Photosynthesis Research (v.94, #2-3)

This mini-review briefly summarizes our current knowledge on the reaction pattern of light-driven water splitting and the structure of Photosystem II that acts as a water:plastoquinone oxidoreductase. The overall process comprises three types of reaction sequences: (a) light-induced charge separation leading to formation of the radical ion pair P680+•Q A −• ; (b) reduction of plastoquinone to plastoquinol at the QB site via a two-step reaction sequence with Q A −• as reductant and (c) oxidative water splitting into O2 and four protons at a manganese-containing catalytic site via a four-step sequence driven by P680+• as oxidant and a redox active tyrosine YZ acting as mediator. Based on recent progress in X-ray diffraction crystallographic structure analysis the array of the cofactors within the protein matrix is discussed in relation to the functional pattern. Special emphasis is paid on the structure of the catalytic sites of PQH2 formation (QB-site) and oxidative water splitting (Mn4O x Ca cluster). The energetics and kinetics of the reactions taking place at these sites are presented only in a very concise manner with reference to recent up-to-date reviews. It is illustrated that several questions on the mechanism of oxidative water splitting and the structure of the catalytic sites are far from being satisfactorily answered.
Keywords: Photosystem II; Water oxidation; Plastoquinone reduction; Structure; Mechanism

The D1 protein, a key subunit of photosystem II reaction center, is synthesized as a precursor form with a carboxyl-terminal extension, in oxygenic photosynthetic organisms with some exceptions. This part of the protein is removed by the action of an endopeptidase, and the proteolytic processing is indispensable for the manifestation of oxygen-evolving activity in photosynthesis. The carboxyl-terminus of mature D1 protein, which appears upon the cleavage, has recently been demonstrated to be a ligand for a manganese atom in the Mn4Ca-cluster, which is responsible for the water oxidation chemistry in photosystem II, based on the isotope-edited Fourier transform infrared spectroscopy and the X-ray crystallography. On the other hand, the structure of a peptidase involved in the cleavage of precursor D1 protein has been resolved at a higher resolution, and the enzyme–substrate interactions have extensively been analyzed both in vivo and in vitro. The present article briefly summarizes the history of research and the present state of our knowledge on the carboxyl-terminal processing of precursor D1 protein in the photosystem II reaction center.
Keywords: Carboxyl-terminal processing; CtpA; D1 protein; Mn4Ca-cluster; Oxygen evolution; Photosystem II; Precursor D1 protein; Processing protease; psbA gene

Recent investigations of photoinhibition have revealed that photodamage to photosystem II (PSII) involves two temporally separated steps: the first is the inactivation of the oxygen-evolving complex by light that has been absorbed by the manganese cluster and the second is the impairment of the photochemical reaction center by light that has been absorbed by chlorophyll. Our studies of photoinhibition in Synechocystis sp. PCC 6803 at various temperatures demonstrated that the first step in photodamage is not completed at low temperatures, such as 10°C. Further investigations suggested that an intermediate state, which is stabilized at low temperatures, might exist at the first stage of photodamage. The repair of PSII involves many steps, including degradation and removal of the D1 protein, synthesis de novo of the precursor to the D1 protein, assembly of the PSII complex, and processing of the precursor to the D1 protein. Detailed analysis of photodamage and repair at various temperatures has demonstrated that, among these steps, only the synthesis of the precursor to D1 appears to proceed at low temperatures. Investigations of photoinhibition at low temperatures have also indicated that prolonged exposure of cyanobacterial cells or plant leaves to strong light diminishes their ability to repair PSII. Such non-repairable photoinhibition is caused by inhibition of the processing of the precursor to the D1 protein after prolonged illumination with strong light at low temperatures.
Keywords: D1 protein; Low temperature; Photodamage; Photoinhibition; Photosynthesis; Photosystem II; Processing; Repair

Engineering model proteins for Photosystem II function by Tom Wydrzynski; Warwick Hillier; Brendon Conlan (225-233).
Our knowledge of Photosystem II and the molecular mechanism of oxygen production are rapidly advancing. The time is now ripe to exploit this knowledge and use it as a blueprint for the development of light-driven catalysts, ultimately for the splitting of water into O2 and H2. In this article, we outline the background and our approach to this technological application through the reverse engineering of Photosystem II into model proteins.
Keywords: Biocatalyst; H2 production; Photosystem II; Protein engineering; Water splitting

Hypothesis: the peroxydicarbonic acid cycle in photosynthetic oxygen evolution by Paul A. Castelfranco; Yih-Kuang Lu; Alan J. Stemler (235-246).
Peroxydicarbonic acid (Podca), a proposed intermediate in photosynthetic oxygen evolution, was synthesized electrochemically. Consistent with literature descriptions of this compound, it was shown to be a highly reactive molecule, spontaneously hydrolyzed to H2O2, as well as susceptible to oxidative and reductive decomposition. In the presence of Mn2+ or Co2+, Podca was quickly broken down with release of O2. The liberation of O2, however, was partially suppressed at high O2 concentrations. In the presence of Ca-washed photosystem II-enriched membranes lacking extrinsic proteins, Podca was decomposed with the release of O2, but only under conditions favoring photosynthetic electron flow (light plus a Hill oxidant). A model is proposed that details how peroxydicarbonic acid could act as an oxygen-evolving intermediate. The hypothesis is consistent with the well-established Kok model and with recent findings related to the chemistry of oxygen evolution.
Keywords: Bicarbonate; Carbonic anhydrase; Kok model; Manganese; Oxygen evolution; Peroxydicarbonic acid; Photosystem II

Interactions of photosystem II with bicarbonate, formate and acetate by Dmitriy Shevela; Vyacheslav Klimov; Johannes Messinger (247-264).
In this study, we probe the effects of bicarbonate (hydrogencarbonate), BC, removal from photosystem II in spinach thylakoids by measuring flash-induced oxygen evolution patterns (FIOPs) with a Joliot-type electrode. For this we compared three commonly employed methods: (1) washing in BC-free medium, (2) formate addition, and (3) acetate addition. Washing of the samples with buffers depleted of BC and CO2 by bubbling with argon (Method 1) under our conditions leads to an increase in the double hit parameter of the first flash (β1), while the miss parameter and the overall activity remain unchanged. In contrast, addition of 40–50 mM formate or acetate results in a significant increase in the miss parameter and to an ∼50% (formate) and ∼10% (acetate) inhibition of the overall oxygen evolution activity, but not to an increased β1 parameter. All described effects could be reversed by washing with formate/acetate free buffer and/or addition of 2–10 mM bicarbonate. The redox potential of the water-oxidizing complex (WOC) in samples treated by Method 1 is compared to samples containing 2 mM bicarbonate in two ways: (1) The lifetimes of the S0, S2, and S3 states were measured, and no differences were found between the two sample types. (2) The S1, S0, S−1, and S−2 states were probed by incubation with small concentrations of NH2OH. These experiments displayed a subtle, yet highly reproducible difference in the apparent Si/S−i state distribution which is shown to arise from the interaction of BC with PSII in the already reduced states of the WOC. These data are discussed in detail by also taking into account the CO2 concentrations present in the buffers after argon bubbling and during the measurements. These values were measured by membrane-inlet mass spectrometry (MIMS).
Keywords: Flash-induced oxygen evolution pattern (FIOPs); Membrane-inlet mass spectrometry (MIMS); S states; Water splitting; Oxygen evolution; Bicarbonate; Hydrogencarbonate; Acetate; Formate

The photosystem II (PSII) double mutant ΔPsbO:ΔPsbU was unable to grow photoautotrophically at pH 7.5, but growth was restored at pH 10. We have isolated a pseudorevertant of this strain, which exhibited photoautotrophic growth at pH 7.5. PSII-specific oxygen evolution and assembled PSII centers in the pseudorevertant and the original ΔPsbO:ΔPsbU strains were similar at pH 7.5. Comparison of global gene expression of the two strains at pH 7.5 revealed that <4% of genes differed. In the pseudorevertant, up-regulated transcripts included stress-responsive genes, many of which were shown previously to be under the control of Hik34. Elevated transcripts included those encoding heat shock proteins (HspA, DnaK2 and HtpG), two Deg proteases (DegP and DegQ), and the orange carotenoid protein (OCP, Slr1963). Up-regulated genes encoded proteins localized to different cell compartments, including the thylakoid, plasma and outer membranes. We suggest that the cell wide up-regulation of stress response genes in the pseudorevertant may limit the impact of PSII instability that is observed in the ΔPsbO:ΔPsbU strain. Futhermore, the OCP has a photoprotective role mediating phycobilisome-associated nonphotochemical quenching, such that increased OCP levels in the pseudorevertant may reduce photons reaching these impaired centers. These two responses, in combination with uncharacterized stress responses, are sufficient to permit the growth of pseudorevertant at pH 7.5.
Keywords: Cyanobacteria; DNA microarray; Oxygen-evolving complex; Histidine kinase; pH; Photosystem II; PsbO; PsbU; Stress response; Synechocystis

The fast and slow kinetics of chlorophyll a fluorescence induction in plants, algae and cyanobacteria: a viewpoint by George C. Papageorgiou; Merope Tsimilli-Michael; Kostas Stamatakis (275-290).
The light-induced/dark-reversible changes in the chlorophyll (Chl) a fluorescence of photosynthetic cells and membranes in the μs-to-several min time window (fluorescence induction, FI; or Kautsky transient) reflect quantum yield changes (quenching/de-quenching) as well as changes in the number of Chls a in photosystem II (PS II; state transitions). Both relate to excitation trapping in PS II and the ensuing photosynthetic electron transport (PSET), and to secondary PSET effects, such as ion translocation across thylakoid membranes and filling or depletion of post-PS II and post-PS I pools of metabolites. In addition, high actinic light doses may depress Chl a fluorescence irreversibly (photoinhibitory lowering; q(I)). FI has been studied quite extensively in plants an algae (less so in cyanobacteria) as it affords a low resolution panoramic view of the photosynthesis process. Total FI comprises two transients, a fast initial (OPS; for Origin, Peak, Steady state) and a second slower transient (SMT; for Steady state, Maximum, Terminal state), whose details are characteristically different in eukaryotic (plants and algae) and prokaryotic (cyanobacteria) oxygenic photosynthetic organisms. In the former, maximal fluorescence output occurs at peak P, with peak M lying much lower or being absent, in which case the PSMT phases are replaced by a monotonous PT fluorescence decay. In contrast, in phycobilisome (PBS)-containing cyanobacteria maximal fluorescence occurs at M which lies much higher than peak P. It will be argued that this difference is caused by a fluorescence lowering trend (state 1 → 2 transition) that dominates the FI pattern of plants and algae, and correspondingly by a fluorescence increasing trend (state 2 → 1 transition) that dominates the FI of PBS-containing cyanobacteria. Characteristically, however, the FI pattern of the PBS-minus cyanobacterium Acaryochloris marina resembles the FI patterns of algae and plants and not of the PBS-containing cyanobacteria.
Keywords: Algae; Chlorophyll fluorescence; Cyanobacteria; Fast fluorescence induction; Higher plants; Kautsky transient; Nonphotochemical quenching; Photochemical quenching; Photoinhibitory fluorescence lowering; Slow fluorescence induction; State transitions

The effects of ultraviolet-B (UV-B) radiation on photosystem II (PS II) were studied in leaves of Chenopodium album. After the treatment with UV-B the damage was estimated using chlorophyll a fluorescence techniques. Measurements of modulated fluorescence using a pulse amplitude modulated fluorometer revealed that the efficiency of photosystem II decreased both with increasing time of UV-B radiation and with increasing intensity of the UV-B. Fluorescence induction rise curves were analyzed using a mechanistic model of energy trapping. It appears that the damage by UV-B radiation occurs first at the acceptor side of photosystem II, and only later at the donor side.
Keywords: Chenopodium album ; Chlorophyll a fluorescence; Photosystem II; UV-B radiation; Photodamage; Photosynthesis

Growth enhancement of soybean (Glycine max) upon exclusion of UV-B and UV-B/A components of solar radiation: characterization of photosynthetic parameters in leaves by Guruprasad Kadur; Bhattacharjee Swapan; Kataria Sunita; Yadav Sanjeev; Tiwari Arjun; Baroniya Sanjay; Rajiv Abhinav; Prasanna Mohanty (299-306).
Exclusion of UV (280–380 nm) radiation from the solar spectrum can be an important tool to assess the impact of ambient UV radiation on plant growth and performance of crop plants. The effect of exclusion of UV-B and UV-A from solar radiation on the growth and photosynthetic components in soybean (Glycine max) leaves were investigated. Exclusion of solar UV-B and UV-B/A radiation, enhanced the fresh weight, dry weight, leaf area as well as induced a dramatic increase in plant height, which reflected a net increase in biomass. Dry weight increase per unit leaf area was quite significant upon both UV-B and UV-B/A exclusion from the solar spectrum. However, no changes in chlorophyll a and b contents were observed by exclusion of solar UV radiation but the content of carotenoids was significantly (34–46%) lowered. Analysis of chlorophyll (Chl) fluorescence transient parameters of leaf segments suggested no change in the F v/F m value due to UV-B or UV-B/A exclusion. Only a small reduction in photo-oxidized signal I (P700+)/unit Chl was noted. Interestingly the total soluble protein content per unit leaf area increased by 18% in UV-B/A and 40% in UV-B excluded samples, suggesting a unique upregulation of biosynthesis and accumulation of biomass. Solar UV radiation thus seems to primarily affect the photomorphogenic regulatory system that leads to an enhanced growth of leaves and an enhanced rate of net photosynthesis in soybean, a crop plant of economic importance. The presence of ultra-violet components in sunlight seems to arrest carbon sequestration in plants.
Keywords: Biomass; Growth; Photosynthesis; Soybean (Glycine max); UV-exclusion; UV-B radiation

Mechanisms for countering environmental stress are essential to photosynthetic organisms. Alteration of the photosynthetic apparatus, a mechanism for balancing the flux of light energy and carbon fixation, can be characterized by fluorescence properties. In this study, we have established a simple protocol to determine the extent of energy-dependent quenching (qE) and quenching by state transition (qT) in Chlamydomonas cells by examining their fluorescence properties under light fluctuations. We identified qE as the uncoupler-sensitive NPQ component that was rapidly relaxed upon transition to dark conditions. We characterized the qT component by determining low-temperature fluorescence spectra and analyzing a state-transition-less mutant. By these methods, we observed that similar abiotic stresses—high light conditions (where excess energy is supplied) and low CO2 conditions (where energy utilization is limited)—induced different types of NPQ. High light conditions induced mainly qE-quenching that increased gradually while low CO2 conditions induced mainly qT-quenching that peaked in 20 min and then decreased gradually. That high light and low carbon signals induced different physiological responses suggests that they triggered different genetic responses, which altered protein expression under each of the conditions.
Keywords: CCM; High light; Light-harvesting complex; NPQ; Photoacclimation; State transition

Previously, it was clarified that phycobilisome (PBS) mobility and energy spillover were both involved in light-to-dark induced state transitions of intact Spirulina platensis cells. In this work, by taking advantage of the characteristic fluorescence spectra of photosystem I (PSI) trimers and monomers as indicators, the relative contributions for the “mobile PBS” and “energy spillover” are quantitatively estimated by separating the fluorescence contribution of PBS mobility from that of PSI oligomeric change. Above the phase transition temperature (T PT) of the membrane lipids, the relative proportion of the contributions is invariable with 65% of “mobile PBS” and 35% of “energy spillover”. Below T PT, the proportion for the “mobile PBS” becomes larger under lowering temperature even reaching 95% with 5% “energy spillover” at 0°C. It is known that lower temperature leads to a further light state due to a more reduced or oxidized PQ pool. Based on the current result, it can be deduced that disequilibrium of the redox state of the PQ pool will trigger PBS movement instead of change in the PSI oligomeric state.
Keywords: Cyanobacterium; State transition; Phycobilisomes; Mobility; Energy spillover; Relative contribution

Trapping of the quenched conformation associated with non-photochemical quenching of chlorophyll fluorescence at low temperature by Petar H. Lambrev; Tsonko Tsonev; Violeta Velikova; Katya Georgieva; Maya D. Lambreva; Ivan Yordanov; László Kovács; Győző Garab (321-332).
The kinetics of non-photochemical quenching (NPQ) of chlorophyll fluorescence was studied in pea leaves at different temperatures between 5 and 25°C and during rapid jumps of the leaf temperature. At 5°C, NPQ relaxed very slowly in the dark and was sustained for up to 30 min. This was independent of the temperature at which quenching was induced. Upon raising the temperature to 25°C, the quenched state relaxed within 1 min, characteristic for qE, the energy-dependent component of NPQ. Measurements of the membrane permeability (ΔA515) in dark-adapted and preilluminated leaves and NPQ in the presence of dithiothreitol strongly suggest that the effect of low temperature on NPQ was not because of limitation by the lumenal pH or the de-epoxidation state of the xanthophylls. These data are consistent with the notion that the transition from the quenched to the unquenched state and vice versa involves a structural reorganization in the photosynthetic apparatus. An eight-state reaction scheme for NPQ is proposed, extending the model of Horton and co-workers (FEBS Lett 579:4201–4206, 2005), and a hypothesis is put forward concerning the nature of conformational changes associated with qE.
Keywords: Chlorophyll a fluorescence; Conformational change; ΔpH; Low temperature; Non-photochemical quenching; qE; Zeaxanthin

Changes in the mode of electron flow to photosystem I following chilling-induced photoinhibition in a C3 plant, Cucumis sativus L. by Sridharan Govindachary; Caroline Bigras; Johanne Harnois; David Joly; Robert Carpentier (333-345).
This study provides evidence for enhanced electron flow from the stromal compartment of the photosynthetic membranes to P700+ via the cytochrome b6/f complex (Cyt b6/f) in leaves of Cucumis sativus L. submitted to chilling-induced photoinhibition. The above is deduced from the P700 oxidation–reduction kinetics studied in the absence of linear electron transport from water to NADP+, cyclic electron transfer mediated through the Q-cycle of Cyt b6/f and charge recombination in photosystem I (PSI). The segregation of these pathways for P700+ rereduction were achieved by the use of a 50-ms multiple turnover white flash or a strong pulse of white or far-red illumination together with inhibitors. In cucumber leaves, chilling-induced photoinhibition resulted in ∼20% loss of photo-oxidizible P700. The measurement of P700+ was greatly limited by the turnover of cyclic processes in the absence of the linear mode of electron transport as electrons were rapidly transferred to the smaller pool of P700+. The above is explained by integrating the recent model of the cyclic electron flow in C3 plants based on the Cyt b6/f structural data [Joliot and Joliot (2006) Biochim Biophys Acta 1757:362–368] and a photoprotective function elicited by a low NADP+/NAD(P)H ratio [Rajagopal et al. (2003) Biochemistry 42:11839–11845]. Over-reduction of the photosynthetic apparatus results in the accumulation of NAD(P)H in vivo to prevent NADP+-induced reversible conformational changes in PSI and its extensive damage. As the ferredoxin:NADP reductase is fully reduced under these conditions, even in the absence of PSII electron transport, the reduced ferredoxin generated during illumination binds at the stromal openings in the Cyt b6/f complex and activates cyclic electron flow. On the other hand, the excess electrons from the NAD(P)H pool are routed via the Ndh complex in a slow process to maintain moderate reduction of the plastoquinone pool and redox poise required for the operation of ferredoxin:plastoquinone reductase mediated cyclic flow.
Keywords: Chilling stress; Cyclic electron transfer; Cytochrome b6/f complex; NADP+/NAD(P)H; P700; Photosystem; Photoinhibition; Plastoquinone

Quantification of cyclic electron flow around Photosystem I in spinach leaves during photosynthetic induction by Da-Yong Fan; Qin Nie; Alexander B. Hope; Warwick Hillier; Barry J. Pogson; Wah Soon Chow (347-357).
The variation of the rate of cyclic electron transport around Photosystem I (PS I) during photosynthetic induction was investigated by illuminating dark-adapted spinach leaf discs with red + far-red actinic light for a varied duration, followed by abruptly turning off the light. The post-illumination re-reduction kinetics of P700+, the oxidized form of the photoactive chlorophyll of the reaction centre of PS I (normalized to the total P700 content), was well described by the sum of three negative exponential terms. The analysis gave a light-induced total electron flux from which the linear electron flux through PS II and PS I could be subtracted, yielding a cyclic electron flux. Our results show that the cyclic electron flux was small in the very early phase of photosynthetic induction, rose to a maximum at about 30 s of illumination, and declined subsequently to <10% of the total electron flux in the steady state. Further, this cyclic electron flow, largely responsible for the fast and intermediate exponential decays, was sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, suggesting an important role of redox poising of the cyclic components for optimal function. Significantly, our results demonstrate that analysis of the post-illumination re-reduction kinetics of P700+ allows the quantification of the cyclic electron flux in intact leaves by a relatively straightforward method.
Keywords: Cyclic electron transport; P700; Photosynthetic induction; Photosystem I

Chloroplast translation regulation by Julia Marín-Navarro; Andrea L. Manuell; Joann Wu; Stephen P. Mayfield (359-374).
Chloroplast gene expression is primarily controlled during the translation of plastid mRNAs. Translation is regulated in response to a variety of biotic and abiotic factors, and requires a coordinate expression with the nuclear genome. The translational apparatus of chloroplasts is related to that of bacteria, but has adopted novel mechanisms in order to execute the specific roles that this organelle performs within a eukaryotic cell. Accordingly, plastid ribosomes contain a number of chloroplast-unique proteins and domains that may function in translational regulation. Chloroplast translation regulation involves cis-acting RNA elements (located in the mRNA 5′ UTR) as well as a set of corresponding trans-acting protein factors. While regulation of chloroplast translation is primarily controlled at the initiation steps through these RNA-protein interactions, elongation steps are also targets for modulating chloroplast gene expression. Translation of chloroplast mRNAs is regulated in response to light, and the molecular mechanisms underlying this response involve changes in the redox state of key elements related to the photosynthetic electron chain, fluctuations of the ADP/ATP ratio and the generation of a proton gradient. Photosynthetic complexes also experience assembly-related autoinhibition of translation to coordinate the expression of different subunits of the same complex. Finally, the localization of all these molecular events among the different chloroplast subcompartments appear to be a crucial component of the regulatory mechanisms of chloroplast gene expression.
Keywords: Chloroplast translation; Photosystem II; Photosystem I; Cytochrome b6/f; Chloroplast ribosome; Light-regulated gene expression

Photodamage occurs when leaves are exposed to light in excess of what can be used for photosynthesis and in excess of the capacity of ancillary photoprotective as well as repair mechanisms. An important site of photodamage is the chloroplast encoded D1 protein, a component of the photosystem II (PSII) reaction center. Even under optimal growth irradiance, D1 is photodamaged necessitating rapid turnover to prevent the accumulation of photodamaged PSII reaction centers and consequent inhibition of photosynthesis. However, this on-going process of D1 turnover and replacement was impeded in the chilling-sensitive tomato (Solanum lycopersicum) plants when exposed to high-growth light at cool temperature. The decrease in D1 turnover and replacement was found not to be due to changes in the steady-state level of the psbA message. While the recruitment of ribosomes to psbA transcript, initiation of D1 translation, and the association of polysomes with the thylakoid membrane occurred normally, chilling temperatures caused ribosomal pausing during D1 peptide elongation in tomato. The pause locations were non-randomly located on the D1 transcript. The interference with translation caused by ribosomal pausing allowed photodamaged PSII centers to accumulate leading to the consequent inhibition of photosynthesis.
Keywords: D1; Environmental stress; PSII; Low temperature; High light; Translation; Polysomes

Chlorophylls, ligands and assembly of light-harvesting complexes in chloroplasts by J. Kenneth Hoober; Laura L. Eggink; Min Chen (387-400).
Chlorophyll (Chl) b serves an essential function in accumulation of light-harvesting complexes (LHCs) in plants. In this article, this role of Chl b is explored by considering the properties of Chls and the ligands with which they interact in the complexes. The overall properties of the Chls, not only their spectral features, are altered as consequences of chemical modifications on the periphery of the molecules. Important modifications are introduction of oxygen atoms at specific locations and reduction or desaturation of sidechains. These modifications influence formation of coordination bonds by which the central Mg atom, the Lewis acid, of Chl molecules interacts with amino acid sidechains, as the Lewis base, in proteins. Chl a is a versatile Lewis acid and interacts principally with imidazole groups but also with sidechain amides and water. The 7-formyl group on Chl b withdraws electron density toward the periphery of the molecule and consequently the positive Mg is less shielded by the molecular electron cloud than in Chl a. Chl b thus tends to form electrostatic bonds with Lewis bases with a fixed dipole, such as water and, in particular, peptide backbone carbonyl groups. The coordination bonds are enhanced by H-bonds between the protein and the 7-formyl group. These additional strong interactions with Chl b are necessary to achieve assembly of stable LHCs.
Keywords: Chlorophyll; Chlorophyll b ; Chlorophyll c ; Chlorophyll molecular axes; Chlorophyllide a oxygenase; Coordination bonds; Dipole moments; Lewis acid; Lewis base; Ligands; Light-harvesting complex; Chloroplast development

Stroma, envelope and thylakoid membranes were prepared from chloroplasts isolated from leaves of Beta vulgaris. Out of total plastidic protochlorophyllide, envelope membranes contained 1.5%, thylakoids had the maximum 98.48% and stroma had a trace fraction of 0.02%. Distribution of the Mg-protoporphyrin IX and its monoester was 89.0% in thylakoids, 10.0% in stroma and 1.0% in envelope. A substantial fraction (33.77%) of plastidic protoporphyrin IX was partitioned into stroma. Envelope contained 0.66% and thylakoids had 65.57% of the total plastidic protoporphyrin IX pool. The proportion of monovinyl and divinyl forms of protochlorophyllide was almost similar in intact plastid, thylakoids, and outer and inner envelope membranes suggesting a tight regulation of vinyl reductase enzyme. The significance of differential distribution of chlorophyll biosynthetic intermediates among thylakoids, envelope and stroma is discussed.
Keywords: Chlorophyll biosynthesis; Chloroplast biogenesis; Cucumis sativus ; Envelope membrane; Mg-protoporphyrin IX monoester; Protoporphyrin IX; Protochlorophyllide

Brownian Dynamics (BD) computer simulations were used to study electrostatic interactions between turnip cytochrome f (cyt f) and spinach plastocyanin (PC). Three different spinach PC structures were studied: The X-ray crystal structure of Xue and coworkers [(1998) Protein Sci 7:2099–2105] and the NMR structure of Musiani et al. [(2005) J Biol Chem 280:18833–18841] and Ubbink and co-workers [(1998) Structure 6:323–335]. Significant differences exist in the backbone conformation between the PC taken from Ubbink and coworkers and the other two PC structures particularly the regions surrounding G10, E59–E60, and D51. Complexes formed in BD simulations using the PC of Ubbink and colleagues had a smaller Cu–Fe distance than the other two. These results suggest that different PC conformations may exist in solution with different capabilities of forming electron-transfer-active docks. All three types of complexes show electrostatic contacts between D42, E43, and D44 on PC and K187 on cyt f as well as between E59 on PC and K58 on cyt f. However, the PC of Ubbink and coworkers reveals additional contacts between D51 and cyt f as a result of the difference in backbone configuration. A second minor complex component was observed for the PC of Ubbink and co-workers and Xue and co-workers which had contacts between K187 on cyt f and E59 and E60 on PC rather than between K187 on cyt f and D42-D44 on PC as observed for the major components. This second type of complex may represent an earlier complex which rearranges to form a final complex capable of electron transfer.
Keywords: Brownian dynamics; Cytochrome f ; Electron transport; Plastocyanin; Protein–protein interactions

The OJIP fast fluorescence rise characterizes Graptophyllum species and their stress responses by Le Buu Thach; Alison Shapcott; Susanne Schmidt; Christa Critchley (423-436).
Causes for rarity in plants are poorly understood. Graptophyllum reticulatum is an endangered endemic species, and it has three close relatives with different conservation status: the vulnerable G. ilicifolium, the rare G. excelsum, and the common G. spinigerum. Applied to the chlorophyll a fluorescence transient of leaves, the JIP test provides a Performance Index (PI) which quantifies the main steps in photosystem II (PSII) photochemistry including light energy absorption, excitation energy trapping, and conversion of excitation energy into electron flow. The PI is calculated from three components which depend on the reaction center density, the trapping efficiency, and the electron transport efficiency. PI was measured in the natural habitats of the four species and under artificially imposed environmental stresses in the glasshouse to determine whether conservation status was related to stress resilience. The results showed that soil type is unlikely to restrict the endangered G. reticulatum, vulnerable G. ilicifolium, or rare G. excelsum because PI was similar in plants grown in diverse soils in the glasshouse. Photoinhibition is likely to restrict the endangered G. reticulatum to shade habitats because PI was significantly reduced when plants were exposed to more than 15% ambient light in controlled experiments. Water availability may determine the location and distribution of the vulnerable G. ilicifolium and common G. spinigerum because PI was reduced more than 60% when plants were exposed to water stress. While the characteristics of their natural habitats correspond to and explain the physiological responses, there was no obvious relationship between conservation status and environmental resilience. PI can be used to monitor vigor and health of populations of plants in the natural habitat. In cultivation experiments PI responds to key environmental variables that affect the distribution of species with conservation significance.
Keywords: Fv/Fm ratio; JIP test; Graptophyllum ; Performance Index; Photosynthesis; Rarity; Stress

Sensitivity to cold and freezing differs between populations within two species of live oaks (Quercus section Virentes Nixon) corresponding to the climates from which they originate. Two populations of Quercus virginiana (originating from North Carolina and north central Florida) and two populations of the sister species, Q. oleoides, (originating from Belize and Costa Rica) were grown under controlled climate regimes simulating tropical and temperate conditions. Three experiments were conducted in order to test for differentiation in cold and freezing tolerance between the two species and between the two populations within each species. In the first experiment, divergences in response to cold were tested for by examining photosystem II (PS II) photosynthetic yield (ΔF/F m′) and non-photochemical quenching (NPQ) of plants in both growing conditions after short-term exposure to three temperatures (6, 15 and 30°C) under moderate light (400 μmol m−2 s−1). Without cold acclimation (tropical treatment), the North Carolina population showed the highest photosynthetic yield in response to chilling temperatures (6°C). Both ecotypes of both species showed maximum ΔF/F m′ and minimum NPQ at their daytime growth temperatures (30°C and 15°C for the tropical and temperate treatments, respectively). Under the temperate treatment where plants were allowed to acclimate to cold, the Q. virginiana populations showed greater NPQ under chilling temperatures than Q. oleoides populations, suggesting enhanced mechanisms of photoprotective energy dissipation in the more temperate species. In the second and third experiments, inter- and intra-specific differentiation in response to freezing was tested for by examining dark-adapted F v/F m before and after overnight freezing cycles. Without cold acclimation, the extent of post-freezing declines in F v/F m were dependent on the minimum freezing temperature (0, −2, −5 or −10°C) for both populations in both species. The most marked declines in F v/F m occurred after freezing at −10°C, measured 24 h after freezing. These declines were continuous and irreversible over the time period. The North Carolina population, however, which represents the northern range limit of Q. virginiana, showed significantly less decline in F v/F m than the north central Florida population, which in turn showed a lower decline in Fv/F m than the two Q. oleoides populations from Belize and Costa Rica. In contrast, after exposure to three months of chilling temperatures (temperate treatment), the two Q. virginiana populations showed no decline in F v/F m after freezing at −10°C, while the two Q. oleoides populations showed declines in F v/F m reaching 0.2 and 0.1 for Costa Rica and Belize, respectively. Under warm growth conditions, the two species showed different F 0 dynamics directly after freezing. The two Q. oleoides populations showed an initial rise in F 0 30 min after freezing, followed by a subsequent decrease, while the Q. virginiana populations showed a continuous decrease in F 0 after freezing. The North Carolina population of Q. virginiana showed a tendency toward deciduousness in response to winter temperatures, dropping 58% of its leaves over the three month winter period compared to only 6% in the tropical treatment. In contrast, the Florida population dropped 38% of its leaves during winter. The two populations of the tropical Q. oleoides showed no change in leaf drop during the 3-months winter (10% and 12%) relative to their leaf drop over the same timecourse in the tropical treatment. These results indicate important ecotypic differences in sensitivity to freezing and cold stress between the two populations of Q. virginiana as well as between the two species, corresponding to their climates of origin.
Keywords: Chilling and freezing sensitivity; Chlorophyll fluorescence; Irreversible decline in F v/F m ; F 0 ; Non-photochemical quenching; Population and species-level variation; Ecotypes; Climatic gradient; Live oaks

Photosynthetic acclimation in the context of structural constraints to carbon export from leaves by William W. Adams III; Amy M. Watson; Kristine E. Mueh; Véronique Amiard; Robert Turgeon; Volker Ebbert; Barry A. Logan; Andrew F. Combs; Barbara Demmig-Adams (455-466).
The potential role of foliar carbon export features in the acclimation of photosynthetic capacity to differences and changes in light environment was evaluated. These features included apoplastic vs. symplastic phloem loading, density of loading veins, plasmodesmatal frequency in intermediary cells, and the ratio of loading cells to sieve elements. In initial studies, three apoplastic loaders (spinach, pea, Arabidopsis thaliana) exhibited a completely flexible photosynthetic response to changing light conditions, while two symplastic loaders (pumpkin, Verbascum phoeniceum), although able to adjust to different long-term growth conditions, were more limited in their response when transferred from low (LL) to high (HL) light. This suggested that constraints imposed by the completely physical pathway of sugar export might act as a bottleneck in the export of carbon from LL-acclimated leaves of symplastic loaders. While both symplastic loaders exhibited variable loading vein densities (low in LL and high in HL), none of the three apoplastic loaders initially characterized exhibited such differences. However, an additional apoplastic species (tomato) exhibited similar differences in vein density during continuous growth in different light environments. Furthermore, in contrast to the other apoplastic loaders, photosynthetic acclimation in tomato was not complete following a transfer from LL to HL. This suggests that loading vein density and loading cells per sieve element, and thus apparent loading surface capacity, play a major role in the potential for photosynthetic acclimation to changes in light environment. Photosynthetic acclimation and vein density acclimation were also characterized in the slow-growing, sclerophytic evergreen Monstera deliciosa. This evergreen possessed a lower vein density during growth in LL compared to HL and exhibited a more severely limited potential for photosynthetic acclimation to increases in light environment than the rapidly-growing, mesophytic annuals.
Keywords: Apoplastic loading; Companion cells; Intermediary cells; Leaf vein density; Phloem; Phloem parenchyma cells; Photosynthetic acclimation; Plasmodesmata; Symplastic loading; Transfer cells