RPT2a, A 26S Proteasome Subunit, Regulates Inflorescence Meristem Maintenance And Architecture In Arabidopsis

Flowering plants display an enormous morphological diversity of the inflorescence architectures,depending on the number of flowers that are formed and the position where flowers are initiated on the inflorescence axis.The development of inflorescence architectures has important effects on fruits and seeds production,and crop yield.Elucidating the genetic networks that control inflorescence development is essential to understanding the evolution of plant form,and molecular breeding in key architectural traits of crops.In this study,we identified RPT2 a which is highly expressed in inflorescence and regulates the maintenance of inflorescence meristem(IM)and inflorescence architecture in Arabidopsis.The main results are as follows:(1)rpt2a-2 mutants display cymose inflorescence in Arabidopsis.Unlike the racemose inflorescence of the Arabidopsis wild-type plants,rpt2a-2 mutants display a kind of cymose inflorescence phenotype.During the development of rpt2a-2inflorescences,the floral meristem(FM)initiated from the apex of the inflorescence stem and developed into a flower.After that,a smaller sized IM was produced from the flanks of the apical FM,continually generated the new apical FM and maintained its meristematic activity at the flanks.This differentiation pattern of IMs repeats for several rounds to form a zigzagshaped inflorescence and eventually terminated with a flower as a determinate pattern.These results indicate that mutation in RPT2 a leads to cymose inflorescence in Arabidopsis.The cymose inflorescence of rpt2a-2 were completely rescued and replaced by the racemose inflorescence in the RPT2apro::RPT2a-GFP rpt2a-2 transgenic lines.The signals of RPT2a-GFP were detected broadly in both the IMs and the FMs.Additionally,RPT2 a m RNA was accumulated at the apex of the inflorescence,including the IMs and the FMs.Thus,RPT2 a expresses in inflorescence and acts as a key regulator that is essential for determining the inflorescence architecture in Arabidopsis.(2)The meristematic activity of IM controlled by TFL1 expression was reduced in rpt2a-2 mutants.We investigated the transcription of the IM identity gene TFL1 and the FM identity gene LFY by the in situ hybridization experiments.Strong TFL1 m RNA was detected in the whole IMs at the apex of the inflorescence stem in wild-type plants.Whereas much weaker signal of TFL1 m RNA was detected in a central subset of cells in the lateral IM of rpt2a-2 inflorescences than that in the wild-type plants.LFY was expressed in FMs initiated on the flanks of the IMs in wild-type inflorescences.In rpt2a-2 mutants,the expression of LFY was localized in the apex of the inflorescence stem when the IM was differentiated into a flower.These results indicate that mutation in RPT2 a leads to abnormal expression of TFL1 and LFY in inflorescence.We further analyzed the inflorescence phenotypes of rpt2a-2 lfy and rpt2a-2 tfl1-11 double mutants.Unlike the cymose inflorescence in rpt2a-2 mutants,rpt2a-2 lfy double mutants showed a typical racemose inflorescence with an IM on the apex of the inflorescence,which are similar to those in the wild-type plants and lfy mutants.Moreover,the phenotypes of early several flowers into inflorescence shoots were also observed on the inflorescence stem of the rpt2a-2 lfy double mutants.The rpt2a-2 tfl1-11 double mutants were phenotypically indistinguishable to single homozygous tfl1-11 inflorescences,indicating the phenotypic epistasis of tfl1-11.As the expression of TFL1 was repressed in the IMs of the rpt2a-2 mutants,we generated p UFO::TFL1 constructs and transformed it into the rpt2a-2 mutants.Overexpressing TFL1 in the rpt2a-2 mutants completely rescued the phenotypes of inflorescence and gave rise to racemose inflorescence.Thus,RPT2 a controls inflorescence architecture by regulating the expression of TFL1 and LFY.(3)TMT-label quantitative proteomics in wild-type and rpt2a-2 inflorescences.To analyze the effects of mutation in RPT2 a on protein accumulation,TMT proteomics analysis was performed between the inflorescence of the wild-type plants and the rpt2a-2mutants.In total,1082 differentially expressed proteins were detected,and they were composed of 744 proteins up-regulated and 338 proteins down-regulated at least 1.5-fold in rpt2a-2mutants compared with the wild-type plants.CC analysis showed that differentially expressed proteins are enriched in proteasome and chromosome;MF analysis showed that differentially expressed proteins are enrich in epigenetic and chromatin binding;BP analysis showed that differentially expressed proteins are enriched in meristem and stem cell development.Many components of ubiquitin-26 S proteasome system and several epigenetic regulators were differentially expressed in rpt2a-2 mutants.(4)Excessively accumulated MET1 protein in rpt2a-2 leads to the mutated inflorescence architecture.TMT-label quantitative proteomics showed that MET1 protein increased significantly in rpt2a-2 mutants.The signal of g MET1-YFP was significantly stronger in rpt2a-2inflorescences than that in the wild-type plants.In addition,the level of MET1 protein in rpt2a-2 inflorescences was noticeably high compared with the wild-type inflorescences using the Western-Blot with the specific antibody of MET1.Although the transcriptional levels of MET1 m RNA in the rpt2a-2 mutants were not significantly different compared with the wild-type plants,excessive accumulation of MET1 might be caused by the mutation of RPT2 a in inflorescence.We crossed the met1-1 into the rpt2a-2 mutants to generate the double mutants and compared the phenotypes of inflorescence architecture between the single and the double mutants.Unlike the cymose inflorescence in rpt2a-2 mutants,rpt2a-2 met1-1 double mutants showed recovered typical racemose inflorescence as those in met1-1 mutants.These results suggested that the cymose inflorescence structure caused by the mutation of RPT2 a is due to the excessive accumulation of MET1.(5)The TFL1 loci was hypermethylation due to the excessive accumulation of MET1 in rpt2a-2 mutants.In rpt2a-2 mutants,excessive accumulation of MET1 can improve the DNA methylation level of downstream genes,and significantly increased the DNA methylation level of TFL1.DNA methylation modification occurs on a transposon in TFL1 promoter(-1276 to-1000).In addition,the expression level of TFL1 increased significantly in met1-1 mutants.These results indicated that the expression of TFL1 was influenced by MET1 mediated DNA methylation.In conclusion,we identified the novel function of RPT2 a for the specific regulation of the inflorescence architectures in Arabidopsis.Mutation of RPT2 a resulted in the changes of the inflorescence architectures from the raceme of Arabidopsis into the cyme,through mediating the expression of TFL1 and LFY.Furthermore,the loss-of-function of RPT2 a involved in the specific downregulation of TFL1 expression in IMs was regulated by DNA hypermethylation.Therefore,RPT2 a acts as a critical factor that is required for regulating the inflorescence architecture in Arabidopsis.Our data provides new information for establishment of inflorescence architecture mechanism.