Molecular Mechanisms Of DEGP Protease Protect PSⅡ From Heat Stress In Arabidopsis Thaliana

Photosystem II ( PSII), which catalyzes light-dependent water oxidation and plastoquinone reduction in chloroplasts, is a large pigment-protein complex in the thylakoid membrane. PSII consists of >20 intrinsic and extrinsic proteins. The PSII reaction center complex is composed of the D1 and D2 proteins, the a- and b-subunits of cytochrome b559, and the PsbI protein. The D1 and D2 heterodimer binds all the essential redox components of PSII required to transfer electrons from the manganese cluster of the water-oxidizing complex to the plastoquinone pool.A distinct feature of PSII is that it is very sensitive to environment, the PSII reaction center D1 protein being the main target for environment stress induced damage among PSII proteins. Damaged D1 proteins are degraded and subsequently replaced with newly synthesized copies. This efficient repair mechanism must be essential to maintain PSII in a functional state. Although turnover of the D1 protein has been extensively studied, it was not clear about the methanism of proteases responsible for the degradation of the damaged D1 protein until now.The widely distributed DEGP proteases play important roles in the degradation of damaged and misfolded proteins. Arabidopsis thaliana contains 16 DEGP-like proteases, four of which are located in the chloroplast. Here, we show that DEG5 and DEG8 form a hexamer in the thylakoid lumen and that recombinant DEG8 is proteolytically active toward both a model substrate (β-casein) and photodamaged D1 protein of photosystem II (PSII) , producing 16-kD N-terminal and 18-kD C-terminal fragments.To study the in vivo function of DEG5 and DEG8, we obtained Arabidopsis lines from the SALK collections in which T-DNA was inserted into DEG5 and DEG8 , the mutants were named deg5 and deg8, respectively. Inactivation of DEG5 and DEG8 resulted in increased sensitivity to heat stress. The degradation of the D1 protein was slower in the mutants than in the WT plants. Furthermore, the levels of other PSII reaction center proteins tested remained relatively stable in the mutant and WT plants following high-temperature treatment. Thus, DEG5 and DEG8 are important for efficient turnover of the D1 protein and for protection against heat stress in vivo. The deg5deg8 double mutant showed increased heat sensitivity and reduced rates of D1 degradation compared with single mutants of deg5 and deg8.A 16-kD N-terminal degradation fragment of the D1 protein was detected in wild-type plants but not in the deg5deg8 mutant following in vivo photoinhibition.Therefore, our results suggest that DEG5 and DEG8 have a synergistic function in the primary cleavage of the CD loop of the PSII reaction center protein D1, thus, they remain the efficient turnover of PSII.