Genome-wide identification and expression analysis of apple PME family during the course of fruit development

【Objective】Pectin methyl esterase (PME) is a key enzyme regulating pectin hydrolysis and metabolism during plant growth and fruit quality formation. In this study, the apple PME gene family was identified through bioinformatics methods, and its expression patterns at different developmental stages of apple fruit were analyzed. These findings would lay a foundation for further research on PME genes.【Methods】Using the 66 Arabidopsis PME protein sequences reported as query sequences, the conserved structural domains of PME proteins, PME domain (PF01095) and PMEI domain (PF04043), were obtained from the Pfam database. The TBtools software was employed to search for members of the apple PME gene family in the apple genome database (GDDH13 v 1.1) using both BLAST and HMM model methods. Using the Protein Parameter Calc tool in TBtools-Ⅱ, the physicochemical properties of apple PME gene family members were analysed. Their gene structures were analysed using software such as NCBI-CDD and SMART. Based on the apple genome gff3 annotation file, the gene structures were visualised using the Gene Structure View (Advanced) tool in TBtools-Ⅱ. The subcellular localisation of apple PME proteins was predicted using Plant- mPLoc. Using MEGA- X software, amino acid sequence alignment was performed on 157 full-length PME protein sequences from apple and other species such as Arabidopsis, tomato, and pear. A phylogenetic tree was constructed using the maximum likelihood (ML) method, with 1000 bootstrap repetitions. The chromosomal locations of apple PME genes were visualised using the Gene Location Visualise from GTF/GFF feature in TBtools-Ⅱ software. The intraspecific collinearity relationships of apple PME genes were analysed using the OneStep MCScanX function in TBtools-Ⅱ software. A 2000 bp region upstream of the translation start site was extracted from apple genome data, and cis-acting elements in the promoter were identified using the Plant-CARE database. Transcriptome data and qRT- PCR were used to analyse the expression patterns of MdPME genes in four different developmental stages of Honeycrisp fruit (young fruit period, expansion period, maturity period, and store at 4 ℃ for 60 days after harvesting). The STRING website was used to predict interacting proteins for the candidate proteins, with corresponding homologous proteins from Arabidopsis as references.【Results】A total of 82 PME gene family members were identified in the apple genome data, unevenly distributed across 15 chromosomes. All members contained the PME conserved domain and encoded 156- 731 amino acids, with protein molecular weights ranging from 17.66 to 82.23 kDa and isoelectric points between 5.02 to 9.79. Most proteins were alkaline and exhibited hydrophilic and good stable properties. The largest proportion of their secondary structure consisted of random coils, and subcellular localisation predictions indicated they were located on the cell wall. The phylogenetic tree analysis divided MdPMEs into four subfamilies, with Group 1 having the most members. The proteins within the same subfamily branch exhibited similar conserved motif arrangements, and most family members contained 2-3 exons. MEME identified 10 conserved motifs in MdPME protein sequences, each with a length of 15-38 amino acids, with the most members containing Motif 1. The intraspecific synteny analysis indicated that whole- genome duplications (WGD) accounted for the highest proportion of the total duplication events at 53.66%, making them the primary driver of expansion in the apple PME gene family. The promoter sequence analysis revealed that the promoter region of the MdPME genes contain 17 cis- acting elements related to growth and development, hormone signalling, and stress response, among them light- responsive elements were the most abundant, accounting for 45.24% of the total cis- acting elements, followed by hormone- regulated responsive elements, including methyl jasmonate, gibberellin, abscisic acid, salicylic acid, and auxin responsive elements. The transcriptome sequencing and qRT- PCR analysis identified 16 MdPME genes expressed in Honeycrisp fruit, and six key candidate genes involved in quality formation during fruit development were screened. Among these, the MdPME40 and MdPME46 exhibited the highest expression levels during fruit development, the MdPME13 and MdPME15 showed a trend of initially increasing and then decreasing during fruit development, with high expression during the fruit ripening stage, while the MdPME42 was upregulated after low-temperature storage of the fruit. The protein interaction network analysis indicated that the candidate genes would interact with other pectin- modifying enzymes, especially pectinase, and jointly participate in the modification or degradation of pectin.【Conclusion】This study systematically identified and analyzed the apple PME gene family at the genomewide level. Six candidate genes related to fruit development were screened out, providing key targets for further analysis of the molecular functions of PME genes in apple fruit ripening and quality formation.