Genomic and Transcriptomic Insights into the Evolution of C4 Photosynthesis in Grasses

C-4 photosynthesis has independently evolved over 62 times within 19 angiosperm families. The recurrent evolution of C-4 photosynthesis appears to contradict the complex anatomical and biochemical modifications required for the transition from C-3 to C-4 photosynthesis. In this study, we conducted an integrated analysis of genomics and transcriptomics to elucidate the molecular underpinnings of convergent C-4 evolution in the grass family. Our genome-wide exploration of C-4-related gene families suggests that the expansion of these gene families may have played an important role in facilitating C-4 evolution in the grass family. A phylogenomic synteny network analysis uncovered the emergence of C-4 genes in various C-4 grass lineages from a common ancestral gene pool. Moreover, through a comparison between non-C-4 and C-4 PEPCs, we pinpointed 14 amino acid sites exhibiting parallel adaptations. These adaptations, occurring post the BEP-PACMAD divergence, shed light on why all C-4 origins in grasses are confined to the PACMAD clade. Furthermore, our study revealed that the ancestor of Chloridoideae grasses possessed a more favorable molecular preadaptation for C-4 functions compared to the ancestor of Panicoideae grasses. This molecular preadaptation potentially explains why C-4 photosynthesis evolved earlier in Chloridoideae than in Panicoideae and why the C-3-to-C-4 transition occurred once in Chloridoideae but multiple times in Panicoideae. Additionally, we found that C-4 genes share similar cis-elements across independent C-4 lineages. Notably, NAD-ME subtype grasses may have retained the ancestral regulatory machinery of the C-4 NADP-ME gene, while NADP-ME subtype grasses might have undergone unique cis-element modifications.