Genome-Wide Identification of Polyamine Oxidase (PAO) Family Genes: Roles of CaPAO2 and CaPAO4 in the Cold Tolerance of Pepper (Capsicum annuum L.)

Abstract

Polyamine oxidases (PAOs), which are flavin adenine dinucleotide-dependent enzymes, catalyze polyamine (PA) catabolism, producing hydrogen peroxide (H₂O₂). Several PAO family members have been identified in plants, but their expression in pepper plants remains unclear. Here, six PAO genes were identified in the 'Zunla-1' pepper genome (named <i>CaPAO1</i>-<i>CaPAO6</i> according to their chromosomal positions). The PAO proteins were divided into four subfamilies according to phylogenetics: CaPAO1 belongs to subfamily I; CaPAO3 and CaPAO5 belong to subfamily III; and CaPAO2, CaPAO4, and CaPAO6 belong to subfamily IV (none belong to subfamily II). <i>CaPAO2</i>, <i>CaPAO4</i>, and <i>CaPAO6</i> were ubiquitously and highly expressed in all tissues, <i>CaPAO1</i> was mainly expressed in flowers, whereas <i>CaPAO3</i> and <i>CaPAO5</i> were expressed at very low levels in all tissues. RNA-seq analysis revealed that <i>CaPAO2</i> and <i>CaPAO4</i> were notably upregulated by cold stress. CaPAO2 and CaPAO4 were localized in the peroxisome, and spermine was the preferred substrate for PA catabolism. <i>CaPAO2</i> and <i>CaPAO4</i> overexpression in <i>Arabidopsis thaliana</i> significantly enhanced freezing-stress tolerance by increasing antioxidant enzyme activity and decreasing malondialdehyde, H₂O₂, and superoxide accumulation, accompanied by the upregulation of cold-responsive genes (<i>AtCOR15A</i>, <i>AtRD29A</i>, <i>AtCOR47</i>, and <i>AtKIN1</i>). Thus, we identified candidate PAO genes for breeding cold-stress-tolerant transgenic pepper cultivars.