Photosynthetic H<Subscript>2</Subscript> metabolism in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> (unicellular green algae)

Unicellular green algae have the ability to operate in two distinctly different environments (aerobic and anaerobic), and to photosynthetically generate molecular hydrogen (H₂). A recently developed metabolic protocol in the green alga Chlamydomonas reinhardtii permitted separation of photosynthetic O₂-evolution and carbon accumulation from anaerobic consumption of cellular metabolites and concomitant photosynthetic H₂-evolution. The H₂ evolution process was induced upon sulfate nutrient deprivation of the cells, which reversibly inhibits photosystem-II and O₂-evolution in their chloroplast. In the absence of O₂, and in order to generate ATP, green algae resorted to anaerobic photosynthetic metabolism, evolved H₂ in the light and consumed endogenous substrate. This study summarizes recent advances on green algal hydrogen metabolism and discusses avenues of research for the further development of this method. Included is the mechanism of a substantial tenfold starch accumulation in the cells, observed promptly upon S-deprivation, and the regulated starch and protein catabolism during the subsequent H₂-evolution. Also discussed is the function of a chloroplast envelope-localized sulfate permease, and the photosynthesis–respiration relationship in green algae as potential tools by which to stabilize and enhance H₂ metabolism. In addition to potential practical applications of H₂, approaches discussed in this work are beginning to address the biochemistry of anaerobic H₂ photoproduction, its genes, proteins, regulation, and communication with other metabolic pathways in microalgae. Photosynthetic H₂ production by green algae may hold the promise of generating a renewable fuel from nature’s most plentiful resources, sunlight and water. The process potentially concerns global warming and the question of energy supply and demand.     

Putative role of the malate valve enzyme NADP–malate dehydrogenase in H2O2 signalling in Arabidopsis

In photosynthetic organisms, sudden changes in light intensity perturb the photosynthetic electron flow and lead to an increased production of reactive oxygen species. At the same time, thioredoxins can sense the redox state of the chloroplast. According to our hypothesis, thioredoxins and related thiol reactive molecules downregulate the activity of H₂O₂-detoxifying enzymes, and thereby allow a transient oxidative burst that triggers the expression of H₂O₂ responsive genes. It has been shown recently that upon light stress, catalase activity was reversibly inhibited in Chlamydomonas reinhardtii in correlation with a transient increase in the level of H₂O₂. Here, it is shown that Arabidopsis thaliana mutants lacking the NADP–malate dehydrogenase have lost the reversible inactivation of catalase activity and the increase in H₂O₂ levels when exposed to high light. The mutants were slightly affected in growth and accumulated higher levels of NADPH in the chloroplast than the wild-type. We propose that the malate valve plays an essential role in the regulation of catalase activity and the accumulation of a H₂O₂ signal by transmitting the redox state of the chloroplast to other cell compartments.     

Resolving diurnal dynamics of the chloroplastic glutathione redox state in Arabidopsis reveals its photosynthetically derived oxidation

Plants are subjected to fluctuations in light intensity, and this might cause unbalanced photosynthetic electron fluxes and overproduction of reactive oxygen species (ROS). Electrons needed for ROS detoxification are drawn, at least partially, from the cellular glutathione (GSH) pool via the ascorbate–glutathione cycle. Here, we explore the dynamics of the chloroplastic glutathione redox potential (chl-E_GSH) using high-temporal-resolution monitoring of Arabidopsis (Arabidopsis thaliana) lines expressing the reduction–oxidation sensitive green fluorescent protein 2 (roGFP2) in chloroplasts. This was carried out over several days under dynamic environmental conditions and in correlation with PSII operating efficiency. Peaks in chl-E_GSH oxidation during dark-to-light and light-to-dark transitions were observed. Increasing light intensities triggered a binary oxidation response, with a threshold around the light saturating point, suggesting two regulated oxidative states of the chl-E_GSH. These patterns were not affected in npq1 plants, which are impaired in non-photochemical quenching. Oscillations between the two oxidation states were observed under fluctuating light in WT and npq1 plants, but not in pgr5 plants, suggesting a role for PSI photoinhibition in regulating the chl-E_GSH dynamics. Remarkably, pgr5 plants showed an increase in chl-E_GSH oxidation during the nights following light stresses, linking daytime photoinhibition and nighttime GSH metabolism. This work provides a systematic view of the dynamics of the in vivo chloroplastic glutathione redox state during varying light conditions.

Monitoring the daily in vivo dynamics of the chloroplastic GSH redox state in light-stressed wild-type plants versus photoprotective mutants provides insight into the photosynthesis-dependent production of oxidants.     

Flux through mitochondrial redox circuits linked to nicotinamide nucleotide transhydrogenase generates counterbalance changes in energy expenditure

Compensatory changes in energy expenditure occur in response to positive and negative energy balance, but the underlying mechanism remains unclear. Under low energy demand, the mitochondrial electron transport system is particularly sensitive to added energy supply (i.e. reductive stress), which exponentially increases the rate of H₂O₂ (JH₂O₂) production. H₂O₂ is reduced to H₂O by electrons supplied by NADPH. NADP⁺ is reduced back to NADPH by activation of mitochondrial membrane potential–dependent nicotinamide nucleotide transhydrogenase (NNT). The coupling of reductive stress-induced JH₂O₂ production to NNT-linked redox buffering circuits provides a potential means of integrating energy balance with energy expenditure. To test this hypothesis, energy supply was manipulated by varying flux rate through β-oxidation in muscle mitochondria minus/plus pharmacological or genetic inhibition of redox buffering circuits. Here we show during both non-ADP– and low-ADP–stimulated respiration that accelerating flux through β-oxidation generates a corresponding increase in mitochondrial JH₂O₂ production, that the majority (∼70–80%) of H₂O₂ produced is reduced to H₂O by electrons drawn from redox buffering circuits supplied by NADPH, and that the rate of electron flux through redox buffering circuits is directly linked to changes in oxygen consumption mediated by NNT. These findings provide evidence that redox reactions within β-oxidation and the electron transport system serve as a barometer of substrate flux relative to demand, continuously adjusting JH₂O₂ production and, in turn, the rate at which energy is expended via NNT-mediated proton conductance. This variable flux through redox circuits provides a potential compensatory mechanism for fine-tuning energy expenditure to energy balance in real time.     

Environmentally modulated phosphorylation and dynamics of proteins in photosynthetic membranes

Recent advances in vectorial proteomics of protein domains exposed to the surface of photosynthetic thylakoid membranes of plants and the green alga Chlamydomonas reinhardtii allowed mapping of in vivo phosphorylation sites in integral and peripheral membrane proteins. In plants, significant changes of thylakoid protein phosphorylation are observed in response to stress, particularly in photosystem II under high light or high temperature stress. Thylakoid protein phosphorylation in the algae is much more responsive to the ambient redox and light conditions, as well as to CO₂ availability. The light-dependent multiple and differential phosphorylation of CP29 linker protein in the green algae is suggested to control photosynthetic state transitions and uncoupling of light harvesting proteins from photosystem II under high light. The similar role for regulation of the dynamic distribution of light harvesting proteins in plants is proposed for the TSP9 protein, which together with other recently discovered peripheral proteins undergoes specific environment- and redox-dependent phosphorylation at the thylakoid surface. This review focuses on the environmentally modulated reversible phosphorylation of thylakoid proteins related to their membrane dynamics and affinity towards particular photosynthetic protein complexes.     

Effect of anaerobiosis on photosynthetic reactions and nitrogen metabolism of algae with and without hydrogenase

Summary The effect of anaerobic (N₂+CO₂) pre-incubation in the dark on photosynthetic reactions (O₂ evolution, measured manometrically and with the oxygraph; fluorescence; and photoproduction of H₂, measured with the mass spectrometer) was studied in algae with hydrogenase (strains of Chlorella fusca, C. kessleri, C. vulgaris f. tertia, and Ankistrodesmus braunii) and in algae without hydrogenase (strains of Chlorella vulgaris, C. saccharophila, and C. minutissima).The inhibition by anaerobic incubation of photosynthetic O₂ evolution is much stronger in algae without hydrogenase than it is in algae with hydrogenase. The effect of anaerobiosis is most pronounced at rather low light intensity (about 1000 lux), in acid medium (pH 4), and after prolonged anaerobic incubation in the dark (about 20 h). These results indicate that the presence of hydrogenase might be ecologically advantageous for algae under certain conditions.Chlorophyll fluorescence showed the fastest response to anaerobic incubation, and the most pronounced difference between algae with and without hydrogenase. After only 30 min under N₂+CO₂, fluorescence in algae with hydrogenase starts with a peak and decreases within 10 to 20 sec to a rather low steady-state level which is only slightly higher than that found under aerobic conditions. In algae without hydrogenase, fluorescence is rather low during the first 1 to 2 sec and then rises to a higher steady-state level which is much higher than that of the aerobic controls. This indicates an inhibition due to anaerobiosis of photosystem II in algae without hydrogenase.Algae with hydrogenase can react in different ways during the first minutes of illumination. In some cases there is an immediate photoproduction of H₂, which is followed after a few minutes by photosynthetic O₂ evolution; in other algae there is a simultaneous production of H₂ and O₂ from the very beginning; in a few experiments there was no photoproduction of H₂ at all, and in this case there was no photosynthetic O₂ evolution either. Thus, photoproduction of H₂ seems to be the process which normally enables algae with hydrogenase to oxidise and thereby activate their photosynthetic electron transport system after anaerobic incubation.A mass spectrometric search for nitrogen fixation (using N₂ and acetylene) in eucaryotic green algae gave negative results, even with species containing hydrogenase and under anaerobic conditions.     

High Yields of Hydrogen Production Induced by Meta-Substituted Dichlorophenols Biodegradation from the Green Alga Scenedesmus obliquus

Hydrogen is a highly promising energy source with important social and economic implications. The ability of green algae to produce photosynthetic hydrogen under anaerobic conditions has been known for years. However, until today the yield of production has been very low, limiting an industrial scale use. In the present paper, 73 years after the first report on H₂-production from green algae, we present a combinational biological system where the biodegradation procedure of one meta-substituted dichlorophenol (m-dcp) is the key element for maintaining continuous and high rate H₂-production (>100 times higher than previously reported) in chloroplasts and mitochondria of the green alga Scenedesmus obliquus. In particular, we report that reduced m-dcps (biodegradation intermediates) mimic endogenous electron and proton carriers in chloroplasts and mitochondria, inhibit Photosystem II (PSII) activity (and therefore O₂ production) and enhance Photosystem I (PSI) and hydrogenase activity. In addition, we show that there are some indications for hydrogen production from sources other than chloroplasts in Scenedesmus obliquus. The regulation of these multistage and highly evolved redox pathways leads to high yields of hydrogen production and paves the way for an efficient application to industrial scale use, utilizing simple energy sources and one meta-substituted dichlorophenol as regulating elements.     

Diurnal changes in the xanthophyll cycle pigments of freshwater algae correlate with the environmental hydrogen peroxide concentration rather than non-photochemical quenching

Background and Aims In photosynthetic organisms exposure to high light induces the production of reactive oxygen species (ROS), such as hydrogen peroxide (H₂O₂), which in part is prevented by non-photochemical quenching (NPQ). As one of the most stable and longest-lived ROS, H₂O₂ is involved in key signalling pathways in development and stress responses, although in excess it can induce damage. A ubiquitous response to high light is the induction of the xanthophyll cycle, but its role in algae is unclear as it is not always associated with NPQ induction. The aim of this study was to reveal how diurnal changes in the level of H₂O₂ are regulated in a freshwater algal community.Methods A natural freshwater community of algae in a temporary rainwater pool was studied, comprising photosynthetic Euglena species, benthic Navicula diatoms, Chlamydomonas and Chlorella species. Diurnal measurements were made of photosynthetic performance, concentrations of photosynthetic pigments and H₂O₂. The frequently studied model organisms Chlamydomonas and Chlorella species were isolated to study photosynthesis-related H₂O₂ responses to high light.Key Results NPQ was shown to prevent H₂O₂ release in Chlamydomonas and Chlorella species under high light; in addition, dissolved organic carbon excited by UV-B radiation was probably responsible for a part of the H₂O₂ produced in the water column. Concentrations of H₂O₂ peaked at 2 µm at midday and algae rapidly scavenged H₂O₂ rather than releasing it. A vertical H₂O₂ gradient was observed that was lowest next to diatom-rich benthic algal mats. The diurnal changes in photosynthetic pigments included the violaxanthin and diadinoxanthin cycles; the former was induced prior to the latter, but neither was strictly correlated with NPQ.Conclusions The diurnal cycling of H₂O₂ was apparently modulated by the organisms in this freshwater algal community. Although the community showed flexibility in its levels of NPQ, the diurnal changes in xanthophylls correlated with H₂O₂ concentrations. Alternative NPQ mechanisms in algae involving proteins of the light-harvesting complex type and antioxidant protection of the thylakoid membrane by de-epoxidized carotenoids are discussed.     

Redox regulation of chloroplast metabolism

Regulation of enzyme activity based on thiol-disulfide exchange is a regulatory mechanism in which the protein disulfide reductase activity of thioredoxins (TRXs) plays a central role. Plant chloroplasts are equipped with a complex set of up to 20 TRXs and TRX-like proteins, the activity of which is supported by reducing power provided by photosynthetically reduced ferredoxin (FDX) with the participation of a FDX-dependent TRX reductase (FTR). Therefore, the FDX–FTR–TRXs pathway allows the regulation of redox-sensitive chloroplast enzymes in response to light. In addition, chloroplasts contain an NADPH-dependent redox system, termed NTRC, which allows the use of NADPH in the redox network of these organelles. Genetic approaches using mutants of Arabidopsis (Arabidopsis thaliana) in combination with biochemical and physiological studies have shown that both redox systems, NTRC and FDX-FTR-TRXs, participate in fine-tuning chloroplast performance in response to changes in light intensity. Moreover, these studies revealed the participation of 2-Cys peroxiredoxin (2-Cys PRX), a thiol-dependent peroxidase, in the control of the reducing activity of chloroplast TRXs as well as in the rapid oxidation of stromal enzymes upon darkness. In this review, we provide an update on recent findings regarding the redox regulatory network of plant chloroplasts, focusing on the functional relationship of 2-Cys PRXs with NTRC and the FDX–FTR–TRXs redox systems for fine-tuning chloroplast performance in response to changes in light intensity and darkness. Finally, we consider redox regulation as an additional layer of control of the signaling function of the chloroplast.

Thiol-dependent redox regulatory and antioxidant systems act concertedly to modulate chloroplast metabolism and signaling function.

Advances

• Plant chloroplasts harbor a complex redox network composed of the FDX–FTR–TRXs pathway, linking redox regulation to light, and NTRC, an NADPH-dependent system required for the activity of TRXs. Both systems adjust chloroplast performance to environmental cues.
• A relevant function of NTRC is redox control of 2-Cys PRXs, which maintains the reductive activity of chloroplast TRXs in the light. The NTRC–2-Cys PRXs redox system helps fine-tune the redox state of chloroplast enzymes thereby adjusting photosynthetic performance to changes in light.
• 2-Cys PRXs participate in the rapid oxidative inactivation of chloroplast enzymes in the dark, mediating the transfer of reducing equivalents from reduced enzymes, via TRXs, to hydrogen peroxide.
• Involvement of redox regulation in chloroplast retrograde signaling modulates early stages of plant development and response to environmental stress.

     

Stromal NADH supplied by PHOSPHOGLYCERATE DEHYDROGENASE3 is crucial for photosynthetic performance

During photosynthesis, electrons travel from light-excited chlorophyll molecules along the electron transport chain to the final electron acceptor nicotinamide adenine dinucleotide phosphate (NADP) to form NADPH, which fuels the Calvin–Benson–Bassham cycle (CBBC). To allow photosynthetic reactions to occur flawlessly, a constant resupply of the acceptor NADP is mandatory. Several known stromal mechanisms aid in balancing the redox poise, but none of them utilizes the structurally highly similar coenzyme NAD(H). Using Arabidopsis (Arabidopsis thaliana) as a C₃-model, we describe a pathway that employs the stromal enzyme PHOSPHOGLYCERATE DEHYDROGENASE 3 (PGDH3). We showed that PGDH3 exerts high NAD(H)-specificity and is active in photosynthesizing chloroplasts. PGDH3 withdrew its substrate 3-PGA directly from the CBBC. As a result, electrons become diverted from NADPH via the CBBC into the separate NADH redox pool. pgdh3 loss-of-function mutants revealed an overreduced NADP(H) redox pool but a more oxidized plastid NAD(H) pool compared to wild-type plants. As a result, photosystem I acceptor side limitation increased in pgdh3. Furthermore, pgdh3 plants displayed delayed CBBC activation, changes in nonphotochemical quenching, and altered proton motive force partitioning. Our fluctuating light-stress phenotyping data showed progressing photosystem II damage in pgdh3 mutants, emphasizing the significance of PGDH3 for plant performance under natural light environments. In summary, this study reveals an NAD(H)-specific mechanism in the stroma that aids in balancing the chloroplast redox poise. Consequently, the stromal NAD(H) pool may provide a promising target to manipulate plant photosynthesis.

PHOSPHOGLYCERATE DEHYDROGENASE 3, an oxidoreductase in leaf chloroplasts with strong preference to reduce the stromal NAD(H) instead of the NADP(H) pool, is required for full photosynthetic capacity.     

Iron supply and hydrogenase activity in green algae

Summary An addition of EDTA to the culture medium considerably increases H₂ metabolism of green algae. However, even under these conditions, several hours of anaerobic incubation (adaptation) are necessary for the optimum of hydrogenase activity to be reached. In addition, the stability of H₂ metabolism during longer periods of anaerobiosis is much better.     

Effect of light fluctuations on photosynthesis and metabolic flux in Synechocystis sp. PCC 6803

In nature, photosynthetic organisms are exposed to fluctuating light, and their physiological systems must adapt to this fluctuation. To maintain homeostasis, these organisms have a light fluctuation photoprotective mechanism, which functions in both photosystems and metabolism. Although the photoprotective mechanisms functioning in the photosystem have been studied, it is unclear how metabolism responds to light fluctuations within a few seconds. In the present study, we investigated the metabolic response of Synechocystis sp. PCC 6803 to light fluctuations using ¹³C-metabolic flux analysis. The light intensity and duty ratio were adjusted such that the total number of photons or the light intensity during the low-light phase was equal. Light fluctuations affected cell growth and photosynthetic activity under the experimental conditions. However, metabolic flux distributions and cofactor production rates were not affected by the light fluctuations. Furthermore, the estimated ATP and NADPH production rates in the photosystems suggest that NADPH-consuming electron dissipation occurs under fluctuating light conditions. Although we focused on the water–water cycle as the electron dissipation path, no growth effect was observed in an flv3-disrupted strain under fluctuating light, suggesting that another path contributes to electron dissipation under these conditions.     

Fluctuating vs. Continuous Exposure to H2O2: The Effects on Mitochondrial Membrane Potential,Intracellular Calcium,and NF-κB in Astroglia

The effects of H₂O₂ are widely studied in cell cultures and other in vitro systems. However, such investigations are performed with the assumption that H₂O₂ concentration is constant, which may not properly reflect in vivo settings, particularly in redox-turbulent microenvironments such as mitochondria. Here we introduced and tested a novel concept of fluctuating oxidative stress. We treated C6 astroglial cells and primary astrocytes with H₂O₂, using three regimes of exposure – continuous, as well as fluctuating at low or high rate, and evaluated mitochondrial membrane potential and other parameters of mitochondrial activity – respiration, reducing capacity, and superoxide production, as well as intracellular ATP, intracellular calcium, and NF-κB activation. When compared to continuous exposure, fluctuating H₂O₂ induced a pronounced hyperpolarization in mitochondria, whereas the activity of electron transport chain appears not to be significantly affected. H₂O₂ provoked a decrease of ATP level and an increase of intracellular calcium concentration, independently of the regime of treatment. However, fluctuating H₂O₂ induced a specific pattern of large-amplitude fluctuations of calcium concentration. An impact on NF-κB activation was observed for high rate fluctuations, whereas continuous and low rate fluctuating oxidative stress did not provoke significant effects. Presented results outline the (patho)physiological relevance of redox fluctuations.     

RNAi Knock-Down of LHCBM1, 2 and 3 Increases Photosynthetic H2 Production Efficiency of the Green Alga Chlamydomonas reinhardtii

Single cell green algae (microalgae) are rapidly emerging as a platform for the production of sustainable fuels. Solar-driven H₂ production from H₂O theoretically provides the highest-efficiency route to fuel production in microalgae. This is because the H₂-producing hydrogenase (HYDA) is directly coupled to the photosynthetic electron transport chain, thereby eliminating downstream energetic losses associated with the synthesis of carbohydrate and oils (feedstocks for methane, ethanol and oil-based fuels). Here we report the simultaneous knock-down of three light-harvesting complex proteins (LHCMB1, 2 and 3) in the high H₂-producing Chlamydomonas reinhardtii mutant Stm6Glc4 using an RNAi triple knock-down strategy. The resultant Stm6Glc4L01 mutant exhibited a light green phenotype, reduced expression of LHCBM1 (20.6% ±0.27%), LHCBM2 (81.2% ±0.037%) and LHCBM3 (41.4% ±0.05%) compared to 100% control levels, and improved light to H₂ (180%) and biomass (165%) conversion efficiencies. The improved H₂ production efficiency was achieved at increased solar flux densities (450 instead of ∼100 µE m⁻² s⁻¹) and high cell densities which are best suited for microalgae production as light is ideally the limiting factor. Our data suggests that the overall improved photon-to-H₂ conversion efficiency is due to: 1) reduced loss of absorbed energy by non-photochemical quenching (fluorescence and heat losses) near the photobioreactor surface; 2) improved light distribution in the reactor; 3) reduced photoinhibition; 4) early onset of HYDA expression and 5) reduction of O₂-induced inhibition of HYDA. The Stm6Glc4L01 phenotype therefore provides important insights for the development of high-efficiency photobiological H₂ production systems.     

H2-Photoproduction by green algae: The significance of anaerobic pre-incubation periods and of high light intensities for H2-photoproductivity ofChlorella fusca

Using sodium-dithionite as an oxygen scavenger, the influences of different light intensities and periods of anaerobic pre-incubation in the dark on H₂-photoproductivity were studied with the green algaChlorella fusca. By measuring hydrogen production in the light using manometric and gas chromatographic methods the effectiveness of sodium dithionite in stabilizing photoproduction was established. For high rates of H₂-photoproduction high light intensities up to 30,000 lux (580 W m^-2) were necessary; these are comparable to those required for light saturation of oxygen photoproduction by this alga. AlthoughChlorella fusca produces H₂ immediately after transition to anaerobic conditions, the optimum rate of H₂ production was reached after a 5 h dark adaptation period only. The results obtained are discussed with respect to characteristics of H₂-photoproduction by green algae: the initial burst kinetics, the light saturation, and the obligate period of anaerobic adaptation. It is concluded that H₂-photoproduction byChlorella is an anaerobic photosynthetic process which occurs in the absence of CO₂ and can be experimentally stabilized by exogenous oxygen scavengers.Abbreviations DCMU (3-(3,4-Dichlorophenyl)-1,1-dimethylurea) - HEPES (2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid)     

Photosynthetic Units

Leaf tissues of aurea mutants of tobacco and Lespedeza have been shown to have higher photosynthetic capacity per molecule of chlorophyll, a higher saturation intensity, a simpler lamellar structure, and the same quantum yield as their dark green parents. Here we report on the values of photosynthetic units for both types of plants and some algae. The unit has been assumed to be about as uniform and steady in the plant world as the quantum efficiency. The number on which all theoretical discussions have been based so far is 2400 per O₂ evolved or CO₂ reduced. With dark green plants and algae our determinations of units by means of 40 µsec flashes superimposed on a steady rate of background photosynthesis at 900 ergs cm^-2 sec^-1 of red light yielded mostly numbers between 2000 and 2700. However, the photosynthetic unit turned out to be very variable, even in these objects. In aurea mutants the unit was distinctly smaller, averaging 600 chl/CO₂. By choosing the right combination of colors for flash and background light, units as low as 300 chl/CO₂ or 40 chl/e^- could be measured consistently. We found five well-defined groups of units composed of multiples of its smallest member. These new findings are discussed in terms of structural entities that double or divide under the influence of far-red light.     

Isotopic relationships between saponifiable lipids and cellulose nitrate prepared from red,brown and green algae

Stable carbon and hydrogen isotope ratios were determined for the saponifiable lipid fraction as well as the cellulose fraction (the latter after nitration to remove exchangeable hydrogens) of several species of red, brown and green algae from three locations. A significant correlation was observed between the hydrogen isotope ratios of cellulose nitrate and saponifiable lipid for red algae, but not for brown or green algae. Carbon-13/carbon-12 ratios for both fractions of red algae were in general lower than those observed for brown and green algae. The results reported here are consistent with the proposals that red algae evolved much earlier than and are metabolically different from the brown and green algae.Abbreviations and symbols CAM Crassulacean acid metabolism - C₃ photosynthetic mode in plants in which CO₂ is fixed into a three-carbon compound - C₄ photosynthetic mode in plants in which CO₂ is fixed into a four-carbon compound - unit used to express isotope ratios Also Archaeology Program, UCLA     

Glutathione regulates enzymatic antioxidant defence with differential thiol content in perennial pepperweed and helps adapting to extreme environment

Perennial pepperweed (Lepidium latifolium Linn.) is a preferred ‘phytofood’ that is available for the longest period of a year in Ladakh. Present study was undertaken to identify the mechanism of redox homeostasis and understand factors responsible for its biochemical superiority during low temperatures. Results reveal that despite the stressful environment at higher altitude, the cellular conditions are more reducing for this plant. The reducing environment is maintained by significant induction of GSH rather than changes in its oxidation state, which changes the redox potential by 12 mV. Lower ratio of NADP⁺/NADPH and induction of new antioxidative isozymes at Leh (3,505 m) suggest crucial role of redox regulation in adaptation. These new proteins have higher thiol content and could provide an efficient redox sensing mechanism in Lepidium latifolium that respond through GSH/NADPH redox buffers. In vitro feeding experiment suggested that GSH plays an important role in induction of antioxidant enzymes, which may not be the direct consequence of H₂O₂ accumulation. It needs to be further investigated whether its responsive redox metabolism has some role in its invasive growth in riparian plains of America.     

Dark Ammonium Assimilation Reduces the Plastoquinone Pool of Photosystem II in the Green Alga Selenastrum minutum

The impact of dark NH₄⁺ and NO₃⁻ assimilation on photosynthetic light harvesting capability of the green alga Selenastrum minutum was monitored by chlorophyll a fluorescence analysis. When cells assimilated NH₄⁺, they exhibited a large decline in the variable fluorescence/maximum fluorescence ratio, the fluorescence yield of photosystem II relative to that of photosystem I at 77 kelvin, and O₂ evolution rate. NH₄⁺ assimilation therefore poised the cells in a less efficient state for photosystem II. The analysis of complementary area of fluorescence induction curve and the pattern of fluorescence decay upon microsecond saturating flash, indicators of redox state of plastoquinone (PQ) pool and dark reoxidation of primary quinone electron acceptor (Q_A), respectively, revealed that the PQ pool became reduced during dark NH₄⁺ assimilation. NH₄⁺ assimilation also caused an increase in the NADPH/NADP⁺ ratio due to the NH₄⁺ induced increase in respiratory carbon oxidation. The change in cellular reductant is suggested to be responsible for the reduction of the PQ pool and provide a mechanism by which the metabolic demands of NH₄⁺ assimilation may alter the efficiency of photosynthetic light harvesting. NO₃⁻ assimilation did not cause a reduction in PQ and did not affect the efficiency of light harvesting. These results illustrate the role of cellular metabolism in the modulating photosynthetic processes.     

Photosynthetic Effect in Selenastrum capricornutum Progeny after Carbon-Ion Irradiation

A large proportion of mutants with altered pigment features have been obtained via exposure to heavy-ion beams, a technique that is efficient for trait improvement in the breeding of plants and algae. However, little is known about the underlying mechanisms by which the photosynthetic pigments are altered by heavy-ion irradiation. In our study, the photosynthetic characteristics of progenies from carbon-ion irradiated Selenastrum capricornutum were investigated. Five progenies deficient in chlorophyll a were isolated after carbon-ion exposure. Photosynthetic characteristics, photoprotection capacity and gene expression of the light-harvesting complex in these progenies were further characterized by the measurement of chlorophyll fluorescence parameters (Fv/Fm, ФPSII, NPQ, ETR), the de-epoxidation state of the xanthophyll cycle, the amount of lutein and quantitative real-time PCR. High maximum quantum yield of photosystem II at day 10 and high thermal dissipation ability were observed in progenies #23 and #37 under normal culture condition. Progenies #18, #19 and #20 showed stronger resistance against high levels of light steps than the control group (612–1077 μmol photons m ^-2 s ^-1, p< 0.05). The progenies #20 and #23 exhibited strong photoprotection by thermal dissipation and quenching of ³Chl* after 24 h of high light treatment. The mRNA levels of Lhcb5, Lhcbm5 and Lhcbm1 of the light-harvesting complex revealed markedly differential expression in the five progenies irradiated by carbon-ion beams. This work indicates that photosynthetic efficiency, photoprotection ability and the expression of light-harvesting antennae in unicellular green algae can be markedly influenced by irradiation. To our knowledge, this is the first report on changes in the photosynthetic pigments of green algae after treatment with carbon-ion beams.