Exploration Of Iron-supply Pathways Of Iron-sulfur Cluster In Chloroplast

Chloroplasts play an important role in plant metabolism and energy conversion,and they are crucial for maintaining the normal growth and development of plants.There are many ironsulfur proteins(Fe-S proteins)in chloroplasts,which play important roles in photosynthesis,chlorophyll metabolism,amino acid metabolism and other pathways.Iron-sulfur clusters(FeS clusters),as the ligands of Fe-S proteins,are necessary for the function of Fe-S proteins.Chloroplast Fe-S clusters are mainly assembled on the SUFBC2D protein complex,but the source of Fe is largely unknown.Until recently,a study found that two similar DNAJ proteins,DJA5 and DJA6,can bind iron and directly transfer Fe to the SUFBC2D complex to participate in the biosynthesis of Fe-S clusters.However,how iron is transported from chloroplast envelope to DJA5/6 remains unclear.This research aims to answer two key questions in this process:first,which of the Fe transporters on the inner chloroplast membrane is upstream of Fe-S cluster synthesis;second,how Fe is delivered from transporter to DJA5/DJA6.Through a series of genetic,physiological and biochemical experiments,this study has obtained the following main research results:(1)The inducible PIC1-RNAi lines were created,and 3-week-old wild type(WT)and PIC1-RNAi plants were sprayed with the inducer estradiol.After 1 week,and the young leaves of the RNAi plants showed yellowing phenotype,but the WT grew green leaves normally.There was no significant difference in the protein content of SUFB and SUFC in yellow and WT green leaves with the same developemtnal stage.This result suggests that PIC1 does not affect the biogenesis of Fe-S clusters within chloroplasts.(2)The abci10 mutant were abtained by using CRISPR/Cas9 to edit ABCI10.The leaves of the abci10 mutant plants showed an albino phenotype.The contents of SUFB and SUFC in albino leaves were significantly lower than that in the same age leaves of WT.This result suggests that ABCI10 on the chloroplast inner envelope provides Fe at the upstream of the SUFBC2D protein complex.(3)To explore how Fe is transferred from ABCI10 to DJA5/DJA6.In this study,proximity labeling technology combined with mass spectrometry were eemployed to find the candidate proteins that can interact with the iron transporter ABCI10.It was found DJA5/DJA6 are the candidates of interactores.(4)To verify the interaction between ABCI10 and DJA5/DJA6,this study further used bimolecular fluorescence complementation(BiFC)experiments to show that ABCI10 interacts with DJA5/DJA6.(5)To further verify the interaction between ABCI10 and DJA5/DJA6,the interaction between ABCI10 and DJA5/DJA6 was proved by yeast two-hybrid(Y2H)experiments.In summary,this study found that the iron transporter ABCI10 on the chloroplast inner envelope is an upstream protein that provide Fe for Fe-S cluster biogenesis in the chloroplast.proximity labeling,BiFC and Y2H experiments showed that ABCI10 could directly interact with DJA5/DJA6 to transfer Fe to DJA5/DJA6,and then the Fe can be delivered to SUFBC2D for the synthesis of Fe-S clusters.