NADPH-dependent thioredoxin reductases (NTRs) are key regulatory enzymes determining the redox state of the thioredoxin system. The Arabidopsis thaliana genome has two genes coding for NTRs (NTRA and NTRB), both of which encode mitochondrial and cytosolic isoforms. Surprisingly, plants of the ntra ntrb knockout mutant are viable and fertile, although with a wrinkled seed phenotype, slower plant growth, and pollen with reduced fitness. Thus, in contrast with mammals, our data demonstrate that neither cytosolic nor mitochondrial NTRs are essential in plants. Nevertheless, in the double mutant, the cytosolic thioredoxin h3 is only partially oxidized, suggesting an alternative mechanism for thioredoxin reduction. Plant growth in ntra ntrb plants is hypersensitive to buthionine sulfoximine (BSO), a specific inhibitor of glutathione biosynthesis, and thioredoxin h3 is totally oxidized under this treatment. Interestingly, this BSO-mediated growth arrest is fully reversible, suggesting that BSO induces a growth arrest signal but not a toxic accumulation of activated oxygen species. Moreover, crossing ntra ntrb with rootmeristemless1, a mutant blocked in root growth due to strongly reduced glutathione synthesis, led to complete inhibition of both shoot and root growth, indicating that either the NTR or the glutathione pathway is required for postembryonic activity in the apical meristem.

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...

The last steps of cysteine synthesis in plants involve two consecutive enzymes. The first enzyme, serine acetyltransferase, catalyses the acetylation of L-serine in the presence of acetyl-CoA to form Oacetylserine. The second enzyme, O-acetylserine (thiol) lyase, converts O-acetylserine to L-cysteine in the presence of sulfide. We have, in the present work, over-produced in Escherichia coli harboring various type of plasmids, either a plant serine acetyltransferase or this enzyme with a plant O-acetylserine (thiol) lyase. The free recombinant serine acetyltransferase (subunit mass of 34 kDa) exhibited a high propensity to form high-molecular-mass aggregates and was found to be highly unstable in solution. However, these aggregates were prevented in the presence of O-acetylserine (thiol) lyase (subunit mass of 36 kDa). Under these conditions homotetrameric serine acetyltransferase associated with two molecules of homodimeric O-acetylserine (thiol) lyase to form a bienzyme complex (molecular mass Ϸ300 kDa) called cysteine synthase containing 4 mol pyridoxal 5′-phosphate/mol complex. O-Acetylserine triggered the dissociation of the bienzyme complex, whereas sulfide counteracted the action of O-acetylserine. ProteinϪprotein interactions within the bienzyme complex strongly modified the kinetic properties of plant serine acetyltransferase : there was a transition from a typical Michaelis-Menten model to a model displaying positive kinetic co-operativity with respect to serine and acetyl-CoA. On the other hand, the formation of the bienzyme complex resulted in a very dramatic decrease in the catalytic efficiency of bound O-acetylserine (thiol) lyase. The latter enzyme behaved as if it were a structural and/or regulatory subunit of serine acetyltransferase. Our results also indicated that bound serine acetyltransferase produces a build-up of Oacetylserine along the reaction path and that the full capacity for cysteine synthesis can only be achieved in the presence of a large excess of free O-acetylserine (thiol) lyase. These findings contradict the widely held belief that such a bienzyme complex is required to channel the metabolite intermediate O-acetylserine.Keywords : cysteine synthase; serine acetyltransferase; O-acetylserine (thiol) lyase; proteinϪprotein interactions; cysteine synthesis in plants.In metabolic networks, a fine tuning of enzyme activities is verted into the final reaction product (P). Here, accumulation of I might be prevented through properly adjusted concentrations required to maintain the rates at which useful end-products are produced with regard to those at which they are needed, but also of E 2 over E 1 , so that, at steady state, the net rate of I transformation through E 2 at least equals that of synthesis through E 1 . This to avoid accumulation of toxic internal metabolites and/or to achieve efficient trapping of highly unstable reaction intermedi-situation is analogous to that encountered in enzyme assays with auxiliary enzymes [1]. ates [1Ϫ3]. Considering the simple case of two enzymes ...