Genome-wide identification of apple <italic>NAC</italic> and responses of <italic>MhNACs</italic> in <italic>Malus hupehensis</italic> to phytohormones

Aims: The NAC (NAM, ATAF1/2, CUC2) gene family is a class of plant-specific transcription factors that can synergize with phytohormones to regulate plant growth, development and stress responses. To explore the functions of NAC genes in Malus species, this study identified NAC family members in the apple genome, analyzed their organ-specific expression patterns and their responses to phytohormones in Malus hupehensis, providing a foundation for subsequent functional studies of this gene family. Methods: Bioinformatic methods were used to identify NAC genes in the apple genome, and a phylogenetic tree was constructed with Arabidopsis NAC homologs. Furthermore, qRT-PCR was performed on Malus hupehensis to analyze the expression patterns of 15 MhNACs in different organs and under various phytohormone treatments. Results: Bioinformatic analysis identified 140 NAC genes in apple genome, which were clustered into 12 subfamilies together with Arabidopsis NAC homologs. The MpNAC family comprises 33 tandemly duplicated and 68 segmentally duplicated genes; Their promoter regions were predicted to contain 539 hormone response elements covering the abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA), auxin (IAA) and gibberellin (GA) signaling pathways. Functional enrichment analysis showed that MpNAC proteins were mainly enriched in DNA-templated transcription regulation, DNA binding and nuclear localization. Based on apple NAC gene sequences, 15 MhNACs with phytohormone-responsive element-rich promoters were selected from Malus hupehensis. Expression pattern analysis indicated that these 15 genes were generally highly expressed in roots and shoots but weakly expressed in leaves and fruits, among which MhNAC3, MhNAC56 and MhNAC75 exhibited high expression levels in both roots and shoots. Phytohormone treatment assays demonstrated that all 15 MhNACs responded to IAA, GA, ABA, SA and MeJA treatments: MhNAC122 showed significant responses to all 5 phytohormones; MhNAC3 specifically responded to IAA, MhNAC121 specifically responded to GA and ABA, and MhNAC45 specifically responded to SA and MeJA; most genes were upregulated after phytohormone treatments, while MhNAC68 and MhNAC104 were significantly downregulated under IAA, GA and MeJA treatments. Conclusion: The apple genome harbors a rich array of NAC gene family members. Fifteen MhNACs from Malus hupehensis exhibit distinct organ-specific expression patterns and generally respond to 5 phytohormones including IAA and GA. This study systematically characterized the features and phytohormone response rules of the apple NAC gene family, which provides theoretical foundations for further investigating the functions of NAC transcription factors in Malus.