Application of low temperatures during photoinhibition allows characterization of individual steps in photodamage and the repair of photosystem II
83
Citation
62
Reference
10
Related Paper
Citation Trend
Keywords:
Photoinhibition
Under light-stress conditions, the photosystem (PS)II reaction center D1 protein is photodamaged. The damage to the D1 protein is induced by singlet oxygen molecules and endogenous free radicals generated by the photochemical reactions of PSII. To maintain PSII activity, the oxidatively damaged D1 protein is replaced by a newly synthesized protein. Thus, degradation and removal of the photodamaged D1 protein in PSII are essential steps for maintaining the viability of PSII. In the present chapter, we describe the method to induce photoinhibition of PSII both in vitro and in vivo, and also the method to assay the processes closely related to the photoinhibition, including degradation of the damaged D1 protein and its crosslinking with the neighboring polypeptides. The method to analyze the protease activity in the stroma that recognizes and digests the crosslinked products of the D1 protein generated by the light stress is also described.
Photoinhibition
Cite
Citations (14)
Photoinhibition of photosystem II (PSII) activity and loss of the D1 reaction center protein were studied in PSII-enriched membrane fragments in which the water-splitting complex was inhibited by depletion of either calcium or chloride or by removing manganese. The Ca2+-depleted PSII was found to be the least susceptible to inhibition by light as reported previously (Krieger, A., and Rutherford, A. W. (1997) Biochim. Biophys. Acta 1319, 91-98). This different susceptibility to light was not reflected in the extent of D1 protein loss. In Mn-depleted PSII the loss of activity and the loss of the D1 protein were correlated, while in Cl-- and Ca2+-depleted PSII, there was very little loss of the D1 protein. The production of free radicals and singlet oxygen was measured by EPR spin-trapping techniques in the different samples. 1O2 and carbon-centered radicals could be detected after photoinhibition of active PSII, while hydroxyl radical formation dominated in all of the other samples. In addition, photoinhibition of PSII was investigated in which the functional Mn cluster was reconstituted (i. e., photoactivated). As expected this led to a protection against photoinhibition. When the photoactivation procedure was done in the absence of Ca2+ no activity was obtained although a nonfunctional Mn cluster was formed. Despite the lack of activity the binding of Mn partially protected against the loss of D1. These data demonstrate that, during photoinhibition, the extent of D1 loss is neither affected by the water-splitting activity of the sample nor correlated to the kinetics of PSII activity loss. D1 loss seems to be independent of the chemical nature of the reactive oxygen species formed during photoinhibition and seems to occur only in the absence of Mn. It is proposed that Mn binding protects against D1 loss by maintaining a protein structure which is not accessible to cleavage.
Photoinhibition
Cite
Citations (55)
P680
Photoinhibition
Pheophytin
Oxygen evolution
Cite
Citations (4)
We have studied photoinhibition of photosynthesis in the cyanobacterium Synechococcus sp. PCC 7942, which possesses two distinct forms of the photosystem II reaction-center protein D1 (D1:1 and D1:2). We report here that when cells adapted to a growth irradiance of 50 mumol.m-2.s-1 are exposed to an irradiance of 500 mumol.m-2.s-1, the normally predominant D1 form (D1:1) is rapidly replaced with the alternative D1:2. This interchange is not only complete within the first hour of photoinhibition but is also fully reversible once cells are returned to 50 mumol.m-2 x s-1. By using a mutant that synthesizes only D1:1, we show that the failure to replace D1:1 with D1:2 during photoinhibition results in severe loss of photosynthetic activity as well as a diminished capacity to recover after the stress period. We believe that this interchange between D1 forms may constitute an active component in a protection mechanism unique among photosynthetic organisms that enables cyanobacteria to effectively cope with and recover from photoinhibition.
Photoinhibition
Cite
Citations (114)
Isolation of photosystem-II reaction centres from pea leaves after photoinhibitory treatment at low temperature (0-1 degrees C) has provided evidence for the mechanism of degradation of the D1 protein in vivo. These isolated reaction centres did not appear to be spectrally distinct from preparations obtained from control leaves that had not been photoinhibited. Breakdown fragments of both the D1 and D2 proteins were, however, found in preparations isolated from photoinhibited leaves, and showed similarities with those detected when isolated reaction centres were exposed to acceptor-side photoinhibition. Analyses of the origin of D1 fragments indicated that the primary cleavage site of this protein was between transmembrane helices IV and V indicative of the acceptor-side mechanism for photoinhibition. The origins of other D1 protein fragments indicate that some donor-side photoinhibition may also have occurred in vivo under the conditions employed. We have shown that the spectral and functional integrating of the isolated photosystem II reaction centre complex is resistant to proteolytic cleavage by trypsin. Use of a more non-specific protease (subtilisin), however, caused significant destabilisation of the special pair of chlorophylls constituting the primary electron donor, P680, with a consequential loss of functional activity. Thus, it is possible that specific cleavage of photosystem-II reaction-centre proteins may occur in vivo following photoinhibitory damage without a significant change in structural integrity, a conclusion supported by the finding that photodamaged and normal reaction centres were isolated together.
Photoinhibition
P680
Cleavage (geology)
Cite
Citations (45)
Photoinhibition
Oxygen evolution
Oxygen-evolving complex
Cite
Citations (306)
Photoinhibition
Plant Physiology
Cite
Citations (58)
Photoinhibition
P680
Oxygen evolution
Oxygen-evolving complex
Cite
Citations (1)
The events of photoinhibition were examined at the molecular level. The evidence presented here suggests that the primary event of photoinhibition involves inactivation of photosystem II reaction centre function. Chlorophyll fluorescence emission and excitation spectra and fluorescence induction transients were suppressed after photoinhibitory treatment. Such fluorescence is directly dependent on photosystem II photochemistry. In addition, photoinhibition caused a decline in charge separation measured by the absorbance change at 320 nm arising from the light-induced reduction of the primary acceptor of photosystem II, QA. Inhibition of this parameter is indicative of damage to a component involved in primary photochemistry. That the effect of high light treatment could not be correlated with any loss of the D1 protein supports the suggestion that a cofactor involved in primary photochemistry is the initial site of photoinhibition. It is possible that D1 may eventually be lost as a result of such damage. The reaction centre chlorophyll of photosystem II, P680, is suggested to be the cofactor involved. Possible mechanisms of damage are discussed with reference to the prosthetic components of the reaction centre of photosynthetic bacteria.
Photoinhibition
P680
P700
Cite
Citations (44)