Genome-wide identification and comparative analyses of key genes involved in C(4) photosynthesis in five main gramineous crops

Compared to C(3) species, C(4) plants showed higher photosynthetic capacity as well as water and nitrogen use efficiency due to the presence of the C(4) photosynthetic pathway. Previous studies have shown that all genes required for the C(4) photosynthetic pathway exist in the genomes of C(3) species and are expressed. In this study, the genes encoding six key C(4) photosynthetic pathway enzymes (β-CA, PEPC, ME, MDH, RbcS, and PPDK) in the genomes of five important gramineous crops (C(4): maize, foxtail millet, and sorghum; C(3): rice and wheat) were systematically identified and compared. Based on sequence characteristics and evolutionary relationships, their C(4) 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 C(3) and C(4) species. Comparisons of expression characteristics confirmed that the expression patterns of non-photosynthetic gene copies were relatively conserved among species, while C(4) gene copies in C(4) species acquired new tissue expression patterns during evolution. Additionally, multiple sequence features that may affect C(4) 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 C(4) photosynthetic pathway and confirmed that the specific high expression in the leaf and appropriate intracellular distribution were the keys to the evolution of C(4) photosynthesis. The results of this study will help determine the evolutionary mechanism of the C(4) photosynthetic pathway in Gramineae and provide references for the transformation of C(4) photosynthetic pathways in wheat, rice, and other major C(3) cereal crops.