Preliminary Study Of Protein Phosphatase TOPP4 Involved On Autophagy In Arabidopsis

Under all kinds of stress conditions,autophagy plays a key role for regulating the survival of plants.Autophagy is a highly conserved protein degradation process dependent on lysosome and vacuole in eukaryotic cell.In plants,this pathway mainly occurs in the vacuole.Intracellular materials and damaged organelles are degraded through vacuole hydrolysis enzyme in order to maintain cell growth and homeostasis.In Arabidopsis thaliana,many key ATG genes have been identified.These genes are essential for autophagosome formation and autophagy regulation.ATG1/ATG13 complex is a key component for autophagy regulation and important for autophagosome being transferred to vacuole for degradation in Arabidopsis.However,post-translational modifications of key ATGs proteins by protein kinases and phosphatases are poorly reported in plants.In search of the interacting proteins of TOPP4,we carried out phosphoproteomics analysis and mass spectrometry identification.Compared with wild type,we found the phosphorylation level of an autophagy-releated protein ATGx in topp4-1 was significantly up-regulated in phosphoproteomics.At the same time,we identified peptide fragments of ATGx by mass spectrometry.Yeast two hybrid experiments demonstrated a strong interactions between ATGx and TOPP4.Through yeast two hybrid and bimolecular fluorescence complementation(Bi FC)experiments,we found that TOPP4 could interact with key proteins of autophagy,including ATGx,ATG8 a,ATG8e and ATGy.The mutant topp4-1 was hypersensitive to inadequate fixed-C and exhibited chlorisis and early senescence,but T-DNA insertion mutant topp4-3 did not have these defective phenotypes.Autophagosome numbers were greatly increased in topp4-1mutant after treated with the inhibitor Con A(Concanamycin A)under the condition of lacking fixed-C and fixed-N.But the autophagosome numbers were still much smaller than that in the wild type,implying that topp4-1 had defect in formation of autophagosomes.Under the condition of lacking fixed-C and fixed-N,usingPro ATG8e-GUS and Pro TOPP4-GUS transgenic plants,we found the expression pattern of TOPP4 was similar to ATG8 e which had strong expression in leaf,hypocotyl and root,indicating both of them had a similar expression pattern under the fixed-carbon or nitrogen starvation.In addition,yeast two hybrid experiments showed that ATGx could interact with some members of PP1 family,mainly including TOPP1,TOPP4,TOPP5,TOPP8,implying that PP1 is involved in plant autophagy processes through ATGx complexs.In conclusion,the above results suggested that PP1 is involved in autophagy processes in Arabidopsis.