Deletion Of PⅡ Increases NDH-1-Dependent Cyclic Electron Transport And Respiration In Synechocystis Sp.Strain PCC 6803

The carbon source and nitrogen source are components that consist of important proteins and nucleic acids in cyanobacteria.Therefore,the proportion of carbon and nitrogen needs to be maintained at a stable level to maintain the normal life activities of cyanobacteria.In order to maintain the balance between carbon and nitrogen,cyanobacteria develop a variety of mechanisms that regulate the balance between carbon and nitrogen,since the external environment for the growth of cyanobacteria is always changing.Cyanobacterial NDH-1 is located on the thylakoid membrane and mediates respiration,cycle electron transport around the photosystem I(PSI),and carbon dioxide uptake.These three functions mainly foucus on carbon metabolism.PⅡ is a signaling protein widely present in bacteria,archaea,and plants and it plays a key regulatory role in nitrogen metabolism.Accoding to previous studies,we have found that PⅡ dephosphorylates in the ndhB-deletion mutant(M55),suggesting that NDH-1not only regulates carbon metabolism,but also regulates nitrogen metabolism.So,can PⅡ protein regulate carbon metabolism by regulating the activity of NDH-1?In this thesis,the PⅡ-deletion mutant(ΔPⅡ)was constructed and its effects on the activity of NDH-1 were detected at the physiological level.The effect ofΔPⅡ on the activity of NDH-1 was verified by constructing double mutants of PⅡ and NDH-1subunits.The main research results were summarized as follows:(1)To test the effect of PⅡ protein on NDH-1 activity,we constructed the PⅡ deletion mutant(ΔPⅡ)and overexpression strain(OX-PⅡ).By monitoring the post-illumination rise in chlorophyll a fluorescence in WT,ΔPⅡ and OX-PⅡ,it was found that deletion of PⅡ can increase the total activity of NDH-1,and overexpression of PⅡ can reduce the total activity of NDH-1.(2)To determine the effect of PⅡ on the activity of NDH-1,we detected P700redox kinetics and re-reduction of P700~+in darkness in WT,ΔPⅡ,and OX-PⅡ.We found that deletion of PⅡ can increase NDH-1-dependent cyclic electron transport(NDH-CET),while overexpression of PⅡ can reduce NDH-CET.By measuring the respiration rate of both WT andΔPⅡ,it was found that deletion of PⅡ can increase NDH-1-dependent respiration rate(NDH-Respiration).(3)To confirm the effect of deletion of PⅡ on the activity of NDH-CET,we knocked out the NDH-1 subunit NdhV,which only affects the activity of NDH-CET,in the background of PⅡ deletion mutant,and measured its NDH-CET activity.The result showed that the NDH-CET activity in the PⅡ and ndhV double mutant(ΔPⅡ/ndhV)was lower than that inΔPⅡ single mutant,which indicated thatΔPⅡ indeed increased the rate of NDH-1-dependent cyclic electron transport.To confirm the effect of PⅡ deletion on NDH-Respiration activity,we knocked out the NDH-1subunit NdhD2,which affects the NDH-Respiration activity,in the background of PⅡ deletion mutant,and measured its NDH-Respiration activity.The result showed that NDH-Respiraton activity in the PⅡ and ndhD2 double mutation(ΔPⅡ/ndhD2)was higher than that inΔPⅡ mutant,indicating thatΔPⅡ indeed increases the respiratory rate mediated by NDH-1.(4)In order to determine the effects of NDH-1-mediated respiration and cyclic electron transport on the growth ofΔPⅡ mutant,we determined the levels of ROS in WT,ΔPⅡ,ΔPⅡ/ndhV,andΔPⅡ/ndhD2.The results showed that the ROS levels inΔPⅡ/ndhV were higher than those in WT,whereas the ROS amounts inΔPⅡ/ndhD2were lower than those in WT.This suggests that the increased activity of NDH-1 inΔPⅡ can reduce ROS content.In summary,the deletion of PⅡ in cyanobacteria increases the activity of NDH-1dependent respiration and cyclic electron transport,and consequently the increase of NDH-1 activity inΔPⅡ helps to reduce ROS accumulation in cyanobacterial cells.