Molecular Mechanism Of OsAUX1,Auxin Influx Carrier,in Tiller Development Of Rice

Rice（Oryza sativa L.）tillering is an important agronomic trait,which is the basis for increasing the number of spikes to improve the harvest and is one of the most important indicators of good or bad rice plant development.There are many factors affecting rice tillering,not only by external environmental factors such as sunlight conditions,nutrients,climate conditions and water,but also by the regulation of the seed itself for fundamental reasons such as plant hormones and varieties.The role of plant hormones in the regulation of tillering in rice continues to be a major focus of research,with new evidence constantly emerging pointing to the role of different hormones in tiller development.Among them,growth hormone was the first plant hormone to be identified,and although growth hormone is widely believed to be involved in the inhibition of tillering mainly through plant apical dominance,its specific mechanism remains unclear and lacks systematic and in-depth studies.To further explore the molecular mechanism of rice tiller development,a semi-dwarf multi-tiller rice mutant htsd1（high tillering and semi-dwarf 1）was screened and identified from the indica rice Xida 1B ethyl methanesulfonate（EMS）mutagenesis library.The mechanism of the mutant gene in rice tillering development was investigated through fine gene localization,phenotypic identification,cytological observation and a series of biological experiments.The main findings are as follows:1.htsd1 is a semi-dwarf multitiller mutantUnder field growing conditions,the mutant htsd1 exhibited significantly more tillers compared to the wild type.And the number of htsd1 tillers showed a continuous increasing trend throughout the reproductive period.Paraffin sections of tiller buds showed that mutant htsd1 did not form more tiller buds,but each tiller bud was longer than the corresponding wild type tiller bud,demonstrating that the inhibition of tiller bud growth of mutant htsd1 was lifted,resulting in an increase in the number of tillers in rice plants.In addition,the mutant htsd1 was shorter than the wild type in all the reproductive stages.It was found by statistical data that the spike length and the length of each internode of the mutant htsd1 were significantly shorter than those of the wild type.The results of paraffin sections and electron microscopy scans confirmed that the width of the mutant htsd1 leaf sheath cells as well as the length and width of the stem cells were shortened compared with the wild type,this indicates that the dwarfism of the mutant htsd1 is caused by the shrinking and shortening of cells.In addition to this,the mutant htsd1 single plant yield increased with the increase of effective spike,and it is an excellent rice germplasm resource.2.HTSD1 is a novel OsAUX1 allele mutant geneBy fine mapping and whole genome sequencing of HTSD1,found a T-to-C base substitution in the third exon of the annotated gene LOC＿Os01g63770/OsAUX1within the localization interval,this resulted in a change of the encoded amino acid from tyrosine（Tyr）in WT to histidine（His）in htsd1.Further using transgenic technology,HTSD1 was verified to be a novel OsAUX1 allelic mutant gene.Bioinformatics analysis revealed that the OsAUX1 family genes in rice and Arabidopsis are highly conserved in structure and both contain an Aa＿trans structural domain in which the mutation site occurs,a result that implies that the alteration of the gene function may be related to the alteration of this structural domain.3.OsAUX1 was highly expressed at tiller budsThe q RT-PCR results showed that although OsAUX1 was expressed at all sites,it was more expressed at the root and tiller bud than at other sites.OsAUX1 own promoter+GUS transgenic plants,again,confirmed that GUS staining could be detected at various plant tissue sites.Using in situ hybridization,OsAUX1 expression was found to be higher at the tiller buds.4.OsAUX1 regulates rice tiller shoot growth through a hormonal pathwayCompared with WT,mutant htsd1 roots exhibited distinct characteristics caused by growth hormone deficiency.By growth hormone treatment,mutant htsd1 was found to require higher concentrations of growth hormone to inhibit its root growth,indicating that htsd1 is a growth hormone-insensitive mutant.In mutant htsd1,the expression levels of many growth hormone-related genes were altered,and external application of growth hormone inhibited its tillerogenesis.the expression levels of SL pathway-related genes were also altered,and OsAUX1 responded to the synthetic analogue of dictamnolide,rac-GR24,signaling and growth hormone signaling.These results imply that OsAUX1 may affect rice tillering by participating in the growth hormone pathway and in conjunction with the dictamnolactone pathway.5.OsAUX1 regulates rice tillering through growth hormone action in TB1The q RT-PCR and in situ hybridization results revealed that TB1 expression was significantly decreased at the rice mutant htsd1 tillers.The TB1 mutant Cas9^TB1,which was created using CRISPR/Cas9 technology,showed a highly significant increase in tiller number and its tiller occurrence was inhibited by external growth hormone application.In addition,TB1 could also respond to IAA and rac-GR24.Based on the above results,it is speculated that OsAUX1 may be involved in the pathway of regulation of tillering in rice by influencing the growth hormone action on TB1.6.OsAUX1 is a target gene of SPL7Five-GTAC-sites are present on the OsAUX1 promoter,which is the core site for Squamosa promoter binding protein（SPL）to bind DNA.Dual luciferase assays and yeast single hybridization experiments demonstrated that SPL7 can bind to and activate the expression of GTAC sequences on the OsAUX1 promoter region.In the mutant htsd1,the expression levels of several SPL family members were reduced,and it is speculated that there may be other SPL family members that act in concert with SPL7 to regulate the expression of OsAUX1.