Molecular Mechanism Of Plastid Translation Elongation Factor Rab8d Regulates The Primary Root Development In Arabidopsis

Plant root system plays a key role in plant absorption of water,nutrients and in material synthesis.Rab8d,a plastid protein translation elongation factor,regulates not only chloroplast development,but also participates in the regulation of plant resistance to high temperature,as well as plasmid activity in pollen grains and plant leaf development.This study found that Rab8d also plays a key role in the primary root development.In this study,we observed and analyzed phenotype of Arabidopsis mutant rab8d.Treatment with chemical drugs,transcriptome analysis,genetic verification and other techniques were employed to explore the molecular mechanism of Rab8d regulating root development.The main results were summarized as follows:1.In this study,three Rab8d T-DNA insertion mutants(rab8d-l,rab8d-2 and rab8d-3)were screened,and the primary root phenotypes of rab8d mutants were analyzed systematically.It was found that the root length was positively correlated with the Rab8d expression level.The decrease number of meristem cells of rab8d mutant is an important reason for the growth retardation of the primary root.The rab8d mutant showed abnormal distribution of root starch granules.Programmed cell death(PCD)in stem cell niche(SCN)and cell division of the quiescent center(QC)were observed in rab8d mutant.In addition,the treatment of lincomycin and spectinomycin results suggest that the roles of Rab8d on root development may not depend on plastid protein translation.2.Construct Rab8d-GFP expression vector driven by Rab8d promoter(Rab8dpro:Rab8d-GFP),and transforming it into Arabidopsis.Then the homozygous transgenic lines that were stably expressing of Rab8dpro:Rab8d-GFP were screened.Confocal microscopy showed that Rab8d protein was evenly distributed in the plastid,and gathered in the plastid under 37? heat shock.Rab8d may also be involved in plastids/chloroplasts maturation during embryonic development and in the systematic distribution of plastids in roots.3.The auxin signal is reduced in the primary root of rab8d-2 mutant decreased,and the maximum concentration of auxin in QC was undetectable.The accumulation of PIN1,PIN2,PIN3 and PIN7 proteins was also significantly reduced in the root of rab8d-2,indicating that the decrease of auxin polarity transport may be an important reason for the reduced of auxin signal in the primary root.The expression levels of transcription factor encoding genes(SCR,SHR,PLT1 and PLT2)that regulate the activity of quiescent centers were all decreased.These results showed that the cell division in QC of rab8d-2 might be related to the impaired auxin,PLT and SHR signaling pathways.Treatment with low concentrations of exogenous auxin or brassinolide did not restore the phenotype of rab8d-2.4.Transcriptome sequencing of the roots of 5 DAG(Days after germination)wild type and rab8d-2 mutant seedlings was performed.Analysis results showed that 2,587 genes of rab8d-2 mutant were up-regulated and 2,149 genes were down-regulated,respectively.The mRNA levels of SCR,SHR,PLT1 and PLT2 were down regulated in rab8d-2 mutant,while the expression of WOX5 was up regulated,and these results were could explain the phenotypes observed by confocal microscopy.5.The expression level of SMR5 in rab8d-2 mutant was increased,and the cell cycle process in root meristem was affected.By constructing smr5 rab8d-2 mutant,it was found that the loss of SMR5 functions can partially restore the size of the meristem of rab8d-2 mutant,thus restoring root length.Results suggest that Rab8d caused reduction in RAM size was mediated by SMR5.6.The contents of H2O2 and superoxide anion in 5 DAG wild-type and rab8d-2 mutant seedlings were detected by DAB and NBT staining,respectively.The results showed that the accumulation level of superoxide anion in root of rab8d-2 mutant was higher than that in wild-type,especially in the elongation zone.However,GSH and DPI treatment had no significant effect on the phenotype of rab8d-2 mutants.These results suggest that induction of SMR5 in rab8d-2 mutants may be independent of ROS.7.The expression level of ERF115 in rab8d-2 mutant was increased in response to root damage signals.By crossed rab8d-2 with a dominant-negative mutant ERF115SRDX,the phenotype analysis showed that the reduced function of ERF 115 could rescue the structure of QC in rab8d-2 mutant.This result suggested that Rab8d may regulate the QC activity through ERF115.