Initially discovered in the context of photosynthesis, regulation by change in the redox state of thiol groups (S-S <--> 2SH) is now known to occur throughout biology. Several systems, each linking a hydrogen donor to an intermediary disulfide protein, act to effect changes that alter the activity of target proteins: the ferredoxin/thioredoxin system, comprised of reduced ferredoxin, a thioredoxin, and the enzyme, ferredoxin-thioredoxin reductase; the NADP/thioredoxin system, including NADPH, a thioredoxin, and NADP-thioredoxin reductase; and the glutathione/glutaredoxin system, composed of reduced glutathione and a glutaredoxin. A related disulfide protein, protein disulfide isomerase (PDI) acts in protein assembly. Regulation linked to plastoquinone and signaling induced by reactive oxygen species (ROS) and other agents are also being actively investigated. Progress made on these systems has linked redox to the regulation of an increasing number of processes not only in plants, but in other types of organisms as well. Research in areas currently under exploration promises to provide a fuller understanding of the role redox plays in cellular processes, and to further the application of this knowledge to technology and medicine.

Thioredoxins are small multifunctional redox active proteins widely if not universally distributed among living organisms. In chloroplasts, two types of thioredoxins ( f and m) coexist and play central roles in regulating enzyme activity. Reduction of thioredoxins in chloroplasts is catalyzed by an iron-sulfur disulfide enzyme, ferredoxin-thioredoxin reductase, that receives photosynthetic electrons from ferredoxin, thereby providing a link between light and enzyme activity. Chloroplast thioredoxins function in the regulation of the Calvin cycle and associated processes. However, the relatively small number of known thioredoxin-linked proteins (about 16) raised the possibility that others remain to be identified. To pursue this opportunity, we have mutated thioredoxins f and m, such that the buried cysteine of the active disulfide has been replaced by serine or alanine, and bound them to affinity columns to trap target proteins of chloroplast stroma. The covalently linked proteins were eluted with DTT, separated on gels, and identified by mass spectrometry. This approach led to the identification of 15 potential targets that function in 10 chloroplast processes not known to be thioredoxin linked. Included are proteins that seem to function in plastid-to-nucleus signaling and in a previously unrecognized type of oxidative regulation. Approximately two-thirds of these targets contained conserved cysteines. We also identified 11 previously unknown and 9 confirmed target proteins that are members of pathways known to be regulated by thioredoxin. In contrast to results with individual enzyme assays, specificity for thioredoxin f or m was not observed on affinity chromatography.

Mitochondria contain thioredoxin (Trx), a regulatory disulfide protein, and an associated flavoenzyme, NADP͞Trx reductase, which provide a link to NADPH in the organelle. Unlike animal and yeast counterparts, the function of Trx in plant mitochondria is largely unknown. Accordingly, we have applied recently devised proteomic approaches to identify soluble Trx-linked proteins in mitochondria isolated from photosynthetic (pea and spinach leaves) and heterotrophic (potato tubers) sources. Application of the mitochondrial extracts to mutant Trx affinity columns in conjunction with proteomics led to the identification of 50 potential Trx-linked proteins functional in 12 processes: photorespiration, citric acid cycle and associated reactions, lipid metabolism, electron transport, ATP synthesis͞ transformation, membrane transport, translation, protein assembly͞ folding, nitrogen metabolism, sulfur metabolism, hormone synthesis, and stress-related reactions. Almost all of these targets were also identified by a fluorescent gel electrophoresis procedure in which reduction by Trx can be observed directly. In some cases, the processes targeted by Trx depended on the source of the mitochondria. The results support the view that Trx acts as a sensor and enables mitochondria to adjust key reactions in accord with prevailing redox state. These and earlier findings further suggest that, by sensing redox in chloroplasts and mitochondria, Trx enables the two organelles of photosynthetic tissues to communicate by means of a network of transportable metabolites such as dihydroxyacetone phosphate, malate, and glycolate. In this way, light absorbed and processed by means of chlorophyll can be perceived and function in regulating fundamental mitochondrial processes akin to its mode of action in chloroplasts.T hioredoxins (Trxs) are small, widely distributed proteins with a redox-active disulfide group. Two types of Trx systems have been described in plants based on the source of reducing power: the ferredoxin͞Trx system located in chloroplasts [in which electrons from ferredoxin reduce Trx by means of the iron-sulfur enzyme ferredoxin͞Trx reductase (1-4)] and the extraplastidic NADP͞Trx system [whereby NADPH reduces Trx by means of the flavoenzyme NADP͞Trx reductase (NTR) (3, 4)].Trx has two main functions by nature of its ability to reduce specific S-S groups: (i) as a substrate for enzymes that catalyze reactions such as the reduction of ribonucleotides, methionine sulfoxide, and hydrogen peroxide; and (ii) as a regulator that alters the activity or other functional properties of interacting target proteins (1-4). The regulatory role of Trx is prominent in plants where the first redox-controlled enzyme was identified in chloroplasts Ͼ25 years ago (1, 2). Since that time, the number of Trx-regulated enzymes has steadily increased (2, 4). Recently developed proteomics-based approaches have accelerated progress in making possible the systematic identification of Trx-linked proteins in complex extracts. With the addition of previously u...

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A growing number of processes throughout biology are regulated by redox via thiol-disulfide exchange. This mechanism is particularly widespread in plants, where almost 200 proteins have been linked to thioredoxin (Trx), a widely distributed small regulatory disulfide protein. The current study extends regulation by Trx to amyloplasts, organelles prevalent in heterotrophic plant tissues that, among other biosynthetic activities, catalyze the synthesis and storage of copious amounts of starch. Using proteomics and immunological methods, we identified the components of the ferredoxin͞Trx system (ferredoxin, ferredoxin-Trx reductase, and Trx), originally described for chloroplasts, in amyloplasts isolated from wheat starchy endosperm. Ferredoxin is reduced not by light, as in chloroplasts, but by metabolically generated NADPH via ferredoxin-NADP reductase. However, once reduced, ferredoxin appears to act as established for chloroplasts, i.e., via ferredoxinTrx reductase and a Trx (m-type). A proteomics approach in combination with affinity chromatography and a fluorescent thiol probe led to the identification of 42 potential Trx target proteins, 13 not previously recognized, including a major membrane transporter (Brittle-1 or ADP-glucose transporter). The proteins function in a range of processes in addition to starch metabolism: biosynthesis of lipids, amino acids, and nucleotides; protein folding; and several miscellaneous reactions. The results suggest a mechanism whereby light is initially recognized as a thiol signal in chloroplasts, then as a sugar during transit to the sink, where it is converted again to a thiol signal. In this way, amyloplast reactions in the grain can be coordinated with photosynthesis taking place in leaves.redox regulation ͉ target proteins ͉ ferredoxin-thioredoxin reductase O ur understanding of the function of the regulatory disulfide protein thioredoxin (Trx) has increased dramatically in the past few years, with the advent of new methodologies. Recent approaches make it possible to identify proteins linked to Trx by combining proteomics with affinity chromatography (1) or thiol probes (2-4). These capabilities have defined an extended role of Trx in chloroplasts (1, 5) and helped elucidate its function in other plant systems: mitochondria (6), seeds (2,3,7,8), and seedlings (4, 9). Currently almost 200 proteins appear to be linked to Trx in plants (10). One major organelle that has been neglected, however, is the amyloplast, a plastid of heterotrophic tissues that performs a wide range of biosynthetic reactions, including the synthesis and storage of abundant quantities of starch.To help fill this gap, we have applied proteomic and immunological approaches to investigate the occurrence and function of Trx in amyloplasts. We now report evidence that amyloplasts isolated from wheat starchy endosperm resemble chloroplasts in containing a complete ferredoxin͞Trx system composed of ferredoxin, ferredoxin-Trx reductase (FTR), and Trx (m-type). Application of affinity chromatography and fl...

The thiol-disulfide oxidoreductase thioredoxin-1 (Trx1) is known to be secreted by leukocytes and to exhibit cytokine-like properties. Extracellular effects of Trx1 require a functional active site, suggesting a redox-based mechanism of action. However, specific cell surface proteins and pathways coupling extracellular Trx1 redox activity to cellular responses have not been identified so far. Using a mechanism-based kinetic trapping technique to identify disulfide exchange interactions on the intact surface of living lymphocytes, we found that Trx1 catalytically interacts with a single principal target protein. This target protein was identified as the tumor necrosis factor receptor superfamily member 8 (TNFRSF8/CD30). We demonstrate that the redox interaction is highly specific for both Trx1 and CD30 and that the redox state of CD30 determines its ability to engage the cognate ligand and transduce signals. Furthermore, we confirm that Trx1 affects CD30-dependent changes in lymphocyte effector function. Thus, we conclude that receptor–ligand signaling interactions can be selectively regulated by an extracellular redox catalyst.

By contrast to chloroplasts, our knowledge of amyloplasts--organelles that synthesize and store starch in heterotrophic plant tissues--is in a formative stage. While our understanding of what is considered their primary function, i.e. the biosynthesis and degradation of starch, has increased dramatically in recent years, relatively little is known about other biochemical processes taking place in these organelles. To help fill this gap, a proteomic analysis of amyloplasts isolated from the starchy endosperm of wheat seeds (10 d post-anthesis) has been conducted. The study has led to the identification of 289 proteins that function in a range of processes, including carbohydrate metabolism, cytoskeleton/plastid division, energetics, nitrogen and sulphur metabolism, nucleic acid-related reactions, synthesis of various building blocks, protein-related reactions, transport, signalling, stress, and a variety of other activities grouped under 'miscellaneous'. The function of 12% of the proteins was unknown. The results highlight the role of the amyloplast as a starch-storing organelle that fulfills a spectrum of biosynthetic needs of the parent tissue. When compared with a recent proteomic analysis of whole endosperm, the current study demonstrates the advantage of using isolated organelles in proteomic studies.

Change in redox status has long been known to link light to the posttranslational regulation of chloroplast enzymes. So far, studies have been conducted primarily with thioredoxin-linked members of the stroma that function in a broad array of biosynthetic and degradatory processes. Consequently, little is known about the role of redox in regulating the growing number of enzymes found to occur in the lumen, the site of oxygen evolution in thylakoid membranes. To help fill this gap, we have studied AtFKBP13, an FKBP-type immunophilin earlier shown to interact with a redoxactive protein of the lumen, and found the enzyme to contain a pair of disulfide bonds in x-ray structural studies. These disulfides, which in protein mutagenesis experiments were shown to be essential for the associated peptidyl-prolyl isomerase activity, are unique to chloroplast FKBPs and are absent in animal and yeast counterparts. Both disulfide bonds were redox-active and were reduced by thioredoxin from either chloroplast or bacterial sources in a reaction that led to loss of enzyme activity. The results suggest a previously unrecognized paradigm for redox regulation in chloroplasts in which activation by light is achieved in concert with oxygen evolution by the oxidation of sulfhydryl groups (conversion of SH to SOS). Such a mechanism, occurring in the thylakoid lumen, is in direct contrast to regulation of enzymes in the stroma, where reduction of disulfides targeted by thioredoxin (SOS converted to SH) leads to an increase in activity in the light.