Enhancing Folate Content in Japonica Rice Through Co-expression of OsADCS and OsGTPCHI Indica Alleles

Abstract

Rice is a poor source of folate, an essential micronutrient for the body. Biofortification offers an effective way to enhance the folate content of rice and alleviate folate deficiencies in humans. In this study, we confirmed that OsADCS and OsGTPCHI, encoding the initial enzymes necessary for folate synthesis, positively regulate folate accumulation in knockout mutants of both japonica and indica rice backgrounds. The folate content in the low-folate japonica variety was slightly increased by the expression of the indica alleles driven by the endosperm-specific promoter. We further obtained co-expression lines by stacking OsADCS and OsGTPCHI genes; the folate accumulation in brown rice and polished rice reached 5.65 μg/g and 2.95 μg/g, respectively, representing 37.9-fold and 26.5-fold increases compared with the wild type. Transcriptomic analysis of rice grains from six transgenic lines showed that folate changes affected biological pathways involved in the synthesis and metabolism of rice seed storage substances, while the expression of other folate synthesis genes was weakly regulated. In addition, we identified Aus rice as a high-folate germplasm carrying superior haplotypes of OsADCS and OsGTPCHI through natural variation. This study provides an alternative and effective complementary strategy for rice biofortification, promoting the rational combination of metabolic engineering and conventional breeding to breed high-folate varieties.