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

Tocopherols are a class of lipid-soluble compounds that have multiple functional roles in plants and exhibit vitamin E activity, an essential nutrient for human and animal health. The tocopherol biosynthetic pathway is conserved across the plant kingdom, but source of the key tocopherol pathway precursor, phytol, is unclear. Two protochlorophyllide reductases (POR1 and POR2) were previously identified as loci controlling the natural variation of total tocopherols in maize grain, a non-photosynthetic tissue. POR1 and POR2 are key genes in chlorophyll biosynthesis yet the contribution of the chlorophyll biosynthetic pathway to tocopherol biosynthesis is still not understood.

We took two approaches to alter the activity of these two POR genes within kernel tissue, physiological treatments and CRISPR/Cas9-mediated knockouts, to determine the role of chlorophyll biosynthesis for tocopherol content. Since light is required for POR enzymatic activity, we imposed a dark treatment on developing kernels, which reduced chlorophyll a and tocopherols levels in embryo tissue by 92-99% and 87-90%, respectively, compared to the light treatment. In CRISPR/Cas9-mediated knockouts, the levels of chlorophyll a and tocopherols in embryos of the por1 por2 double homozygous mutant were reduced by 98-100% and 76-83%, respectively, compared to WT.

These findings demonstrate that tocopherol synthesis in maize grain depends almost entirely on phytol derived from chlorophyll biosynthesis within the embryo. POR1 and POR2 activity play crucial roles in chlorophyll biosynthesis, underscoring the importance of POR alleles and their activity in the biofortification of vitamin E levels in non-photosynthetic grain of maize.