Codon Usage Bias of the Polyphenol Oxidase Genes in Camellia sinensis: A Comprehensive Analysis

Tea, a widely consumed beverage globally, is a vital agricultural product for many countries. Polyphenol oxidases (PPOs), copper-containing enzymes found in plants, fungi, and animals, are essential for physiological metabolism and enzymatic browning in tea plants (<i>Camellia sinensis</i>). Codon usage bias (CUB), a key evolutionary characteristic, offers valuable insights into species evolution and gene function. However, the codon usage patterns of <i>Camellia sinensis</i> polyphenol oxidase (<i>CsPPO</i>) genes remain undocumented. In this study, we conducted, for the first time, a comprehensive analysis of CUB in 24 <i>CsPPO</i> genes, comparing their CUB profiles with those of other <i>Camellia</i> species (<i>Camellia lanceoleosa</i>, <i>Camellia nitidissima</i>, <i>Camellia ptilophylla</i>) and non-<i>Camellia</i> species (<i>Actinidia chinensis</i>, <i>Cornus florida</i>, <i>Rhododendron vialii</i>) to elucidate potential evolutionary relationships and functional constraints influencing CUB. Nucleotide composition analysis revealed an AT-rich bias, with a preference for G/C-ending codons at the third position. Codon usage indices indicated low expression levels and weak CUB. RSCU and RFSC analyses revealed that the preferred and high-frequency codons were mostly G/C-ending. Codon usage frequency analysis suggested <i>Zea mays</i> as a suitable host for <i>CsPPO</i> gene expression. ENC-GC3s, PR2, and neutrality plots showed natural selection had a stronger impact than mutation on CUB. Additionally, measure independent of length and composition (MILC) values confirmed low <i>PPO</i> gene expression levels, and correlation analyses demonstrated that both nucleotide composition and gene expression affect CUB. Overall, codon usage in <i>CsPPO</i> genes is mainly shaped by natural selection, with weak bias and low expression potential, providing useful insights for future genetic engineering and heterologous expression.