The enzyme OsDHFR-TS1 controls leaf senescence during grain filling via folate-dependent chlorophyll biosynthesis in rice

Dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a bifunctional enzyme that catalyzes the conversion of dihydrofolate (DHF) into tetrahydrofolate (THF) by the DHFR domain and dUMP to dTMP as well as 5,10-methylene-THF to DHF by the TS domain. To date, the contributions of DHFR-TS to chlorophyll biosynthesis and plant growth are not well defined. In this study, we isolated a grain-filling-stage premature senescence (fps1) mutant in rice (Oryza sativa), whose causal gene encodes OsDHFR-TS1. In the fps1 mutant, a point mutation in OsDHFR-TS1 resulted in the loss of approximately two-thirds of the TS domain. Interestingly, the DHFR activity was remarkably reduced both in the fps1 mutant and in the OsDHFR-ts1 recombinant protein. Furthermore, DHFR and TS domains within OsDHFR-TS1 mutually enhanced each other's enzymatic activities through their interaction. In fps1 leaves at the mid-to-late stage of grain filling, Mg-protoporphyrin IX accumulated significantly, accompanied by sharp decreases in tetrahydrofolate and chlorophyll levels, which suggests that the methyl groups supplied by tetrahydrofolate become insufficient to maintain normal chlorophyll synthesis. Accordingly, photosensitizing protoporphyrin IX accumulated excessively, promoting the overproduction of reactive oxygen species. These two aspects collectively contributed to the premature senescence phenotype of fps1. This study provides insights into understanding the interrelationship between tetrahydrofolate synthesis, chlorophyll synthesis, and leaf senescence. Additionally, we demonstrated that OsDHFR-TS2, homologous to OsDHFR-TS1, likely plays only a minimal role in tetrahydrofolate synthesis in rice.