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Effects of O 2-removal and cyanide on rapid light-induced quenching of blue-green fluorescence in the presence of HgCl 2. Wildtype cells in the presence of 20 mM HgCl 2. O 2 was removed with the help of the glucose–glucose oxidase trap (3 mM glucose, 30 U ml –1 glucose oxidase). KCN concentration was 1 mM. Other conditions were the same as for Fig. 1.
Source publication
Blue-green fluorescence emission of intact cells of Synechocystis PCC6803 and of its ndhB-defective mutant M55 was measured with a standard pulse-amplitude-modulation chlorophyll fluorometer equipped with a new type of emitter-detector unit featuring pulse-modulated UV-A measuring light and a photomultiplier detector. A special illumination program...
Context in source publication
Context 1
... that the HgCl 2 -effect is not related to the function of NDH, i.e. at least of that type of NDH which is mutated in M55. The fol- lowing observations appear relevant for a tentative explana- tion: the rapid negative transient in the presence of HgCl 2 is substantially slowed down after removal of molecular oxygen and also by addition of KCN (Fig. 5). The data agree with the working hypothesis that HgCl 2 blocks NADP reduction, possi- bly by affecting FNR-activity, thus stimulating O 2 -reduction. The rapid negative transient could reflect oxidation of NADPH by the H 2 O 2 which originates from superoxide formation at the acceptor side of PSI by the Mehler reaction and superoxide ...
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Citations
... At the onset of actinic light, light-induced NADPH fluorescence increased rapidly by 31% in the UV-A-acclimated cells compared to that in the control cells (t-test, P = 0.03), indicating that light-driven generation of NADPH is higher in the UV-A-acclimated cells. Subsequently, the NADPH fluorescence declined in both UV-A-acclimated and control cells due to NADPH oxidation via downstream metabolic pathways and gradually reached a stationary phase, corresponding to the matching rates of light-driven NADP + reduction and NADPH oxidation (43). The NADPH fluorescence declined by 51% and 18%, respectively, in the UV-A-acclimated and control cells (t-test, P = 0.04), indicating that in the UV-A-acclimated cells, the NADPH consumption increases more significantly owing to the downstream metabolic pathways and biomass accumulation. ...
... Finally, NADPH fluorescence increased gradually to a stable value, corresponding to the matching rates of dark oxidation of NADPH and dark reduction of NADP + associated with reductive pentose phosphate cycle. During the dark-light-dark induction transients of NADPH fluorescence, NADPH is primarily consumed by the Calvin cycle (43). Therefore, we assessed the effect of UV-A radiation on the enzyme activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in N. sphaeroides CCNUC1 (Fig. 4C). ...
Ultraviolet (UV) A radiation (315–400 nm) is the predominant component of solar UV radiation that reaches the Earth’s surface. However, the underlying mechanisms of the positive effects of UV-A on photosynthetic organisms have not yet been elucidated. In this study, we investigated the effects of UV-A radiation on the growth, photosynthetic ability, and metabolome of the edible cyanobacterium Nostoc sphaeroides. Exposures to 5–15 W m⁻² (15–46 µmol photons m⁻² s⁻¹) UV-A and 4.35 W m⁻² (20 μmol photons m⁻² s⁻¹) visible light for 16 days significantly increased the growth rate and biomass production of N. sphaeroides cells by 18%–30% and 15%–56%, respectively, compared to the non-UV-A-acclimated cells. Additionally, the UV-A-acclimated cells exhibited a 1.8-fold increase in the cellular nicotinamide adenine dinucleotide phosphate (NADP) pool with an increase in photosynthetic capacity (58%), photosynthetic efficiency (24%), QA re-oxidation, photosystem I abundance, and cyclic electron flow (87%), which further led to an increase in light-induced NADPH generation (31%) and ATP content (83%). Moreover, the UV-A-acclimated cells showed a 2.3-fold increase in ribulose-1,5-bisphosphate carboxylase/oxygenase activity, indicating an increase in their carbon-fixing capacity. Gas chromatography–mass spectrometry-based metabolomics further revealed that UV-A radiation upregulated the energy-storing carbon metabolism, as evidenced by the enhanced accumulation of sugars, fatty acids, and citrate in the UV-A-acclimated cells. Therefore, our results demonstrate that UV-A radiation enhances energy flow and carbon assimilation in the cyanobacterium N. sphaeroides.
IMPORTANCE
Ultraviolet (UV) radiation exerts harmful effects on photo-autotrophs; however, several studies demonstrated the positive effects of UV radiation, especially UV-A radiation (315–400 nm), on primary productivity. Therefore, understanding the underlying mechanisms associated with the promotive effects of UV-A radiation on primary productivity can facilitate the application of UV-A for CO2 sequestration and lead to the advancement of photobiological sciences. In this study, we used the cyanobacterium Nostoc sphaeroides, which has an over 1,700-year history of human use as food and medicine, to explore its photosynthetic acclimation response to UV-A radiation. As per our knowledge, this is the first study to demonstrate that UV-A radiation increases the biomass yield of N. sphaeroides by enhancing energy flow and carbon assimilation. Our findings provide novel insights into UV-A-mediated photosynthetic acclimation and provide a scientific basis for the application of UV-A radiation for optimizing light absorption capacity and enhancing CO2 sequestration in the frame of a future CO2 neutral, circular, and sustainable bioeconomy.
... Green and blue fluorescence emissions from mammalian cells is largely attributed to flavins and NADPH [42]. While NADPH and FAD fluorescence have also been studied in cyanobacteria [49,50], further research is required to determine whether these compounds are contributing to the increased M. aeruginosa green fluorescence detected from UV, violet, and blue laser excitation recorded in this study. ...
M . aeruginosa fluorescent changes were observed using a Cytek Aurora spectral flow cytometer that contains 5 lasers and 64 narrow band detectors located between 365 and 829 nm. Cyanobacteria were treated with different concentrations of H 2 O 2 and then monitored after exposure between 1 and 8 days. The red fluorescence emission derived from the excitation of cyanobacteria with a yellow green laser (550 nm) was measured in the 652–669 nm detector while green fluorescence from excitation with a violet laser (405 nm) was measured in the 532–550 nm detector. The changes in these parameters were measured after the addition of H 2 O 2 . There was an initial increase in red fluorescence intensity at 24 hours. This was followed by a daily decrease in red fluorescence intensity. In contrast, green fluorescence increased at 24 hours and remained higher than the control for the duration of the 8-day study. A similar fluorescence intensity effect as H 2 O 2 on M . aeruginosa fluorescence emissions was observed after exposure to acetylacetone, diuron (DCMU), peracetic acid, and tryptoline. Minimal growth was also observed in H 2 O 2 treated cyanobacteria during exposure of H 2 O 2 for 24 days. In another experiment, H 2 O 2 -treated cyanobacteria were exposed to high-intensity blue (14 mW) and UV (1 mW) lights to assess the effects of light stress on fluorescence emissions. The combination of blue and UV light with H 2 O 2 had a synergistic effect on M . aeruginosa that induced greater fluorescent differences between control and treated samples than exposure to either stimulus individually. These experiments suggest that the early increase in red and green fluorescence may be due to an inhibition in the ability of photosynthesis to process photons. Further research into the mechanisms driving these increases in fluorescence is necessary.
... This could explain higher specific biotransformation rates (and yields) obtained in the presence of D-Glu. It is worth noting that NADH contributes to the NADPH fluorescence signal [32]. ...
Background
Cyanobacteria have emerged as highly efficient organisms for the production of chemicals and biofuels. Yet, the productivity of the cell has been low for commercial application. Cyanobacterial photobiotransformations utilize photosynthetic electrons to form reducing equivalents, such as NADPH-to-fuel biocatalytic reactions. These photobiotransformations are a measure to which extent photosynthetic electrons can be deviated toward heterologous biotechnological processes, such as the production of biofuels. By expressing oxidoreductases, such as YqjM from Bacillus subtilis in Synechocystis sp. PCC 6803, a high specific activity was obtained in the reduction of maleimides. Here, we investigated the possibility to accelerate the NAD(P)H-consuming redox reactions by addition of carbohydrates as exogenous carbon sources such as D-Glucose under light and darkness.
Results
A 1.7-fold increase of activity (150 µmol min ⁻¹ g DCW ⁻¹ ) was observed upon addition of D-Glucose at an OD 750 = 2.5 (DCW = 0.6 g L ⁻¹ ) in the biotransformation of 2-methylmaleimide. The stimulating effect of D-Glucose was also observed at higher cell densities in light and dark conditions as well as in the reduction of other substrates. No increase in both effective photosynthetic yields of Photosystem II and Photosystem I was found upon D-Glucose addition. However, we observed higher NAD(P)H fluorescence when D-Glucose was supplemented, suggesting increased glycolytic activity. Moreover, the system was scaled-up (working volume of 200 mL) in an internally illuminated Bubble Column Reactor exhibiting a 2.4-fold increase of specific activity under light-limited conditions.
Conclusions
Results show that under photoautotrophic conditions at a specific activity of 90 µmol min ⁻¹ g DCW ⁻¹ , the ene-reductase YqjM in Synechocystis sp. PCC 6803 is not NAD(P)H saturated, which is an indicator that an increase of the rates of heterologous electron consuming processes for catalysis and biofuel production will require funnelling further reducing power from the photosynthetic chain toward heterologous processes.
... On the other hand, in M55 cells, full NADP reduction is obtained. This suggests that in M55 cells, ATP limits the activity of the Calvin cycle due to the lack of CET-PSI phosphorylation (Mi, 2000). ...
Light reaction of photosynthesis is efficiently driven by protein complexes arranged in an orderly in the thylakoid membrane. As the 5th complex, NAD(P)H dehydrogenase complex (NDH-1) is involved in cyclic electron flow around photosystem I to protect plants against environmental stresses for efficient photosynthesis. In addition, two kinds of NDH-1 complexes participate in CO2 uptake for CO2 concentration in cyanobacteria. In recent years, great progress has been made in the understanding of the assembly and the structure of NDH-1. However, the regulatory mechanism of NDH-1 in photosynthesis remains largely unknown. Therefore, understanding the regulatory mechanism of NDH-1 is of great significance to reveal the mechanism of efficient photosynthesis. In this mini-review, the author introduces current progress in the research of cyanobacterial NDH-1. Finally, the author summarizes the possible regulatory mechanism of cyanobacterial NDH-1 in photosynthesis and discusses the research prospect.
... An additional difference between GA and pCMB was evident from NADPH fluorescence measurement ( Supplementary Fig. 13); the pCMB-treated cells showed a slowed decay of NADPH fluorescence after switching off the light and NADPH formation in the subsequent dark period was never observed. The slowed NADPH fluorescence decay may be attributable to Calvin cycle inhibition, but the lack of subsequent NADPH formation in the dark suggests the existence of other inhibitory effects of pCMB on the metabolic pathway(s) that reduces NADP, such as the oxidative pentose phosphate pathway 63,64 . We speculate that inhibition of dark NADPH formation changes cellular metabolism to accumulate alternative reducing equivalents instead of NADPH and this may be linked to the enhanced EET activity. ...
Biophotovoltaics (BPV) generates electricity from reducing equivalent(s) produced by photosynthetic organisms by exploiting a phenomenon called extracellular electron transfer (EET), where reducing equivalent(s) is transferred to external electron acceptors. Although cyanobacteria have been extensively studied for BPV because of their high photosynthetic activity and ease of handling, their low EET activity poses a limitation. Here, we show an order-of-magnitude enhancement in photocurrent generation of the cyanobacterium Synechocystis sp. PCC 6803 by deprivation of the outer membrane, where electrons are suggested to stem from pathway(s) downstream of photosystem I. A marked enhancement of EET activity itself is verified by rapid reduction of exogenous electron acceptor, ferricyanide. The extracellular organic substances, including reducing equivalent(s), produced by this cyanobacterium serve as respiratory substrates for other heterotrophic bacteria. These findings demonstrate that the outer membrane is a barrier that limits EET. Therefore, depriving this membrane is an effective approach to exploit the cyanobacterial reducing equivalent(s).
... Thereafter, a post-illumination transient increase in fluorescence intensity was observed in the dark in the wild type (Fig. 2). All of these observations are typical responses for in vivo NAD(P) H fluorescence in cyanobacteria (Mi et al. 2000;Feilke et al. 2017;Kauny and Sétif 2014). Notably, the NAD(P)H fluorescence level was higher before illumination than immediately after the light was turned off (Fig. 2), indicating that the NAD(P)H pool remained partially reduced even in the dark before illumination. ...
... This is consistent with the latest report showing the exogenous glucose effect on the NAD(P)H fluorescence in the wild type and Δgnd of Syn6803 (Ogawa et al. 2021). In addition, a postillumination fluorescence rise, which has been assumed to be derived from the NADPH generation dependent on the accumulation of the CBB cycle intermediates after illumination (Mi et al. 2000), was not detected in either Δzwf or Δgnd. It is plausible from the present results that the OPP pathway was driven by the transiently accumulated CBB cycle intermediates to produce NADPH after the light was turned off. ...
... It is plausible from the present results that the OPP pathway was driven by the transiently accumulated CBB cycle intermediates to produce NADPH after the light was turned off. The NADPH pool was kept reduced in the dark state at the same level as in the illuminated state in the case of the mutant of Syn6803 deficient in NAD(P)H dehydrogenase-1 (NDH-1) complex (Δndh) (Mi et al. 2000;Sétif et al. 2020). In addition, a mutant of Syn6803 that over-expressed tobacco plastid terminal oxidase showed the oxidized pool of NADPH in the dark state, similar to Δzwf and Δgnd (Feilke et al. 2017), which suggests that chlororespiration plays a role in oxidizing the NADPH pool. ...
Live cyanobacteria and algae integrated onto an extracellular electrode can generate a light-induced current (i.e., a photocurrent). Although the photocurrent is expected to be correlated with the redox environment of the photosynthetic cells, the relationship between the photocurrent and the cellular redox state is poorly understood. Here, we investigated the effect of the reduced nicotinamide adenine dinucleotide phosphate [NADP(H)] redox level of cyanobacterial cells (before light exposure) on the photocurrent using several mutants (Δzwf, Δgnd, and ΔglgP) deficient in the oxidative pentose phosphate (OPP) pathway, which is the metabolic pathway that produces NADPH in darkness. The NAD(P)H redox level and photocurrent in the cyanobacterium Synechocystis sp. PCC 6803 were measured noninvasively. Dysfunction of the OPP pathway led to oxidation of the photosynthetic NADPH pool in darkness. In addition, photocurrent induction was retarded and the current density was lower in Δzwf, Δgnd, and ΔglgP than in wild-type cells. Exogenously added glucose compensated the phenotype of ΔglgP and drove the OPP pathway in the mutant, resulting in an increase in the photocurrent. The results indicated that NADPH accumulated by the OPP pathway before illumination is a key factor for the generation of a photocurrent. In addition, measuring the photocurrent can be a non-invasive approach to estimate the cellular redox level related to NADP(H) pool in cyanobacteria.
... Furthermore, upon UV-A excitation, low levels of blue-green fluorescence have been observed not only from isolated chloroplasts (Latouche et al. 2000) but from cyanobacteria (Mi et al. 2000) as well as from algal cells (White et al. 2014). This fluorescence is from NADPH. ...
Oxygenic photosynthesis takes place in thylakoid membranes (TM) of cyanobacteria, algae, and higher plants. It begins with light absorption by pigments in large (modular) assemblies of pigment-binding proteins, which then transfer excitation energy to the photosynthetic reaction centers of photosystem (PS) I and PSII. In green algae and plants, these light-harvesting protein complexes contain chlorophylls (Chls) and carotenoids (Cars). However, cyanobacteria, red algae, and glaucophytes contain, in addition, phycobiliproteins in phycobilisomes that are attached to the stromal surface of TM, and transfer excitation energy to the reaction centers via the Chl a molecules in the inner antennas of PSI and PSII. The color and the intensity of the light to which these photosynthetic organisms are exposed in their environment have a great influence on the composition and the structure of the light-harvesting complexes (the antenna) as well as the rest of the photosynthetic apparatus, thus affecting the photosynthetic process and even the entire organism. We present here a perspective on 'Light Quality and Oxygenic Photosynthesis', in memory of George Christos Papageorgiou (9 May 1933-21 November 2020; see notes a and b). Our review includes (1) the influence of the solar spectrum on the antenna composition, and the special significance of Chl a; (2) the effects of light quality on photosynthesis, measured using Chl a fluorescence; and (3) the importance of light quality, intensity, and its duration for the optimal growth of photosynthetic organisms.
... Actually, reduced NADPH was reported to be accumulated in the dark in the NDH-1 defective mutant of Synechocystis sp. PCC 6803 because of the slow oxidation of NADPH by NDH-1 (Mi et al., 2000). The resulting deficiency of NADP + , the final electron acceptor of photosynthetic electron transport, may 1 http://www.photosynthesis.jp/fluorome/ ...
... We used two cyanobacterial strains, in which the redox states of NADPH are different but those of the PQ pool are similar after dark-acclimation, in order to verify the effect of NADPH on chlorophyll fluorescence. One of the two strains is ΔndhF1, in which the PQ pool is oxidized (Ogawa et al., 2013) while NADPH is reduced in the dark (Mi et al., 2000;Tanaka et al., 2021). The other is the strain with disrupted gnd gene (Δgnd), which encodes 6-phosphogluconate dehydrogenase (Kaneko et al., 1996) in oxidative pentose phosphate (OPP) pathway, the major pathway producing NADPH in the dark (Cheung and Gibbs, 1966;Wolk, 1973). ...
In cyanobacteria, the photosynthetic prokaryotes, direct interaction between photosynthesis and respiration exists at plastoquinone (PQ) pool, which is shared by the two electron transport chains. Another possible point of intersection of the two electron transport chains is NADPH, which is the major electron donor to the respiratory chain as well as the final product of the photosynthetic chain. Here, we showed that the redox state of NADPH in the dark affected chlorophyll fluorescence induction in the cyanobacterium Synechocystis sp. PCC 6803 in a quantitative manner. Accumulation of the reduced NADPH in the dark due to the defect in type 1 NAD(P)H dehydrogenase complex in the respiratory chain resulted in the faster rise to the peak in the dark-to-light induction of chlorophyll fluorescence, while depletion of NADPH due to the defect in pentose phosphate pathway resulted in the delayed appearance of the initial peak in the induction kinetics. There was a strong correlation between the dark level of NADPH determined by its fluorescence and the peak position of the induction kinetics of chlorophyll fluorescence. These results indicate that photosynthesis interacts with respiration through NADPH, which enable us to monitor the redox condition of the acceptor side of photosystem I by simple measurements of chlorophyll fluorescence induction in cyanobacteria.
... Fluorescence detection of NADPH is a representative in vivo method for measuring light-responsive changes in NADPH concentrations. For example, it has been shown that NADPH was produced or consumed in the sub-second order by light-dark transitions (Mi et al. 2000;Kauny and Sétif 2014;Shaku et al. 2016). However, the fluorescent yield of NADPH changes depending on the peripheral environment (Latouche et al. 2000;Kauny and Sétif 2014). ...
... When the light was turned off, the fluorescence intensity decreased and reached a minimum within 5 s, subsequently increasing and stabilizing after 30 s (stage-III). This time-transient behavior is in good agreement with previous reports, and the decrease and increase of the fluorescence level after turning the light off in stage-III are considered to be due to NADPH consumption by the Calvin cycle and NADPH production by the oxidative pentose phosphate pathway (OPPP), respectively (Mi et al. 2000;Kauny and Sétif 2014). The fluorescence-based method can detect NAD(P)H responses to environmental light changes on a time scale of seconds. ...
... Moreover, as shown in Fig. 5b, the total amount of NADP + and NADPH for the mutant was approximately half of that for the WT. On the other hand, since NADH also exhibit fluorescence, it is necessary to verify whether NADH variation also affects typical time transients of NAD(P) H fluorescence shown in Fig. 2a and reported in previous papers (Mi et al. 2000;Kauny and Sétif 2014;Holland et al. 2015;Shaku et al. 2016). In fact, a recent study for chloroplasts in planta showed that NADH also increased by light irradiation (Lim et al. 2020), raising the above possibility. ...
In photosynthetic organisms, it is recognized that the intracellular redox ratio of NADPH is regulated within an appropriate range for the cooperative function of a wide variety of physiological processes. However, despite its importance, there is large variability in the values of the NADPH fraction [NADPH/(NADPH + NADP⁺)] quantitatively estimated to date. In the present study, the light response of the NADPH fraction was investigated by applying a novel NADP(H) extraction method using phenol / chloroform / isoamyl alcohol (PCI) in the cyanobacterium Synechocystis sp. PCC 6803. The light response of NADP(H) observed using PCI extraction was qualitatively consistent with the NAD(P)H fluorescence time course measured in vivo. Moreover, the results obtained by PCI extraction and the fluorescence-based methods were also consistent in a mutant lacking the ability to oxidize NAD(P)H in the respiratory chain, and exhibiting a unique NADPH light response. These observations indicate that the PCI extraction method allowed quantitative determination of NADP(H) redox. Notably, the PCI extraction method showed that not all NADP(H) was oxidized or reduced by light–dark transition. Specifically, the fraction of NADPH was 42% in the dark-adapted cell, and saturated at 68% in light conditions.
... It was found that the concentration of NADPH was higher, and more H 2 O 2 was produced on the acceptor side of PSI, when measuring the NADPH fluorescence kinetics of cyanobacteria NDH mutant (Mi et al., 2000). These observations indicated that NDH-CET plays a key role in the process of antioxidation. ...
Chloroplast NAD(P)H dehydrogenase (NDH) complex, a multiple-subunit complex in the thylakoid membranes mediating cyclic electron transport, is one of the most important alternative electron transport pathways. It was identified to be essential for plant growth and development during stress periods in recent years. The NDH-mediated cyclic electron transport can restore the over-reduction in stroma, maintaining the balance of the redox system in the electron transfer chain and providing the extra ATP needed for the other biochemical reactions. In this review, we discuss the research history and the subunit composition of NDH. Specifically, the formation and significance of NDH-mediated cyclic electron transport are discussed from the perspective of plant evolution and physiological functionality of NDH facilitating plants’ adaptation to environmental stress. A better understanding of the NDH-mediated cyclic electron transport during photosynthesis may offer new approaches to improving crop yield.
