Studies On The Molecular Mechnisms Of MiR1432 In Regulating Rice Grain Filling

Rice grain filling rate contributes largely to grain productivity and accumulation of nutrients.Micro RNAs(mi RNAs)are key regulators of development and physiology in plants and become a novel key target for engineering grain size and crop yield.However,there is little studies,so far,showing the mi RNA regulation of grain filling and rice yield,in consequence.Here,we show that suppressed expression of rice mi R1432(STTM1432)significantly improves grain weight by enhancing grain filling rate and leads to an increase in overall grain yield up to 17.14% in a field trial.Molecular analysis identified rice Acyl-Co A thioesterase(Os ACOT),which is conserved with ACOT13 in other species,as a major target of mi R1432 by cleavage.Moreover,overexpression of mi R1432-resistant form of Os ACOT(OXm ACOT)resembled the STTM1432 plants,that is,a large margin of an increase in grain weight up to 46.69% through improving the grain filling rate.Further study indicated that Os ACOT was involved in biosynthesis of medium chain fatty acids.In addition,RNAseq based transcriptomic analyses of transgenic plants with altered expression of mi R1432 demonstrated that downstream genes of mi R1432-regulated network are involved in fatty acid metabolism and phytohormones biosynthesis and also overlap with the enrichment analysis of co-expressed genes of Os ACOT,which is consistent with the increased levels of auxin and abscisic acid in STTM1432 and OXm ACOT plants.Overall,mi R1432-Os ACOT module plays an important role in grain filling in rice,illustrating its capacity for engineering yield improvement in crops.The results are as follows:In order to investigate the spatial expression patterns of mi R1432 during rice development,q RT-PCR and β-glucuronidase(GUS)activity assays were used to analyse mi R1432 expression of wild type plants.It was found that mi R1432 is preferentially highly expressed in root and developing seeds.And specifically,the transcriptional level of mi R1432 increased along with the seed development.Statistical analysis of characteristics of grains including grain length,grain width and grain thickness of wild type and mi R1432 transgenic plants indicated that STTM1432 transgenic plants got bigger grains.And 1,000-hulled grain weight of STTM1432 transgenic plants significantly increased 18.88%-20.28% compared with wild type.However,1,000-hulled grain weight of OXmi R1432 transgenic plants significantly decreased 23.58%-25.47% compared with wild type.By fitting parameters of grain filling,it was found that the maximum and mean filling rate of STTM1432 transgenic plant increased or decreased of OXmi R1432 transgenic plants without any notable difference in aspects of main panicle features.In addition,the actual yield of field experiments showed that the yield in STTM1432 transgenic plants increased by 13.20%-17.14%,while in OXmi R1432 transgenic plants decreased by 17.94%-29.12%.Therefore,the results indicated that mi R1432 negatively regulated rice grain filling.The downstream targets of mi R1432 were predicted by bioinformatics online.And candidate targets of mi R1432 were selected according to their expression levels in different mi R1432 transgenic plants.A transient expression method,agroinfilitration of Nicotiana benthamiana was used to validate the interaction between mi R1432 and its putative targets in vitro.Os ACOT(LOC_Os04g35590),which encodes thioesterase protein,was validated as the prospective target of mi R1432.And the cleavage sites between mi R1432 and Os ACOT was confirmed by 5’RACE.The results showed that cleavage occurred between the 16 th and 17 th base pair of the mi R1432 target site.To detect the effect of Os ACOT on rice seed development,q RT-PCR was firstly used to analysis the expression patterns of Os ACOT during rice development.The results of q RT-PCR demonstrated that the expression patterns of Os ACOT was similar with that of mi R1432.That is,Os ACOT was highly expressed in root and developing seeds.Therefore,we generated transgenic plants(OXm ACOT)expressing an mi R1432-cleavage resistant gene without any amino acid changes and found that OXm ACOT transgenic plants could phenocopies STTM1432 transgenic plants.Further statistical analysis of characteristics of OXm ACOT transgenic plants grains showed that 1,000-hulled grain weight of OXm ACOT transgenic plants grains increased by 38.91%-46.69%.Meanwhile,by fitting parameters of grain filling,it was found that the maximum and mean filling rate of OXm ACOT transgenic plants increased without any notable difference in aspects of main panicle features.Overall,the results indicated that Os ACOT positively regulated rice grain filling.To further explore the effect of physiological and molecular mechanisms of mi R1432 on regulating downstream genes and rice grain filling,RNA-seq sequencing of 10 DAF endosperm of wild type and mi R1432 transgenic plants was conducted.q RTPCR was used to confirm the RNA-seq data.Go enrichment analysis of differentially expressed genes(DEGs)showed that these DEGs were mainly involved in metabolism and transport of lipids,fatty acid biosynthesis,endomembrane organization,sugar and starch metabolism.Most importantly,biological processes involved in hormone signaling such as auxin(IAA)and abscisic acid(ABA)were also clustered.Moreover,expressions of these DEGs increased in OXm ACOT transgenic plants by q TR-PCR.Co-expressed gene anaylsis was used to clarify the function of Os ACOT in rice grain filling.Together with the chip data of endosperm development,it was found that Os ACOT might be involved in processes such as lipid biosynthesis,endomembrane organization,plant hormone signal and so on,which was consistent with the enrichment analysis of DEGs of mi R1432 transgenic plants.Therefore,contents of fatty acids of wild type and OXm ACOT transgenic plants were measured by GC-MS.By analyzing 111 acids desaturation and elongation pathway,especially from fatty acid C16:0 to C18:2.More important,contents of IAA and ABA significantly increased in STTM1432 and OXm ACOT transgenic plants and decreased in OXmi R1432 transgenic plants respectively.All in all,mi R1432-Os ACOT module may play important role in rice grain filling by affecting the contents of IAA and ABA.