Modulation of Chloroplast Fructose-1,6-bisphosphatase Activity by Light

Inhibitor experiments indicate that light effect mediator_II which is reductively activated by transfer of electrons from the photosynthetic electron transport system at or beyond ferredoxin, is involved in activation by light of fructose-1,6-bisphosphatase in the pea plant. Activation proceeds optimally when the pH is low and Mg²⁺ is 10 millimolar. Modulation by light results in increases in maximal velocity, apparently as a result of changes in enzyme conformation. Pea leaf thylakoids are effective in modulating the activity of glyceraldehyde-3-phosphate dehydrogenase but not of fructose-1,6-bisphosphatase or glucose-6-phosphate dehydrogenase in Kalanchoë stromal extracts. There is apparently species specificity for modulation of some, but not all, of the modulatable enzymes.     

Light modulation of the activity of carbon metabolism enzymes in the crassulacean Acid metabolism plant kalanchoë

When intact Kalanchoë plants are illuminated NADP-linked malic dehydrogenase and three enzymes of the reductive pentose phosphate pathway, ribulose-5-phosphate kinase, NADP-linked glyceraldehyde-3-phosphate dehydrogenase, and sedoheptulose-1,7-diphosphate phosphatase, are activated. In crude extracts these enzymes are activated by dithiothreitol treatment. Light or dithiothreitol treatment does not inactivate the oxidative pentose phosphate pathway enzyme glucose-6-phosphate dehydrogenase. Likewise, neither light, in vivo, nor dithiothreitol, in vitro, affects fructose-1,6-diphosphate phosphatase. Apparently the potential for modulation of enzyme activity by the reductively activated light effect mediator system exists in Crassulacean acid metabolism plants, but some enzymes which are light-dark-modulated in the pea plant are not in Kalanchoë.     

Light modulation of enzyme activity: activation of the light effect mediators by reduction and modulation of enzyme activity by thiol-disulfide exchange

Light and dark modulation experiments with pea (Pisum sativum L.) chloroplast stromal fractions pretreated with dithiothreitol (to reduce protein disulfide bonds) or with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) (to block sulfhydryl groups) suggest that light modulation involves thiol-disulfide exchange on the modulatable stromal enzyme protein. Light-dependent reduction of DTNB involves a photosynthetic electron transport chain component located on the reducing side of photosystem I prior to ferredoxin; DTNB may be acting as a light effect mediator substitute. The thylakoid-bound light effect mediator system, then, in its light-activated reduced form probably catalyzes thiol-disulfide exchange reactions on stromal enzymes.     

Mechanism of reductive photoactivation of enzymes of C4 pathway

Light, besides initiating primary photochemical processes, alters the redox state of soluble components in chloroplast. The present review attempts to cover the mechanism of reductive photoactivation of enzymes of photosynthetic carbon reduction cycle using key enzymes as examples. The reduced soluble components — ferredoxin, thioredoxin and NADPH, in turn, cause the reduction of disulphides to dithiols of chloroplastic enzymes. NADP-malate dehydrogenase is subject to activation by light through changes in NADPH/NADP. The key enzyme of C₄ photosynthesis-PEP carboxylase, though cytosolic, has been shown to be activated by disulphide/sulphhydryl interconversion by reductants generated in light through chloroplast electron transport flow. PyruvateP _i dikinase activity is controlled by the adenylate energy charge. It remains unclear how light controls the activation of cytosolic enzymes.     

Role of Photosynthetic Electron Transfer in Light Activation of Calvin Cycle Enzymes

The effect of light in activating fructose-1,6 biphosphate phosphatase (E.C. 3.1.3.11), sedoheptulose-1,7, biphosphate phosphatase (E.C. 3.1.3.11), ribulose-5 phosphate kinase (E.C. 2.7.1.19), ribulose-1,5 biphosphate carboxylase (E.C. 4.1.1.39) and (NADPH) glyceraldehyde-3 phosphate dehydrogenase (E.C. 1.2.1.13) in intact spinach chloroplasts in the presence of antimycin A, tetramethylethylenediamine (TMEDA) or chlorophenyl-1,1-dimethylurea (CMU) was examined. Antimycin A and TMEDA were added as stimulating agents for photosynthetic electron transfer in intact chloroplasts while CMU was added for its inhibitory characteristics. Light exerted its control through the mediation of the photosynthetic electron transfer. Antimycin A and TMEDA promoted the light activation. CMU nullified the light activation as well as the stimulatory effect of antimycin A and TMEDA. Thus the control by light of the activities of the Calvin cycle enzymes involves a reduced agent formed by the photosynthetic electron transport chain. From the presently available evidence, it seems appropriate to hypothesize that the light activation of the enzymes is not a single mechanism. In fact three types of enzymes can be distinguished: Ru-5 P kinase and (NADPH) G-3 P dehydrogenase, maximal activation of which appears within the first minute of illumination and is promoted by antimycin A and by TMEDA; F-1,6 P2 phosphatase and S-1,7 P2 phosphatase, ferredoxin-dependent enzymes, activation of which is slightly slower but is also promoted by antimycin A and by TMEDA; finally Ru-1,5 P2 carboxylase, activation of which is still slower and characterized by the absence of any response to antimycin A as well as to TMEDA.     

Changing kinetic properties of the two-enzyme phosphoglycerate kinase/NADP-linked glyceraldehyde-3-phosphate dehydrogenase couple from pea chloroplasts during photosynthetic induction

The kinetics of the two enzyme phosphoglycerate kinase (EC 2.7.2.3)/NADP-linked glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) couple are negatively cooperative and will also fit a model for two enzymes acting on one substrate. When the chloroplast is illuminated apparent negative cooperativity is reduced; maximal velocity of only one of the two enzymes in the two-enzyme model is increased. Even after light activation the activity of glyceraldehyde-3-phosphate dehydrogenase appears to be too low to support photosynthesis at calculated levels of glycerate-1,3-bisphosphate in isolated chloroplasts (Marques, I.A., Ford, D.M., Muschinek, G. and Anderson, L.E. (1987) Arch. Biochem. Biophys. 252, 458–466). The activity of the coupled reaction is apparently sufficient to support observed rates of CO₂ fixation, which suggests that glycerate-1,3-bisphosphate may be channeled from the kinase to the dehydrogenase in vivo.     

Isolation and characterization of a thylakoid membrane module showing partial light and dark reactions

A functional thylakoid membrane module of photosynthesis was isolated from cell free extracts of Anacystis nidulans by stepwise sequential ultracentrifugation. The thylakoid membrane fractions sedimenting at 40,000×g, followed by 90,000×g and finally at 150,000×g were collected. These fractions had all the components of electron transport chain, ATP synthase, phycobiliproteins, ferredoxin-NADP reductase but no ferredoxin. Five sequential enzymes of Calvin cycle viz phosphoriboisomerase, phosphoribulokinase, RuBP carboxylase, 3-PGA kinase and glyceraldehyde-3-phosphate dehydrogenase were found to be associated with thylakoid membranes. Among the three different thylakoid fractions, the 150,000×g fraction showed highest activities of these enzymes and also higher rate of whole chain electron transport activity on chlorophyll basis. An important finding was that the 150,000×g fraction showed appreciably higher rate of R-5-P+ADP+Pi dependent CO₂ fixation in light compared to the other two fractions, indicating the efficiency of this fraction in utilizing ATP for Calvin cycle. This thylakoid membrane fraction represents a fully functional module exhibiting a synchronized system of light and dark reactions of photosynthesis. Most of the components of this module remained together even after sucrose density gradient centrifugation. This is the first report on the isolation of a photosynthetic module involving membrane and soluble proteins.     

Biogenesis of chloroplast membranes. I. Plastid dedifferentiation in a dark-grown algal mutant (Chlamydomonas reinhardi)

This paper describes the morphology and photosynthetic activity of a mutant of Chlamydomonas reinhardi (y-1) which is unable to synthesize chlorophyll in the dark. When grown heterotrophically in the light, the mutant is indistinguishable from the wild type Chlamydomonas. When grown in the dark, chlorophyll is diluted through cell division and the photosynthetic activity (oxygen evolution, Hill reaction, and photoreduction of NADP) decays at a rate equal to or faster than that of chlorophyll dilution. However, soluble enzymes associated with the photosynthetic process (alkaline FDPase, NADP-linked G-3-P dehydrogenase, RuDP carboxylase), as well as cytochrome f and ferredoxin, continue to be present in relatively high concentrations. The enzymes involved in the synthesis of the characteristic lipids of the chloroplast (including mono- and digalactoside glycerides, phosphatidyl glycerol, and sulfolipid) are still detectable in dark-grown cells. Such cells accumulate large amounts of starch granules in their plastids. On onset of illumination, dark-grown cells synthesize chlorophyll rapidly, utilizing their starch reserve in the process. At the morphological level, it was observed that during growth in the dark the chloroplast lamellar system is gradually disorganized and drastically decreased in extent, while other subchloroplast components are either unaffected (pyrenoid and its tubular system, matrix) or much less affected (eyespot, ribosomes). It is concluded that the dark-grown mutant possesses a partially differentiated plastid and the enzymic apparatus necessary for the synthesis of the chloroplast membranes (discs). The advantage provided by such a system for the study of the biogenesis of the chloroplast photosynthetic membranes is discussed.     

Photoactivation of NADP-Malate Dehydrogenase by a Heterogeneous Photochemical System in Zea mays

A heterogeneous photochemical electron relay system was constructed, mimicking the chloroplast electron transport reaction in order to activate the NADP-malate dehydrogenase in light. The photocatalyst acridine orange or proflavin sensitized EDTA-dependent reduction of ferredoxin. In a complete system, consisting of a dye donor couple, ferredoxin, thioredoxin and ferredoxin-thioredoxin reductase, light activation of purified NADP-MDH was observed in vitro. The chloroplast mediated redox activation of enzyme essentially required ferredoxin, while heterogeneous photochemical mediated activation of enzyme need not require ferredoxin. The heterogeneus photochemical system activated NADP-MDH by eight fold similar to chloroplasts mediated ferredoxin dependent redox activation but was not affected by the presence of disalicylinden propanediamine-1, 2-disulphonic acid while there was complete inhibition of chloroplasts mediated activation of NADP-MDH in presence of this inhibitor. These observations suggest that a thiol mediator is essential for reductive activation of NADP-MDH and ferredoxin is not required for photochemical activation.     

Isolation and characterization of a thylakoid membrane module showing partial light and dark reactions

A functional thylakoid membrane module of photosynthesis was isolated from cell free extracts of Anacystis nidulans by stepwise sequential ultracentrifugation. The thylakoid membrane fractions sedimenting at 40,000 x g, followed by 90,000 x g and finally at 150,000 x g were collected. These fractions had all the components of electron transport chain, ATP synthase, phycobiliproteins, ferredoxin-NADP reductase but no ferredoxin. Five sequential enzymes of Calvin cycle viz phosphoriboisomerase, phosphoribulokinase, RuBP carboxylase, 3-PGA kinase and glyceraldehyde-3-phosphate dehydrogenase were found to be associated with thylakoid membranes. Among the three different thylakoid fractions, the 150,000 x g fraction showed highest activities of these enzymes and also higher rate of whole chain electron transport activity on chlorophyll basis. An important finding was that the 150,000 x g fraction showed appreciably higher rate of R-5-P+ADP+Pi dependent CO2 fixation in light compared to the other two fractions, indicating the efficiency of this fraction in utilizing ATP for Calvin cycle. This thylakoid membrane fraction represents a fully functional module exhibiting a synchronized system of light and dark reactions of photosynthesis. Most of the components of this module remained together even after sucrose density gradient centrifugation. This is the first report on the isolation of a photosynthetic module involving membrane and soluble proteins.     

Effect of Disalicylidenepropanediamine on the Light-dependent Reduction of Carbon Dioxide and Glycerate 3-Phosphate in Intact Spinach Chloroplasts

Disalicylidenepropanediamine (DSPD) at 0.1 to 1 mm levels inhibited light-dependent (14)CO(2) assimilation in intact spinach chloroplasts about 50 to 80%, and this inhibition was accompanied by an increased ratio of (14)C-glycerate 3-phosphate to (14)C-glyceraldehyde 3-phosphate. Enzymatic analysis established that DSPD also inhibited the light-dependent reduction of glycerate 3-phosphate in intact spinach chloroplasts. DSPD at 0.5 mm did not inhibit ribose 5-phosphate isomerase, ribulose 5-phosphate kinase, glycerate 3-phosphate kinase, NADP(+)-linked glyceraldehyde 3-phosphate dehydrogenase or ribulose 1,5-diphosphate carboxylase. The inhibition of chloroplast (14)CO(2) assimilation by DSPD appeared to be related to the inhibition of the photosynthetic electron transport chain. These observations are consistent with experimental results which demonstrated that DSPD inhibited directly the chloroplast lamellar membrane-mediated, light-dependent reduction of ferredoxin (Trebst, A. and M. Burba, 1967, Z. Pflanzenphysiol. 57: 419-433 and Ben-Amotz, A. and M. Avron, 1972, Plant Physiol. 49: 244-248).     

Regulation of chloroplast photosynthetic activity by exogenous magnesium

Magnesium was most inhibitory to photosynthetic reactions by intact chloroplasts when the magnesium was added in the dark before illumination. Two millimolar MgCl₂, added in the dark, inhibited CO₂-dependent O₂ evolution by Hordeum vulgare L. and Spinacia oleracea L. (C₃ plants) chloroplasts 70 to 100% and inhibited (pyruvate + oxaloacetate)-dependent O₂ evolution by Digitaria sanguinalis L. (C₄ plant) mesophyll chloroplasts from 80 to 100%. When Mg²⁺ was added in the light, O₂ evolution was reduced only slightly. O₂ evolution in the presence of phosphoglycerate was less sensitive to Mg²⁺ inhibition than was CO₂-dependent O₂ evolution.

Magnesium prevented the light activation of several photosynthetic enzymes. Two millimolar Mg²⁺ blocked the light activation of NADP-malate dehydrogenase in D. sanguinalis mesophyll chloroplasts, and the light activation of phosphoribulokinase, NADP-linked glyceraldehyde-3-phosphate dehydrogenase, and fructose 1,6-diphosphatase in barley chloroplasts. The results suggest that Mg²⁺ inhibits chloroplast photosynthesis by preventing the light activation of certain enzymes.

     

Light-induced Enzyme Formation in a Chlorophyll-less Mutant of Euglena gracilis

A mutant strain, Y₉, of Euglena gracilis strain Z that is unable to produce protochlorophyll or chlorophyll has been isolated following treatment of wild type cells with nalidixic acid. Dark-grown cells of the mutant contain proplastids that show only limited ultrastructural development when placed in the light. Treatment of Y₉ cells with ultraviolet light brings about permanent cell bleaching with a target number similar to wild type Euglena, and with a slightly greater sensitivity to ultraviolet. Three enzymes of the reductive pentose phosphate cycle, fructose-1,6-diphosphate aldolase (class I), NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, and 3-phosphoglycerate kinase, are detectable in dark-grown Y₉ cells at the low concentrations characteristic of dark-grown wild type cells, and increase substantially when these cells are exposed to light. The activity of ribulose-1,5-diphosphate carboxylase increases in the light to a lesser extent. Cytochrome 552, a carrier in the photosynthetic electron transport chain, is not present in light-grown cells of Y₉. The significance of this mutant for an understanding of the role of light in Euglena chloroplast development is discussed.     

Selective Inhibition by Actinomycin D of the Synthesis in Photosynthetic and Non-photosynthetic Enzymes During the Greening of Etiolated Bean Leaves

The effect of actinomycin D on the synthesis of the photosynthetic apparatus during illumination of etiolated leaves of Phaseolus vulgaris was studied. The increase of chlorophyll content and of the activities of some photosynthetic enzymes (NADPH diaphorase, ferredoxin, NADP⁺ glyceraldehyde-3-phosphate dehydrogenase) was compared with simultaneous measurements of the level of other enzymes not considered associated with photosynthesis (ornithine transcarbamylase, glucose-6-phosphate dehydrogenase, NAD⁺ glyceraldehyde-3-phosphate dehydrogenase).     

Consequence of restricted mitochondrial oxidative metabolism on photosynthetic carbon assimilation in mesophyll protoplasts: Decrease in light activation of four chloroplastic enzymes

The patterns of light activation of 4 chloroplastic enzymes were examined in mesophyll protoplasts of pea ( Pisum sativum ) in the absence or presence of oligomycin (inhibitor of oxidative phosphorylation) or antimycin A (inhibitor of cytochrome pathway) or salicylhydroxamic acid (SHAM, inhibitor of alternative pathway). The results were compared with those of DCMU (inhibitor of photosynthetic electron transport). The light activation of NADP glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPDH), fructose-1,6-bisphosphatase (FBPase), phosphoribulokinase (PRK) (enzymes of the Calvin cycle) and NADP malate dehydrogenase (NADP-MDH) (reflects chloroplast redox state) was more pronounced at limiting CO₂ (0.1 m M NaHCO₃) than that at optimal CO₂ (1.0 m M NaHCO₃). SHAM decreased markedly (up to 33%) the light activation of all 4 enzymes, while antimycin A or oligomycin exerted only a limited effect (<10% decrease). Antimycin A or oligomycin or SHAM had no significant effect on light activation of these 4 enzymes in isolated chloroplasts. However, DCMU caused a remarkable decrease in light activation of enzymes in both protoplasts (up to 78%) and chloroplasts (up to 69%). These results suggest that the restriction of alternative pathway of mitochondrial metabolism results in a marked decrease in the light activation of key chloroplastic enzymes in mesophyll protoplasts but not in isolated chloroplasts. Such a decrease in the light activation of enzymes could be also a secondary feedback effect because of the restriction on carbon assimilation.     

Photosynthetic carbon metabolism in isolated pea chloroplasts: metabolite levels and enzyme activities

We report here that enzyme activation precedes the rise in metabolite levels, which appear to limit photosynthetic CO2 fixation during induction in pea leaf chloroplasts. Therefore light activation may be required for the build-up of photosynthetic intermediates and hence for photosynthesis in isolated chloroplasts. Analysis of metabolite levels and the known kinetic properties of the chloroplast enzymes indicates that the reductive pentose phosphate cycle is subject to control which fluctuates between several points during induction and when CO2 fixation is maximal. The transketolase-aldolase-catalyzed reactions around sedoheptulose-biphosphatase appear to provide a simple and effective primary control for photosynthetic CO2 fixation. When substrate levels and enzyme active site concentrations are taken into account, there is insufficient glyceraldehyde 3-phosphate dehydrogenase, aldolase, and transketolase activity to support photosynthetic CO2 fixation at observed rates. These results suggest that there may be direct transfer of glyceraldehyde 3-phosphate among these enzymes in the pea chloroplast.     

Heterogeneous photochemical and chloroplasts mediated activation of spinach fructose-1,6-bisphosphatase

A heterogeneous photochemical electron relay system was constructed, mimicking the chloroplast electron transport reaction, in order to activate fructose-1,6-bisphosphatase in light. The photocatalyst acridine orange or proflavin sensitizes EDTA dependent reduction of ferredoxin. In a complete system, consisting of a dye-donor couple, ferredoxin, thioredoxin and ferredoxin-thioredoxin reductase, light activation of purified spinach fructose-1,6-bisphosphatase was observed in vitro. The ferredoxin was not essential for activation of fructose-1,6-bisphosphatase using heterogeneous photochemical system while chloroplasts mediated redox activation essentially required ferredoxin. The heterogeneous photochemical system activated fructose-1,6-bisphosphatase by about 6 fold similar to chloroplasts mediated ferredoxin dependent redox activation. These observations suggest that a thiol mediator is essential for the reductive activation of carboxylating enzymes of photosynthesis. The mechanism of activation is discussed.     

Is Nicotinamide Adenine Dinucleotide Phosphate an Obligatory Intermediate in Photosynthesis?

The site of action of the inhibitors disalicylidenepropanediamine and pyrophosphate was more closely defined as acting on ferredoxin. Three inhibitors which act on the electron transport path between ferredoxin and NADP: disalicylidenepropanediamine, pyrophosphate, and phosphoadenosinediphosphate ribose, had no effect on photosynthesis in cell free preparations of Dunaliela parva at concentrations which completely inhibited the enzymic activity on which each inhibitor acts. The addition of disalicylidenepropanediamine to dark-grown Euglena gracilis cells prevented the light-induced formation of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, but not of photosynthesis, chlorophyll synthesis, or NAD-dependent glyceraldehyde-3-phosphate dehydrogenase.     

Mechanism of light modulation: identification of potential redox-sensitive cysteines distal to catalytic site in light-activated chloroplast enzymes.

Light-dependent reduction of target disulfides on certain chloroplast enzymes results in a change in activity. We have modeled the tertiary structure of four of these enzymes, namely NADP-linked glyceraldehyde-3-P dehydrogenase, NADP-linked malate dehydrogenase, sedoheptulose bisphosphatase, and fructose bisphosphatase. Models are based on x-ray crystal structures from non-plant species. Each of these enzymes consists of two domains connected by a hinge. Modeling suggests that oxidation of two crucial cysteines to cystine would restrict motion around the hinge in the two dehydrogenases and influence the conformation of the active site. The cysteine residues in the two phosphatases are located in a region known to be sensitive to allosteric modifiers and to be involved in mediating structural changes in mammalian and microbial fructose bisphosphatases. Apparently, the same region is involved in covalent modification of phosphatase activity in the chloroplast.     

N-Terminal Structure of Maize Ferredoxin:NADP+ Reductase Determines Recruitment into Different Thylakoid Membrane Complexes

To adapt to different light intensities, photosynthetic organisms manipulate the flow of electrons through several alternative pathways at the thylakoid membrane. The enzyme ferredoxin:NADP(+) reductase (FNR) has the potential to regulate this electron partitioning because it is integral to most of these electron cascades and can associate with several different membrane complexes. However, the factors controlling relative localization of FNR to different membrane complexes have not yet been established. Maize (Zea mays) contains three chloroplast FNR proteins with totally different membrane association, and we found that these proteins have variable distribution between cells conducting predominantly cyclic electron transport (bundle sheath) and linear electron transport (mesophyll). Here, the crystal structures of all three enzymes were solved, revealing major structural differences at the N-terminal domain and dimer interface. Expression in Arabidopsis thaliana of maize FNRs as chimeras and truncated proteins showed the N-terminal determines recruitment of FNR to different membrane complexes. In addition, the different maize FNR proteins localized to different thylakoid membrane complexes on expression in Arabidopsis, and analysis of chlorophyll fluorescence and photosystem I absorbance demonstrates the impact of FNR location on photosynthetic electron flow.