| Chloroplast,as the primary organelles to define plants,are responsible for photosynthesis and numerous essential metabolism processes.Therefore,abnormal chloroplast biogenesis may seriously affect plant growth and development including embryo development.EMB2004 is predicted to be localized in chloroplasts,and the null mutation of EMB2004 is embryo-lethal in Arabidopsis.However,the function of EMB2004 protein,and which machineries mediate this protein are still unknown.In this study,we employed several strategies to create EMB2004 defective lines.With the application of a variety of physiological and biochemical methods,we screened and investigated proteins which interacted with EMB2004.The main results are shown below:1)Due to embryo lethality of null mutation of EMB2004 in Arabidopsis,we firstly created inducible RNAi lines of EMB2004 through inducible RNAi system.Unfortunately,although we chose two different segments from the code sequence of EMB2004 as the targets of RNAi respectively,there was no obvious phenotypical changes in positive plants after spraying with inducer estradiol.2)We subsequently designed two sgRNAs,which were separately used to guide Cas9 to edit different region on the C terminal of EMB2004.After that,we transformed the two vectors into Arabidopsis WT,and expected to obtain knock-down lines of EMB2004.We obtained EMB2004 edited lines,and some of them showed white spots on their cotyledons.The sequence of target edition regions was subsequently checked by PCR and sequencing.The results showed that EMB2004 was indeed edited,but the peaks were overlapped after the edited position.Quantitative reverse transcription(qRT)-PCR analysis revealed the reduction of the expression level of EMB2004.EMB2004 might be function in chloroplast development,because of white spots appeared on the leaves of gene edited lines.3)For obtaining the interactional information of EMB2004 in plants by coimmunoprecipitation(Co-IP),we created Arabidopsis EMB2004-FLAG overexpression lines.We found that several transgenic plants showed yellow phenotype in new boring leaves and stems after 4-leaves stage.Such phenomenon might be resulted from co-suppression of EMB2004,indicating that EMB2004 potentially involved in affecting chloroplast development.4)Transient overexpression of EMB2004-eGFP in the leaves of tobacco shown that the fluorescence of eGFP and chlorophyll were well overlapped,indicating EMB2004 is located in chloroplasts.5)With the utilization of bioinformatics methods,we collected and analyzed proteins,which had the possibility to interact with EMB2004.Thereafter,the interaction between EMB2004 and GET3a was confirmed by using Yeast Two-Hybrid(Y2H),Bimolecular Fluorescence Complementation(BiFC),and Pull-down assays.Considering that GET3a functions in the regulation of guiding the insertion of TA protein into the membrane of organelles and overexpression of GET3a could result in severe growth defects,which suggest that GET3a is involved in the regulation of guiding the correct insertion of EMB2004 into the envelope of chloroplasts.6)In order to figure out more hits about the regulating mechanism and the function of EMB2004 protein in chloroplast,proximity-labeling followed by MS analysis were employed to screen candidate proteins which may interact with EMB2004.The results provide important information for further studies on the function and regulatory mechanisms of EMB2004.In conclusion,we reported an embryo-lethal protein,EMB2004,which located to the chloroplast of Arabidopsis.EMB2004 affected chloroplast development while the function related mechanism is still unknown.Furthermore,the interaction between EMB2004 and GET3a was supported by several protein-protein interaction methods,indicating that the localization of EMB2004 in chloroplast envelope might be related to GET system.Several candidate proteins that might interact with EMB2004 revealed by AirID-MS,which provide hits for further investigation on the function and regulatory mechanisms of EMB2004. |
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