Dynamic changes of genome sizes and gradual gain of cell‐specific distribution of C 4 enzymes during C 4 evolution in genus Flaveria

C 4 plants are believed to have evolved from C 3 plants through various C 3 –C 4 intermediate stages in which a photorespiration‐dependent CO 2 concentration system known as C 2 photosynthesis operates. Genes involved in the C 4 cycle were thought to be recruited from orthologs present in C 3 species and developed cell‐specific expression during C 4 evolution. To understand the process of establishing C 4 photosynthesis, we performed whole‐genome sequencing and investigated expression and mesophyll‐ or bundle‐sheath‐cell‐specific localization of phosphoenolpyruvate carboxylase (PEPC), NADP‐malic enzyme (NADP‐ME), pyruvate, orthophosphate dikinase (PPDK) in C 3 , C 3 –C 4 intermediate, C 4 –like, and C 4 Flaveria species. While genome sizes vary greatly, the number of predicted protein‐coding genes was similar among C 3 , C 3 –C 4 intermediate, C 4 –like, and C 4 Flaveria species. Cell‐specific localization of the PEPC, NADP‐ME, and PPDK transcripts was insignificant or weak in C 3 –C 4 intermediate species, whereas these transcripts were expressed cell‐type specific in C 4 –like species. These results showed that elevation of gene expression and cell‐specific control of pre‐existing C 4 cycle genes in C 3 species was involved in C 4 evolution. Gene expression was gradually enhanced during C 4 evolution, whereas cell‐specific control was gained independently of quantitative transcriptional activation during evolution from C 3 –C 4 intermediate to C 4 photosynthesis in genus Flaveria .