Photosynthesis Research (v.108, #2-3)
Peroxisomal hydroxypyruvate reductase is not essential for photorespiration in Arabidopsis but its absence causes an increase in the stoichiometry of photorespiratory CO2 release by Asaph B. Cousins; Berkley J. Walker; Itsara Pracharoenwattana; Steven M. Smith; Murray R. Badger (91-100).
Recycling of carbon by the photorespiratory pathway involves enzymatic steps in the chloroplast, mitochondria, and peroxisomes. Most of these reactions are essential for plants growing under ambient CO2 concentrations. However, some disruptions of photorespiratory metabolism cause subtle phenotypes in plants grown in air. For example, Arabidopsis thaliana lacking both of the peroxisomal malate dehydrogenase genes (pmdh1pmdh2) or hydroxypyruvate reductase (hpr1) are viable in air and have rates of photosynthesis only slightly lower than wild-type plants. To investigate how disruption of the peroxisomal reduction of hydroxypyruvate to glycerate influences photorespiratory carbon metabolism we analyzed leaf gas exchange in A. thaliana plants lacking peroxisomal HPR1 expression. In addition, because the lack of HPR1 could be compensated for by other reactions within the peroxisomes using reductant supplied by PMDH a triple mutant lacking expression of both peroxisomal PMDH genes and HPR1 (pmdh1pmdh2hpr1) was analyzed. Rates of photosynthesis under photorespiratory conditions (ambient CO2 and O2 concentrations) were slightly reduced in the hpr1 and pmdh1pmdh2hpr1 plants indicating other reactions can help bypass this disruption in the photorespiratory pathway. However, the CO2 compensation points (Γ) increased under photorespiratory conditions in both mutants indicating changes in photorespiratory carbon metabolism in these plants. Measurements of Γ*, the CO2 compensation point in the absence of mitochondrial respiration, and the CO2 released per Rubisco oxygenation reaction demonstrated that the increase in Γ in the hpr1 and pmdh1pmdh2hpr1 plants is not associated with changes in mitochondrial respiration but with an increase in the non-respiratory CO2 released per Rubisco oxygenation reaction.
Keywords: Photosynthesis; Photorespiration; Peroxisomes; Hydroxypyruvate reductase
Phosphorescence study of chlorophyll d photophysics. Determination of the energy and lifetime of the photo-excited triplet state. Evidence of singlet oxygen photosensitization by Konstantin V. Neverov; Stefano Santabarbara; Alexander A. Krasnovsky Jr. (101-106).
Chlorophyll d (Chl d) is the major pigment in both photosystems (PSI and II) of the cyanobacterium Acaryochloris marina, whose pigment composition represents an interesting alternative in oxygenic photosynthesis. While abundant information is available relative to photophysical properties of Chl a , the understanding of Chl d photophysics is still incomplete. In this paper, we present for the first time a characterization of Chl d phosphorescence, which accompanies radiative deactivation of the photoexcited triplet state of this pigment. Reliable information was obtained on the energy and lifetime of the Chl d triplet state in frozen solutions at 77 K using diethyl ether and aqueous dispersions of Triton X100 as solvents. It is shown that triplet Chl d is effectively populated upon photoexcitation of pigment molecules and efficiently sensitizes singlet oxygen phosphorescence in aerobic solutions under ambient conditions. The data obtained are compared with the previous results of the phosphorescence studies of Chl a and Pheo a, and their possible biological implications are discussed.
Keywords: Chlorophyll d ; Phosphorescence; Triplet state; Singlet oxygen
Mutagenesis and phenotypic selection as a strategy toward domestication of Chlamydomonas reinhardtii strains for improved performance in photobioreactors by Giulia Bonente; Cinzia Formighieri; Manuela Mantelli; Claudia Catalanotti; Giovanni Giuliano; Tomas Morosinotto; Roberto Bassi (107-120).
Microalgae have a valuable potential for biofuels production. As a matter of fact, algae can produce different molecules with high energy content, including molecular hydrogen (H2) by the activity of a chloroplastic hydrogenase fueled by reducing power derived from water and light energy. The efficiency of this reaction, however, is limited and depends from an intricate relationships between oxygenic photosynthesis and mitochondrial respiration. The way toward obtaining algal strains with high productivity in photobioreactors requires engineering of their metabolism at multiple levels in a process comparable to domestication of crops that were derived from their wild ancestors through accumulation of genetic traits providing improved productivity under conditions of intensive cultivation as well as improved nutritional/industrial properties. This holds true for the production of any biofuels from algae: there is the need to isolate multiple traits to be combined and produce organisms with increased performances. Among the different limitations in H2 productivity, we identified three with a major relevance, namely: (i) the light distribution through the mass culture; (ii) the strong sensitivity of the hydrogenase to even very low oxygen concentrations; and (iii) the presence of alternative pathways, such as the cyclic electron transport, competing for reducing equivalents with hydrogenase and H2 production. In order to identify potentially favorable mutations, we generated a collection of random mutants in Chlamydomonas reinhardtii which were selected through phenotype analysis for: (i) a reduced photosynthetic antenna size, and thus a lower culture optical density; (ii) an altered photosystem II activity as a tool to manipulate the oxygen concentration within the culture; and (iii) State 1–State 2 transition mutants, for a reduced cyclic electron flow and maximized electrons flow toward the hydrogenase. Such a broad approach has been possible thanks to the high throughput application of absorption/fluorescence optical spectroscopy methods. Strong and weak points of this approach are discussed.
Keywords: Green microalgae; Photosynthesis; Biomass accumulation; Random mutagenesis; Chlamydomonas
New insights into the function of the iron deficiency-induced protein C from Synechococcus elongatus PCC 7942 by Daniel Pietsch; Gábor Bernát; Uwe Kahmann; Dorothee Staiger; Elfriede K. Pistorius; Klaus-Peter Michel (121-132).
Iron limitation has a strong impact on electron transport reactions of the unicellular fresh water cyanobacterium Synechococcus elongatus PCC 7942 (thereafter referred to as S. elongatus). Among the various adaptational processes on different cellular levels, iron limitation induces a strongly enhanced expression of IdiC (iron-deficiency-induced protein C). In this article, we show that IdiC is loosely attached to the thylakoid and to the cytoplasmic membranes and that its expression is enhanced during conditions of iron starvation and during the late growth phase. The intracellular IdiC level was even more increased when additional iron was replenished in the late growth phase. On the basis of its amino acid sequence and of its absorbance spectrum, IdiC can be classified as a member of the family of thioredoxin (TRX)-like (2Fe–2S) ferredoxins. The presence of an iron cofactor in IdiC was detected by inductive coupled plasma optical emission spectrometry (ICP-OES). Comparative measurements of electron transport activities of S. elongatus wild type (WT) and an IdiC-merodiploid mutant called MuD, which contained a strongly reduced IdiC content under iron-sufficient as well as iron-deficient growth conditions, were performed. The results revealed that MuD had a strongly increased light sensitivity, especially under iron limitation. The measurements of photosystem II (PS II)-mediated electron transport rates in WT and MuD strain showed that PS II activity was significantly lower in MuD than in the WT strain. Moreover, P700 + re-reduction rates provided evidence that the respiratory activities, which were very low in the MuD strain in the presence of iron, significantly increased in iron-starved cells. Thus, an increase in respiration may compensate for the drastic decrease of photosynthetic electron transport activity in MuD grown under iron starvation. Based on the similarity of the S. elongatus IdiC to the NuoE subunit of the NDH-1 complex in Escherichia coli, it is likely that IdiC has a function in the electron transport processes from NAD(P)H to the plastoquinone pool. This is in agreement with the up-regulation of IdiC in the late growth phase as well as under stress conditions when PS II is damaged. As absence or high reduction of the IdiC level would prevent or reduce the formation of functional NDH-1 complexes, under such conditions electron transport routes via alternative substrate dehydrogenases, donating electrons to the plastoquinone pool, can be assumed to be up-regulated.
Keywords: Synechococcus elongatus PCC 7942; IdiC; Iron deficiency; Respiration; NDH-1 complex
Effects of the measuring light on the photochemistry of the bacterial photosynthetic reaction center from Rhodobacter sphaeroides by Ivan Husu; Mauro Giustini; Giuseppe Colafemmina; Gerardo Palazzo; Antonia Mallardi (133-142).
The bacterial reaction center (RC) has become a reference model in the study of the diverse interactions of quinones with electron transfer complexes. In these studies, the RC functionality was probed through flash-induced absorption changes where the state of the primary donor is probed by means of a continuous measuring beam and the electron transfer is triggered by a short intense light pulse. The single-beam set-up implies the use as reference of the transmittance measured before the light pulse. Implicit in the analysis of these data is the assumption that the measuring beam does not elicit the protein photochemistry. At variance, measuring beam is actinic in nature at almost all the suitable wavelengths. In this contribution, the analytical modelling of the time evolution of neutral and charge-separated RCs has been performed. The ability of measuring light to elicit RC photochemistry induces a first order growth of the charge-separated state up to a steady state that depends on the light intensity and on the occupation of the secondary quinone (QB) site. Then the laser pulse pumps all the RCs in the charge-separated state. The following charge recombination is still affected by the measuring beam. Actually, the kinetics of charge recombination measured in RC preparation with the QB site partially occupied are two-exponential. The rate constant of both fast and slow phases depends linearly on the intensity of the measuring beam while their relative weights depend not only on the fractions of RC with the QB site occupied but also on the measuring light intensity itself.
Keywords: Rhodobacter sphaeroides ; Reaction center; Electron transfer; Light intensity
The activity of Rubisco’s molecular chaperone, Rubisco activase, in leaf extracts by A. Elizabete Carmo-Silva; Michael E. Salvucci (143-155).
Rubisco frequently undergoes unproductive interactions with its sugar-phosphate substrate that stabilize active sites in an inactive conformation. Restoring catalytic competence to these sites requires the “molecular chiropractic” activity of Rubisco activase (activase). To make the study of activase more routine and physiologically relevant, an assay was devised for measuring activase activity in leaf extracts based on the ATP-dependent activation of inactive Rubisco. Control experiments with an Arabidopsis activase-deficient mutant confirmed that the rate of Rubisco activation was dependent on the concentration of activase in the extracts. Activase catalyzed Rubisco activation at rates equivalent to 9–14% catalytic sites per min in desalted extracts of Arabidopsis, camelina, tobacco, cotton, and wheat. Faster rates were observed in a transgenic line of Arabidopsis that expresses only the β-isoform of activase, whereas no activity was detected in a line that expresses only the α-isoform. Activase activity was also low or undetectable in rice, maize, and Chlamydomonas, revealing differences in the stability of the enzyme in different species. These differences are discussed in terms of the ability of activase subunits to remain associated or to reassociate into active oligomers when the stromal milieu is diluted by extraction. Finally, the temperature response of activase activity in leaf extracts differed for Arabidopsis, camelina, tobacco, and cotton, corresponding to the respective temperature responses of photosynthesis for each species. These results confirmed the exceptional thermal lability of activase at physiological ratios of activase to Rubisco.
Keywords: Rubisco; Molecular chaperone; Heat stress; CO2 assimilation; AAA+ protein; Photosynthesis
Identification of C4 photosynthesis metabolism and regulatory-associated genes in Eleocharis vivipara by SSH by Taiyu Chen; Rongjian Ye; Xiaolei Fan; Xianghua Li; Yongjun Lin (157-170).
This is the first effort to investigate the candidate genes involved in kranz developmental regulation and C4 metabolic fluxes in Eleocharis vivipara, which is a leafless freshwater amphibious plant and possesses a distinct culms anatomy structure and photosynthetic pattern in contrasting environments. A terrestrial specific SSH library was constructed to investigate the genes involved in kranz anatomy developmental regulation and C4 metabolic fluxes. A total of 73 ESTs and 56 unigenes in 384 clones were identified by array hybridization and sequencing. In total, 50 unigenes had homologous genes in the databases of rice and Arabidopsis. The real-time quantitative PCR results showed that most of the genes were accumulated in terrestrial culms and ABA-induced culms. The C4 marker genes were stably accumulated during the culms development process in terrestrial culms. With respect to C3 culms, C4 photosynthesis metabolism consumed much more transporters and translocators related to ion metabolism, organic acids and carbohydrate metabolism, phosphate metabolism, amino acids metabolism, and lipids metabolism. Additionally, ten regulatory genes including five transcription factors, four receptor-like proteins, and one BURP protein were identified. These regulatory genes, which co-accumulated with the culms developmental stages, may play important roles in culms structure developmental regulation, bundle sheath chloroplast maturation, and environmental response. These results shed new light on the C4 metabolic fluxes, environmental response, and anatomy structure developmental regulation in E. vivipara.
Keywords: Eleocharis vivipara ; C4 photosynthesis metabolic fluxes; Kranz anatomy development; Environment and ABA responding; SSH
The effects of moderately high temperature on zeaxanthin accumulation and decay by Ru Zhang; David M. Kramer; Jeffrey A. Cruz; Kimberly R. Struck; Thomas D. Sharkey (171-181).
Moderately high temperature reduces photosynthetic capacities of leaves with large effects on thylakoid reactions of photosynthesis, including xanthophyll conversion in the lipid phase of the thylakoid membrane. In previous studies, we have found that leaf temperature of 40°C increased zeaxanthin accumulation in dark-adapted, intact tobacco leaves following a brief illumination, but did not change the amount of zeaxanthin in light-adatped leaves. To investigate heat effects on zeaxanthin accumulation and decay, zeaxanthin level was monitored optically in dark-adapted, intact tobacco and Arabidopsis thaliana leaves at either 23 or 40°C under 45-min illumination. Heated leaves had more zeaxanthin following 3-min light but had less or comparable amounts of zeaxanthin by the end of 45 min of illumination. Zeaxanthin accumulated faster at light initiation and decayed faster upon darkening in leaves at 40°C than leaves at 23°C, indicating that heat increased the activities of both violaxanthin de-epoxidase (VDE) and zeaxanthin epoxidase (ZE). In addition, our optical measurement demonstrated in vivo that weak light enhances zeaxanthin decay relative to darkness in intact leaves of tobacco and Arabidopsis, confirming previous observations in isolated spinach chloroplasts. However, the maximum rate of decay is similar for weak light and darkness, and we used the maximum rate of decay following darkness as a measure of the rate of ZE during steady-state light. A simulation indicated that high temperature should cause a large shift in the pH dependence of the amount of zeaxanthin in leaves because of differential effects on VDE and ZE. This allows for the reduction in ΔpH caused by heat to be offset by increased VDE activity relative to ZE.
Keywords: Zeaxanthin; Violaxanthin de-epoxidase; Zeaxanthin epoxidase; Moderately high temperature; ΔpH
A novel and mild isolation procedure of chlorosomes from the green sulfur bacterium Chlorobaculum tepidum by Seiji Tokita; Keizo Shimada; Kazuyuki Watabe; Katsumi Matsuura; Mamoru Mimuro (183-190).
In this article, we developed a new and mild procedure for the isolation of chlorosomes from a green sulfur bacterium Chlorobaculum tepidum. In this procedure, Fenna–Matthews–Olson (FMO) protein was released by long cold treatment (6°C) of cells under the presence of a chaotrope (2 M NaSCN) and 0.6 M sucrose. Chlorosomes were released by an osmotic shock of the cold-treated cells after the formation of spheroplasts without mechanical disruption. Chlorosomes were finally purified by a sucrose step-wise density gradient centrifugation. We obtained two samples with different density (20 and 23% sucrose band, respectively) and compared them by SDS-PAGE, absorption spectroscopy at 80 K, fluorescence and CD spectroscopy at room temperature. Cells whose absorption maximum was longer than 750 nm yielded higher amount of the 20% sucrose fraction than those having an absorption maximum shorter than 750 nm.
Keywords: Isolation method; Chlorosomes; FMO protein; Low-temperature absorption; Chlorobaculum tepidum
Adaptation of photosystem II to high and low light in wild-type and triazine-resistant Canola plants: analysis by a fluorescence induction algorithm by Jack J. S. van Rensen; Wim J. Vredenberg (191-200).
Plants of wild-type and triazine-resistant Canola (Brassica napus L.) were exposed to very high light intensities and after 1 day placed on a laboratory table at low light to recover, to study the kinetics of variable fluorescence after light, and after dark-adaptation. This cycle was repeated several times. The fast OJIP fluorescence rise curve was measured immediately after light exposure and after recovery during 1 day in laboratory room light. A fluorescence induction algorithm has been used for resolution and analysis of these curves. This algorithm includes photochemical and photo-electrochemical quenching release components and a photo-electrical dependent IP-component. The analysis revealed a substantial suppression of the photo-electrochemical component (even complete in the resistant biotype), a partial suppression of the photochemical component and a decrease in the fluorescence parameter F o after high light. These effects were recovered after 1 day in the indoor light.
Keywords: Canola (Brassica napus L.); Chlorophyll fluorescence induction algorithm; Photoinhibition; Reaction kinetics; Triazine-resistance
Differential assembly of polypeptides of the light-harvesting 2 complex encoded by distinct operons during acclimation of Rhodobacter sphaeroides to low light intensity by Kamil Woronowicz; Oluwatobi B. Olubanjo; Hee Chang Sung; Joana L. Lamptey; Robert A. Niederman (201-214).
In order to obtain an improved understanding of the assembly of the bacterial photosynthetic apparatus, we have conducted a proteomic analysis of pigment-protein complexes isolated from the purple bacterium Rhodobacter sphaeroides undergoing acclimation to reduced incident light intensity. Photoheterotrophically growing cells were shifted from 1,100 to 100 W/m2 and intracytoplasmic membrane (ICM) vesicles isolated over 24-h were subjected to clear native polyacrylamide gel electrophoresis. Bands containing the LH2 and reaction center (RC)-LH1 complexes were excised and subjected to in-gel trypsin digestion followed by liquid chromatography (LC)-mass spectroscopy (MS)/MS. The results revealed that the LH2 band contained distinct levels of the LH2-α and -β polypeptides encoded by the two puc operons. Polypeptide subunits encoded by the puc2AB operon predominated under high light and in the early stages of acclimation to low light, while after 24 h, the puc1BAC components were most abundant. Surprisingly, the Puc2A polypeptide containing a 251 residue C-terminal extension not present in Puc1A, was a protein of major abundance. A predominance of Puc2A components in the LH2 complex formed at high light intensity is followed by a >2.5-fold enrichment in Puc1B levels between 3 and 24 h of acclimation, accompanied by a nearly twofold decrease in Puc2A levels. This indicates that the puc1BAC operon is under more stringent light control, thought to reflect differences in the puc1 upstream regulatory region. In contrast, elevated levels of Puc2 polypeptides were seen 48 h after the gratuitous induction of ICM formation at low aeration in the dark, while after 24 h of acclimation to low light, an absence of alterations in Puc polypeptide distributions was observed in the upper LH2-enriched gel band, despite an approximate twofold increase in overall LH2 levels. This is consistent with the origin of this band from a pool of LH2 laid down early in development that is distinct from subsequently assembled LH2-only domains, forming the LH2 gel band.
Keywords: Light-harvesting complexes; Light regulation; puc operon; Proteomics; Rhodobacter sphaeroides
Photosynthetic energy storage efficiency in Chlamydomonas reinhardtii, based on microsecond photoacoustics by Chengyi Yan; Oscar Schofield; Zvy Dubinsky; David Mauzerall; Paul G. Falkowski; Maxim Y. Gorbunov (215-224).
Using a novel, pulsed micro-second time-resolved photoacoustic (PA) instrument, we measured thermal dissipation and energy storage (ES) in the intact cells of wild type (WT) Chlamydomonas reinhardtii, and mutants lacking either PSI or PSII reaction centers (RCs). On this time scale, the kinetic contributions of the thermal expansion component due to heat dissipation of absorbed energy and the negative volume change due to electrostriction induced by charge separation in each of the photosystems could be readily distinguished. Kinetic analysis revealed that PSI and PSII RCs exhibit strikingly different PA signals where PSI is characterized by a strong electrostriction signal and a weak thermal expansion component while PSII has a small electrostriction component and large thermal expansion. The calculated ES efficiencies at ~10 μs were estimated to be 80 ± 5 and 50 ± 13% for PSII-deficient mutants and PSI-deficient mutants, respectively, and 67 ± 2% for WT. The overall ES efficiency was positively correlated with the ratio of PSI to PSI + PSII. Our results suggest that the shallow excitonic trap in PSII limits the efficiency of ES as a result of an evolutionary frozen metabolic framework of two photosystems in all oxygenic photoautotrophs.
Keywords: Energy storage efficiency; Photoacoustics; Photosystem I; Photosystem II; Chlamydomonas reinhardtii
A retrieval algorithm to evaluate the Photosystem I and Photosystem II spectral contributions to leaf chlorophyll fluorescence at physiological temperatures by Lorenzo Palombi; Giovanna Cecchi; David Lognoli; Valentina Raimondi; Guido Toci; Giovanni Agati (225-239).
A new computational procedure to resolve the contribution of Photosystem I (PSI) and Photosystem II (PSII) to the leaf chlorophyll fluorescence emission spectra at room temperature has been developed. It is based on the Principal Component Analysis (PCA) of the leaf fluorescence emission spectra measured during the OI photochemical phase of fluorescence induction kinetics. During this phase, we can assume that only two spectral components are present, one of which is constant (PSI) and the other variable in intensity (PSII). Application of the PCA method to the measured fluorescence emission spectra of Ficus benjamina L. evidences that the temporal variation in the spectra can be ascribed to a single spectral component (the first principal component extracted by PCA), which can be considered to be a good approximation of the PSII fluorescence emission spectrum. The PSI fluorescence emission spectrum was deduced by difference between measured spectra and the first principal component. A single-band spectrum for the PSI fluorescence emission, peaked at about 735 nm, and a 2-band spectrum with maxima at 685 and 740 nm for the PSII were obtained. A linear combination of only these two spectral shapes produced a good fit for any measured emission spectrum of the leaf under investigation and can be used to obtain the fluorescence emission contributions of photosystems under different conditions. With the use of our approach, the dynamics of energy distribution between the two photosystems, such as state transition, can be monitored in vivo, directly at physiological temperatures. Separation of the PSI and PSII emission components can improve the understanding of the fluorescence signal changes induced by environmental factors or stress conditions on plants.
Keywords: Chlorophyll; Ficus benjamina L.; Fluorescence emission spectra; Principal component analysis; Photosystem I; Photosystem II
Membrane attachment of Slr0006 in Synechocystis sp. PCC 6803 is determined by divalent ions by Dalton Carmel; Paula Mulo; Natalia Battchikova; Eva-Mari Aro (241-245).
Slr0006 is one of the Synechocystis sp. PCC 6803 proteins strongly induced under carbon limiting conditions. Slr0006 has no predicted transmembrane helices or signal peptide sequence, yet it was exclusively recovered in the membrane fraction of Synechocystis, when the cells were broken in isolation buffers which contain divalent cations and are generally used for photosynthesis studies. Even subsequent washing of the membranes with high salt or various detergents did not release Slr0006, indicating strong binding of the Slr0006 protein to the membranes. Further, DNAse or RNAse treatment did not disturb the tight binding of Slr0006 protein to the membranes. Nevertheless, when the cells were broken in the absence of divalent cations, Slr0006 remained completely soluble. Binding of the Slr0006 to the membrane could not be properly reconstituted if the cations were added after breaking the cells in the absence of divalent ions. This unusual phenomenon has to be considered in identification and localization of other yet uncharacterized cyanobacterial proteins.
Keywords: Divalent ions; Localization; Membrane; Slr0006; Synechocystis
A. Pareek, S. Sopory, H. J. Bohnert and Govindjee (eds): Abiotic stress adaptation in plants: physiological, molecular and genomic foundation by Maitrayee Bhattacharyya-Pakrasi (247-248).