Bao_2025_Plant.Cell_37_

Tocopherol synthesis requires phytyl diphosphate derived from phytol esterified to chlorophyll metabolites. The >600-member Arabidopsis thaliana alpha/beta hydrolase (ABH) gene family contains 4 members that can release phytol from chlorophyll metabolites in vitro; however, only pheophytinase (PPH) affects tocopherol synthesis when mutated, reducing seed tocopherols by 5%. We report the biochemical analysis of 2 previously uncharacterized ABHs, chlorophyll dephytylase 2 (CLD2) and CLD3, and their respective mutants singly and in combinations with pph and cld1 alleles. While all CLDs localized to the thylakoid and could hydrolyze phytol from chlorophylls and Pheophytin a in vitro, CLD3 had the highest in vitro activity and the largest effect on tocopherol synthesis in vivo. The 3 CLDs acted cooperatively to provide phytol for 31% of tocopherols synthesized in light-grown leaf tissue. Dark-induced leaf senescence assays showed PPH is required for 18% of the tocopherols synthesized. Though the cld123 triple mutant had no impact on dark-induced tocopherol content, cld123 in the pph background reduced tocopherol levels by an additional 18%. In seeds, pph and cld123 each reduced tocopherol content by 5% and by 15% in the cld123pph quadruple mutant. VTE7 (ViTamin E7) is an envelope-localized ABH that specifically affects chlorophyll biosynthetic intermediates in vivo and is required for 55% of seed tocopherol synthesis. The introduction of cld123pph into the vte7 background further reduced seed tocopherol levels to 23% of that of the wild type. Our findings demonstrate that phytol provision for tocopherol biosynthesis and homeostasis is a complex process involving the coordinated spatiotemporal expression of multiple ABH family members.