Bertil Andersson

Photoinhibition of Photosystem II. Inactivation, protein damage and turnover.

Even though light is the source of energy for photosynthesis, it can also be harmful to plants. Light-induced damage is targetted mainly to Photosystem II and leads to inactivation of electron transport and subsequent oxidative damage of the reaction centre, in particular to the D1 protein. Inactivation and protein damage can be induced by two different mechanisms, either from the acceptor side or from donor side of P680. The damaged D1 protein is triggered for degradation and digested by at least one serine-type proteinase that is tightly associated with the Photosystem II complex itself. The damaged Photosystem II complex dissociates from the light-harvesting antenna and migrates from appressed to non-appressed thylakoid regions where a new D1 protein is co-translationally inserted into the partially disassembled Photosystem II complex. D1 protein phosphorylation probably allows for coordinated biodegradation and biosynthesis of the D1 protein. After religation of cofactors and assembly of subunits, the repaired Photosystem II complex can again be found in the appressed membrane regions. Various protective mechanisms and an efficient repair cycle of Photosystem II allow plants to survive light stress.

Too much of a good thing: light can be bad for photosynthesis

Even though light is the ultimate substrate for photosynthetic energy conversion, it can also harm plants. This toxicity is targeted to the water-splitting photosystem II and leads to damage and degradation of the reaction centre D1-polypeptide. The degradation of this very important protein appears to be a direct consequence of photosystem II chemistry involving highly oxidizing radicals and toxic oxygen species. The frequency of this damage is relatively low under normal conditions but becomes a significant problem for the plant with increasing light intensity, especially when combined with other environmental stress factors. However, the plant survives this photoinhibition through an efficient repair system which involves an autoproteolytic activity of the photosystem II complex, D1-polypeptide synthesis and reassembly of active complexes.

The Thylakoid FtsH Protease Plays a Role in the Light-Induced Turnover of the Photosystem II D1 Protein

The photosystem II reaction center D1 protein is known to turn over frequently. This protein is prone to irreversible damage caused by reactive oxygen species that are formed in the light; the damaged, nonfunctional D1 protein is degraded and replaced by a new copy. However, the proteases responsible for D1 protein degradation remain unknown. In this study, we investigate the possible role of the FtsH protease, an ATP-dependent zinc metalloprotease, during this process. The primary light-induced cleavage product of the D1 protein, a 23-kD fragment, was found to be degraded in isolated thylakoids in the dark during a process dependent on ATP hydrolysis and divalent metal ions, suggesting the involvement of FtsH. Purified FtsH degraded the 23-kD D1 fragment present in isolated photosystem II core complexes, as well as that in thylakoid membranes depleted of endogenous FtsH. In this study, we definitively identify the chloroplast protease acting on the D1 protein during its light-induced turnover. Unlike previously identified membrane-bound substrates for FtsH in bacteria and mitochondria, the 23-kD D1 fragment represents a novel class of FtsH substrate— functionally assembled proteins that have undergone irreversible photooxidative damage and cleavage.

A chloroplast DegP2 protease performs the primary cleavage of the photodamaged D1 protein in plant photosystem II

Although light is the ultimate substrate in photosynthesis, it can also be harmful and lead to oxidative damage of the photosynthetic apparatus. The main target for light stress is the central oxygen‐evolving photosystem II (PSII) and its D1 reaction centre protein. Degradation of the damaged D1 protein and its rapid replacement by a de novo synthesized copy represent the important repair mechanism of PSII crucial for plant survival under light stress conditions. Here we report the isolation of a single‐copy nuclear gene from Arabidopsis thaliana, encoding a protease that performs GTP‐dependent primary cleavage of the photodamaged D1 protein and hence catalysing the key step in the repair cycle in plants. This protease, designated DegP2, is a homologue of the prokaryotic Deg/Htr family of serine endopeptidases and is associated with the stromal side of the non‐appressed region of the thylakoid membranes. Increased expression of DegP2 under high salt, desiccation and light stress conditions was measured at the protein level.

The dynamic photosynthetic membrane and regulation of solar energy conversion.

Abstract The photosynthetic apparatus of higher plants is remarkably adaptable both to sudden stress conditions and to longer-term changes in light intensity. Clues to the molecular mechanisms involved in the intricate regulatory networks highlight the extraordinarily dynamic nature of the photosynthetic membranes. An intriguing lateral migration of certain proteins and protein complexes between the appressed and nonappressed regions of the membrane maintains a lateral heterogeneity of function between these two regions to optimize photosynthesis and minimize damage to the photosystems.

Identification, Characterization, and Molecular Cloning of a Homologue of the Bacterial FtsH Protease in Chloroplasts of Higher Plants

In an attempt to identify and characterize chloroplast proteases, we performed an immunological analysis of chloroplasts using an antibody against Escherichia coli FtsH protease, which is an ATP-dependent metalloprotease bound to the cytoplasmic membrane. A cross-reacting protein of 78 kDa was found in the thylakoid membrane of spinach, but not in the soluble stromal fraction. Alkali and high salt washes, as well as trypsin treatment of thylakoid membranes, suggest that the chloroplastic FtsH protein is integral to the membrane, with its hydrophilic portion exposed to the stroma. The protein is not bound to any photosynthetic complex and is exclusively located in the stromally exposed regions of the thylakoid membrane. Its expression is dependent on light, as it is present in green pea seedlings, but absent from etiolated ones. An Arabidopsis cDNA was isolated, and the deduced amino acid sequence demonstrated high similarity to the E. coli FtsH protein, especially in the central region of the protein, containing the ATP- and zinc-binding sites. The product of this clone was capable of import into isolated pea chloroplasts, where it was processed to its mature form and targeted to the thylakoid membrane. The trans-bilayer orientation and lateral location of the FtsH protein in the thylakoid membrane suggest its involvement in the degradation of both soluble stromal proteins and newly inserted or turning-over thylakoid proteins.

In vitro studies on light-induced inhibition of Photosystem II and D1-protein degradation at low temperatures

Abstract In order to get information on the molecular background behind the aggrevated photodamage to photosynthesis at low temperatures and to investigate the general mechanism of D1-protein degradation, isolated spinach thylakoids were subjected to photoinhibitory treatment at various temperatures. The results reveal that: (i) the Photosystem II electron transport per se is less sensitive to high light at low temperatures in contrast to the overall photosynthetic process; (ii) the degradation of D1-protein is severely retarded below 7°C; (iii) inhibition of Photosystem II electron transport and D1-protein degradation are separate events the two reactions could be completely separated in time; (iv) D1-protein is degraded by enzymatic proteolysis and not by a direct photocleavage reaction; (v) degradation of the D1-protein readily proceeds in the dark but its triggering for the proteolytic attack requires light; (vi) strong illumination at low temperature does not induce any lateral rearrangement in the location of Photosystem II; and (vii) D1-protein fragments can be identified in vitro and be used to verify the specificity of D1-protein degradation under various experimental conditions.

The initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes

During oxygenic photosynthesis in cyanobacteria and chloroplasts of plants and eukaryotic algae, conversion of light energy to biologically useful chemical energy occurs in the specialized thylakoid membranes. Light-induced charge separation at the reaction centers of photosystems I and II, two multisubunit pigment-protein complexes in the thylakoid membranes, energetically drive sequential photosynthetic electron transfer reactions in this membrane system. In general, in the prokaryotic cyanobacterial cells, the thylakoid membrane is distinctly different from the plasma membrane. We have recently developed a two-dimensional separation procedure to purify thylakoid and plasma membranes from the genetically widely studied cyanobacterium Synechocystis sp. PCC 6803. Immunoblotting analysis demonstrated that the purified plasma membrane contained a number of protein components closely associated with the reaction centers of both photosystems. Moreover, these proteins were assembled in the plasma membrane as chlorophyll-containing multiprotein complexes, as evidenced from nondenaturing green gel and low-temperature fluorescence spectroscopy data. Furthermore, electron paramagnetic resonance spectroscopic analysis showed that in the partially assembled photosystem I core complex in the plasma membrane, the P700 reaction center was capable of undergoing light-induced charge separation. Based on these data, we propose that the plasma membrane, and not the thylakoid membrane, is the site for a number of the early steps of biogenesis of the photosynthetic reaction center complexes in these cyanobacterial cells.

Protein phosphorylation and redox sensing in chloroplast thylakoids

Transduction of light dependent signals to redox sensitive kinases in photosynthetic membranes modulates energy transfer to the photochemical reaction centres and regulates biogenesis, stability and turnover of thylakoid protein complexes. The occupancy of the quinol-oxidation site of the cytochrome bf complex by plastoquinol and the redox state of protein thiol groups act as elements of the signal transducing chains.

Proteomics of Synechocystis sp. Strain PCC 6803 Identification of Plasma Membrane Proteins

Cyanobacteria are unique prokaryotes since they in addition to outer and plasma membranes contain the photosynthetic membranes (thylakoids). The plasma membranes of Synechocystis 6803, which can be completely purified by density centrifugation and polymer two-phase partitioning, have been found to be more complex than previously anticipated, i.e. they appear to be essential for assembly of the two photosystems. A proteomic approach for the characterization of cyanobacterial plasma membranes using two-dimensional gel electrophoresis and mass spectrometry analysis revealed a total of 57 different membrane proteins of which 17 are integral membrane spanning proteins. Among the 40 peripheral proteins 20 are located on the periplasmic side of the membrane, while 20 are on the cytoplasmic side. Among the proteins identified are subunits of the two photosystems as well as Vipp1, which has been suggested to be involved in vesicular transport between plasma and thylakoid membranes and is thus relevant to the possibility that plasma membranes are the initial site for photosystem biogenesis. Four subunits of the Pilus complex responsible for cell motility were also identified as well as several subunits of the TolC and TonB transport systems. Several periplasmic and ATP-binding proteins of ATP-binding cassette transporters were also identified as were two subunits of the F0 membrane part of the ATP synthase.