Primary Exploration Of Mechanisms Underlying Auxin Regulation Of Rice Grain Filling

Rice is one of the most important crops in China, the yield traits of which are always the first goal in rice genetic breeding, where as grain filling and seed setting rate have been considered the basic traits to determine rice yield. Previous work has indicated that auxin IAA has a close relationship with rice grain filling. IAA levels in young spikes rapidly increase after fertilization. Due to the deficiency of photosynthetic capacity resulting in the limitation of rice yield. Exogenous IAA enhances leaf photosynthesis and thereby promotes the transport of photosynthetic products to grains in rice, suggesting that local auxin biosynthesis plays critical roles in rice grain filling. Recent studies have shown that IPA(indole-3-pyruvic acid) pathway is an important branch of IAA biosynthesis, in which TAA1and YUC (YUC) are two key enzyme genes. In this study, using molecular cloning and rice transformation, TAA1and YUC were specifically expressed in the endosperm or the leaf in order to increase their endogenous auxin levels, leading to improving rice grain filling and seed setting rate. The main results in this study are as follows:1. Young spikes and flag leaves were treated with different concentrations of2,4-D, IAA and I-NAA, respectively, in Nipponbare rice (Oryza sativa L. ssp. Japonica cv. Nipponbare), then their seed setting rate,1000-grain weight and single panicle yield were statistically analyzed after harvesting. It was found that treatments with exogenous auxin in young spikes and flag leaves could improve rice setting rate and single panicle yield, but failed to improve thousand-grain weight, indicating that auxin is involved in the regulation of rice grain filling.2. Using molecular cloning techniques, endosperm-specific expression vector OsGt1::AtTAAl:GUS and OsGtl:OsTAA1:GUS, leaf-specific expression vector Atplasto::OsYUC2:GUS were successfully constructed. The above constructs and auxin markers DR5::GFP were tansformed into rice (Nipponbare) via agrobacterium-mediated transformation method, respectively. Analyses of RT-PCR and GUS staining show that transgenic lines of TAA1endosperm-specific expression, YUC leaf-specific expression and DR5::GFP expression were generated.3. The seed setting rate,1000-grain weight, and yield per plant were statistically evaluated in the above transgenic lines and previously obtained homozygous lines. The analysis results showed that TAA1endosperm-specific expression improved seed setting rate and yield per plant of transgenic lines; while TAA1leaf-specific expression improved seed setting rate but did not increase yield per plant. In contrast, YUC leaf-specific expression had a similar effect with TAA1endosperm-specific expression in the transgenic lines; while YUC endosperm-specific expression had a similar effect with TAA1leaf-specific expression. However, both TAA1and YUC tissue-specific expression did not affected1000-grain weight. These results suggest that local auxin biosynthesis in grains and leaves have important biological functions in rice grain filling.4. Real-time PCR was applied to detect the effect of YUC tissue-specific expression on expression levels of auxin responsive genes OsIAAs. The results showed that, compared with nontransgenic lines (NT), YUC endosperm-specific expression increased the expression levels of OsIAA9and OsIAA30in young spikes. YUC leaf-specific expression increased OsIAA1, OsIAA6and OsIAA10expression levels, indicating that YUC endosperm and leaf specific expression did promote auxin responsive gene expression, and that TAA1and YUC tissue-specific expression promoted endogenous IAA levels in rice endosperm and leaf tissue.