Identification of Ascorbate Oxidase Genes and Their Response to Cold Stress in Citrus sinensis

Ascorbate oxidase (AAO) plays an important role in maintaining cellular redox homeostasis, thereby influencing plant growth, development, and responses to both biotic and abiotic stresses. However, there has been no systematic characterization of AAO genes in Citrus, especially their roles in response to cold stress. In the present study, nine AAO genes were identified in C. sinensis through bioinformatics analyses, exhibiting uneven distribution across four chromosomes. All CsAAOs possessed three conserved domains and were predicted to localize in the apoplast. The CsAAO gene family displayed varied intron–exon patterns. Phylogenetic analysis categorized the CsAAO family into three main clades (Clade A–C), suggesting distinct biological functions. Collinearity and Ka/Ks analysis revealed three duplicate gene pairs within the CsAAO gene family, with all duplicated CsAAOs primarily evolving under purifying selection. Analysis of cis-acting elements showed the presence of multiple hormone response elements and stress response elements within the CsAAO promoters. The computational analysis of microRNA target transcripts suggested that CsAAO9 may be a target of csi-miR156. RNA-Seq data demonstrated high expression levels of CsAAOs in roots and young fruits, while qRT-PCR analysis showed significant upregulation of six CsAAOs in response to cold treatment. Furthermore, the activities of CsAAOs exhibited a pattern of initial decrease followed by an increase after exposure to low temperatures. These findings offer important insights into the role of CsAAOs in response to cold stress. Furthermore, AAOs could be target genes for breeding crops with better cold resistance.