Functional Analyze And Regulatory Mechanismof GLW2,a Gene Determining Rice Grain Size

Rice is important food crop in China, the food security is challenged by yield of rice. Yield of rice is determined by four major components directly, effective tillers per plant, the number of grains per panicle, grain weight,and seed setting rate. The grain weight is mainly affected by the grain size and the ratio of filled grains. Although some genes about grain size which numbers rarely have been reported, their regulatory roles in determining grain size or weight is still unclear. An extra-large grain rice line designated 307R, with a 1,000-grain weight (TGW) of 64 g, was identified from our breeding materials in our early experiment. To verify the usefulness of this trait, we crossed 307R with three elite rice restorer lines, IR24, MH63, and 527R. Three near isogenic lines (NILs, NIL-IR24, NIL-MH63, and NIL-527R) were developed from different backgrounds. The large grain gene allele of NILs predominantly promotes cell expansion but also increases cell proliferation. Finally, we limited the GLW2 locus to a 15.3 kb intervalfrom a cross of 307R/IR24, which contains only one candidate the gene enconding OsGRF4 that belongs to growth regulation factor family. So we named the gene GLW2. We analyzed the function of GLW2 based on aforementioned reserach, determined the upstream regulatory factors of the GLW2 and downstream protein interacting with GLW2, and understood the genetic mechanism of GLW2 determining grain size. The following results were obtained:1. To confirm whether OsGRF4 is GLW2, we first generated an overexpression construct in which the OsGRF4 from IR24 background was driven by the 2×35S promoter and introduced it into Nipponbare. Investigation indicated an apparent increase in the grain size and weight of the transgenic plants. And the epidermal cells are bigger than CK's in the transgenic plants'lemma. We next generated two gRNA constructs and introduced it into NIL-527R to knock out (KO) the OsGRF4 gene in a CRISPR/CAS9 strategy. Several independent Bi-allelic or homozygous KO plants were obtained and showed obvious decreases in grain size and weight. These results demonstrate that OsGRF4 is responsible for the grain size and weight phenotype and its elevated expression benefit to a large and heavy grain.2. Transient expression of a GLW2-YFP (yellow fluorescent protein) fusion protein in rice protoplasts showed that GLW2-YFP localized mainly to the nucleus, consistent with that OsGRF4 is a plant-specific transcription factor. We then demonstrated that OsGRF4 hadtranscription activation activity, and the activation domain was located in its C-terminal region according to a series of truncations analysis in yeast cell.3. It is known that the GRF genes are substantially regulated by miR396. Detailed q-PCR analysis indicated that the expression pattern ofGLW2was complementary with that of miR396c during panicle and grain development. situ hybridization results showed that OsGRF4 and miR396c both expressed in rice spikelete hulls. Interestingly, the TC487-488AA mutation of the large grain rice 307R just occurred within the binding site of miR396, which might suggest that the miR396 also directly regulated OsGRF4 but the cleave efficiency was different from the large and small grains.4. An RLM-RACE analysis showed that miR396 could directly cleave OsGRF4 mRNA in vivo at one site within the miR396 pairing region (20/20), however, the 2-bp substitution of GLW2sharply down-regulated the cleave efficiency (2/20). We further demonstrated this by performing reverse transcription PCR (RT-PCR) using mRNAs from the heterozygous (NIL-527R/640A, NIL-MH63/106A) plants. Sequencing results clearly showed that the mRNAs existed predominantly as the NILs forms, suggested that cleavage of the NIL-527R transcripts by miR396 was disrupted, which was in accordance with that OsGRF4 expressed higher in 307R than in Nipponbare.5. To gain more genetic evidence for the regulation of OsmiR396 on OsGRF4, we generated an overexpression construct in which themiR396c was driven by the 2*35S promoter and introduced it into Nipponbare. Investigations indicated an apparent decrease in the grain size and weight of the transgenic plants. The decreased grain size and weight was further confirmed to be a consequence of the overexpression of miR396c, and a resulted substantial decrease in OsGRF4transcripts. To confirm whether perturbation of miR396c regulation on OsGRF4 leads to large grain size, two miR396-resistant variants of OsGRF4 (mOsGRF4) that disrupts the miR396 recognition without changing any amino acid, controlling by the 2×35S promoter, was introduced into Nipponbare. The transgenic plants had an obviously larger grain size and weight, suggested that the blocked down-regulation of OsGRF4 by miR396c cause the large grain phenotype.6.we examined direct interactions between GLW2 and OsGIFl in yeast cells. Further truncations analysis showed that GLW2 interacted with OsGIF1 at their N-terminal domains, consistent with the previous report of GRFs interacted with partners by the QLQ domain. Bimolecular fluorescence complementation (BiFC) assays further revealed that GLW2 interacted with OsGIF1 in planta.Transient expression of aOsGIF1-YFP (yellow fluorescent protein) fusion protein in rice protoplasts showed that OsGIF1-YFP localized mainly to the nucleus.7.we overexpressed OsGIF1, controlling by the 2×35S promoter, in Nipponbare. Investigations showed that transgenic plants overexpressing OsGIFl produced larger and heavier grains than Nipponbare.8.Because all the NILs were generated from the restorer backgrounds, we can directly use them to evaluate whether GLW2 can benefit to the yield of hybrid-rice. We crossed those NILs to two currently used cytoplasmic male sterility lines,106A and 640A, and thus generated several F1 hybrid-rice lines. Field performance indicated that GLW2 locus could significantly increase the plot yield of hybrid-rice lines generated from the NILs when compared to which generated from the recurrent parents (showed a 13.74-28.00% increase), majorly by increasing of the grain length, width, weight and yield per plant of the hybrid-rice. Besides, the NILs generated hybrid-rice was apparently longer than which generated from the recurrent parents in panicle length.9.According to the experiments, we know that OsGRF4'is a key gene determining rice grain size.OsmiR396c directly regulates the OsGRF4 in the upstream and OsGIF1,a pleiotropic gene, interacts with OsGRF4 to control the grain size in the downstream.Then we demonstrate the miR396c-OsGRF4-OsGIF1 regulatory module play a important role in grain size and weight.