Identification of PAT gene family members and analysis of their response to different stresses in Chinese chestnut

Abstract：【Objective】The reversibility of protein palmitoylation is a key mechanism for regulating cellular function. Palmitoyl transferase is an important part of this mechanism, which can connect palmitic acid to the cysteine residue of the target protein. Protein palmitoylation is a post-transcriptional modification that plays a critical role in protein transport and function. Palmitate transferase (PAT) catalyzes the occurrence of acylation. Acylated PAT proteins may be involved in a variety of stress responses in material transport and signal transduction. Palmitate transferase plays an important role in growth, development and response to harmful stresses in plants. However, understanding of PAT genes in chestnut is limited so far. In the experiment, we performed a genome-wide identification of the PAT gene family in chestnut and subsequently analyzed how it responded to various stresses in Chinese chestnut.【Methods】HMMER, NCBI-CDD and SMART searches were performed to identify PAT genes throughout the chestnut genome. The domain information of the candidate gene family members was obtained through preliminary screening, and the protein sequences without the DHHC domain were eliminated. Ultimately, 21 members of the chestnut PAT gene family were identified. The bioinformatics methods were used to study the phylogenetic tree, gene structure and gene motif, protein physicochemical properties, subcellular and chromosomal localization, collinearity and promoter cis- elements of the PAT genes. To check the stress-resistant function of PAT gene family, Yanshanhong cultivar was used as experimental material. For salt treatment, the seedling roots were submerged in a solution containing 200 mmol· L- 1NaCl and samples were collected on the 7th, 10th and 14th days. Drought treatment was also carried out. Samples were collected and tested at different water loss in fresh leaves, specifically at 0%, 5%, 30% and 50%. To treat disease resistance, Chestnut Blight Mycelia Blocks were cultured on PAD medium. After 3 days, the PAD agar blocks containing chestnut blight mycelia were placed on perforated branches. Samples were collected at 0 h, 12 h, 24 h and 3 days after inoculation. Fresh branches that were only treated with punching served as controls. All samples were then harvested, immediately frozen in liquid nitrogen, and stored at -80 ℃. The influence of salt, disease resistance and drought stress on the expression pattern of the PAT gene family was examined using the real-time quantitative fluorescence analysis.【Results】Through a comprehensive genome search and identification, we acquired 21 members of the PAT gene family in the chestnut genome that possessed the DHHC domain. In the phylogenetic evolutionary tree, they grouped into six branches with 24 members of the AtPAT gene family. By analyzing the physical and chemical properties of the protein, it was found that most members of the CmPAT family were hydrophilic, basic and stable proteins, and that their sequence lengths varied significantly. Subcellular localization showed that CmPAT7 was localized in the chloroplast, CmPAT4 in the nucleus, CmPAT23 in the cytoplasm, and the remaining PAT proteins in the plasma membrane. Gene structure and motif visualization showed 10 conserved motifs, and 21 PAT family members all had the DHHC domain, which was the most conserved. Their gene lengths also varied widely, ranging from 3750 to 24 300 bp, and each family member contained 7.28 introns and 8.57 exons. Members of the PAT gene family were unevenly distributed across the nine chromosomes. The CmPAT gene family was more conserved in the process of species differentiation. There were many abiotic stress and hormone response elements in the promoter region of the CmPATs gene. These included basic cis- acting elements, light- response elements, hormone- response elements and many stress- response elements, of which cis- acting and light response elements accounted for the largest proportion. An examination of the transcription factor prediction word cloud identified up to 39 transcription factors related to the control of 21 CmPATs gene. Of these, the Dof protein family was the most abundant at 22%, followed by the AP2/ERF protein family and the BBR-BPC protein family at 17% and 10%, respectively. These protein families may play a crucial role in regulating PAT genes in chestnut. Analysis of the expression pattern revealed that several CmPAT genes were involved to varying degrees in responses to disease resistance, and drought and salt stress.【Conclusion】A total of 21 CmPATs gene were identified. The study found that the expression levels of CmPAT24, CmPAT7 and CmPAT14 significantly increased in response to salt and drought stresses, demonstrating their involvement in the regulation of these stressors. Notably, the CmPAT7 gene showed significant upregulation under salt, drought and disease resistance stresses, indicating its role in regulating all three types of stresses.