Functional Analysis Of GS3, A Gene For Grain Size Regulation In Rice

In rice, yield per plant is determined by three components:number of panicles (tillers) per plant, number of grains per panicle and grain weight. Extensive previous studies showed that grain weight was significant correlated to yield. Grain weight also has high heritability and is determined by grain length, grain width and grain thickness. Much genetic research has identified hundreds of grain weight QTLs (quantitative trait loci) during the last decades. Some of these QTLs have been functional confirmed, which allowed us to know better the mechanism of grain size regulation. A major QTL for grain size (GS3) in rice previously identified in the pericentromeric region of chromosome 3 was map-based cloned recently. It encodes a transmembrane protein with phosphatidylethanolamine-binding protein (PEBP)-like domain at the N-terminus, a TNFR (tumor necrosis factor receptor)/NGFR (nerve growth factor receptor) family cysteine-rich domain and a von Willebrand factor type C (VWFC) in the C-terminal region. By comparative sequencing analysis, a nonsense mutation C to A in the second exon of the putative GS3 shared among all the large-grain varieties tested in comparison to varieties with smaller grains. Based on these results, we confirmed the gene function by transformation, expression analysis, and protein localization assay in this study. In addition, we discovered a unique allele and designed several experiments to confirm the functional domain of the gene. The main results are as follows:1. A construct CT9.8 containing a 9.8-kb genomic DNA fragment encompassing GS3 amplified by PCR from rice cultivar Chuan 7 (short grain) in the binary vector pCAMBIA1301, was transformed into rice cultivar Minghui 63 (long grain). Compared to the wild-type Minghui 63, all the transgenic lines showed highly significant decrease in grain length (more than 20%), together with slight but significant increase in grain width. We performed field-examined T1 families and observed perfect co-segregation between the transgene and the phenotype, which mean the successful complementation test.2. We also used RNA-interference (RNAi) to suppress the expression of GS3 in Chuan 7. As expected, the grain length of the positive segregants was longer than that of negative plants. The correlation between grain length and GS3 expression level was-0.78, significant at P<0.01 (n=14).3. We overexpressed the gene by transforming Minghui 63 with a construct 35SFL containing the full-length cDNA (fl-cDNA, Osigcea013f09t3) of GS3 from the plumule of an indica rice cultivar Guangluai 4 (medium grain). Overexpression of GS3 greatly reduced the plant size including shortened height, leaves and panicles, in addition to short grains. That is, overexpression of GS3 conferred pleiotropic effects to rice plants. Besides, we noticed that GS3 overexpression plants were susceptible to Ustilaginoidea virens takahashi. However, it was not a stable trait which might be affected by environment. The mechanism needs to be further discovered.4. We compared cell numbers and sizes of the glumes between Chuan 7 and Minghui 63 during grain development in the inner cell layer of the palea in successive samples of longitudinal sections. The number of cells of Minghui 63 was greater than Chuan 7. In contrast, the cell sizes of the two varieties were not significantly different in the lower and middle parts of the hull, while the cell size of Minghui 63 was significantly larger than Chuan 7 in the upper part. Taken together, these results demonstrated that the increased grain length resulted from, to a large extent, increase in cell number, and, to a lesser extent, cell size.5. By comparative sequencing, we found that there was not a premature termination in the second exon of GS3 in Chuan 7. However, in the fifth exon, Chuan 7 had a 1-bp deletion, which caused a frameshift mutation in the C-terminus that yielded a truncated 149 amino acid protein. Then, we sequenced the GS3 genomic region and measured grain length for a total of 81 accessions mostly from a core collection of the Chinese cultivated rice germplasm and additional 60 accessions of O. rufipogon and O. nivara from both China and many other countries. We failed to found another accession with GS3 allele like Chuan 7. We conclude that the GS3 allele encoded by Chuan 7 was unique and offered us a precious material to further uncover the gene function.6. The PEBP domain was no longer predicted by the same analysis of the GS3 sequence, while the other domains still remained. Alignment analysis of the previously identified putative PEBP domain of GS3 with the 19 proteins identified as PEBP family members found that the putative GS3 PEBP was only about one-third of the length of the predicted PEBP domain in those proteins, sharing poor similarity in the PEBP region. Lipid binding assay showed that GS3 could not bind to PE. These results indicate that GS3 was not a PEBP protein. We searched databases for homologous sequences of the GS3 N-terminal region, and found an N-terminal motif of 66 amino acids in length (sites 7-72) of GS3 not reported as a conserved domain in the literature, but sharing high similarity with the N-terminal regions of proteins from a number of plants ranging from angiosperm to gymnosperm. However, this domain was not found in animals or other organisms, thus is a plant specific domain. To reflect the function of this protein domain, we tentatively named it the organ size regulation (OSR) domain.7. We evaluated the effects of these alleles with near isogenic lines (NILs) in the background of Minghui 63:NIL(zs97) carrying an introgressed GS3 from Zhenshan 97, and NIL(c7) with the GS3 from Chuan 7. The two NILs were crossed, and self-pollination of the F1 produced three genotypes, NIL(c7), NIL(zs97), and NIL(het). As a result, there was no genetically dominant-recessive relation between the two alleles.8. To investigate the roles of different domains in regulating grain size, we made several domain deletions constructs driven by the GS3 native promoter from Chuan 7, and again delivered to Minghui 63. As a result, we found that all the constructs with OSR domain produced significantly shorter grains while constructs without this domain did not significantly affect grain size. In addition, the TNFR and VWFC domains could to some extent inhibit the effect of OSR in grain size reduction. To further evaluate the effect of the VWFC domain, we overexpressed a truncated cDNA sequence (OMT) with the VWFC domain deleted. Transformants produced much shorter grains and highly dwarfed plants compared with those overexpressing the fl-cDNA, providing further evidence that VWFC has a general role of inhibiting the effect of OSR in regulating plant growth and grain size. 9. By Real-time PCR and RNA in-situ hybridization, to some extent, GS3 showed constitutive expression. It was highly expressed in young panicle, and the signal gradually decreased with panicle development. There was no significant difference between Minghui 63 and NIL(c7), which suggested that the expression level and pattern were not the cause of the loss of function of GS3 in Minghui 63. We performed subcellular localization assay and found that GS3 including four intact domains was localized primarily in cytosol while GS3 encoded by Chuan 7 was mainly on plasma membrane. Several more domain deletion constructs were made and assayed. The results showed that the C-terminal region of GS3 encoded by Chuan 7 was responsible for the plasma membrane localization of the protein, while the previously predicted transmembrane domain did not localize the protein to membrane.10. To gain insight into the GS3 function in controlling rice grain size, we performed whole-genome expression profiling. The results showed that many ribosomal protein genes were depressed in GS3 overexpression plants, which hinted that GS3 might negatively control cell division and grain size by inhibiting protein synthesis.In conclusion, we confirmed the gene function in negatively regulating grain size. Furthermore, we found that the OSR domain is both necessary and sufficient for functioning as a negative regulator. It linked the functional domains of the GS3 protein to natural variation of grain size in rice.