Molecular Mechanism Of NdhP Protein Against High Light Stress In Cyanobacteria

Although it is well known that sunlight is required for the plant growth, excessintensity of light is seriously harmful to the growth. When the amount of light energyabsorbed by light-harvesting systems exceeds the energy consumption of plant cells,the excess light will produce the reactive oxygen species (ROS) molecule. Such activechemical molecule will lead to a severe photoinhibition by attacking photosyntheticapparatus. To adapt various environmental stresses such as high light and alleviate thedamage of photosynthetic apparatus, several strategies have been developed. Amongthem, cyclic electron transport around photosystem I (CET) can alleviate the damageof plants under stress conditions via building the transmembrane proton gradient(pH) and generating the additional ATP molecule.In cyanobacteria, the main route of CET is considered to be NADPHdehydrogenase (NDH-1)-mediated one (NDH-CET). Upon exposure of cells to highlight, certain cyanobacterial NDH-CET defective mutants, such as ndhS and slr1097, were found to have a high light-sensitive growth phenotype. However, themolecular mechanism of NDH-CET underlying the high light-sensitive phenotype isstill poorly understood. In this thesis, a high-light strategy was used to explore themolecular mechanism by screening the mutants from Synechocystis sp. strain PCC6803(hereafter Synechocystis6803) transformed with a transposon-bearing library.Subsequently, the protein that influenced NDH-CET activity was identified andanalyzed using molecular biology, physiological and biochemical approaches. Theserelated results were summarized as follows:(1) Two high light-sensitive mutants were isolated from Synechocystis6803transformed with a transposon-bearing library using a high-light screening strategy.Analyses of the chlorophyll fluorescence and molecular biology indicated that amutation of sml0013(ndhP) gene may impair the NDH-CET activity, therebyproducing the high light-sensitive growth phenotype. To confirm this possibility, weconstructed an ndhP-deletion mutant (ndhP) and analyzed its NDH-CET activity bymeasuring chlorophyll fluorescence and P700oxidation reduction kinetics. The results indicated that the absence of NdhP impairs NDH-CET activity.(2) To reveal how deletion of NdhP impaired NDH-CET, we analyzed theaccumulated level and assembly efficiency of NDH-1complexes in thylakoids. Theresults indicated that the absence of NdhP protein impairs assembly efficiency ofNDH-1L complex, but not accumulated amount of NDH-1complexes in the thylakoidmembrane. We therefore conclude that the disassembly of NDH-1L complex results inimpairing NDH-CET activity.(3) To understand how NdhP stabilized the NDH-1L complex, we investigatedsubcellular localization of NdhP protein. The results indicated that NdhP is a singletransthylakoid protein and its C-terminal tail locates on the lumen side of thethylakoid membranes. Further experimental results from the proteomics and reversegenetics suggested that NdhP is an exclusive subunit for NDH-1L complex andessential to stabilize the complex.(4) To test whether the C-terminus of NdhP is required to stabilize the NDH-1Lcomplex, we constructed an ndhP C-terminal-deletion mutant (ndhP C). TheC-terminus deletion of NdhP resulted in the disassembly of NDH-1L complex,similarly to the ndhP mutant. Furthermore, the N-terminal extension and membranehelix of NdhP did not rescue the disassembly of NDH-1L complex. We thereforeconclude that the C-terminal tail of NdhP is essential for stabilization of the NDH-1Lcomplex.In conclusion, this thesis first identified an exclusive novel subunit of NDH-1Lcomplex, NdhP protein, in a unicellular cyanobacterium Synechocystis6803andrevealed its physiological function and related molecular mechanism underlying thealleviation under high light. These findings will further provide novel insights into theimportant physiological function of NDH-CET and the molecular structure of NDH-1complexes in cyanobacteria.