| The environmental temperature is one of the main factors affecting plant growth and development.Often plants have to suffer the hightemperature stress,which often indirectly causes photooxidative stress.The reactive oxygen species(ROS)molecule will lead to a severe photoinhibition by attacking photosynthetic apparatus.To adapt various environmental stress such as high temperature and alleviate the damage of photosynthetic apparatus,several strategies have been developed around photosystem I(CET)can alleviate the damage of plants under stress conditions via building the transmembrane proton gradient(ApH)and generating the additional ATP molecule.In cyanobacteria,the main route of CET is considered to be NADPH dehydrogenase(NDH-1)-mediated one(NDH-CET).Upon exposure of cells to high light,certain cyanobacterial NDH-CET defective mutants,such as ndhS and ndhV,were found to have a high light-sensitive growth phenotype.The function is determined by the structure of the complex.Unlike photosystem II,cyanobacterial NDH-1L complex possesses a high thermostability,since its expression and assembly amounts can strongly increase under conditions of heat stress.It is evident that such thermostability guarantees a high activity of NDH-CET under high temperature,which is essential to alleviate heat-sensitive growth phenotypes.However,the reason why the cyanobacterial NDH-1L complex has a high thermostability remains elusive.Here,we found that a significant deletion of NdhM,a subunit of cyanobacterial NDH-1L complex,did not have any effect on stabilization of NDH-1L complex under growth temperature but evidently destabilized NDH-1L complex under high temperature.Subsequently the mutant was identified and analyzed using molecular biology,physiological and biochemical approaches.These related results were summarized as follows:(1)To reveal the role of NdhM in stabilizing the NDH-1L under conditions of heat stress,we constructed a ?ndhM mutant by inserting the ndhM coding region using the spectinomycin resistance(SpR)cassette.PCR analysis of the ndhM locus showed an almost complete segregation(more than 90%)of the NdhM-less mutant allele.(2)Although a large complex(NDH-1L)of NDH-1 in cyanobacteria is well known for its high thermostability,the reason remains elusive.In this study,we found that a significant deletion of NdhM subunit did not have any effect on stabilization of NDH-1L complex under growth temperature.We also found that a significant deletion of NdhM subunit evidently destabilized the complex under high temperature.(3)Further researches were conducted regarding whether NdhM regulated assembly of NDH-1 via destroying NDH-1 assembly intermediates(NAIs).And the result show that there is no influence on assembly of NDH-1 complex in NdhM less.(4)During prolonged incubation of the thylakoid membranes of wild type and NdhM-less strains with DM at room temperature,a significant deletion of NdhM markedly accelerated the disassembly of NDH-1L complex.Moreover,in the NdhM-less mutant,disassembling of NdhK was much faster than that of other hydrophilic subunits such as NdhH and NdhI from the NDH-1L complex.It appears that NdhM functions in stabilizing NDH-1L under heat stress conditions possibly via protecting NdhK.(5)To test this possibility,we determined the interaction of NdhM with other 18 Ndhsubunits that had been previously identified in the NDH-1L complex,using a yeast two-hybrid system.A very strong protein-protein interaction was only found between NdhM and NdhK.This interaction was reinforced by the results of GST pull-down analysis and co-immunoprecipitation assay.We therefore conclude that NdhM interacts exclusively with NdhK,which greatly supports this possibility that NdhM protects NdhK under conditions of heat stress,thereby retarding the thermal collapse of NDH-1L complex.(6)To consolidate this possibility,we compared the levels of NDH-1L complex isolated from the thylakoid membranes of the WT,AndhK and NdhM less strains.Deletion of ndhK evidently reduced the assembly of NDH-1L complex and almost abolished the NDH-1M complex as deduced by the protein abundance of NdhH,NdhI and NdhM.The decreasing extent of NDH-1L in AndhK mutant was similar to that in NdhM less mutant grown under conditions of heat stress.And also there are many other similar properties in NdhM less and AndhK.Taking all these results together,In this study,we found that a significant deletion of NdhM subunit did not have any effect on stabilization of NDH-1L complex under growth temperature.But a significant deletion of NdhM subunit evidently destabilized the complex under high temperature.So we propose that NdhM is essential to maintain a high thermostability of NDH-1L complex via protecting NdhK in Synechocystis sp.strain PCC 6803. |
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