Genome-wide identification and expression patterns of AP2 subfamily transcription factor in plums

【Objective】The AP2 subfamily transcription factor represents one of the most important transcription factor families in plants, playing pivotal roles in regulating various biological processes including floral organ morphogenesis, embryogenesis, meristem differentiation, leaf morphology establishment, and biotic and abiotic stress responses. Despite their well- characterised functions in model plants, systematic investigation of AP2 family in economically important fruit crops like Chinese plum (Prunus salicina Lindl.) remains limited. This study presented the first comprehensive genome- wide identification of AP2 transcription factors in three major Chinese plum cultivars Sanyueli, Zhongli No. 6, and Wushancuili.【Methods】Using high-quality genome assemblies of the three plum cultivars, we systematically identified AP2 family members through comprehensive bioinformatics approaches. Multiple sequence alignment and phylogenetic analysis were conducted using MEGA11 with AP2 proteins from Arabidopsis thaliana, Malus domestica, and other related species. Gene structure and conserved motifs were analyzed using SMART and MEME suites. Chromosomal locations and synteny relationships were determined using MCScanX. Cis-acting regulatory elements in promoter regions were predicted using PlantCARE. Expression patterns were investigated through qRT-PCR analysis of six selected AP2 genes (SYPsAP2-5/7, ZLPsAP2-15/16, and WSPsAP2-9/11) across eight fruit developmental stages in Shibanwannai plum.【Results】We identified 20, 22, and 18 AP2 genes in Sanyueli, Zhongli No. 6, and Wushancuili genomes, respectively. The expansion and contraction of the AP2 gene family were likely closely associated with species' evolutionary history and ecological adaptation. The size of the plum AP2 gene family not only reflected shared characteristics within Rosaceae plants, but also demonstrated genus- specific adaptations in Prunus species. The relatively conserved number of AP2 genes in stone fruits may be related to their specific developmental regulatory requirements. The subcellular localization prediction results indicated that the AP2 family members in the three plum varieties were predominantly localized in the nucleus (48 out of 60, 80% ; 16 in Sanyueli, 17 in Zhongli No. 6, and 15 in Wushancuili). A small number of AP2 family members were distributed in organelles such as chloroplasts and peroxisomes. This distribution pattern was consistent with the typical subcellular localization pattern of transcription factors. Referring to the classification criteria of the Arabidopsis thaliana AP2 transcription factor family, the plum AP2 transcription factor family was divided into 13 subfamilies, including the AP2-type, RAP2-7-type, T11A7.19-type, WRI1-type, ADAP-type, ANT-type, AIL5-type, AIL6-type, AIL1-type, BBM-type, PLT1-type, PLT2-type, and TOE-type. From the perspective of species phylogenetic relationships, the AP2 gene members of the three plum varieties clustered together and exhibited closer phylogenetic relationships compared to those of Rosaceae species like apple and pear. This finding is consistent with their systematic taxonomic status. Chromosomal distribution revealed uneven gene dispersion, with Chr1 being a hotspot in Sanyueli (35%). This distribution pattern indicated a clear chromosomal preference. The intraspecies synteny analysis revealed that the genome of Sanyueli contained 3 pairs of SYPsAP2 syntenic genes, while Wushancuili harbored 4 pairs of WSPsAP2 syntenic genes. Among the three plum varieties, Zhongli No. 6 exhibited the most extensive syntenic relationships within the ZLPsAP2 family, with a total of 7 pairs of ZLPsAP2 syntenic genes identified. Motif 2 was conserved across all 60 PsAP2 members and served as the core motif. Motifs 2, 4, 1, and 5 were most frequent, typically arranged orderly and biased toward the 5′ end. Protein domain analysis showed that all PsAP2 members possessed the canonical double AP2 domain. Notably, WSPsAP2-3 contained additional Amb_all and Pec_lyase_N domains, while SYPsAP2-19 harbored a dermokine superfamily domain at its C-terminus. These unique domains suggested potential evolutionary divergence and novel functions for these genes. Cis-acting element analysis of the AP2 gene family promoters in plums revealed significant enrichment of diverse response elements. The three plum varieties shared several core response elements: light-responsive elements (282 in SYPsAP2, 307 in ZLPsAP2, and 226 in WSPsAP2), abscisic acid- responsive elements (66 in SYPsAP2, 62 in ZLPsAP2, and 52 in WSPsAP2), MeJA-responsive elements (40 in SYPsAP2, 48 in ZLPsAP2, and 32 in WSPsAP2), gibberellinresponsive elements (25 in SYPsAP2, 28 in ZLPsAP2, and 23 in WSPsAP2), and anaerobic induction elements (52 in SYPsAP2, 57 in ZLPsAP2, and 49 in WSPsAP2). Each variety had distinct regulatory features. For example, Sanyueli (SYPsAP2) had a higher number of MYBHv1 binding sites (20) compared to Zhongli No. 6 (6) and Wushancuili (8), indicating a potential role in MYBHv1-mediated regulation. Zhongli No. 6 (ZLPsAP2) had the most diverse and abundant cis-acting elements, suggesting a complex regulatory network for its AP2 genes. Wushancuili (WSPsAP2) contained unique wound-responsive elements, likely involved in plant wound response mechanisms. Specific AP2 gene family members may be involved in seed development and light signal transduction. SYPsAP2- 7/11, ZLPsAP2- 3/16, and WSPsAP2-1/11 contained seed-specific regulatory elements. SYPsAP2-2, ZLPsAP2-18, and WSPsAP2-6 contained phytochrome regulatory elements. Six representative genes from the AP2 and BBM clades were selected based on phylogenetic and promoter analyses. Their expression patterns were analyzed across eight developmental stages of Shibanwannai, revealing significant tissue- specific profiles. The gene expression profiling demonstrated that AP2 clade genes (SYPsAP2-5, ZLPsAP2-15, and WSPsAP2- 9) showed peak expression during early fruit development and the BBM clade genes (SYPsAP2-7, ZLPsAP2-16, and WSPsAP2-11) exhibited seed-specific expression, with dramatic upregulation during T4- T8 (from 88 to 148 days after full bloom) stages coinciding with seed abortion events.【Conclusion】 The PsAP2 subfamily genes of Chinese plum were identified and analyzed in detail at the genome-wide level. The temporal-spatial expression characteristic of this subfamily genes was studied, which would establish a foundation for future functional studies and molecular breeding applications targeting fruit quality and seed development traits in plum. The identification of seed-specific PsAP2 regulators offers promising targets for addressing seed abortion issues in plum production.