Total tocopherol levels in maize grain depend on chlorophyll biosynthesis within the embryo

Authors: HERR, SAM - Cornell University; LI, XIAOWEI - Cornell University; WU, DI - Cornell University; Hunter Iii, Charles; MAGALLANES-LUNDBACK, MARIA - Michigan State University; WOOD, JOSHUA - University Of Georgia; KACZMAR, NICHOLAS - Cornell University; DIEPENBROCK, CHRISTINE - University Of California, Davis; BUELL, ROBIN - University Of Georgia; DELLAPENNA, DEAN - Michigan State University; GORE, MICHAEL - Cornell University

Submitted to: BMC Plant Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/16/2025
Publication Date: 3/15/2025
Citation: Herr, S., Li, X., Wu, D., Hunter Iii, C.T., Magallanes-Lundback, M., Wood, J., Kaczmar, N., Diepenbrock, C., Buell, R., Dellapenna, D., Gore, M. 2025. Total tocopherol levels in maize grain depend on chlorophyll biosynthesis within the embryo. BMC Plant Biology. 25,328. https://doi.org/10.1186/s12870-025-06267-6.
DOI: https://doi.org/10.1186/s12870-025-06267-6

Interpretive Summary: Vitamin E deficiency remains a major health concern in parts of the world experiencing food insecurity. Biofortification of staple grains such as maize, which typically have low levels of vitamin E, could help alleviate vitamin E associated health problems. However, accumulation of vitamin E in grains is poorly understood. ARS researchers at the Center for Medical, Agricultural, and Veterinary Entomology, in Gainesville FL, in collaboration with researchers at Cornell University, Michigan State University, University of Georgia, and University of California, Davis, have identified two genes, POR1 and POR2, that are associated with vitamin E content in maize kernels. Removal of these genes by gene editing resulted in almost complete loss of vitamin E content in maize kernels. Interestingly, the researchers were able to replicate the vitamin E deficiency by keeping developing kernels of normal plants in darkness, tying vitamin E synthesis to photosynthesis. The gene edited maize lines developed in this study provide useful tools for continued studies of the roles of POR1 and POR2 and will help facilitate development of crops with higher levels of vitamin E.

Technical Abstract: Tocopherol is a class of lipid-soluble compounds that exhibit vitamin E activity, an essential nutrient for human health and plant fitness. The core tocopherol biosynthetic pathway has been elucidated and is conserved across the plant kingdom but the mechanism(s) providing the key tocopherol pathway precursor, phytol, have not. Two protochlorophyllide reductases (POR1 and POR2) involved in chlorophyll synthesis were identified as large-effect loci controlling the natural variation of total tocopherols in maize grain, a non-photosynthetic tissue with extremely low levels of chlorophyll. We took two approaches to alter the activity of these two POR genes in kernels, physiological treatments and CRISPR/Cas9 knockouts. Embryos deprived of light during development had reduced levels of chlorophyll a and tocopherol levels by 92-99% and 87-90%, respectively, compared to the high-light treatment. In the CRISPR/Cas9 knockout experiment, chlorophyll a and tocopherol levels in the por1 and por2 double mutants were similarily reduced by 98-100% and 76-83% respectively in the embryo, compared to wild type. These findings demonstrate that tocopherol synthesis in the maize grain is largely dependent on phytol provided from a chlorophyll-based biosynthetic cycle within the embryo. Both POR1 and POR2 play a crucial role in facilitating this cycle, underscoring the importance of POR genes in the biofortification of vitamin E levels in non-photosynthetic grain of maize and potentially other cereals.