Activation of Chloroplast NADP-linked Glyceraldehyde-3-Phosphate Dehydrogenase by the Ferredoxin/Thioredoxin System

NADP-glyceraldehyde-3-P dehydrogenase of spinach (Spinacia oleracea) chloroplasts was activated by thioredoxin that was reduced either photochemically with ferredoxin and ferredoxin-thioredoxin reductase or chemically with dithiothreitol. The activation process that was observed with the soluble protein fraction from chloroplasts and with the purified regulatory form of the enzyme was slow relative to the rate of catalysis. The NAD-linked glyceraldehyde-3-P dehydrogenase activity that is also present in chloroplasts and in the purified enzyme preparation was not affected by reduced thioredoxin.

When activated by dithiothreitol-reduced thioredoxin, the regulatory form of NADP-glyceraldehyde-3-P dehydrogenase was partly deactivated by oxidized glutathione. The enzyme activated by photochemically reduced thioredoxin was not appreciably affected by oxidized glutathione. The results suggest that although it resembles other regulatory enzymes in its requirements for light-dependent activation by the ferredoxin/thioredoxin system, NADP-glyceraldehyde-3-P dehydrogenase differs in its mode of deactivation and in its capacity for activation by enzyme effectors independently of thioredoxin.

     

Contrasting modes of photosynthetic enzyme regulation in oxygenic and anoxygenic prokaryotes

Enzymes that are regulated by the ferredoxin/thioredoxin system in chloroplasts — fructose-1,6-bisphosphatase (FBPase), sedoheptulose-1,7-bisphosphatase purified from two different types of photosynthetic prokaryotes (cyanobacteria, purple sulfur bacteria) and tested for a response to thioredoxins. Each of the enzymes from the cyanobacterium Nostoc muscorum, an oxygenic organism known to contain the ferredoxin/thioredoxin system, was activated by thioredoxins that had been reduced either chemically by dithiothreitol or photochemically by reduced ferredoxin and ferredoxin-thioredoxin reductase. Like their chloroplast counterparts, N. muscorum FBPase and SBPase were activated preferentially by reduced thioredoxin f. SBPase was also partially activated by thioredoxin m. PRK, which was present in two regulatory forms in N. muscorum, was activated similarly by thioredoxins f and m. Despite sharing the capacity for regulation by thioredoxins, the cyanobacterial FBPase and SBPase target enzymes differed antigenically from their chloroplast counterparts. The corresponding enzymes from Chromatium vinosum, an anoxygenic photosynthetic purple bacterium found recently to contain the NADP/thioredoxin sytem, differed from both those of cyanobacteria and chloroplasts in showing no response to reduced thioredoxin. Instead, C. vinosum FBPase, SBPase, and PRK activities were regulated by a metabolite effector, 5-AMP. The evidence is in accord with the conclusion that thioredoxins function in regulating the reductive pentose phosphate cycle in oxygenic prokaryotes (cyanobacteria) that contain the ferredoxin/thioredoxin system, but not in anoxygenic prokaryotes (photosynthetic purple bacteria) that contain the NADP/thioredoxin system. In organisms of the latter type, enzyme effectors seem to play a dominant role in regulating photosynthetic carbon dioxide assimilation.     

Studies on the regulatory properties of chloroplast fructose-1,6-bisphosphatase.

The regulatory properties of chloroplast fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, (EC 3.1.3.11) were examined with a homogeneous enzyme preparation isolated from spinach leaves. The activation of the enzyme, that was earlier shown to occur via reduced thioredoxin, was found to be accompanied by a structural change that took place more slowly than the rate of catalysis. The recently found deactivation of the thioredoxin-activated enzyme by physiological oxidants such as oxidized glutathione and dehydroascorbic acid was also slow relative to catalysis. Under the conditions used, the activated enzyme showed a pH optimum of about 8.0, whereas the corresponding value for the non-activated form was pH 8.8. The importance of the thioredoxin-linked mechanism of enzyme regulation that is effected through photoreduced ferredoxin and ferredoxin-thioredoxin reductase is discussed in relation to other light-controlled regulatory agents in chloroplasts.     

Role of ferredoxin in the activation of sedoheptulose diphosphatase in isolated chloroplasts

Sedoheptulose 1,7-diphosphatase activity of isolated spinach chloroplasts shows a requirement for (i) reduced ferredoxin and (ii) a protein factor. Activation by ferredoxin, reduced photochemically by chloroplast fragments, was optimal at pH 7.8 and at a Mg²⁺ concentration of 5 mM. The protein factor needed for activation appears to be the same as that required by the chloroplast fructose-1,6-diphosphatase that is activated by reduced ferredoxin. The results indicate that sedoheptulose-1,7-diphosphatase, like fructose-1,6-diphosphatase, is a regulatory enzyme whose activity in chloroplasts is controlled via ferredoxin by light.     

Regulation of chloroplast phosphoribulokinase by the ferredoxin/thioredoxin system

A newly found form of chloroplast phosphoribulokinase (designated the “regulatory form”) required reduced thioredoxin for activity. A second form of the enzyme (the “nonregulatory form”) was not appreciably affected by thioredoxin. The thioredoxin required for activation of the regulatory enzyme could be reduced (i) photochemically by chloroplast membranes that were supplemented with ferredoxin and ferredoxin-thioredoxin reductase or (ii) chemically in the dark with the sulfhydryl reagent dithiothreitol. Following activation by reduced thioredoxin, phosphoribulokinase was deactivated by the soluble chloroplast oxidants dehydroascorbate and oxidized glutathione. The results suggest that the regulatory form of phosphoribulokinase resembles fructose 1,6-bisphosphatase in its mode of regulation by the ferredoxin/thioredoxin system.     

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.     

Multi‐level regulation of the chloroplast ATP synthase: the chloroplast NADPH thioredoxin reductase C (NTRC) is required for redox modulation specifically under low irradiance

The chloroplast ATP synthase is known to be regulated by redox modulation of a disulfide bridge on the γ‐subunit through the ferredoxin–thioredoxin regulatory system. We show that a second enzyme, the recently identified chloroplast NADPH thioredoxin reductase C (NTRC), plays a role specifically at low irradiance. Arabidopsis mutants lacking NTRC (ntrc) displayed a striking photosynthetic phenotype in which feedback regulation of the light reactions was strongly activated at low light, but returned to wild‐type levels as irradiance was increased. This effect was caused by an altered redox state of the γ‐subunit under low, but not high, light. The low light‐specific decrease in ATP synthase activity in ntrc resulted in a buildup of the thylakoid proton motive force with subsequent activation of non‐photochemical quenching and downregulation of linear electron flow. We conclude that NTRC provides redox modulation at low light using the relatively oxidizing substrate NADPH, whereas the canonical ferredoxin–thioredoxin system can take over at higher light, when reduced ferredoxin can accumulate. Based on these results, we reassess previous models for ATP synthase regulation and propose that NTRC is most likely regulated by light. We also find that ntrc is highly sensitive to rapidly changing light intensities that probably do not involve the chloroplast ATP synthase, implicating this system in multiple photosynthetic processes, particularly under fluctuating environmental conditions.     

Activation of ribulose-1,5-bisphosphate carboxylase by chloroplast metabolites in a reconstituted spinach chloroplast system

In a preparation of soluble components from isolated spinach (Spinecia oleracea L.) chloroplasts, the activity of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) is strongly increased by 6-phosphogluconate or by NADPH at pH 8.0. When the thylakoid system is added to these soluble components (reconstituted chloroplast system) plus ferredoxin, the carboxylase is even more strongly activated in the light. This light activation appears to be due to reduction of endogenous NADP⁺ by electrons from the light reactions transferred via ferredoxin, since NADPH alone can activate the purified enzyme in the dark while reduced ferredoxin does not. The regulatory properties of the enzyme in the reconstituted chloroplast system are compared with those of the isolated enzyme, and their possible physiologic significance is discussed.Abbreviations Fd ferredoxin - PPC pentose phosphate cycle - 6-PGluA 6-phosphogluconate - Rib-5-P ribose-5-phosphate - RuBP ribulose-1,5-bisphosphate     

Light activation of fructose bisphosphatase in isolated spinach chloroplasts and deactivation by hydrogen peroxide

Spinach chloroplast fructose bisphosphatase (EC 3.1.3.11.) exists in both oxidised and reduced forms. Only the latter has the kinetic properties that allow it to function at physiological concentrations of fructose 1,6-bisphosphate and Mg²⁺. Illumination of freshly prepared type A chloroplasts causes a conversion of oxidised to reduced enzyme. The rate of this conversion does not limit the rate of CO₂ fixation. In the dark the reduced enzyme partially reverts back to the oxidised form. If catalase is omitted from the reaction medium the rate of CO₂ fixation by chloroplasts is decreased and seems to be limited by the rate of conversion of the enzyme to the reduced form. The physiological significance of the light dependent generation of dithiol compounds (such as thioredoxin) within chloroplasts is discussed.     

Evidence for function of the ferredoxin/thioredoxin system in the reductive activation of target enzymes of isolated intact chloroplasts

Results obtained with isolated intact chloroplasts maintained aerobically under light and dark conditions confirm earlier findings with reconstituted enzyme assays and indicate that the ferredoxin/thioredoxin system functions as a light-mediated regulatory thiol chain. The results were obtained by application of a newly devised procedure in which a membrane-permeable thiol labeling reagent, monobromobimane (mBBr), reacts with sulfhydryl groups and renders the derivatized protein fluorescent. The mBBr-labeled protein in question is isolated individually from chloroplasts by immunoprecipitation and its thiol redox status is determined quantitatively by combining sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorescence measurements. The findings indicate that each member of the ferredoxin/thioredoxin system containing a catalytically active thiol group is reduced in isolated intact chloroplasts after a 2-min illumination. The extents of reduction were FTR, 38%; thioredoxin m, 75% (11-kDa form) and 87% (13-kDa form); thioredoxin f, 95%. Reduction of each of these components was negligible both in the dark and when chloroplasts were transferred from light to dark conditions. The target enzyme, NADP-malate dehydrogenase, also underwent net reduction in illuminated intact chloroplasts. Fructose-1,6-bisphosphatase showed increased mBBr labeling under these conditions, but due to interfering gamma globulin proteins it was not possible to determine whether this was a result of net reduction as is known to take place in reconstituted assays. Related experiments demonstrated that mBBr, as well as N-ethylmaleimide, stabilized photoactivated NADP-malate dehydrogenase and fructose-1,6-bisphosphatase so that they remained active in the dark. By contrast, phosphoribulokinase, another thioredoxin-linked enzyme, was immediately deactivated following mBBr addition. These latter results provide new information on the relation between the regulatory and active sites of these enzymes.     

Redox regulation in the chloroplast thylakoid lumen: a new frontier in photosynthesis research

Initially linked to photosynthesis, regulation by change in the redox state of thiol groups (S-S<-- -->2SH) is now known to occur throughout biology. Thus, in addition to serving important structural and catalytic functions, it is recognized that, in many cases, disulphide bonds can be broken and reformed for regulation. Several systems, each linking a hydrogen donor to an intermediary disulphide protein, act to effect changes that alter the activity of target proteins by change in the thiol redox state. Pertinent to the present discussion is the chloroplast ferredoxin/thioredoxin system, comprised of photoreduced ferredoxin, a thioredoxin, and the enzyme ferredoxin-thioredoxin reductase, that occur in the stroma. In this system, thioredoxin links the activity of enzymes to light: those enzymes functional in biosynthesis are reductively activated by light via thioredoxin (S-S-->2SH), whereas counterparts acting in degradation are deactivated under illumination conditions and are oxidatively activated in the dark (2SH-->S-S). Recent research has uncovered a new paradigm in which an immunophilin, FKBP13, and potentially other enzymes of the chloroplast thylakoid lumen are oxidatively activated in the light (2SH-->S-S). The present review provides a perspective on this recent work.     

Light and thiol activation of maize leaf glycerate kinase : the stimulating effect of reduced thioredoxins and ATP

Glycerate kinase (EC 2.7.1.31) from maize (Zea mays) leaves was shown to be regulated by light/dark transition. The enzyme more than doubled in activity after either the leaves or isolated mesophyll chloroplasts were illuminated with white light for 10 minutes. Rate of inactivation in the dark was faster in leaves than in the isolated chloroplast fraction. The stimulating effect of light could be mimicked in crude preparations by addition of 10 or 50 millimolar dithiothreitol or 100 millimolar 2-mercaptoethanol. The thiol treatment resulted in 8- to 10-fold activation of glycerate kinase, with the highest rates in the range of 27 to 30 micromoles per mg chlorophyll per hour. Activation was not accompanied by any changes in the apparent M_r value of glycerate kinase as determined by gel filtration (M_r = 47,000). In contrast to maize glycerate kinase, the enzyme from spinach was not affected by either light or thiol exposure.

Partially purified maize glycerate kinase was activated up to 3-fold upon incubation with a mixture of spinach thioredoxins m and f and 5 millimolar dithiothreitol. The thioredoxin and dithiothreitol-treated glycerate kinase could be further stimulated by addition of 2.5 millimolar ATP. The results suggest that glycerate kinase from maize leaves is capable of photoactivation by the ferredoxin/thioredoxin system. The synergistic effect of ATP and thioredoxins in activation of the enzyme supports the earlier expressed view that the ferredoxin/thioredoxin system functions jointly with effector metabolites in light-mediated regulation during photosynthesis.

     

Protein modulase appears to be a complex of ferredoxin, ferredoxin/thioredoxin reductase, and thioredoxin

Protein modulase and ferredoxin/thioredoxin reductase are soluble proteins that have been suggested to catalyze the light-dependent modulation of enzyme activity in the stromal compartment of the chloroplast. Protein modulase is active in vitro without additional ferredoxin and thioredoxin, whereas ferredoxin/thioredoxin reductase requires additional ferredoxin and thioredoxin. We hypothesize that protein modulase is a complex protein composed of ferredoxin/thioredoxin reductase, ferredoxin, and thioredoxin. In reconstituted chloroplast systems, antiserum directed against ferredoxin, at concentrations sufficient to inhibit the photoreduction of NADP, had no effect on light modulation. Antiserum directed against thioredoxin gave variable results: one batch of polyclonal antibodies inhibited light modulation, another was stimulatory, and another was without effect. These results suggest that the ferredoxin and thioredoxin active in light modulation are not free in solution. Furthermore, molecular sieve chromatography of stromal proteins results in the elution of four species that catalyze light modulation. Based on whether or not ferredoxin and/or thioredoxin must be added for activity, these four species have been tentatively identified as protein modulase, a complex of ferredoxin/thioredoxin reductase and ferredoxin, a complex of ferredoxin/thioredoxin reductase and thioredoxin, and ferredoxin/thioredoxin reductase. That is, the four correspond to all the possible combinations of ferredoxin, ferredoxin/thioredoxin reductase, and thioredoxin. We suggest that buffer ionic strength affects the interactions among these proteins and in part determines the fate of the protein modulase complex in vitro.     

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.     

Photoactivation and Regulation of Spinach Leaf Fructose 1,6-bisphosphatase

Fructose 1,6-bisphosphatase, in isolated intact chloroplast from spinach leaves, is photoactivated by ferredoxin/thioredoxin system. The mechanism involved is conversion of enzyme disulfide to sulfhydryl groups as the photoactivation is inhibited by sulfhydryl group modifying agents which are able to penetrate the chloroplast envelope. Reduction of ferredoxin on the reducing side of photosystem I is found to be a key event and active electron flow to ferredoxin must be maintained for keeping the enzyme in activated state. DCMU - a classical electron transport chain inhibitor and other exogenously added electron acceptors, which intercept electrons on or before ferredoxin cause deactivation of fructose 1,6-bisphosphatase in light. The rate of deactivation, in dark, is also enhanced by exogenously added electron acceptors and sulfhydryl group modifying agents. The mechanism of regulation of fructose 1,6-bisphosphatase is discussed.     

A new protein factor functional in the ferredoxin-independent light activation of chloroplast fructose 1,6-bisphosphatase

A protein purified from chloroplasts (the “new protein factor”) activated Fru-P₂ase in a photochemical reaction that depended only on chloroplast membranes. The results suggest that chloroplasts utilize the newly found mechanism for the photoregulation of Fru-P₂ase in addition to the recently described ferredoxin/thioredoxin system.     

Investigating the role of the thiol-regulated enzyme sedoheptulose-1,7-bisphosphatase in the control of photosynthesis

Sedoheptulose-1,7-bisphosphatase (SBPase; EC 3.1.3.37) catalyses the dephosphorylation of sedoheptulose-1,7-bisphosphate in the regenerative phase of the Calvin cycle. Antisense plants with reduced levels of SBPase have decreased photosynthetic capacity and altered carbohydrate status, leading to modifications in growth and development. The catalytic activity of SBPase is regulated by light via the ferredoxin/thioredoxin system. Recently, the amino acids within the SBPase protein involved in this regulatory mechanism have been identified and a deregulated, permanently active form of the enzyme has been produced using site-directed mutagenesis. This paper explores how transgenic Nicotiana tabacum cv. Samsun plants, containing the deregulated form of the SBPase enzyme, may lead to a better understanding of the in vivo role of light activation of this important Calvin cycle enzyme.     

Light activation and molecular-mass changes of NAD(P)-glyceraldehyde 3-phosphate dehydrogenase of spinach and maize leaves

Light modulation of chloroplast glyceraldehyde 3-phosphate dehydrogenase (NAD(P)-GAPDH; EC 1.2.1.13) has been investigated. Complete activation of NADPH-dependent activity is achieved at 25 W.m^–2 photosynthetically active radiation in spinach (Spinacia oleracea L.) and 100 W.m^–2 in maize (Zea mays L.) leaves. Light activation is stronger in spinach (fivefold on average) than in maize (twofold), which shows higher dark activity. The NADH dependent activity does not change appreciably. Several substrate activators can simulate in vitro the light effect with recovery of latent NADPH-dependent activity of spinach enzyme, but they are almost inactive with maize enzyme. A mixture of activators has been devised to fully activate the spinach enzyme under most conditions. The NAD(P)-GAPDH protein can be resolved by rapid gel filtration (fast protein liquid chromatography) into three conformers which have different molecular masses according to the light conditions. Enzyme from darkened leaves or chloroplasts, or dichlorophenyl-1,1-dimethylurea-treated chloroplasts is mainly a 600-kDa regulatory form with low NADPH-dependent activity relative to NADH-activity. Enzyme from spinach leaves or chloroplasts during photosynthesis is mainly a 300-kDa oligomer, which along with the 600-kDa form also occurs in leaves of darkened maize. The conformer of illuminated maize leaves is mainly a 160-kDa species. Results are consistent with a model of NAD(P)-GAPDH freely interconvertible between protomers of the 160-kDa (or 300-kDa intermediate) form with high NADPH-activity, produced in the light by the action of thioredoxin and activating metabolites (spinach only), and a regulatory 600-kDa conformer with lower NADPH-activity produced in darkness or when photosynthesis is inhibited. This behavior is reminiscent of the in-vitro properties of purified enzyme; therefore, it seems unlikely that NAD(P)-GAPDH in the chloroplast is part of a stable multienzyme complex or is bound to membranes.Abbreviations AEM activator equilibrium mixture - Chl chlorophyll - DCMU dichlorophenyl 1,1-dimethylurea - DTT dithiothreitol - FPLC fast protein liquid chromatography - NAD(P)-GAPDH glyceraldehyde 3-phosphate dehydrogenase, NAD(P)-dependent - PAR photosynthetic active radiation - PGK phosphoglycerate kinase - Tricine N-tris(hydroxy-methyl) methyl-glycine This work was supported by grants from the Ministero dell'Università e della Ricerca Seientifica e Tecnologica (40%, years 1990 and 1991).     

Regulation of NADP-Dependent Glyceraldehyde 3-Phosphate Dehydrogenase Activity in Spinach Chloroplasts*

Activation of NAD(P)-glyceraldehyde 3-phosphate dehydrogenase (NADP-GAPDH, EC 1.2.1.13) can be achieved in isolated chloroplasts in the light, or in the dark upon addition of dithiothreitol (DTT) and/or 3-phosphoglycerate plus ATP. Activation in darkened chloroplasts is only partial with DTT or 3-phosphoglycerate plus ATP alone, but complete when both effectors are added. In the light, full activation is only achieved upon addition of ATP. The time-course of activation appears to depend upon the actual concentration of 1,3-bisphosphoglycerate (1,3bisPGA) inside the chloroplasts. The K_a values for 1,3bisPGA are in the same range as has been determined for the purified enzyme, namely around 20 μM for the dark form (in the absence of DTT) and around 1 μM for the light form or in the presence of DTT. In contrast, the K_a value for ATP is 1 to 2 mM for both the oxidized and the reduced enzyme forms. The observed activation of NADP-GAPDH is strongly paralleled by an increase of 3PGA, and consequently of 1,3bisPGA in the illuminated chloroplast, while the ATP level remains constant or declines. Activation by 1,3bisPGA is accompanied by dissociation of the 600 kDa form to the 150 kDa form, while reduction alone does not induce a shift in molecular mass as documented by fast gel filtration on Superdex 200. Thus partial activation by DTT in the dark is due to an increased activity of the 600 kDa form, while the activation state in the light is the result of a partial conversion of the 600 kDa form into the more active 150 kDa form. The principle of this activation is a fast reduction of the enzyme by the ferredoxin/thioredoxin system, resulting in a lowered K_avalue for 1,3bisPGA, and thus adjusting the properties of the enzyme to the stromal 1,3bisPGA level. The occurrence of a 300 kDa oligomer mainly during inactivation has also been observed. From these results a model is constructed that describes the reversible interconversion of various activation and aggregation states of NADP-GAPDH as observed upon light/dark transitions in isolated spinach chloroplasts.     

Crosstalk between chloroplast thioredoxin systems in regulation of photosynthesis

Thioredoxins (TRXs) mediate light‐dependent activation of primary photosynthetic reactions in plant chloroplasts by reducing disulphide bridges in redox‐regulated enzymes. Of the two plastid TRX systems, the ferredoxin‐TRX system consists of ferredoxin‐thioredoxin reductase (FTR) and multiple TRXs, while the NADPH‐dependent thioredoxin reductase (NTRC) contains a complete TRX system in a single polypeptide. Using Arabidopsis plants overexpressing or lacking a functional NTRC, we have investigated the redundancy and interaction between the NTRC and Fd‐TRX systems in regulation of photosynthesis in vivo. Overexpression of NTRC raised the CO₂ fixation rate and lowered non‐photochemical quenching and acceptor side limitation of PSI in low light conditions by enhancing the activation of chloroplast ATP synthase and TRX‐regulated enzymes in Calvin–Benson cycle (CBC). Overexpression of NTRC with an inactivated NTR or TRX domain partly recovered the phenotype of knockout plants, suggesting crosstalk between the plastid TRX systems. NTRC interacted in planta with fructose‐1,6‐bisphosphatase, phosphoribulokinase and CF₁γ subunit of the ATP synthase and with several chloroplast TRXs. These findings indicate that NTRC‐mediated regulation of the CBC and ATP synthesis occurs both directly and through interaction with the ferredoxin‐TRX system and is crucial when availability of light is limiting photosynthesis.