Mutations in the DNA demethylase result in a thickened aleurone and improved nutritional value in rice grains.

The rice endosperm, consisting of an outer single-cell layer aleurone and an inner starchy endosperm, is an important staple food for humans. While starchy endosperm stores mainly starch, the aleurone is rich in an array of proteins, vitamins, and minerals. To improve the nutritional value of rice, we screened for mutants with thickened aleurones using a half-seed assay and identified (), in which the aleurone has 4.8 ± 2.2 cell layers on average. Except for starch, the contents of all measured nutritional factors, including lipids, proteins, vitamins, minerals, and dietary fibers, were increased in grains. Map-based cloning showed that encodes the DNA demethylase OsROS1. A point mutation in the 14th intron of led to alternative splicing that generated an extra transcript, , with a 21-nt insertion from the intron. Genetic analyses showed that the phenotype is inherited with an unusual gametophytic maternal effect, which is caused not by imprinted gene expression but rather by the presence of the transcript. Five additional alleles with the increased aleurone cell layer and different inheritance patterns were identified by TILLING. Genome-wide bisulfite sequencing revealed general increases in CG and CHG methylations in endosperms, along with hypermethylation and reduced expression in two putative aleurone differentiation-related transcription factors. This study thus suggests that OsROS1-mediated DNA demethylation restricts the number of aleurone cell layers in rice and provides a way to improve the nutrition of rice.