Grain yield in many cereal crops is largely determined by grain size. Here we report the genetic and molecular characterization of GS3, a major quantitative trait locus for grain size. It functions as a negative regulator of grain size and organ size. The wild-type isoform is composed of four putative domains: a plant-specific organ size regulation (OSR) domain in the N terminus, a transmembrane domain, a tumor necrosis factor receptor/nerve growth factor receptor (TNFR/NGFR) family cysteine-rich domain, and a von Willebrand factor type C (VWFC) in the C terminus. These domains function differentially in grain size regulation. The OSR domain is both necessary and sufficient for functioning as a negative regulator. The wild-type allele corresponds to medium grain. Loss of function of OSR results in long grain. The C-terminal TNFR/NGFR and VWFC domains show an inhibitory effect on the OSR function; loss-offunction mutations of these domains produced very short grain. This study linked the functional domains of the GS3 protein to natural variation of grain size in rice.grain weight | Oryza sativa L. | protein domain | yield I n recent years, genes for grain and fruit sizes have been isolated from several plant species (1-8), providing opportunities for understanding genetic and molecular mechanisms regulating these traits. In rice, yield per plant is determined by three component traits: number of panicles (tillers) per plant, number of grains per panicle, and grain weight. Extensive genome mapping studies have identified hundreds of QTLs (quantitative trait loci) for yield traits (9). Although a number of QTLs for tillering (10), grain number (11, 12), grain size (8, 13-15), and panicle size and plant architecture (16-18) have been cloned, molecular characterization of these and many more genes is needed to understand the genetic and molecular bases of yield (9).A major QTL for grain size (GS3) in rice was previously identified on chromosome 3 in a number of studies across different genetic backgrounds and environments (19)(20)(21). Fan et al. (8) identified the candidate gene for GS3. By comparative sequencing analysis, they found a nonsense mutation in the second exon of the putative GS3 shared among all of the large-grain varieties tested in comparison with varieties with smaller grains. This mutation caused a 178-aa truncation in the C terminus of the predicted protein, which was widespread in the global rice germplasm collections (22, 23), indicating that this mutation had an ancient origin and played an important role in grain size variation and domestication of the cultivated rice.Here we report the genetic and molecular analysis of GS3, which revealed several important structural and functional features of the GS3 protein in grain size regulation.
ResultsValidation of GS3 on Grain Size Regulation. We validated the effect of GS3 on grain size by transformation. A construct CT9.8 (Fig. S1A), containing a 9.8-kb genomic DNA fragment encompassing GS3 amplified by PCR from rice cultivar Chuan 7 (short grain)...