Analysis Of Chloroplast Transport Mature Protein In Somatic Cells Of Arabidopsis Thaliana

Most chloroplast proteins are encoded by nuclear genes and synthesized in the cytoplasm by precursor proteins and transferred to chloroplasts through the TOC and TIC complexes on the chloroplast membrane.At the N-terminus of the chloroplast precursor protein there is a short peptide of about 20-150 amino acid residues,the transit peptide,which is required for the chloroplast precursor protein to enter the chloroplast.After the chloroplast precursor protein is introduced into the chloroplast,the proteolytic enzyme in the chloroplast matrix cleaves the transit peptide from the precursor protein.The chloroplast precursor protein without the transit peptide then folds and matures in the chloroplast.Because of the small pore diameters of TOC and TIC complexes on chloroplast membranes,many scholars believe that the complexes cannot transport mature proteins,and it is speculated that chloroplast precursor proteins should exist in the cytoplasm as unfolded peptide chains.However,in vitro experiments have shown that chloroplasts can transport mature proteins into the chloroplast.In this study,we provided evidence that chloroplasts can also transport mature proteins in plant somatic cells.Using Arabidopsis thaliana as a research material,we constructed the Pro35S:Atg8a-RbcS-GFP transgenic line to obtain a plant expressing the fusion protein ATG8A-RBCS-GFP.Since the RBCS transit peptide is hidden inside the fusion protein ATG8A-RBCS-GFP,the fusion protein will not be transferred to the chloroplast and will mature in the cytoplasm.The cytoplasmic mature ATG8A-RBCS-GFP protein is cleaved by the ATG4 protein at the C-terminus of the ATG8A protein to produce the cytoplasmic mature fusion protein RBCS-GFP.It was observed that the chloroplasts of the Pro35S Atg8a-RbcS-GFP transgenic line showed bright chloroplast fluorescence,indicating that the mature protein RBCS-GFP can be transferred to the chloroplast.As a control,the transgenic line Pro35S:Atg8aG/A-RbcS-GFP(the Gly mutation of the ATG8A C-terminal cleavage site to ALa,which caused the cleavage to fail to proceed normally)exhibited only weak fluorescence.At the same time,the chloroplasts of the transgenic lines Pro35S:Atg8a-GFP and Pro35S:Atg8aG/A-GFP did not exhibit green fluorescence.Western Blot showed that the cleavage of the ATG8A-RBCS-GFP fusion protein was consistent as expected,whereas the ATG8AG/A-RBCS-GFP fusion protein did not cut as expected,but there was still a small amount of protein cut,which explains Pro35S:Atg8aG/A-RbcS-GFP transgenic lines showed only weak fluorescence.The above results indicate that mature proteins can also be transferred to chloroplasts in plant somatic cells.Using this system,we also studied the function of the phosphorylation site on the chloroplast transit peptide RBCS.Studies have shown that there are Ser sites that can be phosphorylated on many transit peptides.We found that the transgenic lines Pro35S:RbcSAla-GFP-H2B and Pro35S:RbcSAsp-GFP-H2B chloroplasts and nuclear are showing bright green fluorescence,unlike transgenic lines Pro35S:RbcS-GFP-H2B(H2B protein is a nuclear localization).Only chloroplasts have green fluorescence,indicating that mutations at the Ser site affect the efficiency of the precursor proteins input to the chloroplast.Meanwhile.The observation of transgenic lines Pro35S:mcherry-Atg8s-RbcSNT-GFP-H2B,we found only chloroplast showing a bright green fluorescence,while in Pro35S:mcherry-Atg8a-RbcSAla-GFP-H2B and Pro35S:mcherry-Atg8a-RbcSAsp-GFP-H2B transgenic line,only the chloroplast showed a bright green fluorescence,and the nucleus did not show green fluorescence.Because cut from the RBCSAla-GFP-H2B and RBCSAsp-GFP-H2B protein comprises mature RBCSA1a and RBCSAsp transit peptide.Pro35S:RbcSAla-GFP-H2B and Pro35S:RbcSAsp-GFP-H2B transgenic lines have no mature transit peptides.It shows whether the maturation of RBCS protein in the cytoplasm will affect the efficiency of RBCS protein input into the chloroplast,and further reveals that the phosphorylation site of the Ser participates in the maturation of the transit peptide.In a word,our findings provide for the first time evidence that chloroplasts can still transport cytoplasmic mature proteins in plant somatic cells.The phosphorylation site of the Ser on transit peptides is involved in the maturation of transit peptides.These results provide important guidance for the further study of the mechanism of chloroplast protein input into chloroplasts.