Oxygenic photosynthesis produces various radicals and active oxygen species with harmful effects on photosystem II (PSII). Such photodamage occurs at all light intensities. Damaged PSII centres, however, do not usually accumulate in the thylakoid membrane due to a rapid and efficient repair mechanism. The excellent design of PSII gives protection to most of the protein components and the damage is most often targeted only to the reaction centre D1 protein. Repair of PSII via turnover of the damaged protein subunits is a complex process involving (i) highly regulated reversible phosphorylation of several PSII core subunits, (ii) monomerization and migration of the PSII core from the grana to the stroma lamellae, (iii) partial disassembly of the PSII core monomer, (iv) highly specific proteolysis of the damaged proteins, and finally (v) a multi-step replacement of the damaged proteins with de novo synthesized copies followed by (vi) the reassembly, dimerization, and photoactivation of the PSII complexes. These processes will shortly be reviewed paying particular attention to the damage, turnover, and assembly of the PSII complex in grana and stroma thylakoids during the photoinhibition-repair cycle of PSII. Moreover, a two-dimensional Blue-native gel map of thylakoid membrane protein complexes, and their modification in the grana and stroma lamellae during a high-light treatment, is presented.

The thylakoid membrane of photoautotrophic organisms contains the main components of the photosynthetic electron transport chain. Detailed proteome maps of the thylakoid protein complexes of two marine diatoms, Thalassiosira pseudonana and Phaeodactylum tricornutum, were created by means of two-dimensional blue native (BN)/SDS-PAGE coupled with mass spectrometry analysis. One novel diatom-specific photosystem I (PS I)-associated protein was identified. A second plastid-targeted protein with possible PS I interaction was discovered to be restricted to the centric diatom species T. pseudonana. PGR5/PGRL homologues were found to be the only protein components of PS I-mediated cyclic electron transport common to both species. For the first time, evidence for a possible PS I localization of LI818-like light harvesting proteins (Lhcx) is presented. This study also advances the current knowledge on the light harvesting antenna composition and Lhcx expression in T. pseudonana on the protein level and presents details on the molecular distribution of Lhcx in diatoms. Above mentioned proteins and several others with unknown function provide a broad basis for further mutagenesis analysis, aiming toward further understanding of the composition and function of the photosynthetic apparatus of diatoms. The proteomics approach of this study further served as a tool to confirm and improve genome-derived protein models.

To study the synthesis and assembly of multisubunit thylakoid protein complexes, we performed [35S]Met pulse and chase experiments with isolated chloroplasts and intact leaves of spinach (Spinacia oleracea L.), followed by Blue Native gel separation of the (sub)complexes and subsequent identification of the newly synthesized and assembled protein subunits. PSII (photosystem II) core subunits were the most intensively synthesized proteins, particularly in vitro and at high light intensities in vivo, and could be sequestered in several distinct PSII subassemblies. Newly synthesized D1 was first found in the reaction centre complex that also contained labelled D2 and two labelled low-molecular-mass proteins. The next biggest PSII subassembly contained CP47 also. Then PsbH was assembled together with at least two other labelled chloroplast-encoded low-molecular-mass subunits, PsbM and PsbTc, and a nuclear-encoded PsbR. Subsequently, CP43 was inserted into the PSII complex concomitantly with PsbK. These assembly steps seemed to be essential for the dimerization of PSII core monomers. Intact PSII core monomer was the smallest subcomplex harbouring the newly synthesized 33 kDa oxygen-evolving complex protein PsbO. Nuclear-encoded PsbW was synthesized only at low light intensities concomitantly with Lhcb polypeptides and was distinctively present in PSII-LHCII (where LHC stands for light-harvesting complex) supercomplexes. The PsbH protein, on the contrary, was vigorously synthesized and incorporated into PSII core monomers together with the D1 protein, suggesting an intrinsic role for PsbH in the photoinhibition-repair cycle of PSII.

Linkö ping, Sweden (B.A.) Kinetic studies of protein dephosphorylation in photosynthetic thylakoid membranes revealed specifically accelerated dephosphorylation of photosystem II (PSII) core proteins at elevated temperatures. Raising the temperature from 22°C to 42°C resulted in a more than 10-fold increase in the dephosphorylation rates of the PSII reaction center proteins D1 and D2 and of the chlorophyll a binding protein CP43 in isolated spinach (Spinacia oleracea) thylakoids. In contrast the dephosphorylation rates of the light harvesting protein complex and the 9-kD protein of the PSII (PsbH) were accelerated only 2-to 3-fold. The use of a phospho-threonine antibody to measure in vivo phosphorylation levels in spinach leaves revealed a more than 20-fold acceleration in D1, D2, and CP43 dephosphorylation induced by abrupt elevation of temperature, but no increase in light harvesting protein complex dephosphorylation. This rapid dephosphorylation is catalyzed by a PSIIspecific, intrinsic membrane protein phosphatase. Phosphatase assays, using intact thylakoids, solubilized membranes, and the isolated enzyme, revealed that the temperature-induced lateral migration of PSII to the stroma-exposed thylakoids only partially contributed to the rapid increase in the dephosphorylation rate. Significant activation of the phosphatase coincided with the temperature-induced release of TLP40 from the membrane into thylakoid lumen. TLP40 is a peptidylprolyl cis-trans isomerase, which acts as a regulatory subunit of the membrane phosphatase. Thus dissociation of TLP40 caused by an abrupt elevation in temperature and activation of the membrane protein phosphatase are suggested to trigger accelerated repair of photodamaged PSII and to operate as possible early signals initiating other heat shock responses in chloroplasts.

Reactive oxygen species (ROS) are important messengers in eukaryotic organisms, and their production is tightly controlled. Active extracellular ROS production by NADPH oxidases in plants is triggered by receptor-like protein kinase-dependent signaling networks. Here, we show that CYSTEINE-RICH RLK2 (CRK2) kinase activity is required for plant growth and CRK2 exists in a preformed complex with the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) in Arabidopsis (Arabidopsis thaliana). Functional CRK2 is required for the full elicitor-induced ROS burst, and consequently the crk2 mutant is impaired in defense against the bacterial pathogen Pseudomonas syringae pv tomato DC3000. Our work demonstrates that CRK2 regulates plant innate immunity. We identified in vitro CRK2-dependent phosphorylation sites in the C-terminal region of RBOHD. Phosphorylation of S703 RBOHD is enhanced upon flg22 treatment, and substitution of S703 with Ala reduced ROS production in Arabidopsis. Phylogenetic analysis suggests that phospho-sites in the C-terminal region of RBOHD are conserved throughout the plant lineage and between animals and plants. We propose that regulation of NADPH oxidase activity by phosphorylation of the C-terminal region might be an ancient mechanism and that CRK2 is an important element in regulating microbe-associated molecular pattern-triggered ROS production.

Extracellular vesicles released from cells regulate many normal and pathological conditions. Little is known about the role of epidermal keratinocyte microvesicles (KC-MVs) in epithelial-stromal interaction that is essential for wound healing. We investigated, therefore, whether MV-like structures are present in human wounds and whether they affect wound healing-associated gene expression in dermal fibroblasts. In human wounds, MV-like vesicles were observed during active epithelial migration and early granulation tissue formation. When KC-MVs derived from keratinocyte-like cells (HaCaT) were added to fibroblast cultures, expression of 21 genes was significantly regulated (P<0.05) out of 80 genes investigated, including matrix metalloproteinase-1 and -3, interleukin-6 and -8, and genes associated with transforming growth factor-β signaling. Similar changes were observed at the protein level. MVs from normal epidermal keratinocytes showed similar response to HaCaT cells. KC-MVs activated ERK1/2, JNK, Smad, and p38 signaling pathways in fibroblasts with ERK1/2 signaling having the most prominent role in the MV-induced gene expression changes. KC-MVs stimulated fibroblast migration and induced fibroblast-mediated endothelial tube formation but did not affect collagen gel contraction by fibroblasts. The results demonstrate that keratinocyte microvesicles have a strong and a specific regulatory effect on fibroblasts that may modulate several aspects of wound healing.

SummaryHeat treatment of intact spinach leaves was found to induce a unique thylakoid membrane association of an approximately 40 kDa stromal protein. This protein was identi®ed as rubisco activase. Most of the rubisco activase was sequestered to the thylakoid membrane, particularly to the stroma-exposed regions, during the ®rst 10 min of heat treatment at 42°C. At lower temperatures (38±40°C) the association of rubisco activase with the thylakoid membrane occurred more slowly. The temperaturedependent association of rubisco activase with the thylakoid membrane was due to a conformational change in the rubisco activase itself, not to heat-induced alterations in the thylakoid membrane. Association of the 41 kDa isoform of rubisco activase occurred ®rst, followed by the binding of the 45 kDa isoform to the thylakoid membrane. Fractionation of thylakoid membranes revealed a speci®c association of rubisco activase with thylakoid-bound polysomes. Our results suggest a temperaturedependent dual function for rubisco activase. At optimal temperatures it functions in releasing inhibitory sugar phosphates from the active site of Rubisco. During a sudden and unexpected exposure of plants to heat stress, rubisco activase is likely to manifest a second role as a chaperone in association with thylakoid-bound ribosomes, possibly protecting, as a ®rst aid, the thylakoid associated protein synthesis machinery against heat inactivation.

Dephosphorylation of central photosynthetic proteins regulates their turnover in plant thylakoid membranes. A membrane protein phosphatase from spinach thylakoids was purified 13000-fold using detergent-engaged FPLC. The purified enzyme exhibited characteristics typical of eukaryotic Ser/Thr phosphatases of the PP2A family in that it was inhibited by okadaic acid (IC(50) = 0.4 nM) and tautomycin (IC(50) = 25 nM), irreversibly bound to microcystin-agarose, and recognized by a polyclonal antibody raised against a recombinant catalytic subunit of human PP2A. Furthermore, the anti-PP2A antibody inhibited protein dephosphorylation in isolated thylakoids. The phosphatase copurified with TLP40, a cyclophilin-like peptidyl-prolyl isomerase located in the thylakoid lumen. TLP40 could be released from the phosphatase immobilized on microcystin-agarose by high-salt treatment. Binding of cyclosporin A (CsA) to TLP40 led to thylakoid phosphatase activation, while cyclophilin substrates, prolyl-containing oligopeptides, inhibited protein dephosphorylation. This dephosphorylation could be modulated by CsA or oligopeptides only after the thylakoids had been ruptured to expose the lumenal membrane surface where the TLP40 is located. Regulation of the PP2A-like phosphatase at the outer thylakoid surface is likely to operate via reversible binding of TLP40 to the inner membrane surface. This is a first example of transmembrane regulation in which the activity of phosphatase is altered by the binding of a cyclophilin to a site other than the active one. We propose that signaling from TLP40 to the protein phosphatase coordinates dephosphorylation and protein folding, two processes required for protein turnover during the repair of photoinhibited photosystem II reaction centers.