Cecilia Sundby

Temperature-dependent changes in the antenna size of Photosystem II. Reversible conversion of Photosystem IIα to Photosystem IIβ

Abstract Changes in the organization of the chloroplast photosynthetic membrane at moderately elevated temperatures were detected by SDS-polyacrylamide gel electrophoresis, electron transport and fluorescence induction measurements and by subfractionation analyses of heat-treated thylakoids. The results revealed that above 30°C there is a dissociation of peripheral light-harvesting chlorophyll a b complex (LHC II) from Photosystem II. This is followed by a migration of Photosystem II and a portion of its tightly bound LHC II to the Photosystem-I-rich non-appressed thylakoid regions. This lateral migration includes all identifiable Photosystem II polypeptides and not only the reaction centre or core polypeptides. Concomitantly, there is a conversion of Photosystem IIα to Photosystem IIβ as judged from fluorescence induction measurements with the heated thylakoids. These results support the notion of Photosystem IIβ being localized in the non-appressed thylakoids and possessing a smaller antenna size. All temperature-induced changes required a background level of monovalent cations, and were partially reversible upon lowering the temperature. In the 30–40°C temperature range, where most of the changes in the organization and location of Photosystem II occurred, there is very little inhibition of electron transport capacity. We postulate that the temperature-dependent separation of Photosystem II from LHC II is a physiological mechanism to prevent overexcitation and subsequent damage of Photosystem II at high-light intensities that are accompanied by elevated temperatures.

The chloroplast small heat shock protein undergoes oxidation-dependent conformational changes and may protect plants from oxidative stress.

The nuclear-encoded chloroplast-localized Hsp21 is an oligomeric heat shock protein (Hsp), belonging to the protein family of small Hsps and alpha-crystallins. We have investigated the effects of high temperature and oxidation treatments on the structural properties of Hsp21, both in purified recombinant form and in transgenic Arabidopsis thaliana plants engineered to constitutively overexpress Hsp21. A conformational change was observed for the 300 kDa oligomeric Hsp21 protein during moderate heat stress (< or =40 degrees C) of Arabidopsis plants, as judged by a shift to lower mobility in non-denaturing electrophoresis. Similar changes in mobility were observed when purified recombinant Hsp21 protein was subjected to an oxidant. Exposure of Hsp21 protein to temperatures above 70 degrees C led to irreversible aggregation, which was prevented in presence of the reductant dithiothreitol. The transgenic plants that constitutively overexpressed Hsp21 were more resistant to heat stress than were wildtype plants when the heat stress was imposed under high light conditions. These results suggest that the physiological role of Hsp21 involves a response to temperature-dependent oxidative stress.

Characterization of two different subpopulations of spinach light-harvesting chlorophyll ab-protein complex (LHC II): Polypeptide composition, phosphorylation pattern and association with Photosystem II

Abstract The properties of the light-harvesting chlorophyll a b- protein complex of Photosystem II (LHC II) was analysed in subfractions isolated from phosphorylated or heated thylakoids. The results showed that LHC II is heterogenous with respect to polypeptide composition, phosphorylation kinetics and structural association with the core of Photosystem II. One LHC II subpopulation is tightly bound to the core of Photosystem II and contains mainly a slowly phosphorylated 27 kDa polypeptide. The other subpopulation of LHC II is peripherally bound to the core and contains a relatively high proportion of a rapidly phosphorylated 25 kDa polypeptide. The latter pool of LHC II can reversibly detach from Photosystem II in respons to phosphorylation or elevated temperatures. These findings will be accommodated into a model for the dynamic arrangement of the antenna of Photosystem II.

A mechanism for the formation of inside-out membrane vesicles. Preparation of inside-out vesicles from membrane-paired randomized chloroplast lamellae

Inside-out thylakoid membrane vesicles can be isolated by aqueous polymer two-phase partition of Yeda press-fragmented spinach chloroplasts (Andersson, B. and Akerlund, H.-E. (1978) Biochim. Biophys. Acta 503, 462-472). The mechanism for their formation has been investigated by studying the yield of inside-out vesicles after various treatments of the chloroplasts prior to fragmentation. No inside-out vesicles were isolated during phase partitioning if the chloroplasts had been destacked in a low-salt medium prior to the fragmentation. Only in those cases where the chloroplast lamellae had been stacked by cations of membrane-paired by acidic treatment did we get any yield of inside-out vesicles. Thus, the intrinsic properties of chloroplast thylakoids seem to be such that they seal into right-side out vesicles after disruption unless they are in an appressed state. This favours the following mechanism for the formation of inside-out thylakoids. After press treatment, a ruptured membrane still remains appressed with an adjacent membrane. Resealing of such an appressed membrane pair would result in an inside-out vesicle. If the compartmentation of chloroplast lamellae into appressed grana and unappressed stroma lamellae is preserved by cations before fragmentation, the inside-out vesicles are highly enriched in photosystem II. This indicates a granal origin which is consistent with the proposed model outlined. Inside-out vesicles possessing photosystem I and II properties in approximately equal proportions could be obtained by acid-induced membrane-pairing of chloroplasts which had been destacked and randomized prior to fragmentation. Since this new preparation of inside-out thylakoid vesicles also exposes components derived from the stroma lamellae it complements the previous preparation. It is suggested that fragmentation of paired membrane followed by phase partitioning should be a general method of obtaining inside-out vesicles from membranes of various biological sources.

Temperature‐induced reversible migration along the thylakoid membrane of photosystem II regulates it association with LHC‐II

Subfractionation of chloroplast thylakoids at elevated temperatures shows that above 35°C the photosystem II core and some of its light‐harvesting complex (LHC‐II) migrates laterally from the appressed thylakoid regions out to the non‐appressed regions, leaving behind free LHC‐II in the appressions. These structural rearrangements of the thylakoid membrane are largely reversible upon lowering the temperature and may play a physiological role in preventing overexcitation of photosystem II under high light intensities which normally induce elevated leaf temperatures.

Lateral Heterogeneity of Polar Lipids in the Thylakoid Membranes of Spinach Chloroplasts

Isolated appressed and non‐appressed membrane fractions of spinach thylakoids have been subjected to lipid class and fatty acid composition analyses. The ratio of monogalactosyldiacylglycerol to digalactosyldiacylglycerol was much higher in the fractions obtained from the appressed membranes compared with those derived from non‐appressed membranes. Moreover, appressed thylakoids contained a larger amount of anionic lipids in comparison with non‐appressed thylakoids. No major differences were observed in the fatty acid composition between the thylakoid fractions. The results are discussed in terms of the possible significance of lipid heterogeneity in the chloroplast thylakoid membrane.

Difference in sensitivity to photoinhibition between photosystem II in the appressed and non-appressed thylakoid regions

Thylakoid vesicles containing photosystem II from either the appressed or non‐appressed thylakoid region were subjected to photoinhibitory illumination. Photosystem II from the non‐appressed region was found to be less sensitive to photoinhibition compared to photosystem II from the oppressed region under both aerobic and anaerobic conditions.

Characterization of photosystem II in stroma thylakoid membranes

The functional state of the PS II population localized in the stroma exposed non-appressed thylakoid region was investigated by direct analysis of the PS II content of isolated stroma thylakoid vesicles. This PS II population, possessing an antenna size typical for PS IIβ, was found to have a fully functional oxygen evolving capacity in the presence of an added quinone electron acceptor such as phenyl-p-benzoquinone. The sensitivity to DCMU for this PS II population was the same as for PS II in control thylakoids. However, under more physiological conditions, in the absence of an added quinone acceptor, no oxygen was evolved from stroma thylakoid vesicles and their PS II centers were found to be incapable to pass electrons to PS I and to yield NADPH. By comparison of the effect of a variety of added quinone acceptors with different midpoint potentials, it is concluded that the inability of PS II in the stroma thylakoid membranes to contribute to NADPH formation probably is due to that QA of this population is not able to reduce PQ, although it can reduce some artificial acceptors like phenyl-p-benzoquinone. These data give further support to the notion of a discrete PS II population in the non-appressed stroma thylakoid region, PS IIβ, having a higher midpoint potential of QA than the PS II population in the appressed thylakoid region, PS IIα. The physiological significance of a PS II population that does not produce any NADPH is discussed.

The chaperone-like activity of a small heat shock protein is lost after sulfoxidation of conserved methionines in a surface-exposed amphipathic α-helix

The small heat shock proteins (sHsps) possess a chaperone-like activity which prevents aggregation of other proteins during transient heat or oxidative stress. The sHsps bind, onto their surface, molten globule forms of other proteins, thereby keeping them in a refolding competent state. In Hsp21, a chloroplast-located sHsp in all higher plants, there is a highly conserved region forming an amphipathic alpha-helix with several methionines on the hydrophobic side according to secondary structure prediction. This paper describes how sulfoxidation of the methionines in this amphipathic alpha-helix caused conformational changes and a reduction in the Hsp21 oligomer size, and a complete loss of the chaperone-like activity. Concomitantly, there was a loss of an outer-surface located alpha-helix as determined by limited proteolysis and circular dichroism spectroscopy. The present data indicate that the methionine-rich amphipathic alpha-helix, a motif of unknown physiological significance which evolved during the land plant evolution, is crucial for binding of substrate proteins and has rendered the chaperone-like activity of Hsp21 very dependent on the chloroplast redox state.

Substitution of conserved methionines by leucines in chloroplast small heat shock protein results in loss of redox‐response but retained chaperone‐like activity

During evolution of land plants, a specific motif occurred in the N‐terminal domain of the chloroplast‐localized small heat shock protein, Hsp21: a sequence with highly conserved methionines, which is predicted to form an amphipathic α‐helix with the methionines situated along one side. The functional role of these conserved methionines is not understood. We have found previously that treatment, which causes methionine sulfoxidation in Hsp21, also leads to structural changes and loss of chaperone‐like activity. Here, mutants of Arabidopsis thaliana Hsp21 protein were created by site‐directed mutagenesis, whereby conserved methionines were substituted by oxidation‐resistant leucines. Mutants lacking the only cysteine in Hsp21 were also created. Protein analyses by nondenaturing electrophoresis, size exclusion chromatography, and circular dichroism proved that sulfoxidation of the four highly conserved methionines (M49, M52, M55, and M59) is responsible for the oxidation‐induced conformational changes in the Hsp21 oligomer. In contrast, the chaperone‐like activity was not ultimately dependent on the methionines, because it was retained after methionine‐to‐leucine substitution. The functional role of the conserved methionines in Hsp21 may be to offer a possibility for redox control of chaperone‐like activity and oligomeric structure dynamics.