Map-based cloning showed that TA2 encodes a DNA demethylase. This study provides a strategy for enhancing the nutritional value of rice, and possibly of other cereals as well.
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 thick aleurone 2–1 (ta2-1), 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 ta2-1 grains. Map-based cloning showed that TA2 encodes the DNA demethylase OsROS1. A point mutation in the 14th intron of OsROS1 led to alternative splicing that generated an extra transcript, mOsROS1, with a 21-nt insertion from the intron. Genetic analyses showed that the ta2-1 phenotype is inherited with an unusual gametophytic maternal effect, which is caused not by imprinted gene expression but rather by the presence of the mOsROS1 transcript. Five additional ta2 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 ta2-1 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.
Identification and characterization of the ta2-1 mutant. (A) Scheme of the genetic screen for ta mutants in rice using a half-seed assay. (B and C) Transversally (B) and longitudinally (C) sectioned ZH11 and ta2-1dehusked mature grains, stained with the cell viability dye Evans blue. (Scale bar: 0.5 mm.) (D) Morphology of the aleurone at the lateral positions of ZH11 and ta2-1 endosperms. (Scale bar: 50 μm.) (E) Average numbers of aleurone cell layers, calculated in 20-cell file regions at the dorsal, upper lateral, lateral, lower lateral, and ventral positions of ZH11 and ta2-1 endosperms (n = 30). **Significant differences from ZH11 at the 0.01 level according to Student’s t test. (F) Aleurone at the lateral positions of ZH11 and ta2-1 endosperms at 7 and 9 DAP. (Scale bar: 20 μm.) Arrowheads in B and C and orange lines in D and F indicate the aleurone. e, embryo; se, starchy endosperm.