Research Of The Interaction Between GAAP1 And Its Binding Factors In Arabidopsis

Plant has to suffer with several adverse factors during its entire life history. In order to defense these adverse factors and maintain its growth and development, plant will activate several anti-stress responses. Recent researches suggest that anti-apoptotic effects play an important role in plant anti-stress responses. GAAP1 is a family member of TMBIM which could protect plant from stresses and negatively regulates ER-stress induced apoptosis. However, the mechanism of GAAP1-participated anti-stress and anti-apoptotic effects and whether it can be regulated by other factors are still unclear. Protein-protein interaction is one of the important ways for the regulation of protein function. Hence, screening and identification of GAAP1 interaction factors is very necessary for the study of GAAP1 regulatory mechanism. Previously, our lab has identified several potential interaction factors of GAAP1 using yeast two-hybrid assay. Here, the interactions between GAAP1 and these factors were validated by fluorescent microscopic assay and GST-pull down assay. In addition, the function of GAAP1 potential interaction factors in plant anti-stress responses was studied using their mutants. The main results are list as followed:1. Co-localization of GAAP1 and AtWLIM2b/MAPR3/PAS in tobacco leaf cells.For the investigation of subcellular localization of GAAP1 and its potential interaction factors, different fluorescent protein tagged GAAP1, AtWLIM2b, MAPR3 and PAS expression plasmids were transformed into tobacco leaf cells. And fluorescent microscopic results showed that GAAP1 mainly localized in the cell membrane of tobacco leaf cells. Interestingly, AtWLIM2b, MAPR3 and PAS also mainly localized in the cell membrane of tobacco leaf cells and showed co-localization with GAAP1. These results indicate that GAAP1 and AtWLIM2b/MAPR3/PAS may have interactions.2. Purified GST-GAAP1 protein can pull down MAPR3, AtWLIM2b and PAS expressed in tobacco leafs.To validate whether GAAP1 can directly interact with MPAR3 and PAS in vitro, MAPR3-TAP and PAS-TAP expression plasmids were constructed. In addition, I also constructed GST-GAAP1 plasmids that expressed in protease deficient E coli strain BL21 and purified GST-GAAP1 recombinant protein. The results of GST-pull down assay showed that GST-GAAP1 protein can pull down MAPR3-TAP and PAS-TAP, which suggest that GAAP1 can directly interact with MAPR3 and PAS in non-physiological condition in vitro.3. ER-stress inducer TM can influence subcellular localization of GAAP1 and AtWLIM2b in Arabidopsis.GAAP1 mainly localized at the cell membrane of loot cells in Arabidopsis. After TM treatment, the expression level and localization of GAAP1 inloot cells did not significantly changed. In contrast, GAAP1 mainly localized at the chloroplast of mesophyll cells in Arabidopsis. After TM treatment, the expression level of GAAP1 was significantly upregulated and GAAP1 translocated out of the chloroplast in mesophyll cells. These results indicated that GAAP1 could regulate the ER-stress of mesophyll cells in Arabidopsis. AtWLIM2b mainly localized at the cell membrane and nuclear of loot cells in Arabidopsis. After TM treatment, the fluorescent intensity of AtWLIM2b in nuclear was decreased. In contrast, AtWLIM2b mainly localized at the cell membrane and the chloroplast of mesophyll cells in Arabidopsis. TM treatment did not change the fluorescent intensity and localization of the eYFP-AtWLIM2b in mesophyll cells. These results suggest that the sucellular localization and the response to ER-stress of GAAP1 and AtWLIM2b are different, implying the different functions of these two factors.4. Loss of function mutant of AtWLIM2b did not influence the anti-stress responses to NaCl, TM and DTT in Arabidopsis.To investigate the function of AtWLIM2b and MAPR3 in pant anti-stress response, I constructed loss of function Arabidopsis mutant of AtWLIM2b and MAPR3 gene mapr3-land Atwlim2b-1. Results showed that AtWLIM2b mutation did not influence the anti-stress responses to NaCl, TM and DTT in Arabidopsis. In addition, MAPR3 mutation did not influence the anti-stress responses to DTT in Arabidopsis.5. MAPR3 loss of function mutation inhibits main root growth and euphylla genernation in ArabidopsisI constructed loss of function Arabidopsis mutant of MAPR3 gene mapr3-1. Results showed that MAPR3 mutation inhibits main root growth and euphylla genernation in Arabidopsis, suggesting that MAPR3 is involved in the regulation of early development in Arabidopsis.6. AtWLIM2b mutation resucues GAAPl dificiency induced inhibitory effect on early development in Arabidopsis.I constructed and identified GAAP1/AtWLIM2b double mutated Arabidopsis homozygote. Further investigation showed that GAAP1 mutation inhibited the germination of the seeds and euphyllagenernation in Arabidopsis, which suggest that GAAP1 play an important role in regulating early development of Arabidopsis. AtWLIM2b mutation significantly decreased the inhibitory effects of GAAP1 mutation on seed germination and euphyllagenernation. These results imply that AtWLIM2b is an inhibitor of early development in Arabidopsis. GAAP1 can suppress the function of AtWLIM2b via direct interaction which is important for the regulation of early development in Arabidopsis.Taken together, we indenfied the interation between GAAP1 and AtWLIM2b, MAPR3, PAS in plant cells throught fluorescent microscopy and GST-pull down assays. AtWLIM2b and MAPR3 did not significantly affect the anti-stress response in Arabidopsis.MAPR3 and GAAP1 mutation inhibited the early development of Arabidopsis, suggesting that MAPR3 and GAAP1 are involved in the regulation of early development in Arabidopsis. AtWLIM2b mutation rescued the inhibitory effects of GAAP1 mutation on early development in Arabidopsis, suggesting that AtWLIM2b is an inhibitor of early development in Arabidopsis. GAAP1 can suppress the function of AtWLIM2b via direct interaction in Arabidopsis.