Coordination of Chloroplastic Metabolism in N-Limited Chlamydomonas reinhardtii by Redox Modulation (I. The Activation of Phosphoribulosekinase and Glucose-6-Phosphate Dehydrogenase Is Relative to the Photosynthetic Supply of Electrons)

Extraction of Chlamydomonas reinhardtii CW-15 cells by rapid freezing and thawing demonstrates that the in vivo activity of the algal glucose-6-phosphate dehydrogenase (G6PDH) is inhibited by the presence of light and activated in the dark, whereas phosphoribulosekinase (PRK) is light activated and inhibited in the dark. The effects of darkening are reversed by incubation with dithiothreitol (DTT) and mimicked by chemical oxidants, indicating that, as in higher plants, reduction via the ferredoxin-thioredoxin system likely regulates these enzymes. The two enzymes varied in their sensitivity to reduction; the inclusion of 0.5 mM DTT during extraction inhibited G6PDH, whereas PRK required treatment with 40 mM DTT for 1 h to reach maximum activation. The activation change for both enzymes was nearly complete within the 1st min after cells were transferred between light and dark, but the level of activation was relative to the incident light at low intensities; G6PDH activity decreased with increasing light, whereas PRK became more active. The reductive inhibition of G6PDH saturated at very low light, whereas PRK activation kinetics closely followed the increase in photosynthetic oxygen evolution. These results indicate that light-driven redox modulation of G6PDH and PRK is more than an on/off switch, but acts to optimize the reduction and oxidation of carbon in the chloroplast in accordance with the supply of electrons.     

Kinetic and mutational analyses of the regulation of phosphoribulokinase by thioredoxins

Despite little supportive data, differential target protein susceptibility to redox regulation by thioredoxin (Trx) f and Trx m has been invoked to account for two distinct Trxs in chloroplasts. However, this postulate has not been rigorously tested with phosphoribulokinase (PRK), a fulcrum for redox regulation of the Calvin cycle. Prerequisite to Trx studies, the activation of spinach PRK by dithiothreitol, 2-mercaptoethanol, and glutathione was examined. Contrary to prior reports, each activated PRK, but only dithiothreitol supported Trx-dependent activation. Comparative kinetics of activation of PRK showed Trx m to be more efficient than Trx f because of its 40% higher V(max) but similar S(0.5). Activations were insensitive to ribulosebisphosphate carboxylase, which may complex with PRK in vivo. To probe the basis for superiority of Trx m, we characterized site-directed mutants of Trx f, in which unique residues in conserved regions were replaced with Trx m counterparts or deleted. These changes generally resulted in V(max) enhancements, the largest (6-fold) of which occurred with T105I, reflective of substitution in a hydrophobic region that opposes the active site. Inclusive of the present study, activation kinetics of several different Trx-regulated enzymes indicate redundancy in the functions of the chloroplastic Trxs.     

Single cystathionine β-synthase domain-containing proteins modulate development by regulating the thioredoxin system in Arabidopsis

Plant thioredoxins (Trxs) participate in two redox systems found in different cellular compartments: the NADP-Trx system (NTS) in the cytosol and mitochondria and the ferredoxin-Trx system (FTS) in the chloroplast, where they function as redox regulators by regulating the activity of various target enzymes. The identities of the master regulators that maintain cellular homeostasis and modulate timed development through redox regulating systems have remained completely unknown. Here, we show that proteins consisting of a single cystathionine β-synthase (CBS) domain pair stabilize cellular redox homeostasis and modulate plant development via regulation of Trx systems by sensing changes in adenosine-containing ligands. We identified two CBS domain-containing proteins in Arabidopsis thaliana, CBSX1 and CBSX2, which are localized to the chloroplast, where they activate all four Trxs in the FTS. CBSX3 was found to regulate mitochondrial Trx members in the NTS. CBSX1 directly regulates Trxs and thereby controls H(2)O(2) levels and regulates lignin polymerization in the anther endothecium. It also affects plant growth by regulating photosynthesis-related [corrected] enzymes, such as malate dehydrogenase, via homeostatic regulation of Trxs. Based on our findings, we suggest that the CBSX proteins (or a CBS pair) are ubiquitous redox regulators that regulate Trxs in the FTS and NTS to modulate development and maintain homeostasis under conditions that are threatening to the cell.     

Potentiation of acid-sensing ion channels by sulfhydryl compounds

The acid-sensing ion channels (ASICs) are voltage-independent ion channels activated by acidic extracellular pH. ASICs play a role in sensory transduction, behavior, and acidotoxic neuronal death, which occurs during stroke and ischemia. During these conditions, the extracellular concentration of sulfhydryl reducing agents increases. We used perforated patch-clamp technique to analyze the impact of sulfhydryls on H⁺-gated currents from Chinese hamster ovary (CHO) cells expressing human ASIC1a (hASIC1a). We found that hASIC1a currents activated by pH 6.5 were increased almost twofold by the sulfhydryl-containing reducing agents dithiothreitol (DTT) and glutathione. DTT shifted the pH-dose response of hASIC1a toward a more neutral pH (pH_0.5 from 6.54 to 6.69) and slowed channel desensitization. The effect of reducing agents on native mouse hippocampal neurons and transfected mouse ASIC1a was similar. We found that the effect of DTT on hASIC1a was mimicked by the metal chelator TPEN, and mutant hASIC1a channels with reduced TPEN potentiation showed reduced DTT potentiation. Furthermore, the addition of DTT in the presence of TPEN did not result in further increases in current amplitude. These results suggest that the effect of DTT on hASIC1a is due to relief of tonic inhibition by transition metal ions. We found that all ASICs examined remained potentiated following the removal of DTT. This effect was reversed by the oxidizing agent DTNB in hASIC1a, supporting the hypothesis that DTT also impacts ASICs via a redox-sensitive site. Thus sulfhydryl compounds potentiate H⁺-gated currents via two mechanisms, metal chelation and redox modulation of target amino acids. glutathione; DTT; redox; zinc     

Localization of soluble beta-carbonic anhydrase in the marine diatom Phaeodactylum tricornutum. Sorting to the chloroplast and cluster formation on the girdle lamellae

A β-carbonic anhydrase (CA) in the marine diatom Phaeodactylum tricornutum (PtCA1) is encoded by the nuclear genome. This enzyme was previously found to be important for the operation of photosynthesis with a high affinity for dissolved inorganic carbon. A cDNA sequence that encodes PtCA1 (ptca1) was shown to possess a presequence of 138 bp (pre138), which encodes an N-terminal sequence of 46 amino acids (Pre46AA) that does not exist in the mature PtCA1. In this study, pre138 was ligated with the enhanced green fluorescent protein (GFP) gene (egfp), and introduced into P. tricornutum by microprojectile bombardment. Subsequently, the expressed Pre46AA-GFP fusion was shown to be localized in the chloroplast stroma, whereas the expressed GFP without Pre46AA was localized in the cytoplasm. Insertion of the DNA sequence, encoding a mature region of ptca1 (mptca1) between pre138 and egfp, resulted in the formation of particles with concentrated GFP fluorescence in the stroma of P. tricornutum. These particles, 0.3 to 3.0 μm in size, were shown to be distinct from the mitochondria and localized on the surface of the putative girdle lamella. The attachment of the initial one-half of the pre138 to the mptca1-egfp fusion caused the expressed GFP fusion to accumulate in areas surrounding the chloroplast, presumably due to the presence of the endoplasmic reticulum signal encoded by the initial half-sequence and to the absence of the chloroplast transit sequence. These results indicate that PtCA1 is targeted to the stroma by the bipartite sequences of Pre46AA and that the observed GFP particles are formed specifically in the stroma due to the function of the mptca1.     

Regulation of phosphoribulokinase and glyceraldehyde 3‐phosphate dehydrogenase in a freshwater diatom,Asterionella formosa1

The regulation of phosphoribulokinase (PRK) and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) was investigated in a freshwater pennate diatom, Asterionella formosa Hassall, and compared to the well‐studied chlorophyte Chlamydomonas reinhardtii P. A. Dang. As has been reported for a marine centric diatom, in A. formosa, PRK was not regulated by reduction with dithiothreitol (DTT) apart from a weak induction in the presence of NADPH and DTT. However, NADPH‐GAPDH was strongly activated when reduced, in contrast to a previous report on a diatom. Surprisingly, it was inhibited by NADPH, unlike in C. reinhardtii, while NADH‐GAPDH was not affected. NADH‐GAPDH was also strongly activated by DTT in contrast to most other photosynthetic cells. In A. formosa, unlike C. reinhardtii, 1,3‐bisphosphoglycerate, the substrate of GAPDH, activated this enzyme, even in the absence of DTT, when using both NADH and NADPH as cofactors. Some of these kinetic behaviors are consistent with regulation by protein–protein interactions involving CP12, a small protein that links PRK and GAPDH in cyanobacteria, green algae, and higher plants. This conclusion was supported by immunodetection of CP12 in crude extracts of A. formosa, using antibodies raised against CP12 from C. reinhardtii. This is the first report of the existence of CP12 in a diatom, but CP12 may be a common feature of diatoms since a bioinformatic search suggested that it was also present in the Thalassiosira pseudonana Hasle et Heimdal genome v3.0. Despite the presence of CP12, this work provides further support for the differential regulation of Calvin cycle enzymes in diatoms compared to green algae.     

A novel type of thioredoxin dedicated to symbiosis in legumes

Thioredoxins (Trxs) constitute a family of small proteins in plants. This family has been extensively characterized in Arabidopsis (Arabidopsis thaliana), which contains six different Trx types: f, m, x, and y in chloroplasts, o in mitochondria, and h mainly in cytosol. A detailed study of this family in the model legume Medicago truncatula, realized here, has established the existence of two isoforms that do not belong to any of the types previously described. As no possible orthologs were further found in either rice (Oryza sativa) or poplar (Populus spp.), these novel isoforms may be specific for legumes. Nevertheless, on the basis of protein sequence and gene structure, they are both related to Trxs m and probably have evolved from Trxs m after the divergence of the higher plant families. They have redox potential values similar to those of the classical Trxs, and one of them can act as a substrate for the M. truncatula NADP-Trx reductase A. However, they differ from classical Trxs in that they possess an atypical putative catalytic site and lack disulfide reductase activity with insulin. Another important feature is the presence in both proteins of an N-terminal extension containing a putative signal peptide that targets them to the endoplasmic reticulum, as demonstrated by their transient expression in fusion with the green fluorescent protein in M. truncatula or Nicotiana benthamiana leaves. According to their pattern of expression, these novel isoforms function specifically in symbiotic interactions in legumes. They were therefore given the name of Trxs s, s for symbiosis.     

Binding and activation of pea chloroplast fructose-1,6-bisphosphatase by homologous thioredoxins m and f

Fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) binds its putative physiological activator thioredoxin f (Trx f ) at pH 7.9, the pH in the stroma of the illuminated chloroplast. Since Trx m , described as specific in NADP⁺-malate dehydrogenase (NADPMDH) activation, appears in pea (Pisum sativum L.) also to be functional in FBPase modulation, we have here analyzed the effect of pH and the redox status of the chloroplast stroma in the pea FBPase binding of homologous Trx f and m . Both pea Trx were strongly bound by purified FBPase when they were preincubated at pH 7.9 with 2.5 m M dithiothreitol (DTT), but not when the reductant was omitted. As occurs with Trx f the Trx m /FBPase ratio of the complex was 4, but this was only observed with a Trx m /FBPase concentration ratio > 10 in the preincubation mixture. The FBPase-Trx m binding disappeared in the presence of 100 m M NaCl, even with 2.5 m M DTT at pH 7.9, with a concomitant appearance of different aggregation states of the FBPase subunit. A similar FBPase-Trx m complex was detected in the stromal solution when pea chloroplasts were lysed at pH 7.9 in the presence of DTT. No interaction was observed between NADP-MDH and Trx f or m , either in the presence or in the absence of DTT. Pea FBPase showed sigmoidal activation kinetics with pea Trx m , and an S_0.5 of 133 n M versus 6.6 n M with pea Trx f . About 10-fold higher concentration of the former than that of the latter was required for obtaining maximum activity; however, the V_max with Trx f was only 2-fold higher than that with Trx m . We conclude that pea FBPase binds and is activated by the homologous Trx m , even though to a lesser extent than with Trx f . We also deduce that in the light the conditions in the chloroplast stroma are optimal for forming an FBPase-Trx complex.     

Chaperone-like properties of tobacco plastid thioredoxins f and m

Thioredoxins (Trxs) are ubiquitous disulphide reductases that play important roles in the redox regulation of many cellular processes. However, some redox-independent functions, such as chaperone activity, have also been attributed to Trxs in recent years. The focus of our study is on the putative chaperone function of the well-described plastid Trxs f and m. To that end, the cDNA of both Trxs, designated as NtTrxf and NtTrxm, was isolated from Nicotiana tabacum plants. It was found that bacterially expressed tobacco Trx f and Trx m, in addition to their disulphide reductase activity, possessed chaperone-like properties. In vitro, Trx f and Trx m could both facilitate the reactivation of the cysteine-free form of chemically denatured glucose-6 phosphate dehydrogenase (foldase chaperone activity) and prevent heat-induced malate dehydrogenase aggregation (holdase chaperone activity). Our results led us to infer that the disulphide reductase and foldase chaperone functions prevail when the proteins occur as monomers and the well-conserved non-active cysteine present in Trx f is critical for both functions. By contrast, the holdase chaperone activity of both Trxs depended on their oligomeric status: the proteins were functional only when they were associated with high molecular mass protein complexes. Because the oligomeric status of both Trxs was induced by salt and temperature, our data suggest that plastid Trxs could operate as molecular holdase chaperones upon oxidative stress, acting as a type of small stress protein.     

cpFBPaseII, a novel redox-independent chloroplastic isoform of fructose-1,6-bisphosphatase

A full-length FBPase cDNA has been isolated from Fragaria  ×  ananassa (strawberry) corresponding to a novel putative chloroplastic FBPase but lacking the regulatory redox domain, a characteristic of the plastidial isoenzyme (cpFBPaseI). Another outstanding feature of this novel isoform, called cpFBPaseII, is the absence of the canonical active site. Enzymatic assays with cpFBPaseII evidenced clear Mg²⁺-dependent FBPase activity and a K _m for fructose-1,6-bisphosphate (FBP) of 1.3 m m . Immunolocalization experiments and chloroplast isolation confirmed that the new isoenzyme is located in the stroma. Nevertheless, unlike cpFBPaseI, which is redox activated, cpFBPaseII did not increase its activity in the presence of either DTT or thioredoxin f (TRX f ) and is resistant to H₂O₂ inactivation. Additionally, the novel isoform was able to complement the growth deficiency of the yeast FBP1 deletion fed with a non-fermentable carbon source. Furthermore, orthologues are restricted to land plants, suggesting that cpFBPaseII is a novel and an intriguing chloroplastic FBPase that emerged late in the evolution of photosynthetic organisms, possibly because of a pressing need of land plants.     

Evidence for functional convergence of redox regulation in G6PDH isoforms of cyanobacteria and higher plants

In a recent paper (Wenderoth et al., J Biol Chem 272: 26985–26990, 1997) we reported that the positions of the two redox regulatory cysteines identified in a plastidic G6PD isoform from potato (Solanum tuberosum L.) differ substantially from those conserved in cyanobacterial G6PDH sequences. To investigate the origin of redox regulation in G6PDH enzymes from photoautotrophic organisms, we isolated and characterized several G6PD cDNA sequences from higher plants and from a green and a red alga. Alignments of the deduced amino acid sequences showed that the cysteine residues cluster in the coenzyme-binding domain of the plastidic isoforms and are conserved at three out of six positions. Comparison of the mature proteins and the signal peptides revealed that two different plastidic G6PDH classes (P1 and P2) evolved from a common ancestral gene. The two algal sequences branch off prior to this class separation in higher plants, sharing about similar amino acid identity with either of the two plastidic G6PDH classes. The genes for cytosolic plant isoforms clearly share a common ancestor with animal and fungal G6PDH homologues, whereas the cyanobacterial isoforms branch within the eubacterial G6PDH sequences. The data suggest that cysteine-mediated redox regulation arose independently in G6PDH isoenzymes of eubacterial and eukaryotic lineages.     

Activity and subcellular localization of glucose-6-phosphate dehydrogenase in peach fruits

The subcellular distribution and activity of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) were studied in developing peach (Prunus persica L. Batsch cv. Zaoyu) fruit. Fruit tissues were separated by differential centrifugation at 15,000g into plastidic and cytosolic fractions. There was no serious loss of enzyme activity (or activation) during the preparation of fractions. G6PDH activity was found in both the plastidic and cytosolic compartments. Moreover, DTT had no effect on the plastidic G6PDH activities, that is, the redox regulatory mechanism did not play an important role in the peach fleshy tissue. Results from the immunogold electron-microscope localization revealed that G6PDH isoenzymes were mainly present in the cytosol, the secondary wall and plastids (chloroplasts and chromoplasts), but scarcely found in the starch granules or the cell wall. In addition to a decrease in fruit firmness, the G6PDH activity in the cytotolic and plastidic fractions increased, and anthocyanin started to accumulate during fruit maturation. These results suggest that G6PDH, by providing precursors for metabolic processes, might be associated with the red coloration that occurs in peach fruit.     

Binding features of chloroplast fructose-1,6-bisphosphatase-thioredoxin interaction

It has been proposed that a hydrophobic groove surrounded by positively charged amino acids on thioredoxin (Trx) serves as the recognition and docking site for the interaction of Trx with target proteins. This model for Trx-protein interactions fits well with the Trx-mediated fructose-1,6-bisphosphatase (FBPase) activation, where a protruding negatively charged loop of FBPase would bind to this Trx groove, in a process involving both electrostatic and hydrophobic interactions. This model facilitates the prediction of Trx amino acid residues likely to be involved in enzyme binding. Site-directed mutagenesis of some of these amino acids, in conjunction with measurements of the FBPase activation capacity of the wild type and mutated Trxs, was used to check the model and provided evidence that lysine-70 and arginine-74 of pea Trx m play an essential role in FBPase binding. The binding parameters for the interaction between chloroplast FBPase and the wild type pea Trxs f and m, as well as mutated pea Trx m, determined by equilibrium dialysis in accordance with the Koshland-Nemethy-Filmer model of saturation kinetics, provided additional support for the role of these basic Trx residues in the interaction with FBPase. These data, in conjunction with the midpoint redox potential (E(m)) determinations of Trxs, support the hydrophobic groove model for the interaction between chloroplast FBPase and Trx. This model predicts that differences in the FBPase activation capacity of Trxs arise from their different binding abilities.     

A redox signaling mechanism for density-dependent inhibition of cell growth

Reactive oxygen species (ROS) have recently drawn significant attention as putative mitogenic mediators downstream of activated growth factor receptors and oncogenic Ras; however, the possibility that a redox-related mechanism also operates in the negative control of cell proliferation by inhibitory signals has not been investigated thus far. Here we show that the arrest of growth induced by cell confluence ("contact inhibition") is due, at least in part, to a decrease in the steady-state levels of intracellular ROS and the consequent impairment of mitogenic redox signaling. In confluent fibroblast cultures, the decrease in the concentration of oxygen species was associated with diminished activity of the small GTPase Rac-1, a signal transducer directly involved in the ligand-dependent generation of oxygen-derived molecules, and was effectively mimicked by exposure of sparse cultures to dithiothreitol (DTT) and inhibitors of enzymes (phospholipase A2 and lipoxygenase) acting in the arachidonic acid cascade downstream of growth factor receptors and Rac-1. Sparse fibroblasts treated with nontoxic amounts of DTT underwent growth arrest, whereas a low concentration of hydrogen peroxide significantly increased thymidine incorporation in confluent cultures, demonstrating a causal link between redox changes and growth control by cell density. Removal of oxygen species from sparse cultures was accompanied by a drastic decrease of protein tyrosine phosphorylation after epidermal growth factor stimulation, which, at a biochemical level, reproduced the signaling hallmarks of contact inhibition. Moreover, the cytosolic tyrosine phosphatase SHP-2 was identified as a putative target for redox signaling by cell density because the enzyme itself and the associated substrates appear markedly dephosphorylated in both confluent and reductant-treated cells after exposure to epidermal growth factor, and SHP-2 enzymatic activity is strongly activated by DTT in vitro. Taken together, these data support a model in which impaired generation of ROS and increased protein tyrosine phosphatase activity impede mitogenic signaling in contact-inhibited cells.