Identification of a Photosystem II Phosphatase Involved in Light Acclimation in Arabidopsis

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. 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 photo-damage. The rapid reversibility of these acclimatory responses requires the action of protein phosphatases. In a reverse genetic screen we have identified the chloroplast PP2C phosphatase, PBCP (PHOTOSYSTEM II CORE PHOSPHATASE), which is required for efficient de-phosphorylation 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 over-expression 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 PPH1 (TAP38), but their activities may overlap to some degree

Interaction of methyl viologen-induced chloroplast and mitochondrial signalling in Arabidopsis

Reactive oxygen species (ROS) are key signalling intermediates in plant metabolism, defence, and stress adaptation. In plants, both the chloroplast and mitochondria are centres of metabolic control and ROS production, which coordinate stress responses in other cell compartments. The herbicide and experimental tool, methyl viologen (MV) induces ROS generation in the chloroplast under illumination, but is also toxic in non-photosynthetic organisms. We used MV to probe plant ROS signalling in compartments other than the chloroplast. Taking a genetic approach in the model plant Arabidopsis (Arabidopsis thaliana), we used natural variation, QTL mapping, and mutant studies with MV in the light, but also under dark conditions, when the chloroplast electron transport is inactive. These studies revealed a light-independent MV-induced ROS-signalling pathway, suggesting mitochondrial involvement. Mitochondrial Mn SUPEROXIDE DISMUTASE was required for ROS-tolerance and the effect of MV was enhanced by exogenous sugar, providing further evidence for the role of mitochondria. Mutant and hormone feeding assays revealed roles for stress hormones in organellar ROS-responses. The radical-induced cell death1 mutant, which is tolerant to MV-induced ROS and exhibits altered mitochondrial signalling, was used to probe interactions between organelles. Our studies suggest that mitochondria are involved in the response to ROS induced by MV in plants.</p

Arabidopsis RCD1 coordinates chloroplast and mitochondrial functions through interaction with ANAC transcription factors

Reactive oxygen species (ROS)-dependent signaling pathways from chloroplasts and mitochondria merge at the nuclear protein RADICAL-INDUCED CELL DEATH1 (RCD1). RCD1 interacts in vivo and suppresses the activity of the transcription factors ANAC013 and ANAC017, which mediate a ROS-related retrograde signal originating from mitochondrial complex III. Inactivation of RCD1 leads to increased expression of mitochondrial dysfunction stimulon (MDS) genes regulated by ANAC013 and ANAC017. Accumulating MDS gene products, including alternative oxidases (AOXs), affect redox status of the chloroplasts, leading to changes in chloroplast ROS processing and increased protection of photosynthetic apparatus. ROS alter the abundance, thiol redox state and oligomerization of the RCD1 protein in vivo, providing feedback control on its function. RCD1-dependent regulation is linked to chloroplast signaling by 3'-phosphoadenosine 5'-phosphate (PAP). Thus, RCD1 integrates organellar signaling from chloroplasts and mitochondria to establish transcriptional control over the metabolic processes in both organelles.Peer reviewe

Dissecting the interaction of photosynthetic electron transfer with mitochondrial signalling and hypoxic response in the Arabidopsis rcd1 mutant

The Arabidopsis mutant rcd1 is tolerant to methyl viologen (MV). MV enhances the Mehler reaction, i.e. electron transfer from Photosystem I (PSI) to O-2, generating reactive oxygen species (ROS) in the chloroplast. To study the MV tolerance of rcd1, we first addressed chloroplast thiol redox enzymes potentially implicated in ROS scavenging. NADPH-thioredoxin oxidoreductase type C (NTRC) was more reduced in rcd1. NTRC contributed to the photosynthetic and metabolic phenotypes of rcd1, but did not determine its MV tolerance. We next tested rcd1 for alterations in the Mehler reaction. In rcd1, but not in the wild type, the PSI-to-MV electron transfer was abolished by hypoxic atmosphere. A characteristic feature of rcd1 is constitutive expression of mitochondrial dysfunction stimulon (MDS) genes that affect mitochondrial respiration. Similarly to rcd1, in other MDS-overexpressing plants hypoxia also inhibited the PSI-to-MV electron transfer. One possible explanation is that the MDS gene products may affect the Mehler reaction by altering the availability of O-2. In green tissues, this putative effect is masked by photosynthetic O-2 evolution. However, O-2 evolution was rapidly suppressed in MV-treated plants. Transcriptomic meta-analysis indicated that MDS gene expression is linked to hypoxic response not only under MV, but also in standard growth conditions.This article is part of the theme issue 'Retrograde signalling from endosymbiotic organelles'

A Protein Phosphorylation Threshold for Functional Stacking of Plant Photosynthetic Membranes

Phosphorylation of photosystem II (PSII) proteins affects macroscopic structure of thylakoid photosynthetic membranes in chloroplasts of the model plant Arabidopsis. In this study, light-scattering spectroscopy revealed that stacking of thylakoids isolated from wild type Arabidopsis and the mutant lacking STN7 protein kinase was highly influenced by cation (Mg++) concentrations. The stacking of thylakoids from the stn8 and stn7stn8 mutants, deficient in STN8 kinase and consequently in light-dependent phosphorylation of PSII, was increased even in the absence of Mg++. Additional PSII protein phosphorylation in wild type plants exposed to high light enhanced Mg++-dependence of thylakoid stacking. Protein phosphorylation in the plant leaves was analyzed during day, night and prolonged darkness using three independent techniques: immunoblotting with anti-phosphothreonine antibodies; Diamond ProQ phosphoprotein staining; and quantitative mass spectrometry of peptides released from the thylakoid membranes by trypsin. All assays revealed dark/night-induced increase in phosphorylation of the 43 kDa chlorophyll-binding protein CP43, which compensated for decrease in phosphorylation of the other PSII proteins in wild type and stn7, but not in the stn8 and stn7stn8 mutants. Quantitative mass spectrometry determined that every PSII in wild type and stn7 contained on average 2.5±0.1 or 1.4±0.1 phosphoryl groups during day or night, correspondingly, while less than every second PSII had a phosphoryl group in stn8 and stn7stn8. It is postulated that functional cation-dependent stacking of plant thylakoid membranes requires at least one phosphoryl group per PSII, and increased phosphorylation of PSII in plants exposed to high light enhances stacking dynamics of the photosynthetic membranes

High Light Induced Disassembly of Photosystem II Supercomplexes in Arabidopsis Requires STN7-Dependent Phosphorylation of CP29

Photosynthetic oxidation of water and production of oxygen by photosystem II (PSII) in thylakoid membranes of plant chloroplasts is highly affected by changes in light intensities. To minimize damage imposed by excessive sunlight and sustain the photosynthetic activity PSII, organized in supercomplexes with its light harvesting antenna, undergoes conformational changes, disassembly and repair via not clearly understood mechanisms. We characterized the phosphoproteome of the thylakoid membranes from Arabidopsis thaliana wild type, stn7, stn8 and stn7stn8 mutant plants exposed to high light. The high light treatment of the wild type and stn8 caused specific increase in phosphorylation of Lhcb4.1 and Lhcb4.2 isoforms of the PSII linker protein CP29 at five different threonine residues. Phosphorylation of CP29 at four of these residues was not found in stn7 and stn7stn8 plants lacking the STN7 protein kinase. Blue native gel electrophoresis followed by immunological and mass spectrometric analyses of the membrane protein complexes revealed that the high light treatment of the wild type caused redistribution of CP29 from PSII supercomplexes to PSII dimers and monomers. A similar high-light-induced disassembly of the PSII supercomplexes occurred in stn8, but not in stn7 and stn7stn8. Transfer of the high-light-treated wild type plants to normal light relocated CP29 back to PSII supercomplexes. We postulate that disassembly of PSII supercomplexes in plants exposed to high light involves STN7-kinase-dependent phosphorylation of the linker protein CP29. Disruption of this adaptive mechanism can explain dramatically retarded growth of the stn7 and stn7stn8 mutants under fluctuating normal/high light conditions, as previously reported

Genome sequencing and population genomic analyses provide insights into the adaptive landscape of silver birch

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Lack of GLYCOLATE OXIDASE1, but Not GLYCOLATE OXIDASE2, Attenuates the Photorespiratory Phenotype of CATALASE2-Deficient Arabidopsis

The genes coding for the core metabolic enzymes of the photorespiratory pathway that allows plants with C3-type photosynthesis to survive in an oxygen-rich atmosphere, have been largely discovered in genetic screens aimed to isolate mutants that are unviable under ambient air. As an exception, glycolate oxidase (GOX) mutants with a photorespiratory phenotype have not been described yet in C3 species. Using Arabidopsis (Arabidopsis thaliana) mutants lacking the peroxisomal CATALASE2 (cat2-2) that display stunted growth and cell death lesions under ambient air, we isolated a second-site loss-of-function mutation in GLYCOLATE OXIDASE1 (GOX1) that attenuated the photorespiratory phenotype of cat2-2. Interestingly, knocking out the nearly identical GOX2 in the cat2-2 background did not affect the photorespiratory phenotype, indicating that GOX1 and GOX2 play distinct metabolic roles. We further investigated their individual functions in single gox1-1 and gox2-1 mutants and revealed that their phenotypes can be modulated by environmental conditions that increase the metabolic flux through the photorespiratory pathway. High light negatively affected the photosynthetic performance and growth of both gox1-1 and gox2-1 mutants, but the negative consequences of severe photorespiration were more pronounced in the absence of GOX1, which was accompanied with lesser ability to process glycolate. Taken together, our results point toward divergent functions of the two photorespiratory GOX isoforms in Arabidopsis and contribute to a better understanding of the photorespiratory pathway.Peer reviewe

Genome sequencing and population genomic analyses provide insights into the adaptive landscape of silver birch

Silver birch (Betula pendula) is a pioneer boreal tree that can be induced to flower within 1 year. Its rapid life cycle, small (440-Mb) genome, and advanced germplasm resources make birch an attractive model for forest biotechnology. We assembled and chromosomally anchored the nuclear genome of an inbred B. pendula individual. Gene duplicates from the paleohexaploid event were enriched for transcriptional regulation, whereas tandem duplicates were overrepresented by environmental responses. Population resequencing of 80 individuals showed effective population size crashes at major points of climatic upheaval. Selective sweeps were enriched among polyploid duplicates encoding key developmental and physiological triggering functions, suggesting that local adaptation has tuned the timing of and cross-talk between fundamental plant processes. Variation around the tightly-linked light response genes PHYC and FRS10 correlated with latitude and longitude and temperature, and with precipitation for PHYC. Similar associations characterized the growth-promoting cytokinin response regulator ARR1, and the wood development genes KAK and MED5A.Peer reviewe

Genome sequencing and population genomic analyses provide insights into the adaptive landscape of silver birch.

Silver birch (Betula pendula) is a pioneer boreal tree that can be induced to flower within 1 year. Its rapid life cycle, small (440-Mb) genome, and advanced germplasm resources make birch an attractive model for forest biotechnology. We assembled and chromosomally anchored the nuclear genome of an inbred B. pendula individual. Gene duplicates from the paleohexaploid event were enriched for transcriptional regulation, whereas tandem duplicates were overrepresented by environmental responses. Population resequencing of 80 individuals showed effective population size crashes at major points of climatic upheaval. Selective sweeps were enriched among polyploid duplicates encoding key developmental and physiological triggering functions, suggesting that local adaptation has tuned the timing of and cross-talk between fundamental plant processes. Variation around the tightly-linked light response genes PHYC and FRS10 correlated with latitude and longitude and temperature, and with precipitation for PHYC. Similar associations characterized the growth-promoting cytokinin response regulator ARR1, and the wood development genes KAK and MED5A