Identification of histone methylation and demethylation genes and analysis of their responses to different abiotic stresses in kiwifruit

【Objective】Histone methylation and demethylation are the core mechanisms of plant epigenetic regulation. They regulate gene expression by dynamically modifying chromatin structure and play a key role in plant growth and development and responses to abiotic stresses. Histone methyltransferases (HMTs) and demethylases (HDMs) affect the transcriptional activity of stress-responsive genes by modifying lysine/arginine residues in histone tails to form activated or inhibitory chromatin markers. Under abiotic stress (drought, salinity, extreme temperature, etc.), plants reshape chromatin accessibility by dynamically regulating histone modification levels, and activate antioxidant enzymes, osmotic regulation and other pathways to maintain cell homeostasis. Kiwifruit (Actinidia chinensis) is an important economic fruit tree, and its fruit quality and postharvest storability are susceptible to abiotic stress. However, the function of histone modification gene family in the response of kiwifruit to abiotic stress remains unclear. In this study, we performed genome-wide identification of the HMT and HDM gene families in kiwifruit, and then analyzed their responses to various abiotic stresses in kiwifruit.【Methods】The HMTs and HDM genes in the whole kiwifruit genome were identified by HMMER, NCBI, Pfam and kiwifruit genome database search. The conserved domain sequences of kiwifruit were screened by Hmmer search program, and the sequences with e- value less than 10-20 were screened. The selected se-quences were further used to construct a hidden Markov model of the characteristics of the kiwifruit gene family using the Hmmer build program. The final results were obtained by screening the whole genome with the newly constructed kiwifruit's own feature HMM. At the same time, using blast search on NCBI, the database was limited to A. chinensis var. chinensis (taxid: 1590841). The obtained sequences were integrated with HMMER screening results and manually screened to remove redundant sequences. Finally, 83 members of kiwifruit HMT and HDM gene families were identified. The gene structure and motif, protein physicochemical properties, chromosome localization, phylogenetic tree and collinearity of the gene family were studied by bioinformatics methods. In order to verify the involvement of the HMTs and HDMs in stress resistance, the Yuxiang variety was used as the experimental material. Fourweek-old tissue cultured seedlings with good growth were placed in 100 mmol·L-1 PEG4000, 50 mmol·L-1 NaCl, at 38 ℃ or 4 ℃, and sampled at 0 h, 6 h, 12 h and 24 h. The leaves with the similar size and state were frozen in liquid nitrogen and stored in a refrigerator at -80 ℃. The effects of salt, drought, low temperature and high temperature stresses on the expression patterns of HMTs and HDMs were analyzed by real-time fluorescence quantitative technique.【Results】A total of 83 members of the kiwifruit HMT and HDM gene families were identified. Among them, HMTs contained 54 SDGs (containing SET domain) and 1 PRMT, and HDMs contained 8 HDMAs and 20 JMJs (both containing JMJ-C domain). Protein domain analysis showed that the Ⅵ/Ⅶ structure in the SDG subfamily was simple (only containing the SET domain), while the Ⅱ/Ⅳ structure was complex (containing DNA binding domains such as zf- HC5HC2H). KDM4 in the JMJ subfamily contains multiple domains (JMJ-N, JMJ-C, zfC5HC2, etc.), and the structure of KDM3 and KDM5 is relatively simple (zf-CXXC, WRC, etc). Phylogenetic analysis showed that kiwifruit SDG belonged to 7 subfamilies (the highest proportion of V subfamily), and JMJ belonged to 5 subfamilies (the number of JMJD6 / JMJC was very small), which was closely related to the evolution of homologous genes in Arabidopsis thaliana. Specifically, the SDG-V subfamily shares homology with Arabidopsis SuvH genes (regulating heterochromatin formation), while members of the JMJ-KDM5 subfamily (AtJMJ14/15/16) are closely associated with plant stress resistance, participating in systemic resistance establishment, enhanced salt tolerance, and inhibition of leaf senescence, respectively. Collinearity analysis revealed that gene expansion was mainly due to genome-wide replication (68.19% of genome-wide collinearity genes, 30.56% of HMT and HDM collinearity genes), and only 2 cases of tandem duplication events (AcSDG16/AcSDG17 and AcHDMA5/AcHDMA6). Through systematic analysis of the expression patterns of histone methyltransferase (HMTs) and demethylase (HDMs) gene families (including SDGs, HDMAs, and JMJs) in kiwifruit under abiotic stress, it was found that these genes exhibit highly stress-specific regulatory dynamics. Specifically, under salt stress, most genes (AcSDG46, AcHDMA1, etc) exhibited a“rise-fall-rise”cyclic oscillation pattern, reflecting feedback regulation mechanisms. Under drought stress, some genes (AcSDG7, AcSDG9, etc) exhibited a sustained expression pattern forming a basal defense network, while others were upregulated at specific stages [AcSDG4 in stage S2 (6 hours), AcSDG23 in stage S3 (12 hours)]. Cold stress triggered synergistic expression of genes in stage S3 (6 hours), followed by downregulation to facilitate growth recovery, suggesting coordinated activation of early defenses. High-temperature stress induced overall downregulation in the early phase followed by upregulation in the late phase, except for AcSDG29, which exhibited unique preactivation in the early phase, suggesting its role in proactive defense. These findings indicate that HMTs and HDMs, through their spatiotemporally specific expression dynamics, may mediate rapid responses and adaptive remodeling in plants under stresses, providing key targets for elucidating crop stress resistance mechanisms.【Conclusion】A total of 83 HMTs and HDMs were identified. Protein domain analysis showed that the functional differentiation of the subfamily was significant. Synteny analysis showed that gene expansion was mainly due to genome-wide replication. Notably, the histone methyltransferase genes AcSDG35 and AcSDG54 showed significant upregulation under salt stress, drought stress, low-temperature stress, and late- stage high-temperature stress conditions, indicating their crucial role in regulating responses to these four types of stress. Furthermore, only AcSDG29 exhibited upregulation during the early stage of high-temperature stress, suggesting its potential involvement in early active defense regulation.