CPR1 Is Involved In The Pavement Cell Morphogenesis In Arabidopsis And The Preliminary Study On The Functions Of PP1 Family

CPR1 Is Involved in the Pavement Cell Morphogenesis in ArabidopsisThe cytoskeleton plays important roles in the control of cell growth direction and cell expansion.In our study,we performed ethyl methanesulfonate mutagenesis on Col-0 background and identified two mutants,j594 and j2928.Compared with wild-type plants,j594 and j2928 exhibited petite seedlings,narrower leaves,and clustered silique.Closer scrutiny by scanning electron microscopy and quantitative pavement cell(PC)analyses showed that the PCs in j594 and j2928 exhibited less and shorter lobe,smaller area,and larger circularity.These results indicated that the polar growth and cell extension of PCs in j594 and j2928 are seriously inhibited.Through map-based cloning,we found that j594 and j2928 are two new alleles of CPR1(Constitutive Expresser of Pathogenesis Related Genes 1).CPR1 encodes an F-box protein,two CPR1 constructs with mutations in the FBA(F-box associated)domain were overexpressed.The first construct had a point mutation(35S::CPR1I247V)and the second lacked the FBA domain(35S::CPR1?FBA).Transgenic lines of both 35S::CPR1I247V and 35S::CPR1?FBA exhibited similar phenotypes to those observed in cpr1-j594 and cpr1-j2928.However,plants overexpressing the normal CPR1 exhibited phenotypes similar to those observed in wild-type plants.These results suggested that the FBA domain is essential for normal CPR1 function and the mutated cpr1 protein has impact on the normal function of endogenous CPR1.Further investigation on the cytoskeleton showed that the arrangement of the microtubules and microfilaments was disrupted during PC development in cpr1.At the stage II,MT bundles can not arranged at the neck region in cpr1-j594 and cpr1-j2928 PCs and during the transition from stage II to III,the cortical diffuse microfilaments in cpr1-j594 and cpr1-j2928 PCs did not totally disappear and did not form apparent MF networks.These defects of cytoskeleton can cause the abnormal PC morphogenesis.Based on these results,we speculated that CPR1 controls the pavement cell morphogenesis through regulating the cytoskeleton.ROP-GTPases work as key switches that mediate the interactions between the actin and microtubule cytoskeletons,controlling the high interdigitation of PCs.In animal cells,small GTPase proteins,RAC1 and RAC3 are degradated through the ubiquitination pathway.However,yeast two hybrid(Y2H)and bimolecular fluorescence complementation(BiFC)assays showed that CPR1 cannot interact with ROP2,ROP4,and ROP6 directly.Genetic interaction analysis also showed the CPR1 was not involved in ROP-GTPase signaling pathway.It has been reported that CPR1 negatively regulates defense responses through PAD4(Phytoalexin Deficient 4)and EDS1(Enhanced Disease Susceptibility 1),two key components in the plant immune response network.In cpr1 pad4 and cpr1 eds1 double mutants,the cpr1-mediated PC morphogenetic defects were suppressed.In addition,the abnormal PC shapes of cpr1-j594 and cpr1-j2928 were rescued at 28 °C.Therefore,CPR1-mediated control of the cytoskeleton arrangement depends,at least in part,on the plant defense responses.Our results provide further insights into the relationship between the cytoskeleton and PC morphogenesis,and suggest that the cytoskeleton-mediated PC morphogenesis control might be tightly linked to plant defense responses.The Preliminary Study on the Functions of PP1 FamilyPhosphorylation is one of the most important post-translational modifications in regulating the protein functions.Protein kinases and protein phosphatases play major roles during this process.In recent years,researches have paid more attention to protein kinases than protein phosphatases,thus the study on the protein phosphatases is relative hysteresis.In this experiment,we made a primary research on the functions of 9 TOPP members of protein phosphatase 1(PP1)family.Previous researches have discovered that TOPP4 was involved in gibberellin and auxin signaling pathways in Arabidopsis,thus we wondered if the other members of TOPP also have the some corresponding biological functions.We found that each member of TOPP family was mainly localized in the nucleus and the plasma membrane.To some degree,their expression patterns were uniform,but they also exhibited some specificity.These results suggest that TOPP family members are functionally redundant during plant growth and development.The T-DNA insertion lines were obtained,double,triple,or quatruple mutants and TOPP-RNAi transgenic seedlings were constructed,however,all of them exhibited phenotypes similar to those observed in wild-type plants,further suggesting that TOPP family members have functional redundancy.Biochemical analysis showed that certain TOPPs can directly interact with PIN1,RGA and GAI,suggesting that TOPP family might work as a critical junction between gibberellin and auxin signaling pathways.To investigate the biological functions of the other members,we overexpressed other TOPPs in topp4-1 and found that the abnormal phenotypes of topp4-1 were partially rescued.This result indicated that other TOPPs have similar biological functions.The effects of three protein phosphatase inhibitors on root development showed that,only the PP1 specific inhibitor,PPI-2 can seriously inhibit the root growth and reduce the root gravitropism,and decrease the auxin levels in the root.This result further confirmed that PP1 have important biological functions.Based on the above results,we conclude that TOPP family members play important biological roles redundantly.Given the phenotype that PPI-2 seriously inhibited the root growth and gravitropism has not been reported in PP1 mutants,PP1 might be involve in other plant development process.Our researches establish the foundation for further research on the biological function of PP1 in future and also shed light on the role of phosphorylation modification in plant growth and development.