Functional Characterization Of Panicle Architecture Gene OsAFB6 And Tillering Angle Gene LAZY1 In Rice

Rice(Oryza sativa L.)is an important crop and the staple food for almost half of the population in the world.The demand for rice grain yield is gradually increased with the growth of population.Rice grain yield is a complex trait that is controlled by number of effective tillers per unit area,grains per panicle,kilo-grain weight.Among the three components,grains per panicle makes the greatest contribution to grain yield.Plant architecture is characterized by many traits like plant height,tiller angle,leaf angle and panicle morphology,of which tiller angle largely determines the number of effective tillers per unit area,and further regulates grain yield.Tiller angle is the important component of idea plant architecture.Hormones are involved in many processes of plant growth and development,and play important roles in plant architecture.Auxin distributed in plant with concentration gradient through polar,lateral and other transport ways.SCFTIR1/AFBs-Aux/IAAs-ARFs is the core flow of ubiquitin dependent auxin signaling pathway.Cytokinin has antagonistic action with auxin.Both hormones together regulate plant growth and development.The optimal concentrations of cytokinin and auxin are beneficial to crop production.We obtained a late flowering mutant when we developed transgenic plants.The mutants produced more primary branch number,more spikelets per panicle,smaller grains and significantly increased grain yield.Based on the mutant material,we conducted cellular,transcriptional and physiological analyses,and got the following results.1.Flanking sequence isolation and gene expression examination confirmed that the mutation was caused by the insertion of an overexpression vector into the promoter region of OsAFB6,and not by the ectopic overexpression of the target gene in the vector.2.There were many light and auxin responsive cis-elements located in the promoter region of OsAFB6 gene,which was preferred to express in the shoot apical meristem,spikelet primordium and reproductive parts(stigma and ovary).Light induced the expression of OsAFB6.3.RNA-seq and q RT-PCR analysis demonstrated that photoperiod gene Ehd1,florigen genes RFT1 and Hd3 a were all suppressed in the OsAFB6 overexpression mutant,resulted a delayed flowering either in long-day condition or short-day condition.We also found that OsAFB6 gene was involved in auxin transport and signaling pathway as well as cytokinin pathway.Gn1 a,encoding cytokinin oxidase,was downregulated in the OsAFB6 overexpression mutant,which decreased the degradation of cytokinin.4.Microsection and scanned electronic microscope technology analysis discovered that OsAFB6 overexpression made a large diameter of panicle neck with more vascular bundles in the mutant,which strengthened the transport of assimilates.Measurement of auxin and cytokinin concentrations in young panicle showed that the concentration of cytokinin was increased,but auxin concentration was decreased.This result well explained why the mutant had more primary branches and more grains per panicle at hormone level.5.Within a reasonable range,heading dates and grain yield of OsAFB6 overexpression lines were positively related to its expression level.Hence,in future breeding programs,it is encouraged to generate a series of lines with different overexpression levels of OsAFB6 for selection.The most appropriate lines for different regions will be determined with optimal heading date,which makes the plants fully utilize the local sunlight and temperature resources,and maximize grain yield in rice.In another research,we obtained a lazy statured mutant when we generated transgenic plants.Using biological,bioinformatics and genetic methods to analyze the mutant and a germplasm collection,we got the results below.1.A negative transgenic plant showed lazy like phenotype when we generated transgenic plants.But the flanked sequence was failed to isolate in the mutant using TAIL-PCR,which indicated that the lazy like phenotype was caused by mutation occurred in the process of tissue culture rather than the transgenic event.Comparative sequencing showed a 268-bp deletion in LAZY1 gene.The missing 268-bp fragment contained the whole last exon region and part of the previous intron and part of the 3'-UTR,which led to loss-of-function of LAZY1,and resulted in the prostrate phenotype.2.There were many light responsive cis elements in the promoter region of Lazy1,which could be induced under dark condition.Lazy1 gene was proved to strongly expressed in tissues with higher differentiation activities like plumules,young seedlings,and stems in the late stage of vegetative growth.3.According to the analysis of co-expressed genes with LAZY1 and examinations of expression levels of related genes,we found that Lazy1 regulated many auxin related genes,including auxin rapid responsive genes and auxin transporter genes.Therefore,we concluded that Lazy1 might be involved in auxin transport and signaling pathway.4.Sequence comparison showed that natural variation of Lazy1 were abundant among 529 cultivars and 474 wild rice accessions.All insertions/deletions did not cause reading frame shift,and no SNPs caused premature stop codon.Therefore,no non-functional lazy1 alleles existed in the natural resource.The variation of a fragment in exon 3 including 2 3-bp deletions and 2 SNPs was associated with tiller angles,indicated these variations differentiated strong and weak functional LAZY1 alleles.Even in a few wild rice accessions with prostrate growth,LAZY1 was also functional.Hence,we infer that in the natural resource including wild rice,variations of LAZY1 made a quantitative contribution to tiller angle,but not to the prostrate growth.