Functional Analysis Of NdhL On Stabilization Of NDH-1-PSI Supercomplex In Cyanobacteria

It has been found that some of the complexes on the cyanobacterial thylakoid membrane can be moved,resulting in functionally related complexes forming electron-transporting supercomplexes that are beneficial for stabilizing the complexes and exerting related functions,thereby contributing to the adaptation of cyanobacteria to various stress conditions.These supercomplexes include PBS-PSII-PSI,PBS-CpcL-PSI,PSII-PSI and NDH-1-PSI.Among them,the formation of the NDH-1-PSI supercomplex is beneficial for cyanobacteria to carry out cyclic electron transport around photosystem I,which can contribute to the survival of cyanobacteria under stress conditions.Recently,it was found that the deletion of NdhL,a subunit of NDH-1,does not affect the accumulation and assembly of NDH-1,but it leads to the collapse of the NDH-1-PSΙ supercomplex;the reasons for this are not well understood.In this dissertation,in order to understand the functional role of NdhL in stabilizing NDH-1-PSI supercomplex,the localization of NdhL were investigated in various subcomplexes of NDH-1,and the effects of deletion of Ndh L on the accumulation and assembly of each subcomplex and the activity of PSI were measured.The results are as follows:(1)The accumulation of NdhL in ΔndhA,M55 and ΔndhN was investigated by using SDS-PAGE and immunoblotting.It was found that the accumulation of Ndh L on the thylakoid membrane was severely affected only in ΔndhA,whereas in the other two mutants M55 and ΔndhN,the accumulation of NdhL was hardly affected.Since M55 and ΔndhN are mutants of the membrane arm and the hydrophilic arm respectively and ΔndhA is the mutant of the catalytic domain,the above results suggest that Ndh L is located in the catalytic domain,close to the membrane arm side of NdhA,and exists independently of the NDH-1 hydrophilic arm and membrane arm.(2)The accumulations of NdhS and NdhV,subunits of catalytic domain,were detected in ΔndhL and wild type by using BN-PAGE combined with immunoblotting.It was found that the deletion of Ndh L affects the assembly of NdhS and reduces substantially the accumulation of NdhV.Since NdhS and NdhV are located in the catalytic domain of NDH-1,the deletion of Ndh L affects the stability of catalytic domain.(3)The activity of PSΙ and PSΙΙ in ΔndhL and wild type were detected by chlorophyll fluorescence instrument.The results showed that the activity of PSΙ and PSΙΙ in the ΔndhL and the wild type were similar before the linear electron inhibitor DCMU was added,and even the activity of PSΙΙ in ΔndhL was slightly higher than that in the wild type.However,after the addition of DCMU to the cell cultures,the activity of PSΙ in ΔndhL was significantly reduced when compared to the wild-type.This feature resembles to the characteristic of the deletion of CpcG2,which is the linker protein of the NDH-1-PSI supercomplex.Therefore,these results suggest that Ndh L may serve as a linker between NDH-1 and PSI.In summary,the deletion of NdhL affects the stability of the catalytic domain.Meanwhile,Ndh L may serve as an important linking site between NDH-1 and PSI,and its deletion affects the formation of NDH-1-PSI supercomplex and impedes the electron transfer between NDH-1 and PSI,which can suppress an efficient execution of cyclic electron transport around photosystem I.