Abstract:  Rice, one of the primary staple foods, 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 the knockout mutants in both japonica and indica backgrounds. The folate content in the low-folate japonica variety was slightly increased with the expression of the indica alleles driven by the endosperm-specific promoter. We further obtained the 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, which were significantly increased by 37.9-fold and 26.5-fold compared with the wild-type. Transcriptomic analysis of rice grains from six transgenic lines also showed that changes in folate affected biological functional pathways involved in the synthesis and metabolism of rice seed storage substances, and the expression of other folate synthesis genes was weakly regulated. In addition, we also found that Aus rice was a high-folate germplasm carrying superior haplotypes of OsADCS and OsGTPCHI in natural variation. This study provides an alternative and effective complemenary strategy for rice biofortification, promoting the rational combination of metabolic engineering strategies and conventional breeding, which is beneficial for cultivating high-folate varieties.

Key words: biofortification, endosperm-specific expression, OsADCS, OsGTPCHI, folate content, grain, Oryza sativa