The ability of plants to adapt to changing light conditions depends on a protein kinase network in the chloroplast that leads to the reversible phosphorylation of key proteins in the photosynthetic membrane. Phosphorylation regulates, in a process called state transition, a profound reorganization of the electron transfer chain and remodeling of the thylakoid membranes. Phosphorylation governs the association of the mobile part of the light-harvesting antenna LHCII with either photosystem I or photosystem II. Recent work has identified the redox-regulated protein kinase STN7 as a major actor in state transitions, but the nature of the corresponding phosphatases remained unknown. Here we identify a phosphatase of Arabidopsis thaliana, called PPH1, which is specifically required for the dephosphorylation of light-harvesting complex II (LHCII). We show that this single phosphatase is largely responsible for the dephosphorylation of Lhcb1 and Lhcb2 but not of the photosystem II core proteins. PPH1, which belongs to the family of monomeric PP2C type phosphatases, is a chloroplast protein and is mainly associated with the stroma lamellae of the thylakoid membranes. We demonstrate that loss of PPH1 leads to an increase in the antenna size of photosystem I and to a strong impairment of state transitions. Thus phosphorylation and dephosphorylation of LHCII appear to be specifically mediated by the kinase/phosphatase pair STN7 and PPH1. These two proteins emerge as key players in the adaptation of the photosynthetic apparatus to changes in light quality and quantity.Photosynthesis | PP2C phosphatases | thylakoid | plastid P lants are critically dependent on light as a source of energy to drive photosynthesis. However, in natural settings, both the intensity and the spectral quality of light vary extensively, sometimes within very short periods. Photosynthetic organisms posess an arsenal of mechanisms to adapt to such changes in their light environment, optimize photosynthesis, prevent photo-oxidation in excess light, and repair photo-damage (1, 2). These mechanisms operate on different time scales, ranging from seconds to days, and at all levels of organization, from the photosynthetic complexes in the thylakoid membranes to the morphology of the whole plant. Under low light intensity, light harvesting is maximized, but under excess light, acclimation responses lead to reduced light capture and enhanced energy dissipation.Two photosystems, PSII and PSI, together with their associated light-harvesting antennae, function in series to drive linear electron flow in the thylakoid membranes, leading to the production of ATP and reductants such as reduced ferredoxin or NADPH. Cyclic electron flow around PSI allows synthesis of ATP without generating reducing power. Thus, the balance between linear and cyclic electron flow influences the ATP energy charge as well as the redox poise of the plant cell (2). The two photosystems have different light absorption characteristics; depending on the spectral composition of ambient light, a...

Reversible protein phosphorylation plays a major role in the acclimation of the photosynthetic apparatus to changes in light. Two paralogous kinases phosphorylate subsets of thylakoid membrane proteins. STATE TRANSITION7 (STN7) phosphorylates LHCII, the light-harvesting antenna of photosystem II (PSII), to balance the activity of the two photosystems through state transitions. STN8, which is mainly involved in phosphorylation of PSII core subunits, influences folding of the thylakoid membranes and repair of PSII after photodamage. The rapid reversibility of these acclimatory responses requires the action of protein phosphatases. In a reverse genetic screen, we identified the chloroplast PP2C phosphatase, PHOTOSYSTEM II CORE PHOSPHATASE (PBCP), which is required for efficient dephosphorylation of PSII proteins. Its targets, identified by immunoblotting and mass spectrometry, largely coincide with those of the kinase STN8. The recombinant phosphatase is active in vitro on a synthetic substrate or on isolated thylakoids. Thylakoid folding is affected in the absence of PBCP, while its overexpression alters the kinetics of state transitions. PBCP and STN8 form an antagonistic kinase and phosphatase pair whose substrate specificity and physiological functions are distinct from those of STN7 and the counteracting phosphatase PROTEIN PHOSPHATASE1/ THYLAKOID-ASSOCIATED PHOSPHATASE38, but their activities may overlap to some degree.

Circulating mitochondrial DNA (mtDNA) is receiving increasing attention as a danger-associated molecular pattern in conditions such as autoimmunity, cancer, and trauma. We report here that human lymphocytes [B cells, T cells, natural killer (NK) cells], monocytes, and neutrophils derived from healthy blood donors, as well as B cells from chronic lymphocytic leukemia patients, rapidly eject mtDNA as web filament structures upon recognition of CpG and non-CpG oligodeoxynucleotides of class C. The release was quenched by ZnCl2, independent of cell death (apoptosis, necrosis, necroptosis, autophagy), and continued in the presence of TLR9 signaling inhibitors. B-cell mtDNA webs were distinct from neutrophil extracellular traps concerning structure, reactive oxygen species (ROS) dependence, and were devoid of antibacterial proteins. mtDNA webs acted as rapid (within minutes) messengers, priming antiviral type I IFN production. In summary, our findings point at a previously unrecognized role for lymphocytes in antimicrobial defense, utilizing mtDNA webs as signals in synergy with cytokines and natural antibodies, and cast light on the interplay between mitochondria and the immune system.

a b s t r a c tLight-regulated protein kinases STN7 and STN8 phosphorylate thylakoid membrane proteins and also affect expression of several chloroplast proteins via yet unknown mechanisms. Comparative phosphoproteomics of acetic acid protein extracts of chloroplasts from Arabidopsis thaliana wild type, stn7, stn8 and stn7stn8 mutants yielded two previously unknown findings: (i) neither STN7 nor STN8 kinase was required for phosphorylation of Ser-48 in Lhcb1.1-1.3 proteins; and (ii) phosphorylation of Thr-451 in pTAC16 protein was STN7-dependent. pTAC16 was found distributed between thylakoids and nucleoid. Its knockout did not affect the nucleoid protein composition and the Thr-451 phosphorylated protein was excluded from the nucleoid. Thr-451 of pTAC16 is conserved in all studied plants and its phosphorylation may regulate membrane-anchoring functions of the nucleoid. Structured summary with protein interactions:RPOC2, RPOB, pTAC3, emb2746, RPOC1, pTAC2, pTAC12, pTAC16, pTAC5, pTAC14, RPOA, pTAC13, pTAC15 and pTAC6 colocalize by mass spectrometry studies of complexes (View interaction) PsbH, Lhcb4,1, Lhcb4,2, STN7, D1, D2, CP43, TSP9, CaS, PsaP, Alb3, PsbL, Rubisco, Unknown, Unknown, OEP6, pTAC16, PGRL1A, Unknown, Unknown and PsbQ colocalize by mass spectrometry studies of complexes (View interaction)

Chloroplast thylakoid lumen of Arabidopsis thaliana contains 16 immunophilins, five cyclophilins and 11 FK506-binding proteins (FKBPs), which are considered protein folding catalysts, although only two of them, AtFKBP13 and AtCYP20-2, possess peptidyl-prolyl cis/trans isomerase (PPIase) activity. To address the question of the physiological significance of this activity, we obtained and characterized Arabidopsis mutants deficient in the most active PPIase, AtFKBP13, and a double mutant deficient in both AtFKBP13 and AtCYP20-2. Two-dimensional gel electrophoresis of isolated thylakoid lumen, as well as immunoblotting analyses of major photosynthetic membrane protein complexes did not reveal differences in protein composition between the mutants and the wild type. No changes in the relative content of photosynthetic proteins were found by differential stable isotope labeling and liquid chromatography-mass spectrometry (LC-MS) analyses. PPIase activity was measured in vitro in isolated thylakoid lumen samples using two different synthetic peptide substrates. Depending on the peptide substrate used for the assay, the PPIase activity in the thylakoid lumen of the mutants lacking either AtFKBP13 or both AtFKBP13 and AtCYP20-2 was as low as 10 or 2% of that in the wild type. Residual PPIase activity detected in the double mutant originated from AtCYP20-3, a cyclophilin from chloroplast stroma contaminating thylakoid lumen preparations. None of the mutants differed from the wild-type plants when grown under normal, cold stress or high light conditions. It is concluded that cellular functions of immunophilins in the thylakoid lumen of chloroplasts are not related to their PPIase capacity and should be investigated beyond this enzymatic activity.

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How do you wash cells? Three out of four of our colleagues use experimental procedures during everyday lab-bench work that can severely impair data interpretation depending on how cells are handled. We show here that a subpopulation (2-3%) of human leukocytes immediately induce a yet unclassified lytic cell death, concomitant with discharge of chromatin entities and cell elimination, when placed in protein-free solutions (i.e., PBS and HBSS). DNA release was not restricted to hematopoietic cells but occurred also in HEK293T cells. Albumin, fetal bovine serum, polyethylene glycol, and Pluronic F-68 supplements prevented chromatin discharge. Expelled chromatin was devoid of surrounding membranes but maintained its original nuclear shape, although ∼10 times enlarged. These structures differed from DNA appearance after osmotic or detergent-induced cell lysis. Besides sounding a cautionary note to the neutrophil extracellular trap (NET) research community, in which ∼50% of all published studies used protein-free media for NET-formation, our study also provides a rapid tool for analysis of chromatin organization.

This chapter describes different strategies for the study of phosphorylation dynamics and stoichiometry in photosynthetic membranes. Detailed procedures for the detection, large-scale identification, and quantification of phosphorylated proteins optimized for plant thylakoid proteins are given.