Paolo Trost

Dehydroascorbate Influences the Plant Cell Cycle through a Glutathione-Independent Reduction Mechanism

Glutathione is generally accepted as the principal electron donor for dehydroascorbate (DHA) reduction. Moreover, both glutathione and DHA affect cell cycle progression in plant cells. But other mechanisms for DHA reduction have been proposed. To investigate the connection between DHA and glutathione, we have evaluated cellular ascorbate and glutathione concentrations and their redox status after addition of dehydroascorbate to medium of tobacco (Nicotiana tabacum) L. cv Bright Yellow-2 (BY-2) cells. Addition of 1 mm DHA did not change the endogenous glutathione concentration. Total glutathione depletion of BY-2 cells was achieved after 24-h incubation with 1 mm of the glutathione biosynthesis inhibitor l-buthionine sulfoximine. Even in these cells devoid of glutathione, complete uptake and internal reduction of 1 mm DHA was observed within 6 h, although the initial reduction rate was slower. Addition of DHA to a synchronized BY-2 culture, or depleting its glutathione content, had a synergistic effect on cell cycle progression. Moreover, increased intracellular glutathione concentrations did not prevent exogenous DHA from inducing a cell cycle shift. It is therefore concluded that, together with a glutathione-driven DHA reduction, a glutathione-independent pathway for DHA reduction exists in vivo, and that both compounds act independently in growth control.

Redox Regulation of a Novel Plastid-Targeted β-Amylase of Arabidopsis

Nine genes of Arabidopsis (Arabidopsis thaliana) encode for β-amylase isozymes. Six members of the family are predicted to be extrachloroplastic isozymes and three contain predicted plastid transit peptides. Among the latter, chloroplast-targeted β-amylase (At4g17090) and thioredoxin-regulated β-amylase (TR-BAMY; At3g23920; this work) are experimentally demonstrated to be targeted to plastids. Recombinant TR-BAMY was catalytically active only when expressed as a mature protein, i.e. with no transit peptide. Mature TR-BAMY was a monomer of 60 kD, hydrolyzing soluble starch with optimal activity between pH 6.0 and 8.0. The activity of recombinant TR-BAMY was strictly dependent on redox potential with an Em,7.0 of −302 ± 14 mV. Thioredoxins f1, m1, and y1 of Arabidopsis were all able to mediate the reductive activation of oxidized TR-BAMY. Site-specific mutants showed that TR-BAMY oxidative inhibition depended on the formation of a disulfide bridge between Cys-32 and Cys-470. Consistent with TR-BAMY redox dependency, total β-amylase activity in Arabidopsis chloroplasts was partially redox regulated and required reducing conditions for full activation. In Arabidopsis, TR-BAMY transcripts were detected in leaves, roots, flowers, pollen, and seeds. TR-BAMY may be the only β-amylase of nonphotosynthetic plastids suggesting a redox regulation of starch metabolism in these organelles. In leaves, where chloroplast-targeted β-amylase is involved in physiological degradation of starch in the dark, TR-BAMY is proposed to participate to a redox-regulated pathway of starch degradation under specific stress conditions.

Biochemical characterization of glutaredoxins from Chlamydomonas reinhardtii reveals the unique properties of a chloroplastic CGFS-type glutaredoxin.

Glutaredoxins (GRXs) are small ubiquitous disulfide oxidoreductases known to use GSH as electron donor. In photosynthetic organisms, little is known about the biochemical properties of GRXs despite the existence of ∼30 different isoforms in higher plants. We report here the biochemical characterization of Chlamydomonas GRX1 and GRX3, the major cytosolic and chloroplastic isoforms, respectively. Glutaredoxins are classified on the basis of the amino acid sequence of the active site. GRX1 is a typical CPYC-type GRX, which is reduced by GSH and exhibits disulfide reductase, dehydroascorbate reductase, and deglutathionylation activities. In contrast, GRX3 exhibits unique properties. This chloroplastic CGFS-type GRX is not reduced by GSH and has an atypically low redox potential (–323 ± 4 mV at pH 7.9). Remarkably, GRX3 can be reduced in the light by photoreduced ferredoxin and ferredoxin-thioredoxin reductase. Both GRXs proved to be very efficient catalysts of A4-glyceraldehyde-3-phosphate dehydrogenase deglutathionylation, whereas cytosolic and chloroplastic thioredoxins were inefficient. Glutathionylated A4-glyceraldehyde-3-phosphate dehydrogenase is the first physiological substrate identified for a CGFS-type GRX.

Thioredoxin-dependent regulation of photosynthetic glyceraldehyde-3-phosphate dehydrogenase: autonomous vs. CP12-dependent mechanisms

Regulation of the Calvin–Benson cycle under varying light/dark conditions is a common property of oxygenic photosynthetic organisms and photosynthetic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is one of the targets of this complex regulatory system. In cyanobacteria and most algae, photosynthetic GAPDH is a homotetramer of GapA subunits which do not contain regulatory domains. In these organisms, dark-inhibition of the Calvin–Benson cycle involves the formation of a kinetically inhibited supramolecular complex between GAPDH, the regulatory peptide CP12 and phosphoribulokinase. Conditions prevailing in the dark, i.e. oxidation of thioredoxins and low NADP(H)/NAD(H) ratio promote aggregation. Although this regulatory system has been inherited in higher plants, these phototrophs contain in addition a second type of GAPDH subunits (GapB) resulting from the fusion of GapA with the C-terminal half of CP12. Heterotetrameric A2B2-GAPDH constitutes the major photosynthetic GAPDH isoform of higher plants chloroplasts and coexists with CP12 and A4-GAPDH. GapB subunits of A2B2-GAPDH have inherited from CP12 a regulatory domain (CTE for C-terminal extension) which makes the enzyme sensitive to thioredoxins and pyridine nucleotides, resembling the GAPDH/CP12/PRK system. The two systems are similar in other respects: oxidizing conditions and low NADP(H)/NAD(H) ratios promote aggregation of A2B2-GAPDH into strongly inactivated A8B8-GAPDH hexadecamers, and both CP12 and CTE specifically affect the NADPH-dependent activity of GAPDH. The alternative, lower activity with NADH is always unaffected. Based on the crystal structure of spinach A4-GAPDH and the analysis of site-specific mutants, a model of the autonomous (CP12-independent) regulatory mechanism of A2B2-GAPDH is proposed. Both CP12 and CTE seem to regulate different photosynthetic GAPDH isoforms according to a common and ancient molecular mechanism.

Molecular mechanism of thioredoxin regulation in photosynthetic A2B2-glyceraldehyde-3-phosphate dehydrogenase.

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a light-regulated, NAD(P)H-dependent enzyme involved in plant photosynthetic carbon reduction. Unlike lower photosynthetic organisms, which only contain A4–GAPDH, the major GAPDH isoform of land plants is made up of A and B subunits, the latter containing a C-terminal extension (CTE) with fundamental regulatory functions. Light-activation of AB–GAPDH depends on the redox state of a pair of cysteines of the CTE, which can form a disulfide bond under control of thioredoxin f, leading to specific inhibition of the NADPH-dependent activity. The tridimensional structure of A2B2–GAPDH from spinach chloroplasts, crystallized in the oxidized state, shows that each disulfide-containing CTE is docked into a deep cleft between a pair of A and B subunits. The structure of the CTE was derived from crystallographic data and computational modeling and confirmed by site-specific mutagenesis. Structural analysis of oxidized A2B2–GAPDH and chimeric mutant [A+CTE]4–GAPDH revealed that Arg-77, which is essential for coenzyme specificity and high NADPH-dependent activity, fails to interact with NADP in these kinetically inhibited GAPDH tetramers and is attracted instead by negative residues of oxidized CTE. Other subtle changes in catalytic domains and overall conformation of the tetramers were noticed in oxidized A2B2–GAPDH and [A+CTE]4–GAPDH, compared with fully active A4–GAPDH. The CTE is envisioned as a redox-sensitive regulatory domain that can force AB–GAPDH into a kinetically inhibited conformation under oxidizing conditions, which also occur during dark inactivation of the enzyme in vivo.

Spontaneous assembly of photosynthetic supramolecular complexes as mediated by the intrinsically unstructured protein CP12.

CP12 is a protein of 8.7 kDa that contributes to Calvin cycle regulation by acting as a scaffold element in the formation of a supramolecular complex with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) in photosynthetic organisms. NMR studies of recombinant CP12 (isoform 2) of Arabidopsis thaliana show that CP12-2 is poorly structured. CP12-2 is monomeric in solution and contains four cysteines, which can form two intramolecular disulfides with midpoint redox potentials of –326 and –352 mV, respectively, at pH 7.9. Site-specific mutants indicate that the C-terminal disulfide is involved in the interaction between CP12-2 and GAPDH (isoform A4), whereas the N-terminal disulfide is involved in the interaction between this binary complex and PRK. In the presence of NAD, oxidized CP12-2 interacts with A4-GAPDH (KD = 0.18 μm) to form a binary complex of 170 kDa with (A4-GAPDH)-(CP12-2)2 stoichiometry, as determined by isothermal titration calorimetry and multiangle light scattering analysis. PRK is a dimer and by interacting with this binary complex (KD = 0.17 μm) leads to a 498-kDa ternary complex constituted by two binary complexes and two PRK dimers, i.e. ((A4-GAPDH)-(CP12-2)2-(PRK))2. Thermodynamic parameters indicate that assembly of both binary and ternary complexes is exoergonic although penalized by a decrease in entropy that suggests an induced folding of CP12-2 upon binding to partner proteins. The redox dependence of events leading to supramolecular complexes is consistent with a role of CP12 in coordinating the reversible inactivation of chloroplast enzymes A4-GAPDH and PRK during darkness in photosynthetic tissues.

Regulation of Photosynthetic GAPDH Dissected by Mutants

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of higher plants catalyzes an NADPH-consuming reaction, which is part of the Calvin cycle. This reaction is regulated by light via thioredoxins and metabolites, while a minor NADH-dependent activity is constant and constitutive. The major native isozyme is formed by A- and B-subunits in stoichiometric ratio (A2B2, A8B8), but tetramers of recombinant B-subunits (GapB) display similar regulatory features to A2B2-GAPDH. The C-terminal extension (CTE) of B-subunits is essential for thioredoxin-mediated regulation and NAD-induced aggregation to partially inactive oligomers (A8B8, B8). Deletion mutant B(minCTE) is redox insensitive and invariably tetrameric, and chimeric mutant A(plusCTE) acquired redox sensitivity and capacity to aggregate to very large oligomers in presence of NAD. Redox regulation principally affects the turnover number, without significantly changing the affinity for either 1,3-bisphosphoglycerate or NADPH. Mutant R77A of GapB, B(R77A), is down-regulated and mimics the behavior of oxidized GapB under any redox condition, whereas mutant B(E362Q) is constantly up-regulated, resembling reduced GapB. Despite their redox insensitivity, both B(R77A) and B(E362Q) mutants are notably prone to aggregate in presence of NAD. Based on structural data and current functional analysis, a model of GAPDH redox regulation is presented. Formation of a disulfide in the CTE induces a conformational change of the GAPDH with repositioning of the terminal amino acid Glu-362 in the proximity of Arg-77. The latter residue is thus distracted from binding the 2′-phosphate of NADP, with the final effect that the enzyme relaxes to a conformation leading to a slower NADPH-dependent catalytic activity.

Identification of an ascorbate-dependent cytochrome b of the tonoplast membrane sharing biochemical features with members of the cytochrome b561 family

Two membrane-bound, ascorbate-dependent b-type cytochromes were identified in etiolated bean (Phaseolus vulgaris L.) hypocotyls. Following solubilization of microsomal membranes and anion-exchange chromatography at pHxa08.0, two major cytochrome peaks (P-I and P-II) were separated. Both cytochromes were reduced by ascorbate and re-oxidized by monodehydroascorbate, but P-I reduction by ascorbate was higher and saturated at far lower concentrations of ascorbate with respect to P-II. The α-band was symmetrically centered at 561xa0nm in P-I, but it was asymmetric in P-II with a maximum at 562xa0nm and shoulder at 557xa0nm. Ascorbate reduction of P-II, but not P-I, was inhibited by diethyl pyrocarbonate. Reduced P-II but not P-I was readily oxidized by certain ferric chelates, including FeEDTA and Fe-nitrilotriacetic acid. Purified P-I, associated with the plasma membrane, showed up as a 63-kDa glycosylated protein during sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and behaved as a monomer of about 70xa0kDa during size-exclusion chromatography. P-I identified with a previously purified ascorbate-dependent b-type cytochrome of bean hypocotyl plasma membranes [P. Trost et al. (2000) Biochim Biophys Acta 1468:1–5]. Partially purified P-II, on the other hand, correlated with a heme-protein of 27xa0kDa in SDS–PAGE gels, was dimeric (60xa0kDa) during size-exclusion chromatography, and was associated with the tonoplast marker V-ATPase in sucrose gradients. The sequence of a peptide of 11 residues obtained by tryptic digestion of P-II was found to be identical to a segment of a putative cytochromexa0b561 of Zea mays and highly conserved in other related plant sequences, including that of Arabidopsis thaliana cytochromexa0b561-1 (CAA18169). The biochemical features fully support the assignment of P-II cytochrome to the family of cytochromexa0b561, ascorbate-dependent (CYBASC) cytochromes, which also includes cytochromexa0b561 of animal chromaffin granules. The presence of a cytochrome reducing ferric chelates on the tonoplast is consistent with the role of plant vacuoles in iron homeostasis.

Molecular Properties of Chloroplastic CP12 and Its Role in the Assembling of a Supramolecular Complex of Calvin Cycle Enzymes

CP12 is a small regulatory peptide localized n chloroplast stroma, where it leads to the ssembly of a supramolecular complex with glyceraldehyde- -phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK). Once embodied in the complex, both enzymes alter their kinetic properties according to the general regulation of photosynthetic carbon assimilation in land plants (dark-inactivation).

Merging crystallography, site-directed mutagenesis and molecular modelling to unravel the regulatory mechanism of photosynthetic GAPDH

a Dipartimento di Chimica ’’G. Ciamician’’, Università di Bologna Alma Mater Studiorum, via Selmi 2, 40126 Bologna, Italy b Laboratorio di Fisiologia molecolare delle piante, Dipartimento di Biologia Evoluzionistica Sperimentale, Università di Bologna Alma Mater Studiorum, via Irnerio 42,40126 Bologna, Italy. c Laboratorio di Biocomputing, Università di Bologna Alma Mater Studiorum, via San Giacomo 9/2, 40126 Bologna, Italy.