Data on cadmium accumulation in chloroplasts of terrestrial plants are scarce and contradictory. We introduced CdSO4 in hydroponic media to the final concentrations 80 and 250 ?M and studied the accumulation of Cd in chloroplasts of Hordeum vulgare and Zea mays. Barley accumulated more Cd in the chloroplasts as compared to maize, whereas in the leaves cadmium accumulation was higher in maize. The cadmium content in the chloroplasts of two species varied from 49 to 171 ng Cd/mg chlorophyll, which corresponds to one Cd atom per 728–2,540 chlorophyll molecules. Therefore, Mg2+ can be substituted by Cd2+ in a negligible amount of antenna chlorophylls only. The percentage of chloroplastic cadmium can be estimated as 0.21–1.32 % of all the Cd in a leaf. Photochemistry (Fv/Fm, ?PSII, qP) was not influenced by Cd. Non-photochemical quenching of chlorophyll-excited state (NPQ) was greatly reduced in barley but not in maize. The decrease in NPQ was due to its fast relaxing component; the slow relaxing component rose slightly. In chloroplasts, Cd did not affect mRNA levels, but content of some photosynthetic proteins was reduced: slightly in the leaves of barley and heavily in the leaves of maize. In all analyzed C3-species, the effect of Cd on the content of photosynthetic proteins was mild or absent. This is most likely the first evidence of severe reduction of photosynthetic proteins in leaves of a Cd-treated C4-plant.
Chaetoceros gracilis belongs to the centric diatoms, and has recently been used in basic research on photosynthesis. In addition, it has been commercially used in fisheries and is also attracting interest as a feedstock for biofuels production and biorefinery. In this study, we developed an efficient genetic transformation system for C. gracilis. The diatom cells were transformed via multi-pulse electroporation using plasmids containing various promoters to drive expression of the nourseothricin acetyltransferase gene (nat) as a selectable marker. The transformation efficiency reached ~400 positive transgenic clones per 108 recipient cells, which is the first example of successful transformation with electroporation in a centric diatom species. We further produced two expression vectors: the vector pCgLhcr5p contains the light-dependent promoter of a fucoxanthin chlorophyll a/c binding protein gene and the vector pCgNRp contains the inducible promoter of a nitrate reductase gene to drive the expression of introduced genes. In both vectors, an acetyl-CoA acetyltransferase promoter drives nat gene expression for antibiotic selection. Stable integration and expression of reporter genes, such as the firefly luciferase and green fluorescent protein Azami–Green genes, were observed in transformed C. gracilis cells. This efficient and stable transformation system for C. gracilis will enable both functional analysis of diatom-specific genes and strain improvement for further biotechnological applications.
It had been proposed that a loop, typically containing 26 or 27 amino acids, which is only present in monomeric, ferredoxin-dependent, “plant-type” glutamate synthases and is absent from the catalytic ?-subunits of both NADPH-dependent, heterodimeric glutamate synthases found in non-photosynthetic bacteria and NADH-dependent heterodimeric cyanobacterial glutamate synthases, plays a key role in productive binding of ferredoxin to the plant-type enzymes. Site-directed mutagenesis has been used to delete the entire 27 amino acid-long loop in the ferredoxin-dependent glutamate synthase from the cyanobacterium Synechocystis sp. PCC 6803. The specific activity of the resulting loopless variant of this glutamate synthase, when reduced ferredoxin serves as the electron donor, is actually higher than that of the wild-type enzyme, suggesting that this loop is not absolutely essential for efficient electron transfer from reduced ferredoxin to the enzyme. These results are consistent with the results of an in-silico study that suggests that the loop is unlikely to interact directly with ferredoxin in the energetically most favorable model of a 1:1 complex of ferredoxin with the wild-type enzyme.
13C metabolic flux analysis (MFA) has made important contributions to our understanding of the physiology of model strains of E. coli and yeast, and it has been widely used to guide metabolic engineering efforts in these microorganisms. Recent advancements in 13C MFA methodology combined with publicly available software tools are creating new opportunities to extend this approach to examine less characterized microbes. In particular, growing interest in the use of cyanobacteria as industrial hosts for photosynthetic production of biofuels and biochemicals has led to a critical need to better understand how cyanobacterial metabolic fluxes are regulated in response to changes in growth conditions or introduction of heterologous pathways. In this contribution, we review several prior studies that have applied isotopic steady-state 13C MFA to examine heterotrophic or mixotrophic growth of cyanobacteria, as well as recent studies that have pioneered the use of isotopically nonstationary MFA (INST-MFA) to study autotrophic cultures. We also provide recommendations for the design and analysis of INST-MFA experiments in cyanobacteria, based on our previous experience and a series of simulation studies used to assess the selection of measurements and sample time points. We anticipate that this emerging knowledgebase of prior 13C MFA studies, optimized experimental protocols, and public software tools will catalyze increasing use of 13C MFA techniques by the cyanobacteria research community.
Agave salmiana Otto ex Salm-Dyck, a crassulacean acid metabolism plant that is adapted to water-limited environments, has great potential for bioenergy production. However, drought stress decreases the requirement for light energy, and if the amount of incident light exceeds energy consumption, the photosynthetic apparatus can be injured, thereby limiting plant growth. The objective of this study was to evaluate the effects of drought and re-watering on the photosynthetic efficiency of A. salmiana seedlings. The leaf relative water content and leaf water potential decreased to 39.6 % and ?1.1 MPa, respectively, over 115 days of water withholding and recovered after re-watering. Drought caused a direct effect on photosystem II (PSII) photochemistry in light-acclimated leaves, as indicated by a decrease in the photosynthetic electron transport rate. Additionally, down-regulation of photochemical activity occurred mainly through the inactivation of PSII reaction centres and an increased thermal dissipation capacity of the leaves. Prompt fluorescence kinetics also showed a larger pool of terminal electron acceptors in photosystem I (PSI) as well as an increase in some JIP-test parameters compared to controls, reflecting an enhanced efficiency and specific fluxes for electron transport from the plastoquinone pool to the PSI terminal acceptors. All the above parameters showed similar levels after re-watering. These results suggest that the thermal dissipation of excess energy and the increased energy conservation from photons absorbed by PSII to the reduction of PSI end acceptors may be an important acclimation mechanism to protect the photosynthetic apparatus from over-excitation in Agave plants.
William A. Arnold discovered many phenomena in photosynthesis. In 1932, together with Robert Emerson, he provided the first experimental data that led to the concept of a large antenna and a few reaction centers (photosynthetic unit); in 1935, he obtained the minimum quantum requirement of 8–10 for the evolution of one O2 molecule; in 1951, together with Bernard L. Strehler, he discovered delayed fluorescence (also known as delayed light emission) in photosynthetic systems; and in 1956, together with Helen Sherwood, he discovered thermoluminescence in plants. He is also known for providing a solid-state picture of photosynthesis. Much has been written about him and his research, including many articles in a special issue of Photosynthesis Research (Govindjee et al. (eds.) 1996); and a biography of Arnold, by Govindjee and Srivastava (William Archibald Arnold (1904–2001), 2014), in the Biographical Memoirs of the US National Academy of Sciences, (Washington, DC). Our article here offers a glimpse into the everyday life, through stories and photographs, of this remarkable scientist.
This work addresses the question of occurrence and function of photosystem II (PSII) in bundle sheath (BS) cells of leaves possessing NADP-malic enzyme-type C4 photosynthesis (Zea mays). Although no requirement for PSII activity in the BS has been established, several component proteins of PSII have been detected in BS cells of developing maize leaves exhibiting O2-insensitive photosynthesis. We used the basal fluorescence emissions of PSI (F0I) and PSII (F0II) as quantitative indicators of the respective relative photosystem densities. Chl fluorescence induction was measured simultaneously at 680 and 750 nm. In mature leaves, the Fm(680)/F0(680) ratio was 10.5 but less in immature leaves. We propose that the lower ratio was caused by the presence of a distinct non-variable component, Fc, emitting at 680 and 750 nm. After Fc was subtracted, the fluorescence of PSI (F0I) was detected as a non-variable component at 750 nm and was undetectably low at 680 nm. Contents of Chls a and b were measured in addition to Chl fluorescence. The Chl b/(a + b) was relatively stable in developing sunflower leaves (0.25–0.26), but in maize it increased from 0.09 to 0.21 with leaf tissue age. In sunflower, the F0I/(F0I + F0II) was 0.39 ± 0.01 independent of leaf age, but in maize, this parameter was 0.65 in young tissue of very low Chl content (20–50 mg m?2) falling to a stable level of 0.53 ± 0.01 at Chl contents >100 mg m?2. The values of F0I/(F0I + F0II) showed that in sunflower, excitation was partitioned between PSII and PSI in a ratio of 2:1, but the same ratio was 1:1 in the C4 plant. The latter is consistent with a PSII:PSI ratio of 2:1 in maize mesophyll cells and PSI only in BS cells (2:1:1 distribution). We suggest, moreover, that redox mediation of Chl synthesis, rather than protein accumulation, regulates photosystem assembly to ensure optimum excitation balance between functional PSII and PSI. Indeed, the apparent necessity for two Chls (a and b) may reside in their targeted functions in influencing accumulation of PSI and PSII, respectively, as opposed to their spectral differences.
Warwick Hillier (October 18, 1967–January 10, 2014) made seminal contributions to our understanding of photosynthetic water oxidation employing membrane inlet mass spectrometry and FTIR spectroscopy. This article offers a collection of historical perspectives on the scientific impact of Warwick Hillier’s work and tributes to the personal impact his life and ideas had on his collaborators and colleagues.
Govindjee (one name only), who himself is an institution, has been recognized and honored by many in the past for he is a true ambassador of “Photosynthesis Research” to the World. He has been called “Mr. Photosynthesis”, and compared to the Great Wall of China. To us in T?ebo?, he has been a great research collaborator in our understanding of chlorophyll a fluorescence in algae and in cyanobacteria, and more than that a friend of the Czech “Photosynthesis” group, from the time of Ivan Šetlík (1928–2009) and of Zden?k Šesták (1932–2008). Govindjee’s 80th (really 81st) birthday was celebrated by the Institute of Microbiology, Laboratory of Photosynthesis, by toasting him with an appropriate drink of a suspension of green algae grown at the institute itself. After my presentation, on October 23, 2013, of Govindjee’s contributions to photosynthesis, and his intimate association with the photosynthetikers (in Jack Myers’s words) of the Czech Republic, Govindjee gave us his story of how he began research in photosynthesis in the late 1950s. This was followed by a talk on October 25 by him on “Photosynthesis: Stories of the Past.” Everyone enjoyed his animated talk—it was full of life and enjoyment. Here, I present a brief pictorial essay on Govindjee at his 80th (really 81st) birthday in T?ebo? during October 23–25, 2013.
The PsbQ-like protein, termed CyanoQ, found in the cyanobacterium Synechocystis sp. PCC 6803 is thought to bind to the lumenal surface of photosystem II (PSII), helping to shield the Mn4CaO5 oxygen-evolving cluster. CyanoQ is, however, absent from the crystal structures of PSII isolated from thermophilic cyanobacteria raising the possibility that the association of CyanoQ with PSII might not be a conserved feature. Here, we show that CyanoQ (encoded by tll2057) is indeed expressed in the thermophilic cyanobacterium Thermosynechococcus elongatus and provide evidence in support of its assignment as a lipoprotein. Using an immunochemical approach, we show that CyanoQ co-purifies with PSII and is actually present in highly pure PSII samples used to generate PSII crystals. The absence of CyanoQ in the final crystal structure is possibly due to detachment of CyanoQ during crystallisation or its presence in sub-stoichiometric amounts. In contrast, the PsbP homologue, CyanoP, is severely depleted in isolated PSII complexes. We have also determined the crystal structure of CyanoQ from T. elongatus to a resolution of 1.6 Å. It lacks bound metal ions and contains a four-helix up-down bundle similar to the ones found in Synechocystis CyanoQ and spinach PsbQ. However, the N-terminal region and extensive lysine patch that are thought to be important for binding of PsbQ to PSII are not conserved in T. elongatus CyanoQ.
During the early- to mid-twentieth century, a bitter controversy raged among researchers on photosynthesis regarding the minimum number of light quanta required for the evolution of one molecule of oxygen. From 1923 until his death in 1970, Otto Warburg insisted that this value was about three or four quanta. Beginning in the late 1930s, Robert Emerson and others on the opposing side consistently obtained a value of 8–12 quanta. Warburg changed the protocols of his experiments, sometimes in unexplained ways, yet he almost always arrived at a value of four or less, except eight in carbonate/bicarbonate buffer, which he dismissed as “unphysiological”. This paper is largely an abbreviated form of the detailed story on the minimum quantum requirement of photosynthesis, as told by Nickelsen and Govindjee (The maximum quantum yield controversy: Otto Warburg and the “Midwest-Gang”, 2011); we provide here a scientific thread, leaving out the voluminous private correspondence among the principal players that Nickelsen and Govindjee (2011) examined in conjunction with their analysis of the principals’ published papers. We explore the development and course of the controversy and the ultimate resolution in favor of Emerson’s result as the phenomenon of the two-light-reaction, two-pigment-system scheme of photosynthesis came to be understood. In addition, we include a brief discussion of the discovery by Otto Warburg of the requirement for bicarbonate in the Hill reaction.
A chlorosome is an antenna complex located on the cytoplasmic side of the inner membrane in green photosynthetic bacteria that contains tens of thousands of self-assembled bacteriochlorophylls (BChls). Green bacteria are known to incorporate various esterifying alcohols at the C-17 propionate position of BChls in the chlorosome. The effect of these functional substitutions on the biogenesis of the chlorosome has not yet been fully explored. In this report, we address this question by investigating various esterified bacteriochlorophyll c (BChl c) homologs in the thermophilic green non-sulfur bacterium Chloroflexus aurantiacus. Cultures were supplemented with exogenous long-chain alcohols at 52 °C (an optimal growth temperature) and 44 °C (a suboptimal growth temperature), and the morphology, optical properties and exciton transfer characteristics of chlorosomes were investigated. Our studies indicate that at 44 °C Cfl. aurantiacus synthesizes more carotenoids, incorporates more BChl c homologs with unsaturated and rigid polyisoprenoid esterifying alcohols and produces more heterogeneous BChl c homologs in chlorosomes. Substitution of phytol for stearyl alcohol of BChl c maintains similar morphology of the intact chlorosome and enhances energy transfer from the chlorosome to the membrane-bound photosynthetic apparatus. Different morphologies of the intact chlorosome versus in vitro BChl aggregates are suggested by small-angle neutron scattering. Additionally, phytol cultures and 44 °C cultures exhibit slow assembly of the chlorosome. These results suggest that the esterifying alcohol of BChl c contributes to long-range organization of BChls, and that interactions between BChls with other components are important to the assembly of the chlorosome. Possible mechanisms for how esterifying alcohols affect the biogenesis of the chlorosome are discussed.
The arrangement of core antenna complexes (B808-866-RC) in the cytoplasmic membrane of filamentous phototrophic bacterium Chloroflexus aurantiacus was studied by electron microscopy in cultures from different light conditions. A typical nearest-neighbor center-to-center distance of ~18 nm was found, implying less protein crowding compared to membranes of purple bacteria. A mean RC:chlorosome ratio of 11 was estimated for the occupancy of the membrane directly underneath each chlorosome, based on analysis of chlorosome dimensions and core complex distribution. Also presented are results of single-particle analysis of core complexes embedded in the native membrane.
ATP is synthesized using ATP synthase by utilizing energy either from the oxidation of organic compounds, or from light, via redox reactions (oxidative- or photo phosphorylation), in energy-transforming membranes of mitochondria, chloroplasts, and bacteria. ATP synthase undergoes several changes during its functioning. The generally accepted model for ATP synthesis is the well-known rotatory model (see e.g., Junge et al., Nature 459:364–370, 2009; Junge and Müller, Science 333:704–705, 2011). Here, we present an alternative modified model for the coupling of electron and proton transfer to ATP synthesis, which was initially developed by Albert Lester Lehninger (1917–1986). Details of the molecular mechanism of ATP synthesis are described here that involves cyclic low-amplitude shrinkage and swelling of mitochondria. A comparison of the well-known current model and the mechano-chemiosmotic model is also presented. Based on structural, and other data, we suggest that ATP synthase is a Ca2+/H+–K+ Cl?–pump–pore–enzyme complex, in which ?-subunit rotates 360° in steps of 30°, and 90° due to the binding of phosphate ions to positively charged amino acid residues in the N-terminal ?-subunit, while in the electric field. The coiled coil b2-subunits are suggested to act as ropes that are shortened by binding of phosphate ions to positively charged lysines or arginines; this process is suggested to pull the ?3?3-hexamer to the membrane during the energization process. ATP is then synthesized during the reverse rotation of the ?-subunit by destabilizing the phosphated N-terminal ?-subunit and b2-subunits under the influence of Ca2+ ions, which are pumped over from storage—intermembrane space into the matrix, during swelling of intermembrane space. In the process of ATP synthesis, energy is first, predominantly, used in the delivery of phosphate ions and protons to the ?3?3-hexamer against the energy barrier with the help of C-terminal alpha-helix of ?-subunit that acts as a lift; then, in the formation of phosphoryl group; and lastly, in the release of ATP molecules from the active center of the enzyme and the loading of ADP. We are aware that our model is not an accepted model for ATP synthesis, but it is presented here for further examination and test.
Gorgonians are one of the most important benthic components of tropical and temperate areas, and play a fundamental role as ecosystem engineers. Although global warming and pollution increasingly threaten them, the acquisition of nutrients, which is a key process in fitness and stress resistance, has been poorly investigated in such species. This study has thus used an advanced in situ incubation chamber for the first time with gorgonians, to assess the daily acquisition of nutrients and the photophysiology of the Mediterranean symbiotic species, Eunicella singularis. The xanthophyll cycle was assessed in parallel. This work has revealed that E. singularis presents a different functioning than the Mediterranean symbiotic corals. This gorgonian indeed relies on both autotrophy and heterotrophy in summer to optimize its energetic budget, while corals mainly shift to autotrophy for their respiratory needs and tissue growth. In addition, although E. singularis lives in the same depths/locations, and harbours the same symbiont genotype than the corals, the photosynthetic performances of their respective symbionts are significantly different. Indeed, E. singularis acquired 2–3 times less autotrophic carbon from its symbionts than corals, but maintained a positive carbon budget by reducing respiration rates, and by presenting maximal photosynthetic rates throughout the day, suggesting a very efficient light utilization. Almost no photoinhibition was observed under very high light levels, because of the induction of a xanthophyll photoprotection process. These results help understanding why gorgonians often dominate many benthic ecosystems.
Electron-vibrational relaxation in the excited state of the primary electron donor, bacteriochlorophyll dimer P, in the reaction centers (RCs) of purple photosynthetic bacteria Rhodobacter sphaeroides is modeled. A multimode model of three states (i.e., the ground state Pg, initially excited P1*, and relaxed excited P2*) is used to calculate the incoherent dynamics of the difference (?A) spectra on a femtosecond timescale for the YM210 W mutant RCs. The relaxation processes are described by the step-ladder model. The model shows that the electron-vibrational relaxation in the excited state of P is visualized by the transient red shift of the stimulated emission from P*. The dynamics of this shift is observed as a change in the ?A spectrum shape in its red-most part, within a few hundreds of femtoseconds after excitation. As a result, an initial rise in the red-side ?A kinetics is delayed with respect to the blue-side kinetics. The time constant of the P1* ? P2* electronic relaxation (54 fs) and the Pg, P1*, and P2* vibrational relaxations (120 fs), used in the model, provided the best fit of the experimental time-resolved ?A spectra and kinetics at 90 and 293 K. The possible nature of the P1* ? P2* electronic relaxation is discussed.
This study identifies Salsola laricifolia as a C3–C4 intermediate in tribe Salsoleae s.l., Chenopodiaceae, and compares S. laricifolia with the previously described C3–C4 intermediates in Salsoleae. Photosynthetic pathway characteristics were studied in four species of this tribe including S. laricifolia, C3Sympegma regelii, C3–C4S. arbusculiformis, and C4S. arbuscula, using the approaches of leaf anatomy and ultrastructure, activities of ribulose 1-5-bisphosphate carboxylase/oxygenase (Rubisco) and PEP carboxylase (PEPC), CO2 compensation point, and immunolocalization of Rubisco, PEPC, and the P-subunit of glycine decarboxylase (GDC). Salsola laricifolia has intermediate features, with near continuous and distinctive Kranz-like cells (KLCs) compared with the C3-Sympegmoid anatomical type and the C3–C4 intermediate S. arbusculiformis, a relatively low CO2 compensation point (30.4 ?mol mol?1) and mesophyll (M)-to KLC tissue ratio, mitochondria in KLCs primarily occurring along the centripetal wall, and specific localization of P-protein GDC in the KLCs. The C3-type isotope value (?22.4 ‰), the absence of the clear labeling for PEPC in M cells, and the low activity of the PEPC enzyme (61.5 ?mol mg?1 chlorophyll?1 h?1) support the identification of S. laricifolia as a type I C3–C4 intermediate. Although these C3–C4 intermediate species have different structural features, one with discontinuous KL cells and the other with continuous, they have similar characteristics in physiology and biochemistry.
Theoretical prediction of effective mean PAR in optically dense samples is complicated by various optical effects, including light scattering and reflections. Direct information on the mean rate of photon absorption by PS II is provided by the kinetics of the fluorescence rise induced upon onset of strong actinic illumination (O-I1 rise). A recently introduced kinetic multi-color PAM fluorometer was applied to study the relationship between initial slope and cell density in the relatively simple model system of suspensions of Chlorella. Use of a curve fitting routine was made which was originally developed for assessment of the wavelength-dependent absorption cross-section of PS II, ?II(?), in dilute suspensions. The model underlying analysis of the O-I1 rise kinetics is outlined and data on the relationship between fitted values of ?II(?) and PAR in dilute samples are presented. With increasing cell density, lowering of apparent cross-section, <?>(?), with respect to ?II(?), relates to a decrease of effective mean PAR, <PAR>(?), relative to incident PAR(?). When ML and AL are applied in the same direction, the decline of <?>(?)/?II(?) with increasing optical density is less steep than that of the theoretically predicted <PAR>(?)/PAR(?). It approaches a value of 0.5 when the same colors of ML and AL are used, in agreement with theory. These observations open the way for estimating mean PAR in optically dense samples via measurements of <?>(?)/?II(?)).
In this article, we provide a News Report on an international conference “Photosynthesis Research for Sustainability-2014” that was held in honor of Vladimir A. Shuvalov at the Biological Research Center of the Russian Academy of Sciences, in Pushchino, Russia, during June 2–7, 2014 (http://photosynthesis2014.cellreg.org/). We begin this report with a short description of Vladimir A. Shuvalov, the honored scientist. We then provide some information on the conference, and the program. A special feature of this conference was awards given to nine young investigators; they are recognized in this Report. We have also included several photographs to show the pleasant ambiance at this conference. We invite the readers to the next two conferences on ‘‘Photosynthesis Research for Sustainability-2015: the first one to be held in Baku in May or June, 2015, and the second one, which will honor George C. Papageorgiou, will be held in Greece (in Colymbari, near Chania in Crete) during September 21–26, 2015. Information will be posted at: http://photosynthesis2015.cellreg.org/.
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