Identification Of PtoAED3 Interacting Proteins And Its Deubiquitination Activity In Populus Tomentosa

As an important and numerous member of the protease family,aspartic acid protease is widely found in various plants.It involved in the biological processes such as protein storage and degradation,sexual reproduction,aging,programmed cell death and response to adversity stress.Although many aspartic acid proteases have been isolated and identified in model plants such as Arabidopsis thaliana and Nicotiana tobacco,few research have been reported in woody plants,and the mechanism of the regulation on tree growth and development is still unclear.Previous researches in our lab have shown that PtoAED3 hydrolysis proteins of the secondary cell wall and affect the lignin content in xylem.The degree of ubiquitin in transgenic plants was also significantly changed.In this study,the interaction protein of PtoAED3 was screened and deubiquitination activity of PtoAED3 were identified in Populus tomentosa.AED3 interacting proteins were screened by GSTpull-down and mass spectrometry in Populus tomentosa.The results showed that a total of 128 candidate proteins related to various biological processes such as cell process,metabolic process,stress response and developmental process were identified.PtoAED3 interacting proteins were cloned and identified by Y2 H.The results showed that PtoGTPase Era protein could interact with PtoAED3 protein stably in vitro.The subcellular localization of PtoGTPase ERA-GFP was localized in chloroplasts of tobacco.Then PtoAED3 may affect the assembly of ribosome subunit of chloroplasts by interacting with PtoGTPase Era,thus affected the chloroplast and leaf development,gamete formation and other processes in Populus tomentosa.The ubiquitin level of tobacco which overexpressed PtoAED3 by transient expressed was decreased.Ubiquitin substrate was degraded significantly in tobacco leaves expressing only PtoUBQ11 by transient expressed,same substrate was also degraded significantly in tobacco leaves expressing PtoUBQ11 and PtoAED3 protease.There was no significant difference in the degree of degradation between them.In vitro degradation of dimer ubiquitin substrate Di-Ub showed that there was no significant difference between overexpressed PtoAED3 transgenic poplar and wild-type poplar.These results suggest that PtoAED3 cannot directly cleft ε-peptide linked Di-Ub,and the decreased ubiquitination level of OE-PtoAED3 plants may be the indirect result of PtoAED3.