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.     

Redox regulation of chloroplast metabolism

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

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

Advances

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

     

Chloroplasts require glutathione reductase to balance reactive oxygen species and maintain efficient photosynthesis

Thiol‐based redox‐regulation is vital for coordinating chloroplast functions depending on illumination and has been throroughly investigated for thioredoxin‐dependent processes. In parallel, glutathione reductase (GR) maintains a highly reduced glutathione pool, enabling glutathione‐mediated redox buffering. Yet, how the redox cascades of the thioredoxin and glutathione redox machineries integrate metabolic regulation and detoxification of reactive oxygen species remains largely unresolved because null mutants of plastid/mitochondrial GR are embryo‐lethal in Arabidopsis thaliana. To investigate whether maintaining a highly reducing stromal glutathione redox potential (E_GSH) via GR is necessary for functional photosynthesis and plant growth, we created knockout lines of the homologous enzyme in the model moss Physcomitrella patens. In these viable mutant lines, we found decreasing photosynthetic performance and plant growth with increasing light intensities, whereas ascorbate and zeaxanthin/antheraxanthin levels were elevated. By in vivo monitoring stromal E_GSH dynamics, we show that stromal E_GSH is highly reducing in wild‐type and clearly responsive to light, whereas an absence of GR leads to a partial glutathione oxidation, which is not rescued by light. By metabolic labelling, we reveal changing protein abundances in the GR knockout plants, pinpointing the adjustment of chloroplast proteostasis and the induction of plastid protein repair and degradation machineries. Our results indicate that the plastid thioredoxin system is not a functional backup for the plastid glutathione redox systems, whereas GR plays a critical role in maintaining efficient photosynthesis.     

Rice NTRC is a high-efficiency redox system for chloroplast protection against oxidative damage

One of the mechanisms plants have developed for chloroplast protection against oxidative damage involves a 2-Cys peroxiredoxin, which has been proposed to be reduced by ferredoxin and plastid thioredoxins, Trx x and CDSP32, the FTR/Trx pathway. We show that rice (Oryza sativa) chloroplast NADPH THIOREDOXIN REDUCTASE (NTRC), with a thioredoxin domain, uses NADPH to reduce the chloroplast 2-Cys peroxiredoxin BAS1, which then reduces hydrogen peroxide. The presence of both NTR and Trx-like domains in a single polypeptide is absolutely required for the high catalytic efficiency of NTRC. An Arabidopsis thaliana knockout mutant for NTRC shows irregular mesophyll cell shape, abnormal chloroplast structure, and unbalanced BAS1 redox state, resulting in impaired photosynthesis rate under low light. Constitutive expression of wild-type NTRC in mutant transgenic lines rescued this phenotype. Moreover, prolonged darkness followed by light/dark incubation produced an increase in hydrogen peroxide and lipid peroxidation in leaves and accelerated senescence of NTRC-deficient plants. We propose that NTRC constitutes an alternative system for chloroplast protection against oxidative damage, using NADPH as the source of reducing power. Since no light-driven reduced ferredoxin is produced at night, the NTRC-BAS1 pathway may be a key detoxification system during darkness, with NADPH produced by the oxidative pentose phosphate pathway as the source of reducing power.     

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.     

叶绿体硫氧还蛋白系统的调节机制

硫氧还蛋白(Trx)属于巯基-二硫键氧化还原酶家族, 通过作用于底物蛋白侧链2个半胱氨酸残基之间的二硫键(还原、异构和转移)来调控胞内蛋白的结构和功能。叶绿体Trx系统包括Trx及Trx类似蛋白、铁氧还蛋白(Fd)依赖的硫氧还蛋白还原酶(FTR)和还原型烟酰腺嘌呤二核苷磷酸(NADPH)依赖的硫氧还蛋白还原酶C (NTRC)。除了基质蛋白酶类活性变化及叶绿体蛋白的转运受Trx系统调控之外, 在叶绿体中还存在1条跨类囊体膜的还原势传递途径, 把基质Trx的还原势经跨膜转运蛋白介导, 最终传递给类囊体腔蛋白。FTR和NTRC共同作用维持叶绿体的氧化还原平衡。该文对叶绿体硫氧还蛋白系统的调节机制进行了综述, 同时讨论了叶绿体硫氧还蛋白系统对维持植物光合效率的重要意义。     

Chloroplast redox homeostasis is essential for lateral root formation in Arabidopsis

Redox regulation based on dithiol-disulphide interchange is an essential component of the control of chloroplast metabolism. In contrast to heterotrophic organisms, and non-photosynthetic plant tissues, chloroplast redox regulation relies on ferredoxin (Fd) reduced by the photosynthetic electron transport chain, thus being highly dependent on light. The finding of the NADPH-dependent thioredoxin reductase C (NTRC), a chloroplast-localized NTR with a joint thioredoxin domain, showed that NADPH is also used as source of reducing power for chloroplast redox homeostasis. Recently we have found that NTRC is also in plastids of non-photosynthetic tissues. Because these non-green plastids lack photochemical reactions, their redox homeostasis depends exclusively on NADPH produced from sugars and, thus, NTRC may play an essential role maintaining the redox homeostasis in these plastids. The fact that redox regulation occurs in any type of plastids raises the possibility that the functions of chloroplasts and non-green plastids, such as amyloplasts, are integrated to harmonize the growth of the different organs of the plant. To address this question, we generated Arabidopsis plants the redox homeostasis of which is recovered exclusively in chloroplasts, by leaf-specific expression of NTRC in the ntrc mutant, or exclusively in amyloplasts, by root-specific expression of NTRC. The analysis of these plants suggests that chloroplasts exert a pivotal role on plant growth, as expected because chloroplasts constitute the major source of nutrients and energy, derived from photosynthesis, for growth of heterotrophic tissues. However, NTRC deficiency causes impairment of auxin synthesis and lateral root formation. Interestingly, recovery of redox homeostasis of chloroplasts, but not of amyloplasts, was sufficient to restore wild type levels of lateral roots, showing the important signaling function of chloroplasts for the development of heterotrophic organs.     

Plant responses to fungal volatiles involve global posttranslational thiol redox proteome changes that affect photosynthesis

Microorganisms produce volatile compounds (VCs) that promote plant growth and photosynthesis through complex mechanisms involving cytokinin (CK) and abscisic acid (ABA). We hypothesized that plants' responses to microbial VCs involve posttranslational modifications of the thiol redox proteome through action of plastidial NADPH‐dependent thioredoxin reductase C (NTRC), which regulates chloroplast redox status via its functional relationship with 2‐Cys peroxiredoxins. To test this hypothesis, we analysed developmental, metabolic, hormonal, genetic, and redox proteomic responses of wild‐type (WT) plants and a NTRC knockout mutant (ntrc) to VCs emitted by the phytopathogen Alternaria alternata. Fungal VC‐promoted growth, changes in root architecture, shifts in expression of VC‐responsive CK‐ and ABA‐regulated genes, and increases in photosynthetic capacity were substantially weaker in ntrc plants than in WT plants. As in WT plants, fungal VCs strongly promoted growth, chlorophyll accumulation, and photosynthesis in ntrc–Δ2cp plants with reduced 2‐Cys peroxiredoxin expression. OxiTRAQ‐based quantitative and site‐specific redox proteomic analyses revealed that VCs promote global reduction of the thiol redox proteome (especially of photosynthesis‐related proteins) of WT leaves but its oxidation in ntrc leaves. Our findings show that NTRC is an important mediator of plant responses to microbial VCs through mechanisms involving global thiol redox proteome changes that affect photosynthesis.     

NTRC new ways of using NADPH in the chloroplast

Despite being the primary source of energy in the biosphere, photosynthesis is a process that inevitably produces reactive oxygen species. Chloroplasts are a major source of hydrogen peroxide production in plant cells; therefore, different systems for peroxide reduction, such as ascorbate peroxidase and peroxiredoxins (Prxs), are found in this organelle. Most of the reducing power required for hydrogen peroxide reduction by these systems is provided by Fd reduced by the photosynthetic electron transport chain; hence, the function of these systems is highly dependent on light. Recently, it was described a novel plastidial enzyme, stated NTRC, formed by a thioredoxin reductase (NTR) domain at the N-terminus and a thioredoxin (Trx) domain at the C-terminus. NTRC is able to conjugate both NTR and Trx activities to efficiently reduce 2-Cys Prx using NADPH as a source of reducing power. Based on these results, it was proposed that NTRC is a new pathway to transfer reducing power to the chloroplast detoxification system, allowing the use of NADPH, besides reduced Fd, for such function. In this article, the most important features of NTRC are summarized and the implications of this novel activity in the context of chloroplast protection against oxidative damage are discussed.     

Chloroplast phenylalanine ammonia-lyase from spinach leaves

Phenylalanine ammonia-lyase (PAL) from spinach (Spinacia oleracea L.) leaves was resolved into three forms by diethyl-aminoethyl(DEAE)-cellulose chromatography. Two forms were found in isolated chloroplasts, and the third form (the major component) was located outside of the chloroplasts. One of the chloroplast forms of the enzyme (designated the regulatory form) was activated by reduced thioredoxin. Neither the other chloroplast form nor the extra-chloroplast form showed a response to thioredoxin. After further purification by hydroxyapatite column chromatography and gel filtration, the regulatory form of chloroplast PAL was stimulated approximately 3-fold by thioredoxin reduced either photochemically by chloroplast membranes, via ferredoxin and ferredoxin-thioredoxin reductase, or chemically by dithiothreitol. Once activated, the enzyme required an added oxidant for deactivation. Physiological oxidants-oxidized glutathione (GSSG) and dehydroascorbate-as well as nonphysiological oxidants-sodium tetrathionate and diamide-were effective in deactivation. The results indicate that chloroplast PAL is regulated by light via the ferredoxin/thioredoxin system in a manner similar to that described for regulatory enzymes of CO₂ assimilation. The extra-chloroplast form of the enzyme, by contrast, appears to be regulated by light via the earlier-described phytochrome-linked system.     

Ferredoxin/thioredoxin system plays an important role in the chloroplastic NADP status of Arabidopsis

NADP is a key electron carrier for a broad spectrum of redox reactions, including photosynthesis. Hence, chloroplastic NADP status, as represented by redox status (ratio of NADPH to NADP⁺) and pool size (sum of NADPH and NADP⁺), is critical for homeostasis in photosynthetic cells. However, the mechanisms and molecules that regulate NADP status in chloroplasts remain largely unknown. We have now characterized an Arabidopsis mutant with imbalanced NADP status (inap1), which exhibits a high NADPH/NADP⁺ ratio and large NADP pool size. inap1 is a point mutation in At2g04700, which encodes the catalytic subunit of ferredoxin/thioredoxin reductase. Upon illumination, inap1 demonstrated earlier increases in NADP pool size than the wild type did. The mutated enzyme was also found in vitro to inefficiently reduce m‐type thioredoxin, which activates Calvin cycle enzymes, and NADP‐dependent malate dehydrogenase to export reducing power to the cytosol. Accordingly, Calvin cycle metabolites and amino acids diminished in inap1 plants. In addition, inap1 plants barely activate NADP‐malate dehydrogenase, and have an altered redox balance between the chloroplast and cytosol, resulting in inefficient nitrate reduction. Finally, mutants deficient in m‐type thioredoxin exhibited similar light‐dependent NADP dynamics as inap1. Collectively, the data suggest that defects in ferredoxin/thioredoxin reductase and m‐type thioredoxin decrease the consumption of NADPH, leading to a high NADPH/NADP⁺ ratio and large NADP pool size. The data also suggest that the fate of NADPH is an important influence on NADP pool size.     

A comparative analysis of the NADPH thioredoxin reductase C-2-Cys peroxiredoxin system from plants and cyanobacteria

Redox regulation based on disulfide-dithiol conversion catalyzed by thioredoxins is an important component of chloroplast function. The reducing power is provided by ferredoxin reduced by the photosynthetic electron transport chain. In addition, chloroplasts are equipped with a peculiar NADPH-dependent thioredoxin reductase, termed NTRC, with a joint thioredoxin domain at the carboxyl terminus. Because NADPH can be produced by the oxidative pentose phosphate pathway during the night, NTRC is important to maintain the chloroplast redox homeostasis under light limitation. NTRC is exclusive for photosynthetic organisms such as plants, algae, and some, but not all, cyanobacteria. Phylogenetic analysis suggests that chloroplast NTRC originated from an ancestral cyanobacterial enzyme. While the biochemical properties of plant NTRC are well documented, little is known about the cyanobacterial enzyme. With the aim of comparing cyanobacterial and plant NTRCs, we have expressed the full-length enzyme from the cyanobacterium Anabaena species PCC 7120 as well as site-directed mutant variants and truncated polypeptides containing the NTR or the thioredoxin domains of the protein. Immunological and kinetic analysis showed a high similarity between NTRCs from plants and cyanobacteria. Both enzymes efficiently reduced 2-Cys peroxiredoxins from plants and from Anabaena but not from the cyanobacterium Synechocystis. Arabidopsis (Arabidopsis thaliana) NTRC knockout plants were transformed with the Anabaena NTRC gene. Despite a lower content of NTRC than in wild-type plants, the transgenic plants showed significant recovery of growth and pigmentation. Therefore, the Anabaena enzyme fulfills functions of the plant enzyme in vivo, further emphasizing the similarity between cyanobacterial and plant NTRCs.     

Structural analysis revealed a novel conformation of the NTRC reductase domain from Chlamydomonas reinhardtii

In plant chloroplasts, thiol regulation is driven by two systems. One relies on the activity of thioredoxins through their light dependent reduction by ferredoxin via a ferredoxin-thioredoxin reductase (FTR). In the other system, a NADPH-dependent redox regulation is driven by a NADPH-thioredoxin reductase C (NTRC). While the thioredoxin system has been deeply studied, a more thorough understanding of the function of this plant specific NTRC is desirable. NTRC is a single polypeptide harbouring a thioredoxin domain (Trx) at the C-terminus of a NADPH-dependent Thioredoxin reductase (TrxR). To provide functional and structural insights, we studied the crystal structure of the TrxR domain of the NTRC from Chlamydomonas reinhardtii (CrNTRC, Cre01.g054150.t1.2) and its Cys136Ser (C136S) mutant, which is characterized by the mutation of the resolving cysteine in the active site of the TrxR domain. Furthermore, we confirmed the role of NTRC as electron donor for 2-Cys peroxiredoxin (PRX) also in C. reinhardtii. The structural data of TrxR were employed to develop a scheme of action which addresses electron transfer between TrxR and Trx of NTRC and between NTRC and its substrates.     

Redox regulation of Arabidopsis 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase

The cDNA for 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase of Arabidopsis encodes a polypeptide with an amino-terminal signal sequence for plastid import. A cDNA fragment encoding the processed form of the enzyme was expressed in Escherichia coli. The resulting protein was purified to electrophoretic homogeneity. The enzyme requires Mn(2+) and reduced thioredoxin (TRX) for activity. Spinach (Spinacia oleracea) TRX f has an apparent dissociation constant for the enzyme of about 0.2 microM. The corresponding constant for TRX m is orders of magnitude higher. In the absence of TRX, dithiothreitol partially activates the enzyme. Upon alkylation of the enzyme with iodoacetamide, the dependence on a reducing agent is lost. These results indicate that the first enzyme in the shikimate pathway of Arabidopsis appears to be regulated by the ferredoxin/TRX redox control of the chloroplast.     

Enzyme Regulation in Crassulacean Acid Metabolism Photosynthesis : Studies on the Ferredoxin/Thioredoxin System of KalanchoE daigremontiana

Cell-free preparations of the Crassulacean acid metabolism (CAM) plant, Kalanchoë daigremontiana, were analyzed for thioredoxins and ferredoxin-thioredoxin reductase. Three distinct forms of thioredoxin were identified in Kalanchoë leaves, two of which specifically activated fructose 1,6-bisphosphatase (designated f₁ and f₂) and a third which activated NADP-malate dehydrogenase (thioredoxin m). The apparent molecular weight of both forms of thioredoxin f was 11,000 and that of thioredoxin m was 10,000. In parallel studies, ferredoxin and ferredoxin-thioredoxin reductase were purified from Kalanchoë leaf preparations. Kalanchoë ferredoxin-thioredoxin reductase was similar to that of C₃ and C₄ plants in molecular weight (31,000) and immunological cross-reactivity. Kalanchoë ferredoxin-thioredoxin reductase exhibited an affinity for ferredoxin as demonstrated by its binding to an immobilized ferredoxin affinity column. The purified components of the Kalanchoë ferredoxin-thioredoxin system could be recombined to function in the photoregulation of chloroplast enzymes. The data suggest that the ferredoxin/thioredoxin system plays a role in enzyme regulation of all higher plants irrespective of whether they show C₃, C₄, or CAM photosynthesis.     

Crystal structure of chloroplastic thioredoxin z defines a type-specific target recognition

Thioredoxins (TRXs) are ubiquitous disulfide oxidoreductases structured according to a highly conserved fold. TRXs are involved in a myriad of different processes through a common chemical mechanism. Plant TRXs evolved into seven types with diverse subcellular localization and distinct protein target selectivity. Five TRX types coexist in the chloroplast, with yet scarcely described specificities. We solved the crystal structure of a chloroplastic z-type TRX, revealing a conserved TRX fold with an original electrostatic surface potential surrounding the redox site. This recognition surface is distinct from all other known TRX types from plant and non-plant sources and is exclusively conserved in plant z-type TRXs. We show that this electronegative surface endows thioredoxin z (TRXz) with a capacity to activate the photosynthetic Calvin–Benson cycle enzyme phosphoribulokinase. The distinct electronegative surface of TRXz thereby extends the repertoire of TRX–target recognitions.     

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.