Roberto Barbato

A complex containing PGRL1 and PGR5 is involved in the switch between linear and cyclic electron flow in Arabidopsis.

During photosynthesis, two photoreaction centers located in the thylakoid membranes of the chloroplast, photosystems I and II (PSI and PSII), use light energy to mobilize electrons to generate ATP and NADPH. Different modes of electron flow exist, of which the linear electron flow is driven by PSI and PSII, generating ATP and NADPH, whereas the cyclic electron flow (CEF) only generates ATP and is driven by the PSI alone. Different environmental and metabolic conditions require the adjustment of ATP/NADPH ratios and a switch of electron distribution between the two photosystems. With the exception of PGR5, other components facilitating CEF are unknown. Here, we report the identification of PGRL1, a transmembrane protein present in thylakoids of Arabidopsis thaliana. Plants lacking PGRL1 show perturbation of CEF, similar to PGR5-deficient plants. We find that PGRL1 and PGR5 interact physically and associate with PSI. We therefore propose that the PGRL1-PGR5 complex facilitates CEF in eukaryotes.

Role of Plastid Protein Phosphatase TAP38 in LHCII Dephosphorylation and Thylakoid Electron Flow

Regulation of photosynthesis efficiency involves reversible phosphorylation of the light-harvesting complex through the activity of the newly identified phosphatase TAP38.

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

Abstract. 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.

Degradation of Photosystem II reaction center D1-protein induced by UVB radiation in isolated thylakoids. Identification and characterization of C- and N-terminal breakdown products

Abstract The effects of UVB radiation on the stability of Photosystem II reaction center D1-protein in isolated thylakoids are investigated. A new C-terminal degradation product has been identified and characterized, produced by cleavage in the second transmembrane segment. Immunological evidence for the presence of N-terminal fragments corresponding to the remaining part of D1-protein is also provided. The appearance of these fragments is not affected by lowering the temperature from 22°C to 4°C, changing the pH from 6 to 8, adding soybean trypsin inhibitor or excluding oxygen from the thylakoid suspension.

New evidence suggests that the initial photoinduced cleavage of the D1-protein may not occur near the PEST sequence

When isolated reaction centres of photosystem 2 from pea or wheat are exposed to photoinhibitory illumination in the presence of an electron acceptor, breakdown products of the D1‐protein are observed having molecular masses ranging from about 24 to 10 kDa. By using antibodies raised to the C‐terminal or N‐terminal portions of D1 it was shown that the major breakdown fragment of 24 kDa was derived from the C‐terminus. This conclusion was supported by phosphorylation studies and from the digestion pattern obtained by lysine specific endoprotease‐induced proteolysis. The complementary N‐terminal breakdown fragment was found to have an apparent molecular mass of 10 kDa. The implications of these data are discussed in terms of the possible relationship between the 24 kDa C‐terminal fragment and the 23.5 kDa breakdown fragment detected in vivo by Greenberg et al. [1987, EMBO J. 6, 2865–2869] and it is suggested, based on limited proteolysis using papain, that the latter may not be derived from the N‐terminus as previously thought but also originates from the C‐terminus.

Crystal structure of the PsbQ protein of photosystem II from higher plants

The smallest extrinsic polypeptide of the water‐oxidizing complex (PsbQ) was extracted and purified from spinach (Spinacia oleracea) photosystem II (PSII) membranes. It was then crystallized in the presence of Zn2+ and its structure was determined by X‐ray diffraction at 1.95‐Å resolution using the multi‐wavelength anomalous diffraction method, with the zinc as the anomalous scatterer. The crystal structure shows that the core of the protein is a four‐helix bundle, whereas the amino‐terminal portion, which possibly interacts with the photosystem core, is not visible in the crystal. The distribution of positive and negative charges on the protein surface might explain the ability of PsbQ to increase the binding of Cl− and Ca2+ and make them available to PSII.

Light-harvesting chlorophyll a/b proteins (LHCII) populations in phosphorylated membranes

The properties of the light-harvesting chlorophyll a/b-protein complex of Photosystem II (LHCII) have been analyzed, in thylakoids and PS II membrane after phosphorylation. Using a newly developed fractionation method, by flat-bed electrofocusing in granulated gel, seven Chl a/b proteins have been separated from thylakoids. Their phosphorylation level and polypeptide composition have been evaluated. PhosphoLHCII represent only 30% of the total Chl a/b proteins in thylakoids and the ‘mobile’ fraction binds 20% of the total LHCII chlorophyll. Tightly bound LHCII differs from the mobile fraction for the presence of a 26 kDa polypeptide characterized by the absence of the N-terminal LHCII proteolytic fragment which is phosphorylated during state transition.

Characterization of a 41 kDa photoinhibition adduct in isolated photosystem II reaction centres

When isolated reaction centres of photosystem II are subjected to photoinhibitory illumination, a 41 kDa SDS‐PAGE band is observed under all experimental conditions. The same band is also found, together will lower molecular weight fragments of the D1 protein, in whole thylakoids and in all PSII sub‐particles investigated up to now. In the case of isolated reaction centres the 41 kDa band is represented by a heterodimer of the D1 polypeptide and the α‐subunit of cytochrome b 559. The cross‐linkage between D1 and α‐cyl b 559 involves a region on D1 between the N‐terminal residue and Arg·225, and is an early event in photo‐induced damage to the D1 protein.

Inactivation of photosynthetic oxygen evolution by UV-B irradiation: A thermoluminescence study

The influence of UV-B irradiation on photosynthetic oxygen evolution by isolated spinach thylakoids has been investigated using thermoluminescence measurements. The thermoluminescence bands arising from the S2QB- (B band) and S2QA− (Q band) charge recombination disappeared with increasing UV-B irradiation time. In contrast, the C band at 50°C, arising from the recombination of QA- with an accessory donor of Photosystem II, was transiently enhanced by the UV-B irradiation. The efficiency of DCMU to block QA to QB electron transfer decreased after irradiation as detected by the incomplete suppression of the B band by DCMU. The flash-induced oscillatory pattern of the B band was modified in the UV-B irradiated samples, indicating a decrease in the number of centers with reduced QB. Based on the results of this study, UV-B irradiation is suggested to damage both the donor and acceptor sides of Photosystem II. The damage of the water-oxidizing complex does not affect a specific S-state transition. Instead, charge stabilization is enhanced on an accessory donor. The acceptor-side modifications decrease the affinity of DCMU binding. This effect is assumed to reflect a structural change in the QB/DCMU binding site. The preferential loss of dark stable QB- may be related to the same structural change or could be caused by the specific destruction of reduced quinones by the UV-B light.

The role of the light harvesting complex and photosystem II in thylakoid stacking in thechlorina-f2 barley mutant

The stacking behaviour of isolated thylakoids of the chlorophyllb-less barley mutantchlorina-f2, which is highly deficient in the light harvesting complex (LHCH), was investigated using electron microscopy. Pre-treatment with EDTA prevented thylakoid vesiculation during de-stacking, and was essential for the induction of re-stacking, which required a higher Mg++ concentration (25 mM) than for wild type (5 mM). The stability of re-stackedchlorina-f2 thylakoids allowed their fractionation into photosystem I and photosystem II by treatment with Triton X-100 or β-octyl glucoside, demonstrating thylakoid lateral heterogeneity in the absence of LHCII. An Mg++-dependent aggregation of detergent-solubilized photosystem II fromchlorina-f2 occurred at the same concentration (25 mM) as the one needed for the re-stacking of isolated thylakoids. We propose that thylakoid stacking and lateral heterogeneity inchlorina-f2 is not mediated by residual LHCII polypeptides, but by a component of photosystem II. This mechanism may also be involved in stacking of wild type thylakoids.A simple method is described for the one-step isolation fromchlorina-f2 of pure photosystem I and photosystem II preparations free of their respective chlorophyll b-containing light harvesting antenna proteins.