Study On The Function Of MicroRNAs Involved In Rice Grain Filling

Rice (Oryza sativa L.) is one of the most important food crops in the world, providing calories for over 21% of global population and 76% of South East Asian. To increase yield, a series of potential high yield rice varieties, such as New Plant Type rice and ’super’ rice, have been selected and cultivated successively by traditional breeding technology. Although these cultivars generally have a higher yield potential compared with conventional varieties, often this high yield potential is usually not fully realized completely because of poor grain-filling of inferior spikelets. To study the relationship among grain weight, grain locations on the panicle and flowering order, rice grain weight and their flowering order in the same panicle were investigated. High through-put sequencing and miRNA down-regulation techniques were applied to analyze miRNAs’ expressional patterns during superior and inferior spikelets filling. The functions of differentially expressed miRNAs between superior and inferior spikelets in rice were further studied in detail. The results are as follows:To determine the accurate relationship between superior spikelets and the flowering order in rice panicle development, we localized all the seeds in a panicle in four distinct rice species and systematically documented the rice flowering order, grain locations, and the final grain weight for their relationships. Our results demonstrated that the grain weight is more heavily determined by the position of the seeds than by the flowering order. Despite earlier flowering has a positive correlation with the grain yield in general, grains from flowers blooming on the second day after anthesis generally gained the highest weight. This suggests earlier flowers may not result in superior seeds. Based on our observations, we concluded that superior grains, commonly determined by grain weight, are not fully correlated with the flowering order in rice. Following the order of the grain weight, we can attribute the superior seeds to the heaviest grains occupying 20% of the total weight of the entire panicle seeds, while the inferior seeds to the lightest grains consisting of 20% of the total panicle seed weight. The remaining seeds of 60% total weight are between the superior and inferior seeds. By these standards, the superior seeds are generally localized at the middle and lower parts of the primary branches, whereas inferior spikelets were mainly on the last two secondary branches at the lower half part of the panicle.In order to analyze the expression patterns of miRNAs during rice grain filling, and to figure out the differentially expressed miRNAs between superior and inferior spikelets, high through-put sequencing were conducted on grains from five distinct developmental stages. Totally,457 known miRNAs and 80 novel miRNAs were identified, with most known miRNAs up-regulated along with both superior and inferior spikelets filling processes. In addition to that,141 known miRNAs expressed higher in superior grains than inferior ones during early grain filling. Furthermore, 662 targets of the 263 known miRNAs were discovered through degradome sequencing. Among them, those targets of the differentially expressed miRNAs, including the targets of miR156, miR164, miR167, miR397, miR1861, and miR1867 were found to play roles in regulating plant hormone homeostasis and starch accumulation. Our data established a complicated link between miRNA dynamics and the traditional role of hormones in grain filling and development, providing new insights into the widely accepted concepts of the so-called superior and inferior spikelets in rice production.Despite 713 miRNAs have been reported in Oryza sativa (miRBase release 20.0), regulatory roles of these miRNAs, especially those of rice-specific miRNAs were poorly understood. In order to study the function of the differentially expressed miRNAs between superior and inferior spikelets during rice grain filling, and to investigate other miRNAs deposited in miRBase, Short Tandem Target Mimic (STTM) was employed. So far 8 MIM,11 STTM constructs were used to specifically down regulate miRNAs’expression in rice endosperm, and 35 STTMs were constructed to analyze the function of miRNA in rice development and stress response etc. All these materials can be used to further research the function of miRNAs in rice development, especially for rice grain development, stress response and so on.MiR167 is a typical miRNA that differential expressed in superior and inferior spikelets. Its expressional level also negatively correlated with rice grain filling rate of both superior and inferior spikelets during rice grain development. To further study the function of miR167 in rice grain filling, endosperm-specific promoter Gtl3a was used. Gtl3a-driven Target Mimic (MIM) and Short Tandem Target Mimic (STTM) were applied to down-regulate miR167’s expression specific in rice endosperm during rice grain plumping. Our results indicated that knocking down miR167 in rice endosperm by both MIM167 and STTM167 methods can significantly increase brown rice weight comparing to the untreated wild-type. What different from expectation was that the expression of miR167’s targets were lower expressed in STTM167 and MIM167 as well. The ratio of ARF8/miR167 was significantly negatively associated with brown rice weight, whereas the ratio of ARF17/miR167 and ARF25/miR167 were positively related with brown rice weight. Furthermore, Auxin response elements (AuxREs) were demonstrated to frequently present in the promoters of all miR167 family members. Last but not least, the expression of auxin biosynthesis related genes were also down regulated in the endosperm of MIM 167 and STTM 167 transgenic lines, indicating a complex regulation network among miR167, miR167’s targets, and auxin in rice endosperm. Their interactions might play important roles in controlling rice grain filling and determining the final grain weight of rice.