Regulation And Molecular Mechanism Of GS5, A Minor QTL For Grain Size In Rice

Rice is one of the most important food crops worldwide. Grain yield in rice is determined by the number of panicles, the number of grains per panicle and grain weight. Grain weight is determined by grain size and the degree of grain filling, thus grain size is one of the important yield-related traits, which is measured by grain length, width and thickness. GS5 is a minor QTL on chromosome 5 for grain width, filling and weight. It is a positive regulator of grain size, and higher expression level is correlated with increased grain width. Previous work suggested that the 18 polymorphisms between the wide-grain allele GS5-ZS97 and the narrow-grain allele GS5-H94 in the promoter region might be responsible for the variation in grain size. GS5 functions putatively as a positive modulator upstream of cell cycle genes, and its overexpression may result in an increase in cell numbers by promoting mitotic division. To further investigate the regulation and molecular mechanism of GS5, we need to figure out some more questions: What will result from the polymorphisms in the promoter region? How do the polymorphisms in the promoter region influence the expression of GS5? Why is the expression level of GS5 correlated with grain size? What happened to GS5 during the rice domestication? The results are as follow. 1. In general, GS5 had much higher expression level in green tissues than in non-green tissues. In non-green tissues, the highest expression level of GS5 was in young panicles. Throughout the young panicle development, the GS5-ZS97 transcript in the developing panicle was more abundant than GS5-H94, while in leaves, the GS5-ZS97 expression had a lower level than GS5-H94, implying that the expression of GS5 was differentially regulated in green leaves and developing panicles by different regulatory elements. 2. Plenty of light responsive elements were identified in the promoter of GS5 and the polymorphisms between the two GS5 alleles caused different number of light responsive elements. GS5-H94 has more light responsive elements than GS5-ZS97. Results confirmed that the expression of GS5 in leaves, which had circadian rhythms, was induced by light and the up-regulation of GS5-H94 was more dramatic. 3. Using a series of 5’ deletions and site-directed mutated promoter fragments fused with the β-Glucuronidase(GUS) reporter gene, we found that the polymorphisms in promoter fraction B(–1139 bp to –931 bp) were critical for the transcriptional regulation of GS5. Comparison of the two alleles with the third one GS5-ZH11 from Zhonghua 11, another wide grain variety, revealed three SNPs(SNP_–1109, SNP_–1032 and SNP_–825) occurred between GS5-H94 and the other two in promoter fractions B and C(–1139 bp to –604 bp). Mutation of SNP_–1109 and SNP_–1032 of GS5-ZS97 into GS5-H94 genotype brought the GS5-ZS97 promoter strength to GS5-H94 level. 4. SNP_–1109 and SNP_–1032 in fraction B lied in the flanking sequence of a putative gibberellin(GA) responsive element and SNP_–825 in fraction C resulted in change of a light responsive element. Mutated GA response element resulted in increased GUS activity, while the mutated light response element resulted in reduced activity, suggesting the GA response element may function as a transcription repressor while the light response element was an activator. When both elements were mutated, increased GUS activity was observed, suggesting the GA response element in fraction B may be the limiting factor in young panicles. Results showed that both GS5-ZS97 and GS5-H94 failed to respond to GA. However, the expression of GS5-H94, but not GS5-ZS97, was obviously suppressed by ABA, resulting in a lower level than GS5-ZS97. 5. GS5 encoded a serine carboxypeptidase-like protein(SCPL). No functional difference between GS5-ZS97 and GS5-H94 was observed; overexpression of either protein increased grain width. GS5 was secreted to the cell surface and probably had some link with the plasma membrane. Excessive GS5 proteins might get stuck in the endoplasmic reticulum(ER). 6. Yeast two hybrid assay revealed the interaction between GS5 and the extracellular LRR(Leucine-Rich Repeat) domain of OsBAK1-7, a BAK1(BRI1-Associatied Kinase1) homologue. As in Arabidopsis, a MSBP1(Membrane Steroid-Binding Protein1) homologue OsMSBP1 interacted with the LRR domain of OsBAK1-7 thus accelerated the endocytosis of Os BAK1-7. Enhanced expression of GS5 competitively inhibited the interaction between OsBAK1-7 and OsMSBP1 by occupying the extracellular LRR domain of OsBAK1-7 thus preventing OsBAK1-7 from endocytosis, which could explain the fact that grain size is quantitatively regulated by the expression level of GS5. 7. The expression of both GS5 alleles was suppressed by brassinosteroid(BR). Overexpression of BRS1 in rice, a SCPL involved in the Arabidopsis BR signaling pathway, increased grain width, and the grain width was positively correlated with the expression level of BRS1. These results, together with the interaction between GS5 and OsBAK1-7, suggested a possible link between GS5 and the BR signaling pathway. Overexpressed OsBAK1-7 produced semi-dwarf rice and thinner culms, while T-DNA insertion mutant osbak1-7 showed higher plant height and thicker culms, but neither of them changed grain size; T-DNA insertion mutant osmsbp1 showed blocked growth and reduced plant size including smaller grains. From these preliminary results we speculated that OsBAK1-7 negatively regulated plant growth while the constitutive expressed OsMSBP1 was indispensable to rice, and the GS5-regulation of grain size was achieved by regulating the balance of their interaction. 8. The GS5 locus spanning a 6.4-kb region from the 2-kb promoter to the 3’-flanking region was sequenced in the 527 accessions of Oryza sativa germplasm core collection and 17 wild rice accessions. In total, 130 SNPs and 27 InDels were detected within the entire GS5 region. More than 70% of the polymorphisms were located in the 2-kb promoter. According to these polymorphisms, 49 haplotypes of GS5 were identified, and 12 haplogroups were constructed. Ten types of GS5 protein were identified in all rice accessions, and no premature stop codon was detected in the coding region. Results illustrated that GS5-P2(GS5-H94) protein was the likely ancestral type, and GS5-P1(GS5-ZS97) was derived from it by several mutational steps. 9. Among the four most prevalent haplogroups, almost all accessions of the GS5-1-type(GS5-ZS97) and GS5-2-type(GS5-H94) haplogroups were found in indica; GS5-3-type(GS5-ZH11) haplogroup was largely represented by japonica varieties; and most accessions of the GS5-4-type haplogroup existed in aus. Because of the complex population structure, the associations of GS5-3-type and GS5-4-type haplogroups with grain width were hard to determine. No difference in grain width was observed between GS5-1-type and GS5-2-type accessions in indica carrying the same qSW5/GW5 genotype, a major grain width QTL on the same chromosome, implying that the small effect of GS5 in indica might be masked by the complicated genetic background. 10. In IndI subpopulation, the Tajima’s D values reached a significant negative level both in promoter and the entire genomic region of GS5, and extremely low values of π across the whole region of GS5 were observed, suggesting the prevalent haplogroup GS5-1-type in Ind I might result from artificial selection. However, it was not possible to be noticed or selected since its inconspicuous effect. The frequency of GS5-1-type remained roughly equal to that of GS5-2-type after the rc and wx mutations occurred, but increased sharply once qSW5/GW5-Kas mutated to qsw5/gw5-IndII, which gave rise to obvious wider grains. Since there was a LD(linkage disequilibrium) block detected between GS5 and qSW5/GW5, we conclused that GS5 was probably under the hitchhiking effect of positively selected qsw5/gw5-IndII, and the qsw5/gw5-IndII mutation might have occurred in a GS5-1 background.