Identificationof The Inflorescence Architecture Gene DLB1 And Its Molecular Mechanism Onpanicle Axillary Meristem Development In Rice

Rice is one of the most important food crops and an ideal model plant of monocotyledons for genomic researches.Rice yield mainly depends on the development of axillary meristem,which determines the plant architecture and panicle shape.LAX1(LAX Panicle1)is a major gene controlling the development of axillary meristem in rice,but the molecular networkneeds to be studied deeply,in particular,little is known how spatiotemporal expression and transcriptional activity of LAX1 controls the development of axillary meristem.In this study,we identified a sparse-panicle mutant dlb1-D(defective lateral branching1 in panicle-Dominant),through genetics,cell biology and biochemical analysis,a molecular model of DLB1 and LAX1 transcription complexes cooperative regulation of panicle axillary meristemdevelopment was established.The major results are as follows:1.We identified a dominant mutation of a single gene line that exhibits sparse-panicle from our rice T-DNA insertional library.The phenotype of dlb1-D mutant was caused by T-DNA insertion in the promoter region of DLB1,that increasing its expression level.Overexpress DLB1 can reproduce the sparse-panicle phenotype.2.DLB1 encodes a nuclear transcriptional repressor containing an EAR motif(ERF-associated amphiphilic repression motif).Phylogenetic analysis showed that the DLB1 is classified into a distinct clade of PCF5(Proliferating Cell Factor5)transcription factor,their single mutant panicle is normal,but dlb1 pcf5 double mutants displayed sparse-panicle phenotype,which indicates that DLB1 and PCF5 functional redundancy in the regulation of panicle development.3.RNAin situ hybridization showed that DLB1 m RNA was detected in developedaxillary meristem and the expression of LAX1 is priority over DLB1 in the formation of axillary meristem.Immunostainingresultsshowed that LAX1 and DLB1 accumulation overlap in the developed axillary meristem.4.In vitropull-down assay and LCI(Luciferase Complementation Image)assayverified DLB1 physical interacts with LAX1.DLB1 interacts with ASP1(Aberrant Spikelet and Panicle1)through EAR motif by confirmed by yeast two-hybrid and LCI assay.5.Transcriptional activity assay showed that DLB1 represses the transcriptional activity of LAX1 via recruits transcriptional corepressor ASP1.Furthermore,Genetic analysis verified that DLB1 inhibits LAX1 transcriptional activity is necessary for axillary meristem formation.6.Using SELEX(Systematic Evolution of Ligands by EXponential enrichment)method to identified the LAX1 DNA-binding components and determine the core sequence of TTTTGCT/A/CNNNA.KEGG analysis of the down-regulated genes in RNA-seq data of lax1-7 and dlb1-D mutant showed that LAX1 transcription complexesmay involved in plant hormone signal transduction pathway.7.We adopt bioinformatics approach to screen the TTTTGCT/A/CNNNA core motif in rice genome;Os PID,Os PIN1 c,Os PIN1 d,Os IAA7 and LOGmay be thedirectly target genes of LAX1.QRT-PCR analyses verified that the expression level of Os PID,Os PIN1 c,Os PIN1 d,Os IAA7 and LOG in lax1-7 and dlb1-D mutant was decreased.EMSA(Electrophoretic Mobility Shift Assay)assays verified LAX1 could bind to the promoter region of Os PID,Os PIN1 c,Os PIN1 d,Os IAA7 and LOG.8.Ch IP-q PCR with acetylated histone H3 antibody showed that the acetylated histone level at Os PID,Os PIN1 c,Os PIN1 d,Os IAA7 and LOG were significantly increased in the dlb1 pcf5 double mutant compared with the wild type,that is consistent with the elevated expression levels of Os PID,Os PIN1 c,Os PIN1 d,Os IAA7 and LOG in the dlb1 pcf5 double mutant.Consistent with the reduced expression level of Os PID,Os PIN1 c,Os PIN1 d,Os IAA7 and LOG in the dlb1-D mutant,acetylated histone level at Os PID,Os PIN1 c,Os PIN1 d,Os IAA7 and LOG in the dlb1-D were also decreased,compared with the wild type.9.To speculate a molecular model of DLB1 and LAX1 transcription complexes cooperative regulation of panicle axillary meristemdevelopment: In inflorescence meristem,LAX1 m RNA is specifically expressed in the boundary region between the initiating axillary meristem and the inflorescence meristem,then,LAX1 protein is trafficked form the boundary region to the developing axillary meristem,and activatedthe downstream target gene(Os PID,Os PIN1 c,Os PIN1 d,Os IAA7 and LOG)expression,initiation of axillary meristem development.In the developed axillary meristem,DLB1 m RNA is expressed and DLB1 protein physically interacts with LAX1 through C terminal,and through the EAR motif recruits ASP1 to form a repressor complex,and then decrease the histone acetylation at the LAX1 target genes to repress its transcription,maintainthe normal development of axillary meristem.