Heterogeneity and Photoinhibition of Photosystem II Studied with Thermoluminescence1

Thermoluminescence (TL) signals were recorded from grana stacks, margins, and stroma lamellae from fractionated, dark-adapted thylakoid membranes of spinach (Spinacia oleracea L.) in the absence and in the presence of 2,6-dichlorphenylindophenol (DCMU). In the absence of DCMU, the TL signal from grana fractions consisted of a homogenous B-band, which originates from recombination of the semi-quinone QB− with the S2 state of the water-splitting complex and reflects active photosystem II (PSII). In the presence of DCMU, the B-band was replaced by the Q-band, which originates from an S2QA− recombination. Margin fractions mainly showed two TL-bands, the B- and C-bands, at approximately 50°C in the absence of DCMU, and Q- and C-bands in the presence of DCMU. The C-band is ascribed to a TyrD+-QA− recombination. In the absence of DCMU, the fractions of stromal lamellae mainly gave rise to a TL emission at 42°C. The intensity of this band was independent of the number of excitation flashes and was shifted to higher temperatures (52°C) after the addition of DCMU. Based on these observations, this band was considered to be a C-band. After photoinhibitory light treatment of uncoupled thylakoid membranes, the TL intensities of the B- and Q-bands decreased, whereas the intensity at 45°C (C-band) slightly increased. It is proposed that the 42 to 52°C band that was observed in marginal and stromal lamellae and in photoinhibited thylakoid membranes reflects inactive PSII centers that are assumed to be equivalent to inactive PSII QB-nonreducing centers.

The Small, Methionine-Rich Chloroplast Heat-Shock Protein Protects Photosystem II Electron Transport during Heat Stress1

Evidence suggests that the small chloroplast heat-shock protein (Hsp) is involved in plant thermotolerance but its site of action is unknown. Functional disruption of this Hsp using anti-Hsp antibodies or addition of purified Hsp to chloroplasts indicated that (a) this Hsp protects thermolabile photosystem II and, consequently, whole-chain electron transport during heat stress; and (b) this Hsp completely accounted for heat acclimation of electron transport in pre-heat-stressed plants. Therefore, this Hsp is a major adaptation to acute heat stress in plants.

Excited-state dynamics in photosystem II: Insights from the x-ray crystal structure

The heart of oxygenic photosynthesis is photosystem II (PSII), a multisubunit protein complex that uses solar energy to drive the splitting of water and production of molecular oxygen. The effectiveness of the photochemical reaction center of PSII depends on the efficient transfer of excitation energy from the surrounding antenna chlorophylls. A kinetic model for PSII, based on the x-ray crystal structure coordinates of 37 antenna and reaction center pigment molecules, allows us to map the major energy transfer routes from the antenna chlorophylls to the reaction center chromophores. The model shows that energy transfer to the reaction center is slow compared with the rate of primary electron transport and depends on a few bridging chlorophyll molecules. This unexpected energetic isolation of the reaction center in PSII is similar to that found in the bacterial photosystem, conflicts with the established view of the photophysics of PSII, and may be a functional requirement for primary photochemistry in photosynthesis. In addition, the model predicts a value for the intrinsic photochemical rate constant that is 4 times that found in bacterial reaction centers.

Quenching of chlorophyll fluorescence in the major light-harvesting complex of photosystem II: a systematic study of the effect of carotenoid structure.

The role of carotenoids in quenching of chlorophyll fluorescence in the major light-harvesting complex of photosystem II has been studied with a view to understanding the molecular basis of the control of photoprotective nonradiative energy dissipation by the xanthophyll cycle in vivo. The control of chlorophyll fluorescence quenching in the isolated complex has been investigated in terms of the number of the conjugated double bonds for a series of carotenoids ranging from n = 5-19, giving an estimated first excited singlet state energy from 20,700 cm-1 to 10,120 cm-1. At pH 7.8 the addition of exogenous carotenoids with >=10 conjugated double bonds (including zeaxanthin) stimulated fluorescence quenching relative to the control with no added carotenoid, whereas those with n photosystem II was induced by a lowering of pH to 5.5, carotenoids with n photosystem II could only be reversed by violaxanthin. These results are discussed in terms of the two theories developed to explain the role of zeaxanthin and violaxanthin in nonphotochemical quenching of chlorophyll fluorescence.

Destruction of a single chlorophyll is correlated with the photoinhibition of photosystem II with a transiently inactive donor side.

Pigments destroyed during photoinhibition of water-splitting photosystem II core complexes from the green alga Chlamydomonas reinhardtii were studied. Under conditions of a transiently inactivated donor side, illumination leads to an irreversible inhibition of the electron transfer at the donor side that is paralleled by the destruction of chlorophylls a absorbing maximally around 674 and 682 nm. The observed stochiometry of 1 +/- 0.1 destroyed chlorophyll per inhibited photosystem II suggests that chlorophyll destruction could be the primary photodamage causing the inhibition of photosystem II under these conditions.

Molecular characterization of PsbW, a nuclear-encoded component of the photosystem II reaction center complex in spinach.

We describe the isolation and characterization of cDNAs encoding the precursor polypeptide of the 6.1-kDa polypeptide associated with the reaction center core of the photosystem II complex from spinach. PsbW, the gene encoding this polypeptide, is present in a single copy per haploid genome. The mature polypeptide with 54 amino acid residues is characterized by a hydrophobic transmembrane segment, and, although an intrinsic membrane protein, it carries a bipartite transit peptide of 83 amino acid residues which directs the N terminus of the mature protein into the chloroplast lumen. Thylakoid integration of this polypeptide does not require a delta pH across the membrane, nor is it azide-sensitive, suggesting that the polypeptide chain inserts spontaneously in an as yet unknown way. The PsbW mRNA levels are light regulated. Similar to cytochrome b559 and PsbS, but different from the chlorophyll-complexing polypeptides D1, D2, CP43, and CP47 of photosystem II, PsbW is present in etiolated spinach seedlings.

A multimer model for P680, the primary electron donor of photosystem II.

We consider a model of the photosystem II (PS II) reaction center in which its spectral properties result from weak (approximately 100 cm-1) excitonic interactions between the majority of reaction center chlorins. Such a model is consistent with a structure similar to that of the reaction center of purple bacteria but with a reduced coupling of the chlorophyll special pair. We find that this model is consistent with many experimental studies of PS II. The similarity in magnitude of the exciton coupling and energetic disorder in PS II results in the exciton states being structurally highly heterogeneous. This model suggests that P680, the primary electron donor of PS II, should not be considered a dimer but a multimer of several weakly coupled pigments, including the pheophytin electron acceptor. We thus conclude that even if the reaction center of PS II is structurally similar to that of purple bacteria, its spectroscopy and primary photochemistry may be very different.

Detection of one slowly exchanging substrate water molecule in the S3 state of photosystem II.

The exchangeability of the substrate water molecules at the catalytic site of water oxidation in photosystem II has been probed by isotope-exchange measurements using mass spectrometric detection of flash-induced oxygen evolution. A stirred sample chamber was constructed to reduce the lag time between injection of H2(18)O and the detecting flash by a factor of more than 1000 compared to the original experiments by R. Radmer and O. Ollinger [(1986) FEBS Lett. 195, 285-289]. Our data show that there is a slow (t1/2 approximately 500 ms, 10 degrees C) and a fast (t1/2 <25 ms, 10 degrees C) exchanging substrate water molecule in the S3 state of photosystem II. The slow exchange is coupled with an activation energy of about 75 kJ/mol and is discussed in terms of a terminal manganese oxo ligand, while the faster exchanging substrate molecule may represent a water molecule not directly bound to the manganese center.

Identification of a Ca2+/H+ Antiport in the Plant Chloroplast Thylakoid Membrane1

To assess the availability of Ca2+ in the lumen of the thylakoid membrane that is required to support the assembly of the oxygen-evolving complex of photosystem II, we have investigated the mechanism of 45Ca2+ transport into the lumen of pea (Pisum sativum) thylakoid membranes using silicone-oil centrifugation. Trans-thylakoid Ca2+ transport is dependent on light or, in the dark, on exogenously added ATP. Both light and ATP hydrolysis are coupled to Ca2+ transport through the formation of a transthylakoid pH gradient. The H+-transporting ionophores nigericin/K+ and carbonyl cyanide 3-chlorophenylhydrazone inhibit the transport of Ca2+. Thylakoid membranes are capable of accumulating up to 30 nmol Ca2+ mg−1 chlorophyll from external concentrations of 15 μm over the course of a 15-min reaction. These results are consistent with the presence of an active Ca2+/H+ antiport in the thylakoid membrane. Ca2+ transport across the thylakoid membrane has significant implications for chloroplast and plant Ca2+ homeostasis. We propose a model of chloroplast Ca2+ regulation whereby the activity of the Ca2+/H+ antiporter facilitates the light-dependent uptake of Ca2+ by chloroplasts and reduces stromal Ca2+ levels.

In vitro reconstitution of the photosystem I light-harvesting complex LHCI-730: Heterodimerization is required for antenna pigment organization

Here we describe the in vitro reconstitution of photosystem I light-harvesting complexes with pigments and proteins (Lhca1 and Lhca4) obtained by overexpression of tomato Lhca genes in Escherichia coli. Using Lhca1 and Lhca4 individually for reconstitution results in monomeric pigment-proteins, whereas a combination thereof yields a dimeric complex. Interactions of the apoproteins is highly specific, as reconstitution of either of the two constituent proteins in combination with a light-harvesting protein of photosystem II does not result in dimerization. The reconstituted Lhca1/4, but not complexes obtained with either Lhca1 or Lhca4 alone, closely resembles the native LHCI-730 dimer from tomato leaves with regard to spectroscopic properties, pigment composition, and stoichiometry. Monomeric complexes of Lhca1 or Lhca4 possess lower pigment/protein ratios, indicating that interactions of the two subunits not only facilitates pigment reorganization but also recruitment of additional pigments. In addition to higher averages of chlorophyll a/b ratios in monomeric complexes than in LHCI-730, comparative fluorescence and CD spectra demonstrate that heterodimerization involves preferential ligation of more chlorophyll b.

The medial-Golgi Ion Pump Pmr1 Supplies the Yeast Secretory Pathway with Ca2+ and Mn2+ Required for Glycosylation, Sorting, and Endoplasmic Reticulum-Associated Protein Degradation

The yeast Ca2+ adenosine triphosphatase Pmr1, located in medial-Golgi, has been implicated in intracellular transport of Ca2+ and Mn2+ ions. We show here that addition of Mn2+ greatly alleviates defects of pmr1 mutants in N-linked and O-linked protein glycosylation. In contrast, accurate sorting of carboxypeptidase Y (CpY) to the vacuole requires a sufficient supply of intralumenal Ca2+. Most remarkably, pmr1 mutants are also unable to degrade CpY*, a misfolded soluble endoplasmic reticulum protein, and display phenotypes similar to mutants defective in the stress response to malfolded endoplasmic reticulum proteins. Growth inhibition of pmr1 mutants on Ca2+-deficient media is overcome by expression of other Ca2+ pumps, including a SERCA-type Ca2+ adenosine triphosphatase from rabbit, or by Vps10, a sorting receptor guiding non-native luminal proteins to the vacuole. Our analysis corroborates the dual function of Pmr1 in Ca2+ and Mn2+ transport and establishes a novel role of this secretory pathway pump in endoplasmic reticulum-associated processes.

Modification of Carbon Partitioning, Photosynthetic Capacity, and O2 Sensitivity in Arabidopsis Plants with Low ADP-Glucose Pyrophosphorylase Activity1

Wild-type Arabidopsis plants, the starch-deficient mutant TL46, and the near-starchless mutant TL25 were evaluated by noninvasive in situ methods for their capacity for net CO2 assimilation, true rates of photosynthetic O2 evolution (determined from chlorophyll fluorescence measurements of photosystem II), partitioning of photosynthate into sucrose and starch, and plant growth. Compared with wild-type plants, the starch mutants showed reduced photosynthetic capacity, with the largest reduction occurring in mutant TL25 subjected to high light and increased CO2 partial pressure. The extent of stimulation of CO2 assimilation by increasing CO2 or by reducing O2 partial pressure was significantly less for the starch mutants than for wild-type plants. Under high light and moderate to high levels of CO2, the rates of CO2 assimilation and O2 evolution and the percentage inhibition of photosynthesis by low O2 were higher for the wild type than for the mutants. The relative rates of 14CO2 incorporation into starch under high light and high CO2 followed the patterns of photosynthetic capacity, with TL46 showing 31% to 40% of the starch-labeling rates of the wild type and TL25 showing less than 14% incorporation. Overall, there were significant correlations between the rates of starch synthesis and CO2 assimilation and between the rates of starch synthesis and cumulative leaf area. These results indicate that leaf starch plays an important role as a transient reserve, the synthesis of which can ameliorate any potential reduction in photosynthesis caused by feedback regulation.

Stepwise Photoinhibition of Photosystem II1 : Studies with Synechocystis Species PCC 6803 Mutants with a Modified D-E Loop of the Reaction Center Polypeptide D1

Several mutant strains of Synechocystis sp. PCC 6803 with large deletions in the D-E loop of the photosystem II (PSII) reaction center polypeptide D1 were subjected to high light to investigate the role of this hydrophilic loop in the photoinhibition cascade of PSII. The tolerance of PSII to photoinhibition in the autotrophic mutant ΔR225-F239 (PD), when oxygen evolution was monitored with 2,6-dichloro-p-benzoquinone and the equal susceptibility compared with control when monitored with bicarbonate, suggested an inactivation of the QB-binding niche as the first event in the photoinhibition cascade in vivo. This step in PD was largely reversible at low light without the need for protein synthesis. Only the next event, inactivation of QA reduction, was irreversible and gave a signal for D1 polypeptide degradation. The heterotrophic deletion mutants ΔG240-V249 and ΔR225-V249 had severely modified QB pockets, yet exhibited high rates of 2,6-dichloro-p-benzoquinone-mediated oxygen evolution and less tolerance to photoinhibition than PD. Moreover, the protein-synthesis-dependent recovery of PSII from photoinhibition was impaired in the ΔG240-V249 and ΔR225-V249 mutants because of the effects of the mutations on the expression of the psbA-2 gene. No specific sequences in the D-E loop were found to be essential for high rates of D1 polypeptide degradation.

A chloroplast processing enzyme functions as the general stromal processing peptidase

A highly specific stromal processing activity is thought to cleave a large diversity of precursors targeted to the chloroplast, removing an N-terminal transit peptide. The identity of this key component of the import machinery has not been unequivocally established. We have previously characterized a chloroplast processing enzyme (CPE) that cleaves the precursor of the light-harvesting chlorophyll a/b binding protein of photosystem II (LHCPII). Here we report the overexpression of active CPE in Escherichia coli. Examination of the recombinant enzyme in vitro revealed that it cleaves not only preLHCPII, but also the precursors for an array of proteins essential for different reactions and destined for different compartments of the organelle. CPE also processes its own precursor in trans. Neither the recombinant CPE nor the native CPE of chloroplasts process a preLHCPII mutant with an altered cleavage site demonstrating that both forms of the enzyme are sensitive to the same structural modification of the substrate. The transit peptide of the precursor of ferredoxin is released by a single cleavage event and found intact after processing by recombinant CPE and a chloroplast extract as well. These results provide the first direct demonstration that CPE is the general stromal processing peptidase that acts as an endopeptidase. Significantly, recombinant CPE cleaves in the absence of other chloroplast proteins, and this activity depends on metal cations, such as zinc.

Addition of destabilizing poly(A)-rich sequences to endonuclease cleavage sites during the degradation of chloroplast mRNA

In this work, we report the posttranscriptional addition of poly(A)-rich sequences to mRNA in chloroplasts of higher plants. Several sites in the coding region and the mature end of spinach chloroplast psbA mRNA, which encodes the D1 protein of photosystem II, are detected as polyadenylylated sites. In eukaryotic cells, the addition of multiple adenosine residues to the 3′ end of nuclear RNA plays a key role in generating functional mRNAs and in regulating mRNA degradation. In bacteria, the adenylation of several RNAs greatly accelerates their decay. The poly(A) moiety in the chloroplast, in contrast to that in eukaryotic nuclear encoded and bacterial RNAs, is not a ribohomopolymer of adenosine residues, but clusters of adenosines bounded mostly by guanosines and rarely by cytidines and uridines; it may be as long as several hundred nucleotides. Further analysis of the initial steps of chloroplast psbA mRNA decay revealed specific endonuclease cleavage sites that perfectly matched the sites where poly(A)-rich sequences were added. Our results suggest a mechanism for the degradation of psbA mRNA in which endonucleolytic cleavages are followed by the addition of poly(A)-rich sequences to the upstream cleavage products, which target these RNAs for rapid decay.