Genome-wide identification of the NAC gene family and its expression profiling during fruit development in Chinese cherry

【Objective】The NAC transcription factor family represents one of the largest plant-specific transcription factor families, playing pivotal roles in diverse biological processes including growth, development, response to biotic and abiotic stresses, and fruit quality formation. Chinese cherry (Prunus pseudocerasus L.) is an important native fruit tree species in China, valued for its early ripening, ornamental value, and nutritional fruit. To elucidate the potential functions of NAC genes in Chinese cherry, particularly during fruit development, a systematic genome- wide analysis was conducted. The objectives of this study were to: 1) identify all putative NAC gene family members in the Chinese cherry genome; 2) characterize their basic physicochemical properties, chromosomal distributions, and phylogenetic relationships; 3) analyze their gene structures, conserved protein motifs, and promoter cis-acting elements; 4) investigate the evolutionary mechanisms through collinearity and duplication event analysis; and 5) profile their expression patterns across different cultivars and key fruit developmental stages using transcriptomics and RT-qPCR.【Methods】The study utilized the genome and protein sequences of Chinese cherry Zhuji Duanbing. NAC gene family members were identified through a dual- ap-proach: BlastP analysis using Arabidopsis NAC proteins as queries (E-value ＜ 1e- 5 ), and an HMM search for the NAM domain (PF02365; E-value ＜ 1e- 10). Redundant candidates were merged and removed. Genes were systematically renamed PpseNAC001 to PpseNAC114 according to chromosomal position. Physicochemical parameters were computed with TBtools, subcellular localizations predicted by WoLF PSORT, and chromosomal locations mapped. A phylogenetic tree was constructed from aligned PpseNAC and AtNAC proteins using the neighbor-joining method (JTT+G model; 1000 bootstraps) and classified into subfamilies based on established systems. Gene structures, conserved motifs, and domains were analyzed using genome annotations, MEME, and NCBI CD-Search. Putative cis-elements were identified by scanning 2- kb promoter regions with PlantCARE. MCScanX (TBtools) was employed for intra- genomic collinearity and duplication analysis; interspecific synteny was assessed with Arabidopsis thaliana and Prunus avium genomes. Ka/Ks ratios were calculated for duplicated pairs. For expression analysis, RNA was extracted from three cultivars at three fruit developmental stages (S2, S5, S7) and sequenced (Illumina). Expression levels (FPKM) were log2-transformed for heatmap visualization. Four candidate genes were validated by RT-qPCR using β-actin as the reference and the 2-ΔCt method for quantification.【Results】A total of 114 non-redundant NAC genes were identified and systematically characterized. These genes were unevenly distributed across all eight chromosomes and nine unanchored scaffolds, with chromosomes 2 (25 genes) and 4 (19 genes) being the most gene-rich. The predicted proteins exhibited considerable diversity in physicochemical properties: amino acid length varied from 152 (PpseNAC077) to 680 (PpseNAC098), molecular weight from 17.38 kDa (PpseNAC074) to 77.00 kDa (PpseNAC098), and theoretical pI from 4.55 (PpseNAC114, acidic) to 9.82 (PpseNAC010, basic). Subcellular localization prediction indicated that 96 members (84.2%) were localized to the nucleus, while the others were predicted to reside in chloroplasts, cytoplasm, extracellular space, and other organelles. Phylogenetic analysis classified the 219 NAC proteins (114 PpseNACs + 105 AtNACs) into 18 distinct subfamilies, with the TIP (22 members), OsNAC7 (12 members), ANAC063-B (11 members), and NAC2-A (10 members) subfamilies being the most populous. Sixteen PpseNAC genes formed a distinct clade and were designated as unclassified. Gene structure analysis revealed a range of 1 to 9 CDS per gene, with the majority (88 genes, 77.2%) containing 2 to 4 CDS. Motif analysis identified 10 conserved motifs; Motif 2, 3, and 6 were present in over 88% of all members and constituted the core NAM domain. The number of motifs per protein ranged from 3 to 10, and closely related members within the same phylogenetic clade generally shared similar motif compositions. Promoter analysis revealed a vast number of cis-acting elements. Hormone- responsive elements were abundant. 90 genes were found to have abscisic acid response elements, 78 with methyl jasmonate-responsive elements, and 68 with salicylic acid response elements. Stress-responsive elements were also prevalent, with 60 genes containing low- temperature responsiveness elements and 39 containing defense and stress responsiveness elements. Notably, seven genes (PpseNAC010, PpseNAC049, PpseNAC055, PpseNAC069, PpseNAC077, PpseNAC089, and PpseNAC111) harbored MYB binding sites implicated in flavonoid biosynthesis. Collinearity analysis identified 9 segmental duplication pairs and 16 tandem duplication pairs, indicating both mechanisms contributed to family expansion. Chromosome 2 was a hot spot for tandem duplication, containing 7 of the 16 pairs. The Ka/Ks ratios for all 25 duplicated pairs were significantly less than 1 (ranging from 0.092 to 0.818), strongly suggesting purifying selection has been the dominant evolutionary force. Interspecific synteny analysis identified 70 collinear blocks between Chinese cherry and Arabidopsis, and 100 collinear blocks between Chinese cherry and sweet cherry, indicating a closer evolutionary relationship with the latter. Expression profiling across three cultivars and three fruit developmental stages revealed highly divergent expression patterns. Sixteen genes showed no expression in any sample. Several genes, including PpseNAC044, PpseNAC067, and PpseNAC071, exhibited a consistent and significant upregulation during fruit maturation (from S5 to S7) in all three cultivars. Conversely, PpseNAC076 showed highest expression at the early green fruit stage (S2) and decreased thereafter. Some genes, like PpseNAC010, PpseNAC036, and PpseNAC063, displayed distinct expression trajectories among the different cultivars, suggesting potential roles in cultivar- specific traits. PpseNAC005 was constitutively highly expressed across all stages and cultivars. The RT-qPCR results for the four selected key genes perfectly corroborated the RNA-seq expression trends, validating the reliability of the transcriptome data.【Conclusion】This study provides a comprehensive genomic identification and characterization of the NAC transcription factor family in Chinese cherry, revealing 114 PpseNAC genes. The analysis delineates their chromosomal distribution, phylogenetic relationships, gene structures, protein motifs, and promoter regulatory elements. The expansion of this gene family was driven by both segmental and tandem duplication events, with all duplicated pairs being under strong purifying selection. The prevalence of hormone-, stress-, and development-related cis-elements in their promoters suggests diverse regulatory roles. Expression profiling further uncovered dynamic and sometimes cultivar-specific expression patterns during fruit development, pinpointing several candidate genes, such as PpseNAC044, PpseNAC067, PpseNAC071 (potentially associated with maturation), and PpseNAC076 (potentially associated with early development), for future functional studies. This work establishes a foundational resource for understanding the roles of NAC transcription factors in Chinese cherry biology and offers valuable candidate genes for molecular breeding efforts aimed at improving fruit quality traits.