Genome-wide identification and comparative analyses of key genes involved in C₄ photosynthesis in five main...

Compared to C₃ species, C₄ plants showed higher photosynthetic capacity as well as water and nitrogen use efficiency due to the presence of the C₄ photosynthetic pathway. Previous studies have shown that all genes required for the C₄ photosynthetic pathway exist in the genomes of C₃ species and are expressed. In this study, the genes encoding six key C₄ photosynthetic pathway enzymes (β-CA, PEPC, ME, MDH, RbcS, and PPDK) in the genomes of five important gramineous crops (C₄: maize, foxtail millet, and sorghum; C₃: rice and wheat) were systematically identified and compared. Based on sequence characteristics and evolutionary relationships, their C₄ functional gene copies were distinguished from non-photosynthetic functional gene copies. Furthermore, multiple sequence alignment revealed important sites affecting the activities of PEPC and RbcS between the C₃ and C₄ species. Comparisons of expression characteristics confirmed that the expression patterns of non-photosynthetic gene copies were relatively conserved among species, while C₄ gene copies in C₄ species acquired new tissue expression patterns during evolution. Additionally, multiple sequence features that may affect C₄ gene expression and subcellular localization were found in the coding and promoter regions. Our work emphasized the diversity of the evolution of different genes in the C₄ photosynthetic pathway and confirmed that the specific high expression in the leaf and appropriate intracellular distribution were the keys to the evolution of C₄ photosynthesis. The results of this study will help determine the evolutionary mechanism of the C₄ photosynthetic pathway in Gramineae and provide references for the transformation of C₄ photosynthetic pathways in wheat, rice, and other major C₃ cereal crops.