Functional Analysis Of Heading Date Related Genes OsLHY And Se14 In Rice

1 Molecular mechanism of the circadian clock gene OsLHY regulates the critical day length for heading in riceFlowering time(Heading date)of rice(Oryza sativa)significance affects its development,reproduction,yield,and adaptability.It is mainly regulated by endogenous genetic factors and environmental components.The circadian clock is an endogenous timing system that controls the rhythmic expression of biological processes in most organisms,and its core regulatory system is mainly constituted by a series of transcription translation feedback loop(TTFL),which enables organisms to anticipate and adapt to photoperiodic changes in the environment.Present study shows that plants respond to light signals through the biological clock to adapt to the dynamic changes of photoperiod and accurately control their flowering time.However,there are few studies on this mechanism in short day plant rice.It is of great significance to explore the interaction mechanism between photoperiod and circadian clock in heading regulation of rice.In previous work,the rice circadian clock core gene OsLHY was cloned by Mut Map and identified by genetically validated.It was found that the heading date of mutant oslhy was reversed under different sunshine lengths,and the heading was late at day longer than 12 h,and heading early at day length less than 11 h.Gene expression analysis showed that OsLHY might play a role by regulating OsGI-Hd1 pathway.On this basis,this study conducted a more in-depth study on the mechanism of OsLHY regulating heading date from biochemical and genetic perspectives,the results are as follows:(1)RNA-seq comparisons for global transcriptome showed that the DEGs enriched in the circadian pathway in both comparison groups(Nipponbare & oslhy,lvp1 & lem1).The results showed that OsLHY participates in the circadian rhythm pathway.(2)In order to find out the regulatory mechanism of OsLHY on OsGI,we first verified the direct binding of oslhy to the promoter of OsGI was by one-hybrid assays in yeast.Then DNA affinity purification-q PCR(DAP-q PCR)of OsGI gene loci showed that OsLHY binding to the promoter region of OsGI was predominantly in the region containing the CBS(CCA1 binding site)element.Furtherly,Electrophoretic mobility shift assays(EMSA)confirmed the specificity of this OsLHY binding CBS element and was dependent on the core DNA base of OsGI CBS element.(3)Using CRISPR/Cas9,we constructed oslhy osgi double mutants that were sown at different day lengths under natural conditions to investigate the heading stage,and we found that oslhy still plays a dual role in regulating the heading date in the osgi mutant background.The critical day length of the double mutant oshy(~ 13.5 h)was altered compared with that of the single gene mutant oslhy osgi(11 h ~ 12 h),indicating that the critical day length for OsLHY formation is OsGI dependent and that the OsGI-Hd1 pathway can regulate the critical day length in rice.(4)Moreover,the double mutant oslhy hd1 showed the same heading date as hd1 at 11.5 h,13.5 h and 14 h of day length,indicating that the dual function of OsLHY is entirely Hd1 dependent.Together,both biochemical molecular and genetic evidence show that OsLHY can fine tune critical day length by directly regulating OsGI,and Hd1 acts as the final effector of critical day length downstream of OsLHY.Our study elucidates a novel regulatory mechanism between the circadian clock and photoperiodic flowering.2 Histone demethylase Se14 affects brassinolide synthesis pathway in riceBrassinosteroids(BRS)are an important class of plant hormones that play important roles in normal plant growth,development,and adaptation to biotic and abiotic stresses.BRS in rice are responsible for controlling many important agronomic traits,such as plant type,flowering time,and seed yield.The genetic regulation of BR synthesis and signal transduction thus provides an avenue for crop improvement.Histone methylation,which plays an important role in chromatin structure and gene expression regulation,is involved in regulating many biological processes,but in plants,histone methylation regulation of BR biosynthesis and signal transduction has been rarely reported..In the previous work,a late heading date mutant lvp1(long vegetative phase 1)was induced by ethyl methanesulfonate(EMS)mutagenesis,and an early heading mutant em61 was obtained.Through Mut Map cloning technology,from which we identified a candidate gene,Se14(Photothermal Sensitivity 14).In addition to earlier heading date,both se14 and CRISPR/Cas9 knockout lines showed dwarf and erect leaves,the same as BR-deficient mutant phenotypes.Here,we investigated the mechanism by which Se14 is involved in the biological pathway of BR,as follows:(1)Lamina joint bending assay revealed that mutant se14 was hypersensitive to BR.Expression analysis of BR-related genes showed that BR synthesis gene D11 was significantly reduced in the mutant,whereas the BR signaling pathway genes GSK2,Os BZR1 were altered accordingly.(2)By in vitro one-hybrid assays in yeast and EMSA assays,we found that Se14 recruited D11 CTCTGYTY motifs by sequence-specific DNA-binding activity of the C2H2-Zn F domains.(3)Chromatin immunoprecipitation(Ch IP)analysis showed that the that H3K27me2 levels of se14 was specifically increased at the upstream region of the D11 chromatin.(4)Gene expression analysis after BR treatment showed that the pathway regulating D11 by negative feedback of BR signaling was disrupted in the mutant se14,(5)The expression level of Se14 was upregulated in both two allelic mutants of Os BZR1,osbzr1-1 and osbzr1-2,leading to the speculation that Os BZR1 may feedback regulate br synthesis by inhibiting the expression of Se14.Together,our results suggest that Se14 is a histone H3K27 demethylates,and its function during growth and development is diverse in rice.Se14 is recruited to D11 loci by the C2H2-Zn F domains directly binding CTCTGYTY motif,to modulate H3K27me2 levels and activate D11 expression,thus regulating BR biosynthesis.In addition,Se14 is possible target genes of Os BZR1 and was suppressed expression by it.This feedback regulatory mechanism maintains BR homeostasis in rice.