Functional Study Of OsbZIPX,A New Transcription Factor Highly Expressed In Flower In Rice

Rice is most self-pollinating crop.During anthesis,lodicule swelling is the driving force for the opening of a rice floret.Rice floret opening comprises several processes,the swelled lodicule levering away the lemma and palea,anthers exposure to outside by filament elongation and subsequent dehiscence for spreading the pollens,meanwhile two pistils bend out ready for fertilizing.Rice floret opening is strictly controlled genetically,and can be easily affected by environmental factors such as light and temperature.Rice anthesis at a proper time,for example,avoiding high temperature of noon,is critical for a good yield and hybrid rice breeding.Studies showed that jasmonic acid(JAs),soluble sugar,inorganic iron movement and accumulation of carbon dioxide all contribute to rice floret opening.However,the molecular mechanism is still remaining obscured,as lacking of ideal rice cleistogamy mutant.OsbZIPX is a flowering stage high expressing transcription factor,bzipx T-DNA insertion mutant of OsbZIPX showed cleistogamy at anthesis.Besides,other agronomic traits were only slightly affected.Thus,bzipx provide a desirable object for search the molecular mechanism of rice flower opening.In this study,biological informatics,modern molecular biology,reverse genetics were used,to find out the molecular mechanism of OsbZIPX mediated rice anthesis.Results were showed as below:1.bzipx showed cleistogamy phenotype.OsbZIPX is a panicle highly expressed transcription factor.Phenotype investigation revealed that The T-DNA insertion mutant bzipx plant is cleistogamy in the flowering stage.Furthermore,the plant height is slightly reduced,and the plant leaves are more erect compared with wildtype(WT)plants.Although florets of bzipx remained closure during anthesis,other processes of flowering such as filament elongation,anther dehiscence were not defected.Thus,the time point of anther reaching the top of glume shell of bzipx floret was used as the index of ‘anthesis’,called floret opening time(FOT)in this study.Phenotype evaluation showed that the floret opening time(FOT)of bzipx was delayed by 2 hours compared with WT.Besides,the lodicule volume、fresh weight of bzipx at FOT were decreased in bzipx.The bzipx lodicule is partially dysfunction in swelling.Scanning electron microscope(SEM)showed that lodicule epidermal cells of WT were fully expanded while the mutant did not.Histological paraffin section of lodicule confirmed these observations.Results showed that the lodicule of WT is much bigger and thicker than that of bzipx mutant and some parts of them even swell enough to embed in between lemma and palea during anthesis.All these results indicated that the deficient in lodicule development and swelling during anthesis might be the major cause of cleistogamy in bzipx mutant.2.OsbZIPX is mainly expressed in spikelet and may act as a transcrip tion activator.RT-qPCR showed OsbZIPX is expressing throughout the panicle developmental stage,and has a high level at early stage(0-2cm length panicl e).GUS staining showed that OsbZIPX promoter(0-4Kbps)drived OsbZIPX an d GUS fusion protein was highly detected in anther and lodicule.These results showed that OsbZIPX is a spikelet highly expressed gene.Furthermore,OsbZI PX protein can be detected mainly in nuclei by using GFP fusion and its trans activation activity can be also detected in yeast one hybrid assay.These results suggested that OsbZIPX protein may act as a tanscription activator.3.OsbZIPX maybe involved in BR pathway.Ectopic expressing OsbZIPX in rice Nipponbare under constitutive expression promoter,resulted in larger leaf angle compared to WT,which is thought to be related with BR pathway.Realtime quantitative PCR showed the OsbZIPX OE plants had a high expression level of OsCPD1 which is an important BR biosynthase gene.Paraffin section showed the lodicule cell of OsbZIPX OE lines is larger than WT.These results indicating OsbZIPX may regulate rice development through BR pathway.4.OsbZIPX promotes JAZs expression.Jasmonic acid can induce rice anthesis.JAZs play an important role in JA signaling.Both before and during anthesis,qRT-PCR showed the expression of several JAZs were significantly upregulated in the florets of OsbZIPX OE plants,while downregulated in the bzipx mutant flowers compared to that in WT.Furthermore,dual luciferase assay in rice protoplast also showed OsbZIPX can promote the expression of JAZs.5.OsbZIPX interaction with OsDLT in yeast.As full length OsbZIPX has strong transcription activation activity in yeast.Therefore,N terminal of OsbZIPX(0-265 amino acid residues)was used as a bait to screen rice panicle cDNA library.The BR signaling protein OsDLT was isolated in the yeast two-hybrid assay.These results indicating OsbZIPX might enhance BR pathway through OsDLT protein.Of course,these interactions need be further verified in the future.In conclusion,OsbZIPX may be involved in regulation of floret open by both BR and JA pathways.OsbZIPX may controlthe tissue or cell size,especially lodicule development through BR pathway while it could alsopromot anthesis by enhanceing JA signaling.More efforts are needed to verify this conclusion in future work.