Cloning And Functional Analysis Of The TAC3 Gene Controlling Tiller Angle In Rice

Rice plant architecture including plant height,tiller angle,tiller number and panicle morphology is closely related to rice yield.Isolating genes which control rice architecture and studying the function and regulation mechanism of their proteins will be helpful to understand the genetic mechanism of plant development,and provide the theoretical basis for regulating rice plant architecture and molecular breeding.In this study,we identified a tac3 mutant which displayed an increased tiller angle from the ethyl methane sulphonate(EMS)-mutagenized population of the line SH55 with compact plant architecture.The TAC3 gene was successfully isolated using the map-based cloning approach.The main results were as follows:Genetic analysis indicated that the mutant trait was controlled by a single recessive gene.Using a F2 population from a cross between tac3 and a japonica cultivar Shuiyuan392,the TAC3 gene was mapped within a 2400 kb genomic region on the centromere region of chromosome 2.After sequencing 78 predicted genes,we found a single nucleotide change,G to A,at position+2028 bp in exon of LOC_Os02g25230,resulting in a premature stop codon in the tac3 mutant.To confirm whether the mutant phenotype was caused by the truncated mutation in the LOC_Os02g25230 gene,we performed a genetic complementation analysis by over-expressing the LOC_Os02g25230 gene of wild type in the mutant,and the transgenic plants showed a complementation of the tac3 phenotypes.Furthemore,we transformed the japonica cultivar Nipponbare with the TAC3 RNA interfering(RNAi)construct,and most RNAi plants dispalyed the mutant phenotype.The genetic transformation evidences demonstrated that the LOC_Os02g25230 gene corresponded to TAC3.To understand the temporal and spatial expression pattern of TAC3,we performed quantitative RT-PCR(qRT-PCR)assay.The qRT-PCR results showed that TAC3 was transcribed in all tested organs with different abundance.To visualize the expression pattern of TAC3y we constructed a pTAC3::GUS vector which fused a 2175-bp TAC3 promoter.The result of GUS staining was consistent with those of qRT-PCR.Quantitative RT-PCR results also showed that transcription of TAC3 was not induced by auxin,cytokinin,gibberellins and diffbrent illumination conditions.RNA in situ hybridization analysis showed that the expression of TAC3 was dectected in tiller primordium and tiller bud.TAC3 protein is localized in the nucleus with unknown function.In addition,we can't search out any known domain in TAC3 protein.We tested whether TAC3 had the ability of transcriptional activation or transcriptional repression,the results showed that TAC3 protein might not be a transcription factor even though it was localized in the nucleus,and its biochemical function needed further study.We compared the gravitropism between the wild type and tac3 mutant.The results revealed that the mutation of TAC3 could affect the gravitropism of rice plants,suggesting that TAC3 affects tiller angle by influencing gravitropism in rice.Detection of auxin content showed that the auxin content of coleoptile was decreased in tac3 mutant,and the expression of auxin biosynthesis-related genes was decreased,these results indicated that TAC3 might affect auxin content by affecting the expression of auxin biosynthesis-related genes.In addition,we found that the auxin content of stem base was decreased in the mutant,especially in the outside half of stem base,leading to the decreased gravitropism and increased tiller angle in the mutant.In summary,tac3 could affect the distribution of auxin in the stem base by affecting the synthesis of auxin,which finally leads to a decrease gravitropism and increasing tiller angle of rice.