Structural characterization of NDH-1 complexes of Thermosynechococcus elongatus by single particle electron microscopy

The structure of the multifunctional NAD(P)H dehydrogenase type 1 (NDH-1) complexes from cyanobacteria was investigated by growing the wild type and specific ndh His-tag mutants of Thermosynechococcus elongatus BP-1 under different CO(2) conditions, followed by an electron microscopy (EM) analysis of their purified membrane protein complexes. Single particle averaging showed that the complete NDH-1 complex (NDH-1L) is L-shaped, with a relatively short hydrophilic arm. Two smaller complexes were observed, differing only at the tip of the membrane-embedded arm. The smallest one is considered to be similar to NDH-1M, lacking the NdhD1 and NdhF1 subunits. The other fragment, named NDH-1I, is intermediate between NDH-1L and NDH-1M and only lacks a mass compatible with the size of the NdhF1 subunit. Both smaller complexes were observed under low- and high-CO(2) growth conditions, but were much more abundant under the latter conditions. EM characterization of cyanobacterial NDH-1 further showed small numbers of NDH-1 complexes with additional masses. One type of particle has a much longer peripheral arm, similar to the one of NADH: ubiquinone oxidoreductase (complex I) in E. coli and other organisms. This indicates that Thermosynechococcus elongatus must have protein(s) which are structurally homologous to the E. coli NuoE, -F, and -G subunits. Another low-abundance type of particle (NDH-1U) has a second labile hydrophilic arm at the tip of the membrane-embedded arm. This U-shaped particle has not been observed before by EM in a NDH-I preparation.     

Photosystem II organisation in chloroplasts of <Emphasis Type="Italic">Arum italicum</Emphasis> leaf depends on tissue location

The growth of plants under stable light quality induces long-term acclimation responses of the photosynthetic apparatus. Light can even cause variations depending on the tissue location, as in Arum italicum leaf, where chloroplasts are developed in the lamina and in the entire thickness of the petiole. We addressed the question whether differences in plastids can be characterised in terms of protein–protein interactions in the thylakoid membranes. Thylakoid assembly was studied in the palisade and spongy tissue of the lamina and in the outer parenchyma and inner aerenchyma of the petiole of the mature winter leaf of Arum italicum. The chlorophyll–protein complexes were analysed by means of blue-native-PAGE and fluorescence emission spectra. The petiole chloroplasts differ from those in the lamina in thylakoid composition: (1) reaction centres are scarce, especially photosystem (PS) I in the inner aerenchyma; (2) light-harvesting complex (LHC) II is abundant, (3) the relative amount of LHCII trimers increases, but this is not accompanied by increased levels of PSII–LHCII supercomplexes. Nevertheless, the intrinsic PSII functionality is comparable in all tissues. In Arum italicum leaf, the gradient in thylakoid organisation, which occurs from the palisade tissue to the inner aerenchyma of the petiole, is typical for photosynthetic acclimation to low-light intensity with a high enrichment of far-red light. The results obtained demonstrate a high plasticity of chloroplasts even in an individual plant. The mutual interaction of thylakoid protein complexes is discussed in relation to the photosynthetic efficiency of the leaf parts and to the ecodevelopmental role of light.     

Localisation and processing of the precursor form of photosystem II protein D1 inSynechocystis 6803

Pure plasma membrane and thylakoid membrane fractions from Synechocystis 6803 were isolated to study the localisation and processing of the precursor form of the D1 protein (pD1) of photosystem II (PSII). PSII core proteins (D1, D2 and cytb559) were localised both to plasma and thylakoid membrane fractions, the majority in thylakoids. pD1 was found only in the thylakoid membrane where active PSII is known to function. Membrane fatty acid unsaturation was shown to be critical in processing of pD1 into mature D1 protein. This was concluded from pulse-labelling experiments at low temperature using wild type and a mutant Synechocystis 6803 with a low level of membrane fatty acid unsaturation. Further, pD1 was identified as two distinct bands, an indication of two cleavage sites in the precursor peptide or, alternatively, two different conformations of pD1. Our results provide evidence for thylakoid membranes being a primary synthesis site for D1 protein during its light-activated turnover. The existence of the PSII core proteins in the plasma membrane, on the other hand, may be related to the biosynthesis of new PSII complexes in these membranes.     

Single particle analysis of thylakoid proteins from Thermosynechococcus elongatus and Synechocystis 6803: localization of the CupA subunit of NDH-1

The larger protein complexes of the cyanobacterial photosynthetic membrane of Thermosynechoccus elongatus and Synechocystis 6803 were studied by single particle electron microscopy after detergent solubilization, without any purification steps. Besides the "standard" L-shaped NDH-1L complex, related to complex I, large numbers of a U-shaped NDH-1MS complex were found in both cyanobacteria. In membranes from Synechocystis DeltacupA and DeltacupA/cupB mutants the U-shaped complexes were absent, indicating that CupA is responsible for the U-shape by binding at the tip of the membrane-bound arm of NDH-1MS. Comparison of membranes grown under air levels of CO(2) or 3% CO(2) indicates that the number of NDH-1MS particles is 30-fold higher under low-CO(2).     

Deletion of the tobacco plastid psbA gene triggers an upregulation of the thylakoid-associated NAD(P)H dehydrogenase complex and the plastid terminal oxidase (PTOX)

We have constructed a tobacco psbA gene deletion mutant that is devoid of photosystem II (PSII) complex. Analysis of thylakoid membranes revealed comparable amounts, on a chlorophyll basis, of photosystem I (PSI), the cytochrome b6f complex and the PSII light-harvesting complex (LHCII) antenna proteins in wild-type (WT) and Δ psbA leaves. Lack of PSII in the mutant, however, resulted in over 10-fold higher relative amounts of the thylakoid-associated plastid terminal oxidase (PTOX) and the NAD(P)H dehydrogenase (NDH) complex. Increased amounts of Ndh polypeptides were accompanied with a more than fourfold enhancement of NDH activity in the mutant thylakoids, as revealed by in-gel NADH dehydrogenase measurements. NADH also had a specific stimulating effect on P700⁺ re-reduction in the Δ psbA thylakoids. Altogether, our results suggest that enhancement of electron flow via the NDH complex and possibly other alternative electron transport routes partly compensates for the loss of PSII function in the Δ psbA mutant. As mRNA levels were comparable in WT and Δ psbA plants, upregulation of the alternative electron transport pathways (NDH complex and PTOX) occurs apparently by translational or post-translational mechanisms.     

Influence of protein phosphorylation on the electron-transport properties of Photosystem II

Many of the core proteins in Photosystem II (PS II) undergo reversible phosphorylation. It is known that protein phosphorylation controls the repair cycle of Photosystem II. However, it is not known how protein phosphorylation affects the partial electron transport reactions in PS II. Here we have applied variable fluorescence measurements and EPR spectroscopy to probe the status of the quinone acceptors, the Mn cluster and other electron transfer components in PS II with controlled levels of protein phosphorylation. Protein phosphorylation was induced in vivo by varying illumination regimes. The phosphorylation level of the D1 protein varied from 10 to 58% in PS II membranes isolated from pre-illuminated spinach leaves. The oxygen evolution and Q_A ⁻ to Q_B(Q_B ⁻) electron transfer measured by flash-induced fluorescence decay remained similar in all samples studied. Similar measurements in the presence of DCMU, which reports on the status of the donor side in PS II, also indicated that the integrity of the oxygen-evolving complex was preserved in PS II with different levels of D1 protein phosphorylation. With EPR spectroscopy we examined individual redox cofactors in PS II. Both the maximal amplitude of the charge separation reaction (measured as photo-accumulated pheophytin⁻) and the EPR signal from the Q_A ⁻ Fe²⁺ complex were unaffected by the phosphorylation of the D1 protein, indicating that the acceptor side of PS II was not modified. Also the shape of the S₂ state multiline signal was similar, suggesting that the structure of the Mn-cluster in Photosystem II did not change. However, the amplitude of the S₂ multiline signal was reduced by 35% in PS II, where 58% of the D1 protein was phosphorylated, as compared to the S₂ multiline in PS II, where only 10% of the D1 protein was phosphorylated. In addition, the fraction of low potential Cyt b ₅₅₉ was twice as high in phosphorylated PS II. Implications from these findings, were precise quantification of D1 protein phosphorylation is, for the first time, combined with high-resolution biophysical measurements, are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Suppression of a key gene involved in chlorophyll biosynthesis by means of virus-inducing gene silencing

The ChlH gene coding the H subunit of magnesium chelatase, an enzyme involved in chlorophyll biosynthesis, was silenced in Nicotiana benthamiana plants by infection with tobacco mosaic virus vectors (pTMV-30b) containing 67, 214 or 549 nt long ChlH inserts. Silencing of the nuclear ChlH gene induced a chimeric phenotype with green and yellow/white leaves associated with alterations of chloroplast shape and ultrastructure. The symptoms became first evident around veins of young leaves, and only later in the mesophyll tissues. The efficiency of gene silencing was not dependent on the insert orientation, but was strongly correlated with the size of the ChlH insert, providing a flexible method to modulate the level of gene suppression. Silencing efficiency seemed to be strongly dependent on endogenous ChlH mRNA level of the target tissue. Silencing of the ChlH gene with the longest fragment of 549 nt also lowered the accumulation of ChlD and chlorophyll synthetase mRNAs, i.e. other genes involved in chlorophyll biosynthesis.     

Regulation of D1-protein degradation during photoinhibition of photosystem II in vivo: Phosphorylation of the D1 protein in various plant groups

Photoinhibition of PSII and turnover of the D1 reaction-centre protein in vivo were studied in pumpkin leaves (Cucurbita pepo L.) acclimated to different growth irradiances and in low-light-grown moss, (Ceratodon purpureus) (Hedw.) Brid. The low-light-acclimated pumpkins were most susceptible to photoinhibition. The production rate of photoinhibited PSII centres (k_PI), determined in the presence of a chloroplast-encoded protein-synthesis inhibitor, showed no marked difference between the high- and low-light-grown pumpkin leaves. On the other hand, the rate constant for the repair cycle (k_REC) of PSII was nearly three times higher in the high-light-grown pumpkin when compared to low-light-grown pumpkin. The slower degradation rate of the damaged D1 protein in the low-light-acclimated leaves, determined by pulsechase experiments with [³⁵S]methionine suggested that the degradation of the Dl protein retards the repair cycle of PSII under photoinhibitory light. Slow degradation of the D1 protein in low-light-grown pumpkin was accompanied by accumulation of a phosphorylated form of the D1 protein, which we postulate as being involved in the regulation of D1-protein degradation and therefore the whole PSII repair cycle. In spite of low growth irradiance the repair cycle of PSII in the moss Ceratodon was rapid under high irradiance. When compared to the high- or low-light-acclimated pumpkin leaves, Ceratodon had the highest rate of D1-protein degradation at 1000 mol photons m^–2 s^–1. In contrast to the higher plants, the D1 protein of Ceratodon was not phosphorylated either under high irradiance in vivo or under in-vitro conditions, which readily phosphorylate the D1 protein of higher plants. This is consistent with the rapid degradation of the D1 protein in Ceratodon. Screening experiments indicated that D1 protein can be phosphorylated in the thylakoid membranes of angiosperms and conifers but not in lower plants. The postulated regulation mechanism of D1-protein degradation involving phosphorylation and the role of thylakoid organization in the function of PSII repair cycle are discussed.Abbreviations Chl Chlorophyll - D1^* phosphorylated form of D1 protein - F_max and F_v maximal and variable fluorescence respectively - k_PJ and k_REC rate constants of photoinhibition and concurrent recovery respectively - LHCII lightharvesting chlorophyll a/bprotein of PSII - PFD photon flux density Dr. R. Barbato (Dipartimento di Biologia, Universita di Padova, Padova, Italy), Prof. P. Böger (Lehrstuhl fur Physiologie und Biochemie der Pflanzen, Universität Konstanz, Konstanz, Germany), Prof. A. Melis (Department of Plant Biology, University of California, Berkeley, USA), Prof. I. Ohad (Department of Biological Chemistry, Hebrew University, Jerusalem, Israel) and Mr. A. Soitamo (Department of Biology, University of Turku, Turku, Finland) are gratefully acknowledged for the D1-protein-specific antibodies. The authors thank Ms. Virpi Paakkarinen for excellent technical assistance. This work was supported by the Academy of Finland and the Foundation of the University of Turku.