Map-based Cloning And Functional Characterizations Of OsSHI1 And PFP_? In Rice

Tillering or branching is a common biological phenomenon and important determinant for plant architecture and yield.The final yield of cereal crops,especially for rice(Oryza sativa),wheat(Triticum aestivum),barley(Hordeum vulgare)and sorghum(Sorghum bicolor)is determined by three components:panicle number,grain number per panicle and grain weight,which are largely affected by tiller and panicle branch numbers.Therefore,the illumination of the regulatory mechanisms for tiller and panicle development is of great values for our understanding of plant morphogenesis and the future crop yield improvement.In this study,we obtained a rice mutant shil with dramatical variations in plant architecture,cloned the candidate gene and demonstrated its roles in regulating tiller and panicle development.The main contents and conclusions are as follows:1.The shil mutant was initially isolated by screening our 9311 60Co irradiation-mutation pool.Compared with the wild type,the tiller numbers of shil are significantly reduced.EdU staining and paraffin section observations revealed that the axilllary bud initiation is normal but its outgrowth was greatly retarded.In addition,compared with the wild type,shil mutant has dense and erect panicles accompanied with substantially increased primary and secondary branch numbers,its culm diameters are significantly increased with enhanced lodging resistance and it has shortened and widened dark-green leaves with elevated chlorophyll contents.These phenotypes of shil mutant indicated that the candidate gene plays important and pleiotropic roles in regulating tiller,panicle and leaf development.2.Genetic analysis of two F2 populations derived from reciprocal crosses between shil and WT(9311)suggested that the shil mutant phenotype was controlled by one recessive nuclear locus,given that the segregation ratio between wild type and mutant type individuals is quite close to 3:1.To identify the mutant gene,a F2 population was generated from a cross between the shil mutant and a rice Japanica cv.02428.Map-based cloning anchored the mutant locus to a 50 kb region on the long arm of chromosome 9 with 4 ORFs.Sequencing,RT-PCR and Western blot analysis revealed that ORF2 was deleted in the shil mutant.The genome fragment containing the wild-type ORF2 was transformed into the shil mutant and the obtained transgenic lines can complements the shil mutant phenotypes.ORF2 knockout transgenic plants were further generated by CRISPR-Cas9 genome editing approach.Compared with the wild-type plants,tiller numbers of ORF2 knockout transgenic lines were reduced while panicle secondary branch numbers were significantly increased,reminiscent of the phenotypes of shil mutant.Taken together,these analyses confirmed that ORF2 was indeed responsible for the mutant phenotypes.Sequence analysis revealed that ORF2 encodes a homologous transcription factor of the Arabidopsis short internode(SHI)family with the same C3HC3H zinc finger domain and the SHI family-specific IGGH domain.No SHI-like gene was reported in rice and ORF2 is hereafter referred to as OsSH11.Consistent with its function as a transcription factor,transient expression analysis in rice protoplasts showed that OsSHI1-GFP fusion protein was predominantly located in the nucleus.Yeast two-hybrid assays indicated that OsSHI1 exhibits quite weak transcriptional activation activity and can form homodimer via its C terminus.qRT-PCR and Western blot analyses validated that both the OsSHI1 mRNA and OsSHI1 protein are mainly expressed in axillary bud and young panicle tissues,consisting with its roles in regulating the tiller and panicle development.3.Yeast two-hybrid screening assay identified IPA1 as an interaction protein of OsSHIl.Detailed dissection of the interaction domains showed that both the N and C terminal regions of OsSHI1 can interact with C-terminus of IPA1.Further,bimolecular fluorescence complementation(BiFC)and Pull-down assays confirmed that OsSHI1 physically interacted with IPA1 both in vitro and in vivo.IPA1 regulates tiller and panicle development by directly activating the expression of OsTB1 and OsDEP1,and two and one putative SHI-recognition T/GCTCTAC motifs were found in the promoter regions of OsTBl and OsDEP1,respectively.Yeast one-hybrid assays suggested that OsSHI1 bound directly to the F4 promoter region of OsTBl and F3 promoter region of OsDEPl where the OsSHI1 recognition cis-elements reside.Electrophoretic mobility shift assays(EMSA)showed that OsSHI1 could bind specifically to these motifs and none-labeled competition probes could effectively reduce the binding ability of OsSHI1 in a dosage-dependent manner.Further,the mutation of core motif(T/GCTCTAC mutated to T/GAAAAAC)almost abolished the formation of the OsSHI1-DNA complex.Furthermore,specific anti-OsSHIl antibody was generated against the peptide of SRDPTKRPRARPSATTP.Followingly,Chromatin immunoprecipitation assay(ChIP)assay revealed that OsSHI1 could be specifically recruited to the P3 promoter regions of OsTB1 and OsDEPl.To further evaluate the DNA binding domain of OsSHI1,truncated versions of OsSHI1 protein were generated and we found that N terminal region conferred the DNA binding ability and pointed mutations of the conserved cysteines and histidine within the C3HC3H domain almost abolished the binding ability of OsSHIl to the promoter of OsTB1 or OsDEP1.Taken together,these results demonstrated that OsSHIl binds directly to the promoter region of OsTBl and OsDEP1.4.qRT-PCR analysis revealed that the expression levels of OsTBl and OsDEP1 were significantly upregulated in shil mutant compared to the wild-type.Transient expression assays in rice protoplasts showed that IPA1 greatly enhanced the transcriptional activity of OsTBl promoter.However,when co-expressed with OsSHIl,the transcriptional activation activity of IPA1 was significantly repressed.EMSA assays revealed that IPA1 could bind directly to the GTAC motifs in the OsTBl and OsDEP1 promoters and no shifted bands were observed for OsSHIl to the GTAC motifs.However,the binding ability of IPA1 to target probes was repressed gradually by increasing amounts of OsSHIl protein in the IPA1-DNA reactions.Furthermore,ChIP-qPCR assay with 35S:IPA1-Flag transgenic young seedlings indicated that IPA1-Flag was predominantly enriched to the P3 promoter region of OsTBl.However,due to the presence of excess OsSHIl protein in Actin1:OsSHI1/35S:IPA1-Flag transgenic young seedlings,the enrichment of IPA1-Flag protein to the P3 promoter region was significantly reduced.These results supported the speculation that OsSHI1 antagonizes the IPA1-promoted expression of OsTBl and OsDEPl by repressing the DNA binding affinity of IPA1.Compared with WT,the tiller number of 35S:IPA1-Flag transgenic plants was dramatically reduced due to the excess accumulation of IPA1-Flag proteins which can evidently enhance the OsTB1 expression.In contrast to the repressed tillering of 35S:IPA1-Flag transgenic plants,the Actin1:OsSHI1/35S:IPA1-Flag transgenic plants display obviously reduced plant height and somewhat recovered tillering ability.The expression level of OsTBl in Actin1:OsSHI1/35S:IPA1-Flag transgenic plants was significantly reduced compared with that in 35S:IPA1-Flag transgenic plants,even though still higher than that in the WT.Moreover,we downregulated the transcript abundance of IPA1 in shil mutant by RNA-interference approach.Obtained positive transgenic plants exhibit dwarfism,dramatically increased tiller number and decreased panicle size accompanied with remarkably reduced primary and secondaiy branch numbers,reriniscent of the phenotypes previously reported in IPA1-RNAi plants.Consistently,qRT-PCR analysis revealed that expression levels of OsTB1 and OsDEP1 were significantly reduced in IPA1-RNAi plants.Taken together,we concluded that OsSHI1 acts antagonistically on IPA1 to regulate plant morphogenesis in rice.5.Besides,we characterized two allelic rice mutants,pfp1-1 and pfp1-2 with floury endosperm consisting of loosely packed starch granules.Map-based cloning revealed that the mutant phenotypes were controlled by a gene encoding pyrophosphate:fructose-6-phosphate 1-phosphotransferase(PFP,EC 2.7.1.90)p subunit(PFP?).The identity of PFP? was further confirmed by genetic complementation test qRT-PCR analysis indicated that PFP? was ubiquitously expressed in various tissues and from the early to late stages of seed development.Further analyses showed that grain thickness and starch contents were significantly decreased while soluble sugar contents were evidently increased in pfp1-1 and pfp1-2 compared to WT.Our results suggest that cytosolic PFPp play an important tole in modulating carbon metabolism during grain filling in rice.