| Heading date is an important agronomic trait of rice which determines the planting area and sowing time of rice.Appropriate flowering time is crucial for the formation of crop yields,so it is of great significance to study the heading date of rice and make it flowering and fruiting at a suitable time.Histone methylation modification is involved in diverse biological processes of organisms,including flowering of plants.The methylation of histone H3K4 is catalyzed by a high evolutionarily conserved multi-protein complex,which is named COMPASS or COMPASS-Like complex according to member species and the function of methyltransferase.The function of each member in the complex had been revealed in Arabidopsis thaliana,while only OsWDR5 a and OsTrx1 were reported in rice.In this study,we identified two kinds of new members of the rice COMPASS-Like complex,OsRBL and OsASHLs(OsASHL1 and OsASHL2),and emphatically explored the functional mechanism of OsASHLs,the results are as follows:1.Yeast two-hybrid assay,luciferase complementation assay,bimolecular fluorescence complementation assay,and coimmunoprecipitation assay(Co-IP)in rice protoplast confirmed that OsRBL interacted with OsWDR5 a,OsASHL1 and OsASHL2,but OsWDR5 a did not interact with OsASHL1 and OsASHL2,indicating that OsRBL plays a “ bridge ” role between OsWDR5 a and OsASHLs.2.Subcellular localization experiment in tobacco transient expression system showed that OsWDR5 a and OsRBL were localized in the nucleus and cytoplasm,while OsASHL1 and OsASHL2 were only localized in the nucleus,which not only provides a spatial basis for their interaction,but also means that they may have different functions.3.osashl1 and osashl2 double knockout mutants(named osashls double mutants)were generated via CRISPR-Cas9 technology.Phenotypic analysis and statistical results showed that the osashls double mutants delayed heading date under both short day and long day conditions,interestingly,the difference between the heading date of osashls double mutants and the wild type was amplified under an extremely long day.This indicated that OsASHL1 and OsASHL2 could regulate the heading date of rice not only depending on photoperiod pathway but also involving in affecting the basic vegetative phase.In addition,osashls double mutants were more dwarf than wild type during the whole growth period of rice,according to transcriptome data,it should be presumed that OsASHL1 and OsASHL2 regulate plant height of rice by affecting BR biosynthesis and GA metabolic process.4.Western Blot experiment showed that the methylation levels of H3K4me1,H3K4me2 and H3K4me3 in osashls double mutants were significantly lower than those in wild type,indicating that OsASHL1 and OsASHL2 regulate mono-methylation,dimethylation,and tri-methylation of histone H3K4.5.RT-q PCR and Ch IP-q PCR experiment showed that H3K4me3 levels were lower at the OsVIL4 locus in osashls double mutants compared to the wild type,resulting in relative OsVIL4 transcript levels decreasing,therefore delayed the vegetative growth phase.6.OsWDR5 a overexpression transgenic rice was obtained by agrobacteriummediated method.Phenotypic analysis and statistical results showed that overexpression of OsWDR5 a led to late heading date of rice under short day and long day conditions,indicating that OsWDR5 a could regulate heading date of rice which not only depends on photoperiod pathway but also involves in effecting the basic vegetative phase.This is similar to the effect of osashls double mutant on heading date,suggesting that each member of the complex regulates heading date of rice through the same pathway in vivo.In conclusion,there is a COMPASS-Like complex composed of OsASHL1,OsASHL2,OsRBL,and OsWDR5 a in rice,and OsRBL plays a “ bridge ” role to assemble OsASHLs and OsWDR5 a.The COMPASS-Like complex regulates heading date by affecting the basic vegetative phase and photoperiod sensitive phase of rice,and other development progresses of rice through affecting BR biosynthesis and GA metabolic process. |
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