI) were predicted using ExPASy. Plant-mPLoc was used for predicting protein localization and SignalP 5.0 was applied to identify potential signal peptides. MEGA11.0 and chiplot were used for multiple sequence alignment of XTH proteins from different species and phylogenetic tree visualization. Gene structure, conserved motif identification and promoter cis-acting element prediction were conducted by GSDS 2.0, MEME Suite 5.5.7, and PlantCARE, respectively. To analyze the expression patterns of CrXTHs in response to low temperature, one-year-old seedlings of Chongyi wild mandarin were subjected to cold stress at 0 ℃, and leaves were sampled after 0 d, 1 d, and 3 d for RNA extraction and subsequent real- time quantitative PCR analysis. The 1500 bp promoter fragments of three CrXTH genes were fused to the GUS reporter gene to assess their responsiveness to low temperature treatment.【Results】A total of 27 XTH family genes (CrXTH1-CrXTH27) were identified in the Chongyi wild mandarin genome. All CrXTHs gene family members contained two conserved domains: Glyco_hydro_16 and XET_C. The protein lengths of CrXTHs ranged from 267 to 356 amino acids, with molecular weights spanning 30.66 kDa (CrXTH12) to 40.46 kDa (CrXTH6). The isoelectric points (pI) varied from 4.91 (CrXTH9) to 9.2 (CrXTH27), with 57% of proteins being basic (pI＞7.0). Subcellular localization predicted that CrXTHs were primarily located in the cell wall, with 14 members also detected in the cytoplasm. Signal peptide prediction analysis revealed that CrXTH5, CrXTH16, and CrXTH26 exhibited Sec/SPI scores below the threshold of 0.5, suggesting these members likely lack typical signal peptides. In contrast, all other CrXTH proteins contained high-confidence Sec signal peptides. Based on phylogenetic analysis of Chongyi wild mandarin, Arabidopsis thaliana, and Oryza sativa, the XTH gene family was classified into four distinct clades: Group Ⅰ/Ⅱ, Group Ⅲ-A, Group Ⅲ-B, and the Ancestral group. Among the Chongyi wild mandarin XTH members, GroupⅠ/Ⅱ represented the largest proportion (19 members, 70.37% ), followed by Group Ⅲ-B (4 members, 14.81% ), with both Group Ⅲ-A and the Ancestral group containing 2 members each (7.41% respectively). Chromosomal distribution showed an uneven distribution across nine chromosomes (Chr) of Chongyi wild mandarin, with Chr4 harboring the most genes (13) and Chr1 and Chr9 containing only one each. Collinearity analysis identified four gene pairs between Chongyi wild mandarin and rice, and 25 pairs between Chongyi wild mandarin and Arabidopsis thaliana, indicating closer evolutionary relationships with dicots. Cisacting element analysis of CrXTHs highlighted abundant light responsive elements, followed by five types of hormone- related elements including 77 abscisic acid response elements, 72 methyl jasmonate response elements, 30 gibberellin response elements, nine auxin response elements, and seven salicylic acid response elements. The main abiotic stress response elements were anaerobic induction, drought, and low temperature. Regulatory growth and development response elements mainly included meristem expression, metabolic regulation, and seed-specificity. These results suggested that CrXTHs might play important roles in photoregulation, hormone signaling, abiotic stress responses, and developmental regulation. Under cold stress, CrXTHs exhibited four types of expression patterns: (1) Sustained induction type, including 14 genes (CrXTH1, CrXTH3, CrXTH6, CrXTH8, CrXTH9, CrXTH10, CrXTH11, CrXTH12, CrXTH14, CrXTH15, CrXTH18, CrXTH21, CrXTH23, and CrXTH27), which exhibited 4- to 57-fold upregulation after 1 d and 3 d of cold stress; (2) Late-phase upregulation type, including 9 genes (CrXTH2, CrXTH5, CrXTH7, CrXTH13, CrXTH17, CrXTH19, CrXTH20, CrXTH24, and CrXTH25), whose expression showed no significant change or even a decline at 1 d but increased significantly (P＜ 0.05) by 3 d; (3) Suppression type, including CrXTH4 and CrXTH26, whose expression decreased by 3- to 10-fold compared to the control under cold stress; (4) Non-responsive type, including CrXTH16 and CrXTH22, which displayed no significant expression changes at either 1 d or 3 d. The qRT-PCR results indicated that most CrXTHs were involved in cold response of Chongyi wild mandarin. The GUS staining and quantitative analysis showed that the promoter activities of CrXTH10, CrXTH11, and CrXTH12 were significantly induced under low temperature conditions compared to the control, consistent with the previously observed upregulation of their corresponding gene transcripts.【Conclusion】Twenty-seven CrXTH genes were identified in Chongyi wild mandarin, demonstrating uneven chromosomal distribution and evolutionary conservation with dicotyledonous plants. The promoter regions of CrXTHs contained abundant light-responsive elements and stress-related hormone response cis-acting elements, particularly for ABA and MeJA. Under cold conditions, 92.6% of CrXTHs showed significant upregulation, indicating their crucial role in cold response of citrus.