Towards understanding the functional difference between the two PsbO isoforms in Arabidopsis thaliana—insights from phenotypic analyses of psbo knockout mutants

The extrinsic PsbO subunit of the water-oxidizing photosystem II (PSII) complex is represented by two isoforms in Arabidopsis thaliana, namely PsbO1 and PsbO2. Recent analyses of psbo1 and psbo2 knockout mutants have brought insights into their roles in photosynthesis and light stress. Here we analyzed the two psbo mutants in terms of PsbOs expression pattern, organization of PSII complexes and GTPase activity. Both PsbOs are present in wild-type plants, and their expression is mutually controlled in the mutants. Almost all PSII complexes are in the monomeric form not only in the psbo1 but also in the psbo2 mutant grown under high-light conditions. This results either from an enhanced susceptibility of PSII to photoinactivation or from malfunction of the repair cycle. Notably, the psbo1 mutant displays such problems even under growth-light conditions. These results together with the finding that PsbO2 has a threefold higher GTPase activity than PsbO1 have significance for the turnover of the PSII D1 subunit in Arabidopsis.     

AtCYP38 ensures early biogenesis, correct assembly and sustenance of photosystem II

AtCYP38 is a thylakoid lumen protein comprising the immunophilin domain and the phosphatase inhibitor module. Here we show the association of AtCYP38 with the photosystem II (PSII) monomer complex and address its functional role using AtCYP38-deficient mutants. The dynamic greening process of etiolated leaves failed in the absence of AtCYP38, due to specific problems in the biogenesis of PSII complexes. Also the development of leaves under short-day conditions was severely disturbed. Detailed biophysical and biochemical analysis of mature AtCYP38-deficient plants from favorable growth conditions (long photoperiod) revealed: (i) intrinsic malfunction of PSII, which (ii) occurred on the donor side of PSII and (iii) was dependent on growing light intensity. AtCYP38 mutant plants also showed decreased accumulation of PSII, which was shown not to originate from impaired D1 synthesis or assembly of PSII monomers, dimers and supercomplexes as such but rather from the incorrect fine-tuning of the oxygen-evolving side of PSII. This, in turn, rendered PSII centers extremely susceptible to photoinhibition. AtCYP38 deficiency also drastically decreased the in vivo phosphorylation of PSII core proteins, probably related to the absence of the AtCYP38 phosphatase inhibitor domain. It is proposed that during PSII assembly AtCYP38 protein guides the proper folding of D1 (and CP43) into PSII, thereby enabling the correct assembly of the water-splitting Mn₄–Ca cluster even with high turnover of PSII.     

Expression of protein complexes and individual proteins upon transition of etioplasts to chloroplasts in pea (Pisum sativum)

The protein complexes of pea (Pisum sativum L.) etioplasts,etio-chloroplasts and chloroplasts were examined using 2D BlueNative/SDS–PAGE. The most prominent protein complexesin etioplasts were the ATPase and the Clp and FtsH proteasecomplexes which probably have a crucial role in the biogenesisof etioplasts and chloroplasts. Also the cytochrome b₆f (Cytb₆f) complex was assembled in the etioplast membrane, as wellas Rubisco, at least partially, in the stroma. These complexesare composed of proteins encoded by both the plastid and nucleargenomes, indicating that a functional cross-talk exists betweenpea etioplasts and the nucleus. In contrast, the proteins andprotein complexes that bind chlorophyll, with the PetD subunitand the entire Cyt b₆f complex as an exception, did not accumulatein etioplasts. Nevertheless, some PSII core components suchas PsbE and the luminal oxygen-evolvong complex (OEC) proteinsPsbO and PsbP accumulated efficiently in etioplasts. After 6h de-etiolation, a complete PSII core complex appeared with40% of the maximal photochemical efficiency, but a fully functionalPSII was recorded only after 24 h illumination. Similarly, thecore complex of PSI was assembled after 6 h illumination, whereasthe PSI–light-harvesting complex I was stably assembledonly in chloroplasts illuminated for 24 h. Moreover, a batteryof proteins responsible for defense against oxidative stressaccumulated particularly in etioplasts, including the stromaland thylakoidal forms of ascorbate peroxidase, glutathione reductaseand PsbS.     

Mechanism of copper-enhanced photoinhibition in thylakoid membranes

The effect of copper on photoinhibition of photosystem II (PSII) in vitro was studied in bean ( Phaseolus vulgaris L. cv. Dufrix) and pumpkin ( Cucurbita pepo L.) thylakoids. The thylakoids were illuminated at 200–2 000 μmol photons m⁻² s⁻¹ in the presence of 70–1 830 added Cu²⁺ ions per PSII. Three lines of evidence show that the irreversible damage of PSII caused by illumination of thylakoids in the presence of Cu²⁺ was mainly due to donor-side photoinhibition resulting from inhibition of the PSII donor side by Cu²⁺. First, addition of an artificial electron donor partially restored PSII activity of thylakoids that had been illuminated in the presence of Cu²⁺. Second, already moderate light was enough to cause rapid inhibition of PSII, and the inhibition could be saturated by light. Third, the extrinsic polypeptides of the oxygen-evolving complex were found to become oxidized by the combined effect of Cu²⁺ and light. The presence of oxygen was not necessary for the copper-induced enhancement of photoinhibition of PSII. When the illumination was prolonged, copper caused a gradual collapse of the thylakoid structure by increasing degradation of thylakoid proteins.     

Dissecting a cyanobacterial proteolytic system: efficiency in inducing degradation of the D1 protein of photosystem II in cyanobacteria and plants

A chromatography fraction, prepared from isolated thylakoids of a fatty acid desaturation mutant (Fad6/desA Colon, two colons Km(r)) of the cyanobacterium Synechocystis 6803, could induce an initial cleavage of the D1 protein in Photosystem II (PSII) particles of Synechocystis 6803 mutant and Synechococcus 7002 wild type as well as in supercomplexes of PSII-light harvesting complex II of spinach. Proteolysis was demonstrated both in darkness and in light as a reduction in the amount of full-length D1 protein or as a production of C-terminal initial degradation fragments. In the Synechocystis mutant, the main degradation fragment was a 10-kDa C-terminal one, indicating an initial cleavage occurring in the cytoplasmic DE-loop of the D1 protein. A protein component of 70-90 kDa isolated from the chromatographic fraction was found to be involved in the production of this 10-kDa fragment. In spinach, only traces of the corresponding fragment were detected, whereas a 24-kDa C-terminal fragment accumulated, indicating an initial cleavage in the lumenal AB-loop of the D1 protein. Also in Synechocystis the 24-kDa fragment was detected as a faint band. An antibody raised against the Arabidopsis DegP2 protease recognized a 35-kDa band in the proteolytically active chromatographic fraction, suggesting the existence of a lumenal protease that may be the homologue DegP of Synechocystis. The identity of the other protease cleaving the D1 protein in the DE-loop exposed on the stromal (cytoplasmic) side of the membrane is discussed.     

TLP18.3, a novel thylakoid lumen protein regulating photosystem II repair cycle

A proteome analysis of Arabidopsis thaliana thylakoid-associated polysome nascent chain complexes was performed to find novel proteins involved in the biogenesis, maintenance and turnover of thylakoid protein complexes, in particular the PSII (photosystem II) complex, which exhibits a high turnover rate. Four unknown proteins were identified, of which TLP18.3 (thylakoid lumen protein of 18.3 kDa) was selected for further analysis. The Arabidopsis mutants (SALK_109618 and GABI-Kat 459D12) lacking the TLP18.3 protein showed higher susceptibility of PSII to photoinhibition. The increased susceptibility of DeltaTLP18.3 plants to high light probably originates from an inefficient reassembly of PSII monomers into dimers in the grana stacks, as well as from an impaired turnover of the D1 protein in stroma exposed thylakoids. Such dual function of the TLP18.3 protein is in accordance with its even distribution between the grana and stroma thylakoids. Notably, the lack of the TLP18.3 protein does not lead to a severe collapse of the PSII complexes, suggesting a redundancy of proteins assisting these particular repair steps to assure functional PSII. The DeltaTLP18.3 plants showed no clear visual phenotype under standard growth conditions, but when challenged by fluctuating light during growth, the retarded growth of DeltaTLP18.3 plants was evident.     

The PsbZ subunit of Photosystem II in Synechocystis sp. PCC 6803 modulates electron flow through the photosynthetic electron transfer chain

The psbZ gene of Synechocystis sp. PCC 6803 encodes the ∼6.6 kDa photosystem II (PSII) subunit. We here report biophysical, biochemical and in vivo characterization of Synechocystis sp. PCC 6803 mutants lacking psbZ. We show that these mutants are able to perform wild-type levels of light-harvesting, energy transfer, PSII oxygen evolution, state transitions and non-photochemical quenching (NPQ) under standard growth conditions. The mutants grow photoautotrophically; however, their growth rate is clearly retarded under low-light conditions and they are not capable of photomixotrophic growth. Further differences exist in the electron transfer properties between the mutants and wild type. In the absence of PsbZ, electron flow potentially increased through photosystem I (PSI) without a change in the maximum electron transfer capacity of PSII. Further, rereduction of P700⁺ is much faster, suggesting faster cyclic electron flow around PSI. This implies a role for PsbZ in the regulation of electron transfer, with implication for photoprotection.     

Role of phosphorylation in the repair cycle and oligomeric structure of photosystem II

The role of PSII protein phosphorylation in the oligomeric structure of the complex and in the repair of photodamaged PSII centers was studied with intact thylakoids and thylakoid membrane subfractions isolated from differentially light-treated pumpkin (Cucurbita pepo L.) leaves. A combination of sucrose gradient fractionation of thylakoid protein complexes and immunodetection with phosphothreonine and protein-specific antibodies was used. We report in this study that the extent of phosphorylation of PSII core proteins is equivalent in dimers and monomers, and directly depends on light intensity. Phosphorylated PSII monomers migrate to the stroma-exposed thylakoids, probably following damage of the D1 protein and the dissociation of the light-harvesting complex of PSII. Once in the stroma lamellae, monomers are gradually dephosphorylated to allow the reparation of the complex. First, CP43 is dephosphorylated and as a consequence of this modification it detaches from the PSII core. In addition to D1, D2 is also thereafter dephosphorylated. Phosphorylation of PSII core polypeptides probably ensures the integrity of the monomers until repair can proceed. Dephosphorylation, on the other hand, might serve the need for opening the complex and coordinating D1 proteolysis and the attachment of ribosomes.     

True oxygen reduction capacity during photosynthetic electron transfer in thylakoids and intact leaves

Reactive oxygen species (ROS) are generated in electron transport processes of living organisms in oxygenic environments. Chloroplasts are plant bioenergetics hubs where imbalances between photosynthetic inputs and outputs drive ROS generation upon changing environmental conditions. Plants have harnessed various site-specific thylakoid membrane ROS products into environmental sensory signals. Our current understanding of ROS production in thylakoids suggests that oxygen (O₂) reduction takes place at numerous components of the photosynthetic electron transfer chain (PETC). To refine models of site-specific O₂ reduction capacity of various PETC components in isolated thylakoids of Arabidopsis thaliana, we quantified the stoichiometry of oxygen production and consumption reactions associated with hydrogen peroxide (H₂O₂) accumulation using membrane inlet mass spectrometry and specific inhibitors. Combined with P700 spectroscopy and electron paramagnetic resonance spin trapping, we demonstrate that electron flow to photosystem I (PSI) is essential for H₂O₂ accumulation during the photosynthetic linear electron transport process. Further leaf disc measurements provided clues that H₂O₂ from PETC has a potential of increasing mitochondrial respiration and CO₂ release. Based on gas exchange analyses in control, site-specific inhibitor-, methyl viologen-, and catalase-treated thylakoids, we provide compelling evidence of no contribution of plastoquinone pool or cytochrome b6f to chloroplastic H₂O₂ accumulation. The putative production of H₂O₂ in any PETC location other than PSI is rapidly quenched and therefore cannot function in H₂O₂ translocation to another cellular location or in signaling.

Photosynthetically derived H₂O₂ only accumulates at Photosystem I and may trigger cooperation with mitochondria during stress.