Feedback regulation of photosynthetic electron transport by NADP(H) redox poise

When plants experience an imbalance between the absorption of light energy and the use of that energy to drive metabolism, they are liable to suffer from oxidative stress. Such imbalances arise due to environmental conditions (e.g. heat, chilling or drought), and can result in the production of reactive oxygen species (ROS). Here, we present evidence for a novel protective process — feedback redox regulation via the redox poise of the NADP(H) pool. Photosynthetic electron transport was studied in two transgenic tobacco (Nicotiana tabacum) lines — one having reduced levels of ferredoxin NADP⁺-reductase (FNR), the enzyme responsible for reducing NADP⁺, and the other reduced levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), the principal consumer of NADPH. Both had a similar degree of inhibition of carbon fixation and impaired electron transport. However, whilst FNR antisense plants were obviously stressed, with extensive bleaching of leaves, GAPDH antisense plants showed no visible signs of stress, beyond having a slowed growth rate. Examination of electron transport in these plants indicated that this difference is due to feedback regulation occurring in the GAPDH but not the FNR antisense plants. We propose that this reflects the occurrence of a previously undescribed regulatory pathway responding to the redox poise of the NADP(H) pool.     

Energy dissipation is an essential mechanism to sustain the viability of plants: The physiological limits of improved photosynthesis

In bright sunlight photosynthetic activity is limited by the enzymatic machinery of carbon dioxide assimilation. This supererogation of energy can be easily visualized by the significant increases of photosynthetic activity under high CO₂ conditions or other metabolic strategies which can increase the carbon flux from CO₂ to metabolic pools. However, even under optimal CO₂ conditions plants will provide much more NADPH + H⁺ and ATP that are required for the actual demand, yielding in a metabolic situation, in which no reducible NADP⁺ would be available. As a consequence, excited chlorophylls can activate oxygen to its singlet state or the photosynthetic electrons can be transferred to oxygen, producing highly active oxygen species such as the superoxide anion, hydroxyl radicals and hydrogen peroxide. All of them can initiate radical chain reactions which degrade proteins, pigments, lipids and nucleotides. Therefore, the plants have developed protection and repair mechanism to prevent photodamage and to maintain the physiological integrity of metabolic apparatus. The first protection wall is regulatory energy dissipation on the level of the photosynthetic primary reactions by the so-called non-photochemical quenching. This dissipative pathway is under the control of the proton gradient generated by the electron flow and the xanthophyll cycle. A second protection mechanism is the effective re-oxidation of the reduction equivalents by so-called “alternative electron cycling” which includes the water-water cycle, the photorespiration, the malate valve and the action of antioxidants. The third system of defence is the repair of damaged components. Therefore, plants do not suffer from energy shortage, but instead they have to invest in proteins and cellular components which protect the plants from potential damage by the supererogation of energy. Under this premise, our understanding and evaluation for certain energy dissipating processes such as non-photochemical quenching or photorespiration appear in a quite new perspective, especially when discussing strategies to improve the solar energy conversion into plant biomass.     

Differential uptake of photosynthetic and non-photosynthetic proteins by pea root plastids

The photosynthetic proteins RuBiSCO, ferredoxin I and ferredoxin NADP(+)-oxidoreductase (pFNR) were efficiently imported into isolated pea chloroplasts but not into pea root plastids. By contrast non-photosynthetic ferredoxin III and heterotrophic FNR (hFNR) were efficiently imported into both isolated chloroplasts and root plastids. Chimeric ferredoxin I/III (transit peptide of ferredoxin I attached to the mature region of ferredoxin III) only imported into chloroplasts. Ferredoxin III/I (transit peptide of ferredoxin III attached to the mature region of ferredoxin I) imported into both chloroplasts and root plastids. This suggests that import depends on specific interactions between the transit peptide and the translocon apparatus.     

Activation of Ferredoxin-NADP+Oxidoreductase in Vicia fabaLeaves Induced by a Short-Term Increase in Irradiance

The effect of a short-term increase in growth irradiance (I) by 1.5–5 times on the rate of the photosynthetic electron transport and the activity of ferredoxin-NADP⁺oxidoreductase (FNR) in the leaves of broadbean (Vicia fabaL.) plants grown under an irradiance of 8 W/m²was studied. NADPH-diaphorase and cytochrome creductase activities of FNR were determined in isolated chloroplasts and leaf homogenates. The duration of the plant exposure to a higher I varied from 1–30 min to 2 or 24 h. The rate of noncyclic electron transport from water to NADP⁺and the NADPH-diaphorase activity of FNR increased significantly 15 min after a twofold increase in the I. FNR activation was also found after a short-term (1 min) increase in growth I by 1.5 times. The degree of light-induced activation of FNR was dependent on the light intensity, the duration of plant exposure, and the leaf age. The activation of FNR induced by a short-term increase in the I was reversible. However, inactivation of FNR proceeded more slowly than its light-induced activation. Thus, a relatively small change in the I was sufficient to induce the adaptive response of the photosynthetic apparatus at the level of the electron-transport chain. The results obtained confirm a conclusion made previously that a rapid activation of FNR induced by an increase in the I occurs in the absence of de novoprotein synthesis.     

Biochemical and genetic engineering strategies to enhance hydrogen production in photosynthetic algae and cyanobacteria

As an energy carrier, hydrogen gas is a promising substitute to carbonaceous fuels owing to its superb conversion efficiency, non-polluting nature, and high energy content. At present, hydrogen is predominately synthesized via chemical reformation of fossil fuels. While various biological methods have been extensively explored, none of them is justified as economically feasible. A sustainable platform for biological production of hydrogen will certainly impact the biofuel market. Among a selection of biological systems, algae and cyanobacteria have garnered major interests as potential cell factories for hydrogen production. In conjunction with photosynthesis, these organisms utilize inexpensive inorganic substrates and solar energy for simultaneous biosynthesis and hydrogen evolution. However, the hydrogen yield associated with these organisms remains far too low to compete with the existing chemical systems. This article reviews recent advances of biochemical, bioprocess, and genetic engineering strategies in circumventing technological limitations to hopefully improve the applicative potential of these photosynthetic hydrogen production systems.     

Characterization and responses to environmental cues of a photosynthetic antenna-deficient mutant of the filamentous cyanobacterium Anabaena sp. PCC 7120

The cyanobacterial phycobilisome (PBS) is a giant pigment-protein complex which harvests light energy for photosynthesis and comprises two structures: a core and peripheral rods. Most studies on PBS structure and function are based on mutants of unicellular strains. In this report, we describe the phenotypic and genetic characterization of a transposon mutant of the filamentous Anabaena sp. strain PCC 7120, denoted LC1, which cannot synthesize the phycobiliprotein phycocyanin (PC), the main component of the rods; in this mutant, the transposon had inserted into the cpcB gene (orf alr0528) which putatively encodes PC-β chain. Mutant LC1 was able to synthesize phycoerythrocyanin (PEC), a phycobiliprotein (PBP) located at the terminal region of the rods; but in the absence of PC, PEC did not attach to the PBSs that only retained the allophycocyanin (APC) core; ferredoxin: NADP⁺-oxidoreductase (FNR) that is associated with the PBS in the wild type, was not found in isolated PBSs from LC1. The performance of the mutant exposed to different environmental conditions was evaluated. The mutant phenotype was successfully complemented by cloning and transfer of the wild type complete cpc operon to mutant LC1. Interestingly, LC1 compensated its mutation by significantly increasing the number of its core-PBS and the effective quantum yield of photosystem II (PSII) photochemistry; this feature suggests a more efficient energy conversion in the mutant which may be useful for biotechnological applications.     

Competition between linear and cyclic electron flow in plants deficient in Photosystem I

Photosynthetic electron transport can involve either a linear flow from water to NADP, via Photosystems (PS) II and I or a cyclic flow just involving PSI. Little is known about factors regulating the relative flow through each of these pathways. We have examined photosynthetic electron transport through each system in plants of Arabidopsis thaliana in which either the PSI-D1 or PSI-E1 subunits of PSI have been knocked out. In both cases, this results in an imbalance in the turnover of PSI and PSII, such that PSII electron transport is limited by PSI turnover. Phosphorylation of light-harvesting complex II (LHCII) and its migration to PSI is enhanced but only partially reversible and not sufficient to balance photosystem turnover. In spite of this, cyclic electron flow is able to compete efficiently with PSI across a range of conditions. In dark-adapted leaves, the efficiency of cyclic relative to linear flow induced by far-red light is increased, implying that the limiting step of cyclic flow lies in the re-injection of electrons into the electron transport chain. Illumination of leaves with white light resulted in transient induction of a significant non-photochemical quenching in knockout plants which is probably high energy state quenching induced by cyclic electron flow. At high light and at low CO₂, non-photochemical quenching was greater in the knockout plants than in the wildtype. Comparison of PSI and PSII turnover under such conditions suggested that this is generated by cyclic electron flow around PSI. We conclude that, when the concentration of PSI is limiting, cyclic electron flow is still able to compete effectively with linear flow to maintain a high ΔpH to regulate photosynthesis.     

The role of PGR5 in the redox poising of photosynthetic electron transport

The pgr5 mutant of Arabidopsis thaliana has been described as being deficient in cyclic electron flow around photosystem I, however, the precise role of the PGR5 protein remains unknown. To address this issue, photosynthetic electron transport was examined in intact leaves of pgr5 and wild type A. thaliana. Based on measurements of the kinetics of P700 oxidation in far red light and re-reduction following oxidation in the presence of DCMU, we conclude that this mutant is able to perform cyclic electron flow at a rate similar to the wild type. The PGR5 protein is therefore not essential for cyclic flow. However, cyclic flow is affected by the pgr5 mutation under conditions where this process is normally enhanced in wild type leaves, i.e. high light or low CO₂ concentrations resulted in enhancement of cyclic electron flow. This suggests a different capacity to regulate cyclic flow in response to environmental stimuli in the mutant. We also show that the pgr5 mutant is affected in the redox poising of the chloroplast, with the electron transport chain being substantially reduced under most conditions. This may result in defective feedback regulation of photosynthetic electron transport under some conditions, thus providing a rationale for the reduced efficiency of cyclic electron flow.     

Dissipation of excess energy triggered by blue light in cyanobacteria with CP43′ (isiA)

The chlorophyll-protein CP43′ (isiA gene) induced by stress conditions in cyanobacteria is shown to serve as an antenna for Photosystem II (PSII), in addition to its known role as an antenna for Photosystem I (PSI). At high light intensity, this antenna is converted to an efficient trap for chlorophyll excitations that protects system II from photo-inhibition. In contrast to the ‘energy-dependent non-photochemical quenching’ (NPQ) in chloroplasts, this photoprotective energy dissipation in cyanobacteria is triggered by blue light. The induction is proportional to light intensity. Induction and decay of the quenching exhibit the same large temperature-dependence.     

Photosynthetic activity of far-red light in green plants

We have found that long-wavelength quanta up to 780 nm support oxygen evolution from the leaves of sunflower and bean. The far-red light excitations are supporting the photochemical activity of photosystem II, as is indicated by the increased chlorophyll fluorescence in response to the reduction of the photosystem II primary electron acceptor, Q_A. The results also demonstrate that the far-red photosystem II excitations are susceptible to non-photochemical quenching, although less than the red excitations. Uphill activation energies of 9.8 ± 0.5 kJ mol⁻¹ and 12.5 ± 0.7 kJ mol⁻¹ have been revealed in sunflower leaves for the 716 and 740 nm illumination, respectively, from the temperature dependencies of quantum yields, comparable to the corresponding energy gaps of 8.8 and 14.3 kJ mol⁻¹ between the 716 and 680 nm, and the 740 and 680 nm light quanta. Similarly, the non-photochemical quenching of far-red excitations is facilitated by temperature confirming thermal activation of the far-red quanta to the photosystem II core. The observations are discussed in terms of as yet undisclosed far-red forms of chlorophyll in the photosystem II antenna, reversed (uphill) spill-over of excitation from photosystem I antenna to the photosystem II antenna, as well as absorption from thermally populated vibrational sub-levels of photosystem II chlorophylls in the ground electronic state. From these three interpretations, our analysis favours the first one, i.e., the presence in intact plant leaves of a small number of far-red chlorophylls of photosystem II. Based on analogy with the well-known far-red spectral forms in photosystem I, it is likely that some kind of strongly coupled chlorophyll dimers/aggregates are involved. The similarity of the result for sunflower and bean proves that both the extreme long-wavelength oxygen evolution and the local quantum yield maximum are general properties of the plants.     

Alternative photosynthetic electron flow to oxygen in marine Synechococcus

Cyanobacteria dominate the world's oceans where iron is often barely detectable. One manifestation of low iron adaptation in the oligotrophic marine environment is a decrease in levels of iron-rich photosynthetic components, including the reaction center of photosystem I and the cytochrome b₆f complex [R.F. Strzepek and P.J. Harrison, Photosynthetic architecture differs in coastal and oceanic diatoms, Nature 431 (2004) 689-692.]. These thylakoid membrane components have well characterised roles in linear and cyclic photosynthetic electron transport and their low abundance creates potential impediments to photosynthetic function. Here we show that the marine cyanobacterium Synechococcus WH8102 exhibits significant alternative electron flow to O₂, a potential adaptation to the low iron environment in oligotrophic oceans. This alternative electron flow appears to extract electrons from the intersystem electron transport chain, prior to photosystem I. Inhibitor studies demonstrate that a propyl gallate-sensitive oxidase mediates this flow of electrons to oxygen, which in turn alleviates excessive photosystem II excitation pressure that can often occur even at relatively low irradiance. These findings are also discussed in the context of satisfying the energetic requirements of the cell when photosystem I abundance is low.     

Functional aspects of the photosynthetic light reactions in heat stressed Arabidopsis deficient in digalactosyl-diacylglycerol

Plants are often submitted, in their natural environment, to various abiotic stresses such as heat stress. However, elevated temperature has a detrimental impact on overall plant growth and development. We have examined the physiological response of the dgd1-2 and dgd1-3 Arabidopsis mutants lacking 30-40% of digalactosyl-diacylglycerol (DGDG) exposed to heat constraint. These mutants, which grow similarly to wild type under normal conditions, were previously reported to be defective in basal thermotolerance as measured by cotyledon development. However their functional properties were not described. Chlorophyll fluorescence measurements and absorbance changes at 820 nm were used to monitor photosystem II (PSII) and PSI activity, respectively. It was observed that both mutants have similar photosystem activities with some differences. The mutants were less able to use near saturation light energy and elicited higher rates of cyclic PSI electron flow compare to wild type. Arabidopsis leaves exposed to short-term (5 min) mild (40 °C) or strong (44 °C) heat treatment have shown a decline in the operating effective quantum yield of PSII and in the proportion of active PSI reaction centers. However, cyclic PSI electron flow was enhanced. The establishment of the energy-dependent non-photochemical quenching of chlorophyll fluorescence was accelerated but its decline under illumination was inhibited. Furthermore, heat stress affected the process implicated in the redistribution of light excitation energy between the photosystems known as the light state transitions. All the effects of heat stress mentioned above were more intense in the mutant leaves with dgd1-3 being even more susceptible. The decreased DGDG content of the thylakoid membranes together with other lipid changes are proposed to influence the thermo-sensitivity of the light reactions of photosynthesis towards heat stress.     

Improved survival of very high light and oxidative stress is conferred by spontaneous gain-of-function mutations in Chlamydomonas

Investigations into high light and oxidative stress in photosynthetic organisms have focussed primarily on genetic impairment of different photoprotective functions. There are few reports of “gain-of-function” mutations that provide enhanced resistance to high light and/or oxidative stress without reduced productivity. We have isolated at least four such very high light resistant (VHL^R) mutations in the green alga, Chlamydomonas reinhardtii, that permit near maximal growth rates at light intensities lethal to wild type. This resistance is not due to an alteration in electron transport rate or quantity and functionality of the two photosystems that could have enhanced photochemical quenching. Nor is it due to reduced excitation pressure by downregulation of the light harvesting antennae or increased nonphotochemical quenching. In fact, photosynthetic activity is unaffected in more than 30 VHL^R isolates. Instead, the basis of the VHL^R phenotype is a combination of traits, which appears to be dominated by enhanced capacity to tolerate reactive oxygen species generated by excess light, methylviologen, rose bengal or hydrogen peroxide. This is further evidenced in lower levels of ROS after exposure to very high light in the VHL^R-S9 mutant. Additionally, the VHL^R phenotype is associated with increased zeaxanthin accumulation, maintenance of fast synthesis and degradation rates of the D1 protein, and sustained balanced electron flow into and out of PSI under very high light. We conclude that the VHL^R mutations arose from a selection pressure that favors changes to the regulatory system(s) that coordinates several photoprotective processes amongst which repair of PSII and enhanced detoxification of reactive oxygen species play seminal roles.     

Excess iron-induced changes in the photosynthetic characteristics of sweet potato

Iron (Fe) is an essential nutrient for plant growth and development. In plant tissues, approximately 80% of Fe is found in photosynthetic cells. This study was carried out to determine the effect of different iron concentrations on the photosynthetic characteristics of sweet potato plants. The fluorescence transient of chlorophyll a (OJIP), chlorophyll index and gas exchange were measured in plants grown for seven days in Hoagland solution containing an iron concentration of 0.45, 0.90, 4.50 or 9.00 mM Fe (as Fe-EDTA). The initial and maximum fluorescence increased in the plants receiving 9.00 mM Fe. In the analysis of the fluorescence kinetic difference, L- and K-bands appeared in all of the treatments, but the amplitude was higher in plants receiving 4.50 or 9.00 mM Fe. In plants grown in 9.00 mM Fe, the parameters of the JIP-Test indicated a better efficiency in the capture, absorption and use of light energy, and although the chlorophyll index was higher, the net photosynthesis was lower. The overall data showed that sweet potato plants subjected to high iron concentrations may not exhibit the toxicity symptoms, but the light reactions of photosynthesis can be affect, which may result in a declining net assimilation rate.     

Drought tolerance through over-expression of the expansin gene TaEXPB23 in transgenic tobacco

Expansins are proteins that are the key regulators of wall extension during plant growth. To investigate the role of TaEXPB23, a wheat expansin gene, we analyzed TaEXPB23 mRNA expression levels in response to water stress in wheat and examined the drought resistance of transgenic tobaccos over-expressing TaEXPB23. We found that the expression of TaEXPB23 corresponded to wheat coleoptile growth and the response to water stress. The results also indicated that the transgenic tobacco lines lost water more slowly than the wild-type (WT) plants under drought stress; their cells could sustain a more integrated structure under water stress than that of WT. Other physiological and biochemical parameters under water stress, such as electrolyte leakage, malondialdehyde (MDA) level, photosynthetic rate, F_v/F_m and ΦPSII, also suggested that the transgenic tobaccos were more drought resistant than WT plants.     

NADPH fluorescence in the cyanobacterium Synechocystis sp. PCC 6803: A versatile probe for in vivo measurements of rates,yields and pools

We measured the kinetics of light-induced NADPH formation and subsequent dark consumption by monitoring in vivo its fluorescence in the cyanobacterium Synechocystis PCC 6803. Spectral data allowed the signal changes to be attributed to NAD(P)H and signal linearity vs the chlorophyll concentration was shown to be recoverable after appropriate correction. Parameters associated to reduction of NADP⁺ to NADPH by ferredoxin–NADP⁺-oxidoreductase were determined: After single excitation of photosystem I, half of the signal rise is observed in 8 ms; Evidence for a kinetic limitation which is attributed to an enzyme bottleneck is provided; After two closely separated saturating flashes eliciting two photosystem I turnovers in less than 2 ms, more than 50% of the cytoplasmic photoreductants (reduced ferredoxin and photosystem I acceptors) are diverted from NADPH formation by competing processes. Signal quantitation in absolute NADPH concentrations was performed by adding exogenous NADPH to the cell suspensions and by estimating the enhancement factor of in vivo fluorescence (between 2 and 4). The size of the visible (light-dependent) NADP (NADP⁺ + NADPH) pool was measured to be between 1.4 and 4 times the photosystem I concentration. A quantitative discrepancy is found between net oxygen evolution and NADPH consumption by the light-activated Calvin–Benson cycle. The present study shows that NADPH fluorescence is an efficient probe for studying in vivo the energetic metabolism of cyanobacteria which can be used for assessing multiple phenomena occurring over different time scales.     

Photoelectrochemical control of the balance between cyclic- and linear electron transport in photosystem I. Algorithm for P700 induction kinetics

Redox transients of chlorophyll P700, monitored as absorbance changes ΔA₈₁₀, were measured during and after exclusive PSI excitation with far-red (FR) light in pea (Pisum sativum, cv. Premium) leaves under various pre-excitation conditions. Prolonged adaptation in the dark terminated by a short PSII + PSI− exciting light pulse guarantees pre-conditions in which the initial photochemical events in PSI RCs are carried out by cyclic electron transfer (CET). Pre-excitation with one or more 10 s FR pulses creates conditions for induction of linear electron transport (LET). These converse conditions give rise to totally different, but reproducible responses of P700⁻ oxidation. System analyses of these responses were made based on quantitative solutions of the rate equations dictated by the associated reaction scheme for each of the relevant conditions. These provide the mathematical elements of the P700 induction algorithm (PIA) with which the distinguishable components of the P700⁺ response can be resolved and interpreted. It enables amongst others the interpretation and understanding of the characteristic kinetic profile of the P700⁺ response in intact leaves upon 10 s illumination with far-red light under the promotive condition for CET. The system analysis provides evidence that this unique kinetic pattern with a non-responsive delay followed by a steep S-shaped signal increase is caused by a photoelectrochemically controlled suppression of the electron transport from Fd to the PQ-reducing Q_r site of the cytb₆f complex in the cyclic pathway. The photoelectrochemical control is exerted by the PSI-powered proton pump associated with CET. It shows strong similarities with the photoelectrochemical control of LET at the acceptor side of PSII which is reflected by release of photoelectrochemical quenching of chlorophyll a fluorescence.