Generation of vitamin C enriched rice to improve iron bioavailability

Broad RC1, Bonneau JP1, O'Brien M1, Beasley JT1, Roden SM2, Eftekhari FN2, Hellens RP2 and Johnson AAT1

  1. School of Biosciences, The University of Melbourne, VIC 3010, Australia.
  2. Centre for Tropical Crops and Biocommodities, Queensland University of Technology, QLD 4001, Australia.

Breeding strategies to produce iron (Fe) biofortified crops typically focus on increasing Fe density and/or reducing inhibitors of Fe absorption, with less attention given to compounds that enhance Fe absorption. Ascorbate, most commonly known as vitamin C, is a potent enhancer of Fe bioavailability in human digestion. Because many staple crops have little to no ascorbate, enriching crops with ascorbate could provide a novel means of improving human Fe nutrition. This study aims to investigate the effects of increased ascorbate biosynthesis in rice on grain Fe bioavailability and plant growth. Rice plants genetically engineered to constitutively overexpress the rice GDP-L-galactose phosphorylase (OsGGP) gene, encoding the key rate-limiting enzyme involved in ascorbate biosynthesis, contained 8.7-fold more ascorbate in germinated grain, 1.8-fold more ascorbate in seedling shoots, and 4.5-fold more ascorbate in seedling roots, relative to wild-type rice. At maturity, however, the OsGGP overexpressing rice plants contained less ascorbate in shoots and had reduced expression of the endogenous OsGGP gene, correlated with significant yield penalties, indicating that OsGGP overexpression has several pleiotropic effects on rice physiology. Therefore, as an alternative strategy to increase ascorbate biosynthesis in rice, the CRISPR/Cas9 gene-editing system has been used to induce mutations in the OsGGP upstream open reading frame (uORF), a negative translational regulator of the OsGGP coding sequence. Here we present findings from several molecular and phenotypic analyses of the OsGGP overexpressing rice plants, including an Fe bioavailability assessment of ascorbate-enriched, germinated grain, and summarize findings of CRISPR/Cas9-induced mutations in the OsGGP uORF sequence.