Aleurone forms the outermost layer of the rice endosperm and plays a critical role in apoplastic nutrient uptake during endosperm development. Thickening the aleurone layer has been proposed to significantly increase the nutrient content of rice grains. In this study, we used a CRISPR/Cas9-mediated precise base editing method to targetOsROS1, a gene associated with aleurone thickness, in the background of thejaponicaglutinous rice cultivar Zhennuo 19. We successfully generated a transgenefree elite, homozygous base-edited line named T85-11, with a C8-to-T8 transition that led to the S1357F mutation in OsROS1.Cytohistological analysis revealed that the aleurone layer of T85-11 grains was noticeably thickened compared with that of the wild type, while there was no significant difference in endosperm appearance between T85-11 and the wild type. As a result, the nutrient contents of T85-11 grains were significantly enhanced compared with those of Zhennuo 19 grains. Furthermore,T85-11 exhibited agronomic performance similar to the wild type. These findings suggested that base editing ofOsROS1can be a highly efficient approach to generate novel mutants with improved nutritional quality without compromising agronomic traits.

The endosperm of cereal grains, rich in starch and other nutrients, serves as one of the main energy sources for humans(do Nascimento et al, 2022). The aleurone layer, responsible for accumulating a variety of storage proteins, lipids, vitamins, and minerals, is an important structural component of triploid cereal endosperm (Becraft and Yi, 2011; Bai et al, 2016; Wu et al,2016; Yang et al, 2018). Thickening the aleurone layer through molecular manipulation of genes, regulating its formation and development, is considered as a feasible strategy to improve the nutritional quality of cereal grains (Liu et al, 2018). In rice, the aleurone covers outer surface of the endosperm and consists of a single layer of cells in most regions, with a small region near the dorsal vascular bundle of the endosperm, exhibiting a thickened structure with three to four cell layers (Wu et al,2016). Several genes, such asRISBZ1,RPBF,ADL1,OsCR4,andOsROS1, have been reported to be involved in aleurone formation and development (Hibara et al, 2009; Kawakatsu et al,2009; Pu et al, 2012; Liu et al, 2018). Among them,OsROS1is a rice homolog ofArabidopsis ROS1and encodes a bi-functional DNA demethylase that removes 5-methylcytosine and nicks double-stranded DNA (Gong et al, 2002; Ono et al, 2012; Tang et al, 2016; Du et al, 2023). Previous research has shown that the accumulation of an alternatively splicedOsROS1transcript or point mutations at specific sites withinOsROS1, as observed in mutantsta2-1tota2-6, leads to an obviously thickened aleurone layer and a significant increase in the nutritional content of rice grains (Liu et al, 2018). Genome editing has proved to be a powerful tool for improving crop agronomic traits (Gao, 2021). Furthermore, glutinous rice, as a special type of rice, generally has higher commercial value than conventional cultivars. Thus, we hypothesized that, by editing theOsROS1gene in glutinous rice cultivars, the nutritional content of the grains can be efficiently improved, thereby significantly increasing the value of rice products.

In our previous study, we found that knocking outOsROS1using CRISPR/Cas9 in thejaponicaglutinous rice cultivar Zhennuo 19, which possesses the wild typeOsROS1gene, cannot thicken the aleurone or promote nutritional quality. Instead, it leads to defects in pollen and embryo sac development,ultimately resulting in seedlessness (Xu et al, 2020). In this study, we generated a glutinous rice mutant with a thickened aleurone layer using a base editing strategy by mimicking the point mutation (S1357F) ofOsROS1, which has been previously reported in theta2-4mutant (Liu et al, 2018), to enhance nutritional content of rice grains.

Based on the mutations responsible for altered aleurone layers observed in theta2mutants (Liu et al, 2018) and the requirements of cytidine base editors (CBEs) (Zong et al, 2017),we attempted to mimic the point mutations reported in theta2-6orta2-4mutants by designing two sgRNAs targeting the first (target site 1, TS1) or third (target site 2, TS2) exon ofOsROS1(Fig. 1-A). The two sgRNAs were individually cloned into the pH-nCas9-PBE vector to generate BE-TS1 and BE-TS2 vectors. These vectors were then introduced into Zhennuo 19 throughAgrobacterium-mediated transformation.For TS1, we identified 23 independent T0hygromycin-positive plants, none of which displayed mutations in the target site(Table S1). For TS2, we generated 24 independent T0transgenic lines, of which, 19 lines were the wild type and the remaining 5 lines (T85-11, T85-12, T85-13, T85-17, and T85-23) were heterozygous mutants containing the same mutation within the editing window. This mutation was characterized by a C8-to-T8 transition, leading to the S1357F amino acid mutation, as previously observed inta2-4(Table S1). These five T0lines were then propagated to the T1generation. The point mutation was faithfully passed onto the T1progeny derived from the five T0lines. The T1progenies were screened for transgene-free homozygous mutants, and one such mutant (named T85-11 as it was derived from T0line of T85-11) was identified and used for further phenotypic analysis (Fig. 1-B). During the heading stage, the appearance of the T1plants for the T85-11 mutant(Fig. 1-C) was indistinguishable from that of the wild type Zhennuo 19. The appearance of the T2milled rice grains from the mutant (Fig. 1-D), which appeared opaque due to its glutinous rice background, was also similar to that of the wild type. In addition, no mutations were found in any of the potential off-target sites in the T85-11 mutant (Fig. 1-E).

Fig. 1. Precise base editing of OsROS1 gene thickens aleurone layer in rice grains.

To explore whether the aleurone layer in the T85-11 mutant was thickened, several histological analyses were performed.Hand-cut section observations showed no detectable differences in the endosperm appearance between T85-11 and Zhennuo 19(Fig. 1-F), consistent with the previous finding that the grain traits of theta2-4mutant were similar to those of the wild type(Liu et al, 2018). However, a thickened aleurone layer in T85-11 was evident in both transverse (Fig. 1-G) and longitudinal (Fig. 1-H) sections of grains stained with Evans blue, a dye that stains the starchy endosperm blue but not the aleurone, and was further confirmed by scanning electron microscope (SEM) observation (Fig. 1-I). Furthermore, we also prepared semi-thin sections of developing endosperms at 9, 12,and 15 d after flowering (DAF). Toluidine blue staining revealed that the aleurone layers of the wild type Zhennuo 19 consisted of 1 to 3 cell layers, while those of T85-11 exhibited 3 to 5 cell layers (Fig. 1-J to -O). This observation was further validated by SEM analysis of mature grains (Fig. 1-M). These results indicated that T85-11 developed more aleurone layers,which could potentially enhance the nutritional content of the grains.

To determine whether the increased number of aleurone cell layers in T85-11 resulted in improved nutritional value, the contents of various nutrients in the dehusked mature grains of Zhennuo 19 and T85-11 were compared. The results showed that the contents of total proteins, total lipids, zinc, calcium,and total phenolic, as well as the levels of vitamin B1, B3, B6,C, and E were significantly higher in T85-11 grains than in Zhennuo 19 grains (Fig. 2-A to -J). However, the total starch and the apparent amylose contents (AAC) were lower in T85-11 grains than in Zhennuo 19 grains (Fig. 2-K and -L).

Fig. 2. Nutritional contents of dehusked mature grains in Zhennuo 19 (ZN19) and T85-11 mutant.

To investigate the effects of the S1357F mutation in OsROS1 on important agronomic traits, we analyzed the progeny of T85-11 by planting them in the greenhouse in Nanjing city,Jiangsu Province, China, in 2021. We found that T85-11 was indistinguishable from Zhennuo 19 in terms of tiller number per plant, heading date, number of grains per panicle,seed-setting rate, 1000-grain weight, grain yield per plant, and actual yield per plot, although the height of T85-11 plants was reduced by about 5 cm compared with that of the wild type(Table S2). These results suggested that the S1357F allele of OsROS1 was a valuable resource for developing elite rice varieties with high nutritional quality.

As a specialized product, functional rice is an important component of nutrition-oriented agriculture and functional agriculture, garnering significant attention from consumers.Over the past decades, researchers have proved that grain quality-related traits, such as the contents of flavonoid, folate,γ-aminobutiric acid, and resistant starch, which are controlled by single genes, could be enhanced in rice by manipulating the expression levels of these genes (Reddy et al, 2007;Storozhenko et al, 2007; Nagasawa et al, 2013; Xu et al, 2015;Guo et al, 2020). In this study, we demonstrated that multiple nutritional indices of rice grains could be fortified by thickening the aleurone layer via base-editing-mediated S1357F mutation of OsROS1 (Fig. 2-A to -J). This strategy has wider implications for efficiently improving the nutritional quality of rice grains. In addition, we observed that the contents of total starch and the apparent amylose were lower in T85-11 than in the wild type (Fig. 2-K and -L). In general, the quality of the milled rice appearance (especially the transparency of grains) is negatively correlated with AAC (Li and Gilbert,2018). The milled rice grains of the conventional rice cultivars with an AAC ＞ 14% are translucent, while the glutinous rice grains with an AAC ＜ 2% are opaque. Thus, if we attempt to createOsROS1mutants in the background of conventional rice varieties in the future, we need to pay more attention to the trait of grain transparency. However, this may not be a concern in this case due to the glutinous rice background.

As shown in a previous study, non-frameshift mutants, such asta2-1tota2-6, exhibit varying degrees of aleurone thickening phenotype due to the mutations at different functional sites of OsROS1 (Liu et al, 2018). The most dramatic aleurone thickening was observed inta2-1, but several important agronomic traits ofta2-1were compromised, particularly the 1000-grain weight,seed-setting rate, yield per plant, and yield per plot, which limits its practical application. In contrast, mutants with moderately thickened aleurone (about twice thicker compared with the wild type), such as theta2-4mutant and T85-11 generated in this study (Fig. 1-O), resembled most agronomic traits of the wild type (Table S2), and could be more suitable for breeding purpose. Furthermore, we anticipate that with the development of genome editing technology, more powerful editing tools will become available for directed evolution ofOsROS1to generate novel alleles contributing to thickened aleurone and improved nutritional quality without adversely affecting important agronomic traits (Packer and Liu, 2015;Engqvist and Rabe, 2019; Li et al, 2020).

In summary, our study demonstrated the feasibility of base editing in generatingOsROS1mutants with thickened aleurone layer and enhanced nutrient contents. These mutants can be directly applied in commercial production or serve as novel germplasms for breeding high-quality new rice varieties.

ACKNOWLEDGEMENTS

This study was supported by the Ministry of Agriculture and Rural Affairs of China, the Jiangsu Provincial Key Research and Development Program (Grant No. BE2022383), Key Laboratory of Jiangsu Province for Agrobiology, China (Grant No. JKLA2021-ZD01), and the Exploratory Project of the Jiangsu Academy of Agricultural Sciences, China (Grant No.ZX(21)1201). We thank Prof. GAO Caixia of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, for providing the base editing vector. We thank Prof.REN Yulong, Mrs. WEI Qian and Mrs. WU Jia’nan of the Core Facility Platform, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, for their assistance in performing semi-thin sections.

SUPPLEMENTAL DATA

The following materials are available in the online version of this article at http://www.sciencedirect.com/journal/rice-science;http://www.ricescience.org.

File S1. Methods.

Table S1. Sequencing results of T0transgenic lines generated by base editing.

Table S2. Agronomic trait analysis.