The circadian night depression of photosynthesis analyzed in a herb, Pulmonaria vallarsae. Day/night quantitative relationships.

Although many photosynthesis related processes are known to be controlled by the circadian system, consequent changes in photosynthetic activities are poorly understood. Photosynthesis was investigated during the daily cycle by chlorophyll fluorescence using a PAM fluorometer in Pulmonaria vallarsae subsp. apennina, an understory herb. A standard test consists of a light induction pretreatment followed by light response curve (LRC). Comparison of the major diagnostic parameters collected during day and night showed a nocturnal drop of photosynthetic responses, more evident in water-limited plants and consisting of: (i) strong reduction of flash-induced fluorescence peaks (FIP), maximum linear electron transport rate (Jmax, ETREM) and effective PSII quantum yield (ΦPSII); (ii) strong enhancement of nonphotochemical quenching (NPQ) and (iii) little or no change in photochemical quenching qP, maximum quantum yield of linear electron transport (Φ), and shape of LRC (θ). A remarkable feature of day/night LRCs at moderate to high irradiance was their linear-parallel course in double-reciprocal plots. Photosynthesis was also monitored in plants subjected to 2-3 days of continuous darkness ("long night"). In such conditions, plants exhibited high but declining peaks of photosynthetic activity during subjective days and a low, constant value with elevated NPQ during subjective night tests. The photosynthetic parameters recorded in subjective days in artificial darkness resembled those under natural day conditions. On the basis of the evidence, we suggest a circadian component and a biochemical feedback inhibition to explain the night depression of photosynthesis in P. vallarsae.

Photosynthetic properties of spring geophytes assessed by chlorophyll fluorescence analysis.

Since spring ephemerals are credited to be all "sun" species with unusually elevate photosynthesis, in contrast to shade-tolerant trees and understory geophytes with a long aboveground cycle, we examined the photosynthetic efficiency of 6 woody species, 9 long-cycle geophytes, and 8 spring ephemeral geophytes using blue flashes of increasing energy with the Imaging PAM fluorometer. Several parameters were obtained: quantum yield of electron transport (ΦETR) or of PSII (ΦPSII), maximum measured photosynthesis rate (ETRhv), maximum extrapolated rate of photosynthesis (ETRem), half-saturating photon flux density (KPAR), and in some cases photochemical (qP) and non-photochemical quenching (NPQ). Results confirm the ecological consistency of the three plant groups, with internal differences. Woody species have low ETRem and KPAR values with good ΦETR; long-cycle herbs have low ETRem and ΦETR and moderate KPAR values; spring ephemerals have elevate ΦETR, ETRem and KPAR values. The mean ETRem of ephemerals of 91 µmol m-2 s-1 exceeds that of long-cycle herbs 2.9-fold and woody species 4.8-fold, and corresponds to 19 µmol CO2 m-2 s-1 by assuming an ETR/ΦCO2 ratio of 4.7. Highest photosynthesis rates and KPAR were exhibited by five ephemerals (Eranthis, Erythronium, Narcissus, Scilla, Tulipa) with peak ETRem values equivalent to ∼40 µmol CO2 m-2 s-1 or ∼60 µmol CO2 (g Chl)-1 s-1 ("sun" species). According to a new, fluorescence based heliophily index, all trees and five long-cycle herbs were definitely "shade" species, while four long-cycle herbs and three ephemerals were intermediate shade-tolerant.

Erythronium dens-canis L. (Liliaceae): an unusual case of change of leaf mottling.

Erythronium dens-canis is an early-flowering understory lily of southern Europe with two leaves and a single flower, although a number of plants have only one leaf and do not flower. The leaves are mottled with silvery flecks and brown patches, that gradually vanish turning to a lively green color. The nature and function of this striking variegation pattern were investigated in differently colored leaf parts following the springtime color change. Tissue organization was examined by light and electron microscopy; photosynthetic pigments were analyzed by spectrophotometry and HPLC; chlorophyll fluorescence parameters were evaluated by MINI-PAM. The results showed that brown patches originated in vacuolar anthocyanins in the subepidermal cell layer while air spaces between the upper epidermis and underlying chlorenchyma resulted in silvery flecks. The two leaf areas did not differ in photosynthetic pigments, chloroplast organization and photosynthetic parameters (F(v)/F(m), NPQ, rETR). Greening of brown patches due to anthocyanin resorption was faster in non-flowering plants than in flowering ones, occurring only when young fruits were developing. Anthocyanin disappearance did not change the structural-functional features of photosynthetic tissues. As a whole the results suggest that the anthocyanin pigmentation of E. dens-canis leaves does not affect the photosynthetic light use and has no photoprotective function. It is proposed that the complex leaf color pattern may act as a camouflage to escape herbivores, while the reflective silvery spots may have a role in attracting pollinators of this early-flowering species.

Variegation in Arum italicum leaves. A structural-functional study.

The presence of pale-green flecks on leaves (speckling) is a frequent character among herbaceous species from shady places and is usually due to local loosening of palisade tissue (air space type of variegation). In the winter-green Arum italicum L. (Araceae), dark-green areas of variegated leaf blades are ca. 400 µm thick with a chlorophyll content of 1080 mg m⁻² and a palisade parenchyma consisting of a double layer of oblong cells. Pale-green areas are 25% thinner, have 26% less chlorophyll and contain a single, loose layer of short palisade cells. Full-green leaves generally present only one compact layer of cylindrical palisade cells and the same pigment content as dark-green sectors, but the leaf blade is 13% thinner. A spongy parenchyma with extensive air space is present in all leaf types. Green cells of all tissues have normal chloroplasts. Assays of photosynthetic activities by chlorophyll fluorescence imaging and O2 exchange measurements showed that variegated pale-green and dark-green sectors as well as full-green leaves have comparable photosynthetic activities on a leaf area basis at saturating illumination. However, full-green leaves require a higher saturating light with respect to variegated sectors, and pale-green sectors support relatively higher photosynthesis rates on a chlorophyll basis. We conclude that i) variegation in this species depends on number and organization of palisade cell layers and can be defined as a "variable palisade" type, and ii) the variegated habit has no limiting effects on the photosynthetic energy budget of A. italicum, consistent with the presence of variegated plants side by side to full-green ones in natural populations.

Thioredoxin-regulated beta-amylase (BAM1) triggers diurnal starch degradation in guard cells, and in mesophyll cells under osmotic stress.

BAM1 is a plastid-targeted ß-amylase of Arabidopsis thaliana specifically activated by reducing conditions. Among eight different chloroplast thioredoxin isoforms, thioredoxin f1 was the most efficient redox mediator, followed by thioredoxins m1, m2, y1, y2, and m4. Plastid-localized NADPH-thioredoxin reductase (NTRC) was also able partially to restore the activity of oxidized BAM1. Promoter activity of BAM1 was studied by reporter gene expression (GUS and YFP) in Arabidopsis transgenic plants. In young (non-flowering) plants, BAM1 was expressed both in leaves and roots, but expression in leaves was mainly restricted to guard cells. Compared with wild-type plants, bam1 knockout mutants were characterized by having more starch in illuminated guard cells and reduced stomata opening, suggesting that thioredoxin-regulated BAM1 plays a role in diurnal starch degradation which sustains stomata opening. Besides guard cells, BAM1 appears in mesophyll cells of young plants as a result of a strongly induced gene expression under osmotic stress, which is paralleled by an increase in total ß-amylase activity together with its redox-sensitive fraction. Osmotic stress impairs the rate of diurnal starch accumulation in leaves of wild-type plants, but has no effect on starch accumulation in bam1 mutants. It is proposed that thioredoxin-regulated BAM1 activates a starch degradation pathway in illuminated mesophyll cells upon osmotic stress, similar to the diurnal pathway of starch degradation in guard cells that is also dependent on thioredoxin-regulated BAM1.

Structure of photosynthetic glyceraldehyde-3-phosphate dehydrogenase (isoform A4) from Arabidopsis thaliana in complex with NAD.

The crystal structure of the A(4) isoform of photosynthetic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Arabidopsis thaliana, expressed in recombinant form and complexed with NAD, is reported. The crystals, which were grown in 2.4 M ammonium sulfate and 0.1 M sodium citrate, belonged to space group I222. The asymmetric unit includes ten subunits, i.e. two independent tetramers plus a dimer that generates a third tetramer by a crystallographic symmetry operation. The crystal structure was solved by molecular replacement and refined to an R factor of 23.7% and an R(free) factor of 28.9% at 2.6 A resolution. In the final model, each subunit binds one NAD(+) molecule and two sulfates, which occupy the P(s) and the P(i) anion-binding sites. Detailed knowledge of this structure is instrumental for structural investigation of supramolecular complexes of A(4)-GAPDH, phosphoribulokinase and CP12, which are involved in the regulation of photosynthesis in the model plant A. thaliana.

In vitro characterization of Arabidopsis CP12 isoforms reveals common biochemical and molecular properties.

In oxygenic photosynthetic organisms, the activities of two Calvin cycle enzymes (glyceraldehyde-3-phosphate dehydrogenase, GAPDH and phosphoribulokinase, PRK) are regulated by CP12-mediated complex formation. The Arabidopsis genome contains three genes encoding different CP12 isoforms (CP12-1, At2g47400; CP12-2, At3g62410 and CP12-3, At1g76560), all plastid-targeted, as demonstrated by localization in the chloroplast stroma of CP12 precursor sequences fused with the green fluorescence protein (GFP). The disorder predictor PONDR classified Arabidopsis CP12s as largely disordered proteins, and circular dichroism spectra confirmed these predictions. Based on sequence similarity, 66 CP12s from different organisms were identified and clustered in six types, with CP12-1 and -2 grouping together with other largely disordered sequences (Type I), while a lower level of disorder was predicted within the cluster including CP12-3 (Type II). The three Arabidopsis CP12 isoforms were expressed as mature recombinant forms and purified to homogeneity. Redox titrations demonstrated that the four conserved cysteines of each CP12 isoform could form two internal disulfide bridges with different midpoint redox potentials (E(m,7.9) -326 mV and -350 mV in both CP12-1 and CP12-2; E(m,7.9) -332 mV and -373 mV in CP12-3). In agreement with their similar redox properties, all CP12 isoforms formed, in vitro, a supramolecular complex with GAPDH and PRK, with comparable inhibitory effects on both enzyme activities. In order to test whether CP12 isoforms might have broader regulatory functions than regulating Calvin cycle enzymes, CP12 proteins were analyzed for their capacity to bind plastidial glycolytic GAPDH (GapCp). To this purpose, the mature form of Arabidopsis GapCp2 was cloned, expressed in recombinant form and purified to homogeneity. However, contrary to expectations, no CP12 isoform was able to bind GapCp2 under any of the conditions tested.

Prompt and easy activation by specific thioredoxins of calvin cycle enzymes of Arabidopsis thaliana associated in the GAPDH/CP12/PRK supramolecular complex.

The Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) can form under oxidizing conditions a supramolecular complex with the regulatory protein CP12. Both GAPDH and PRK activities are inhibited within the complex, but they can be fully restored by reduced thioredoxins (TRXs). We have investigated the interactions of eight different chloroplast thioredoxin isoforms (TRX f1, m1, m2, m3, m4, y1, y2, x) with GAPDH (A(4), B(4), and B(8) isoforms), PRK and CP12 (isoform 2), all from Arabidopsis thaliana. In the complex, both A(4)-GAPDH and PRK were promptly activated by TRX f1, or more slowly by TRXs m1 and m2, but all other TRXs were ineffective. Free PRK was regulated by TRX f1, m1, or m2, while B(4)- and B(8)-GAPDH were absolutely specific for TRX f1. Interestingly, reductive activation of PRK caged in the complex was much faster than reductive activation of free oxidized PRK, and activation of A(4)-GAPDH in the complex was much faster (and less demanding in terms of reducing potential) than activation of free oxidized B(4)- or B(8)-GAPDH. It is proposed that CP12-assembled supramolecular complex may represent a reservoir of inhibited enzymes ready to be released in fully active conformation following reduction and dissociation of the complex by TRXs upon the shift from dark to low light. On the contrary, autonomous redox-modulation of GAPDH (B-containing isoforms) would be more suited to conditions of very active photosynthesis.

Auxin-responsive genes AIR12 code for a new family of plasma membrane b-type cytochromes specific to flowering plants.

We report here on the identification of the major plasma membrane (PM) ascorbate-reducible b-type cytochrome of bean (Phaseolus vulgaris) and soybean (Glycine max) hypocotyls as orthologs of Arabidopsis (Arabidopsis thaliana) AIR12 (for auxin induced in root cultures). Soybean AIR12, which is glycosylated and glycosylphosphatidylinositol-anchored to the external side of the PM in vivo, was expressed in Pichia pastoris in a recombinant form, lacking the glycosylphosphatidylinositol modification signal and purified from the culture medium. Recombinant AIR12 is a soluble protein predicted to fold into a beta-sandwich domain and belonging to the DOMON (for dopamine beta-monooxygenase N terminus) domain superfamily. It is shown to be a b-type cytochrome with a symmetrical alpha-band at 561 nm, fully reduced by ascorbate, and fully oxidized by monodehydroascorbate radical. AIR12 is a high-potential cytochrome b showing a wide bimodal dependence from the redox potential between +80 mV and +300 mV. Optical absorption and electron paramagnetic resonance analysis indicate that AIR12 binds a single, highly axial low-spin heme, likely coordinated by methionine-91 and histidine-76, which are strongly conserved in AIR12 sequences. Phylogenetic analyses reveal that the auxin-responsive genes AIR12 represent a new family of PM b-type cytochromes specific to flowering plants. Circumstantial evidence suggests that AIR12 may interact with other redox partners within the PM to constitute a redox link between cytoplasm and apoplast.

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 A(4)), 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 A(4)-GAPDH (K(D) = 0.18 microm) to form a binary complex of 170 kDa with (A(4)-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 (K(D) = 0.17 microm) leads to a 498-kDa ternary complex constituted by two binary complexes and two PRK dimers, i.e. ((A(4)-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 A(4)-GAPDH and PRK during darkness in photosynthetic tissues.

Redox regulation of a novel plastid-targeted beta-amylase of Arabidopsis.

Nine genes of Arabidopsis (Arabidopsis thaliana) encode for beta-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 beta-amylase (At4g17090) and thioredoxin-regulated beta-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 beta-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 beta-amylase of nonphotosynthetic plastids suggesting a redox regulation of starch metabolism in these organelles. In leaves, where chloroplast-targeted beta-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.

Reconstitution and properties of the recombinant glyceraldehyde-3-phosphate dehydrogenase/CP12/phosphoribulokinase supramolecular complex of Arabidopsis.

Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) form together with the regulatory peptide CP12 a supramolecular complex in Arabidopsis (Arabidopsis thaliana) that could be reconstituted in vitro using purified recombinant proteins. Both enzyme activities were strongly influenced by complex formation, providing an effective means for regulation of the Calvin cycle in vivo. PRK and CP12, but not GapA (A(4) isoform of GAPDH), are redox-sensitive proteins. PRK was reversibly inhibited by oxidation. CP12 has no enzymatic activity, but it changed conformation depending on redox conditions. GapA, a bispecific NAD(P)-dependent dehydrogenase, specifically formed a binary complex with oxidized CP12 when bound to NAD. PRK did not interact with either GapA or CP12 singly, but oxidized PRK could form with GapA/CP12 a stable ternary complex of about 640 kD (GapA/CP12/PRK). Exchanging NADP for NAD, reducing CP12, or reducing PRK were all conditions that prevented formation of the complex. Although GapA activity was little affected by CP12 alone, the NADPH-dependent activity of GapA embedded in the GapA/CP12/PRK complex was 80% inhibited in respect to the free enzyme. The NADH activity was unaffected. Upon binding to GapA/CP12, the activity of oxidized PRK dropped from 25% down to 2% the activity of the free reduced enzyme. The supramolecular complex was dissociated by reduced thioredoxins, NADP, 1,3-bisphosphoglycerate (BPGA), or ATP. The activity of GapA was only partially recovered after complex dissociation by thioredoxins, NADP, or ATP, and full GapA activation required BPGA. NADP, ATP, or BPGA partially activated PRK, but full recovery of PRK activity required thioredoxins. The reversible formation of the GapA/CP12/PRK supramolecular complex provides novel possibilities to finely regulate GapA ("non-regulatory" GAPDH isozyme) and PRK (thioredoxin sensitive) in a coordinated manner.

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.

Co-ordinated gene expression of photosynthetic glyceraldehyde-3-phosphate dehydrogenase, phosphoribulokinase, and CP12 in Arabidopsis thaliana.

Photosynthetic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) interact in the chloroplast stroma through the action of the small peptide CP12. This supramolecular complex concurs with the light-dependent modulation in vivo of GAPDH and PRK activities. The expression patterns of several genes potentially involved in the formation of the complex have been studied. The genome of Arabidopsis thaliana includes seven genes for phosphorylating GAPDH isozymes, one PRK gene, and three genes for CP12. The expression of four GAPDH genes was analysed, i.e. GapA-1 and GapB for photosynthetic GAPDH of chloroplasts (NAD(P)-dependent), GapC-1 for cytosolic GAPDH, and GapCp-1 for plastid GAPDH (both NAD-dependent). A similar analysis was performed with PRK and two CP12 genes (CP12-1, CP12-2). The expression of GapA-1, GapB, PRK, and CP12-2 was found to be co-ordinately regulated with the same organ specificity, all four genes being mostly expressed in leaves and flower stalks, less expressed in flowers, and little or not expressed in roots and siliques. The expression of all these genes in leaves was terminated during prolonged darkness or following sucrose treatments, and their transcripts decayed with similar kinetics. At variance with CP12-2, gene CP12-1 appeared to be expressed more in flowers, it was totally insensitive to darkness, and less affected by sucrose. The expression of glycolytic GapC was strong and ubiquitous, insensitive to dark treatments, and unaffected by sucrose. GapCp transcripts were also found to be ubiquitous at lower levels, slowly decreasing in the dark and stable in sucrose-treated leaves. The co-ordinated expression of genes GapA-1, GapB, PRK, and CP12-2 is consistent with their specific involvement in the formation of the photosynthetic regulatory complex of chloroplasts.

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 pH 8.0, 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 alpha-band was symmetrically centered at 561 nm in P-I, but it was asymmetric in P-II with a maximum at 562 nm and shoulder at 557 nm. 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 70 kDa during size-exclusion chromatography. P-I identified with a previously purified ascorbate-dependent b-type cytochrome of bean hypocotyl plasma membranes. Partially purified P-II, on the other hand, correlated with a heme-protein of 27 kDa in SDS-PAGE gels, was dimeric (60 kDa) 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 cytochrome b561 of Zea mays and highly conserved in other related plant sequences, including that of Arabidopsis thaliana cytochrome b561-1 (CAA18169). The biochemical features fully support the assignment of P-II cytochrome to the family of cytochrome b561, ascorbate-dependent (CYBASC) cytochromes, which also includes cytochrome b561 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.

Coenzyme site-directed mutants of photosynthetic A4-GAPDH show selectively reduced NADPH-dependent catalysis, similar to regulatory AB-GAPDH inhibited by oxidized thioredoxin.

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of higher plants uses both NADP(H) and NAD(H) as coenzyme and consists of one (GapA) or two types of subunits (GapA, GapB). AB-GAPDH is regulated in vivo through the action of thioredoxin and metabolites, showing higher kinetic preference for NADPH in the light than in darkness due to a specific effect on kcat(NADPH). Previous crystallographic studies on spinach chloroplast A4-GAPDH complexed with NADP or NAD showed that residues Thr33 and Ser188 are involved in NADP over NAD selectivity by interacting with the 2'-phosphate group of NADP. This suggested a possible involvement of these residues in the regulatory mechanism. Mutants of recombinant spinach GapA (A4-GAPDH) with Thr33 or Ser188 replaced by Ala (T33A, S188A and double mutant T33A/S188A) were produced, expressed in Escherichia coli, and compared to wild-type recombinant A4-GAPDH, in terms of crystal structures and kinetic properties. Affinity for NADPH was decreased significantly in all mutants, and kcat(NADPH) was lowered in mutants carrying the substitution of Ser188. NADH-dependent activity was unaffected. The decrease of kcat/Km of the NADPH-dependent reaction in Ser188 mutants resembles the behaviour of AB-GAPDH inhibited by oxidized thioredoxin, as confirmed by steady-state kinetic analysis of native enzyme. A significant expansion of size of the A4-tetramer was observed in the S188A mutant compared to wild-type A4. We conclude that in the absence of interactions between Ser188 and the 2'-phosphate group of NADP, the enzyme structure relaxes to a less compact conformation, which negatively affects the complex catalytic cycle of GADPH. A model based on this concept might be developed to explain the in vivo light-regulation of the GAPDH.

Dual coenzyme specificity of photosynthetic glyceraldehyde-3-phosphate dehydrogenase interpreted by the crystal structure of A4 isoform complexed with NAD.

Photosynthetic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Spinacia oleracea belongs to a wide group of GAPDHs found in most organisms displaying oxygenic photosynthesis, including cyanobacteria, green and red algae, and higher plants. As a major catalytic difference with respect to glycolytic GAPDH, photosynthetic GAPDH exhibits dual cofactor specificity toward pyridine nucleotides with a preference for NADP(H). Here we report the crystal structure of NAD-complexed recombinant A(4)-GAPDH (NAD-A(4)-GAPDH) from Spinacia oleracea, expressed in Escherichia coli. Its superimposition onto native A(4)-GAPDH complexed with NADP (NADP-A(4)-GAPDH) pinpoints specific conformational changes resulting from cofactor replacement. In photosynthetic NAD-A(4)-GAPDH, the side chain of Asp32 is oriented toward the coenzyme to interact with the adenine ribose diol, similar to glycolytic GAPDHs (NAD-specific). On the contrary, in NADP-A(4)-GAPDH Asp32 moves away to accommodate the additional 2'-phosphate group of the coenzyme and to minimize electrostatic repulsion. Asp32 rotation is allowed by the presence of the small residue Ala40, conserved in most photosynthetic GAPDHs, replacing bulky amino acid side chains in glycolytic GAPDHs. While in NADP-A(4)-GAPDH two amino acids, Thr33 and Ser188, are involved in hydrogen bonds with the 2'-phosphate group of NADP, in the NAD-complexed enzyme these interactions are lacking. The crystallographic structure of NAD-A(4)-GAPDH highlights that four residues, Thr33, Ala40, Ser188, and Ala187 (Leu, Leu, Pro, and Leu respectively, in glycolytic Bacillus stearothermophilus GAPDH sequence) are of primary importance for the dual cofactor specificity of photosynthetic GAPDH. These modifications seem to trace the minimum evolutionary route for a primitive NAD-specific GAPDH to be converted into the NADP-preferring enzyme of oxygenic photosynthetic organisms.

The C-terminal extension of glyceraldehyde-3-phosphate dehydrogenase subunit B acts as an autoinhibitory domain regulated by thioredoxins and nicotinamide adenine dinucleotide.

The regulatory isoform of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a light-activated enzyme constituted by subunits GapA and GapB. The NADPH-dependent activity of regulatory GAPDH from spinach chloroplasts was affected by the redox potential (E(m,7.9), -353 +/- 11 mV) through the action of thioredoxin f. The redox dependence of recombinant GapB (E(m,7.9), -347 +/- 9 mV) was similar to native GAPDH, whereas GapA was essentially redox-insensitive. GapB mutants having one or two C-terminal cysteines mutated into serines (C358S, C349S, C349S/C358S) were less redox-sensitive than GapB. Different mutants with other cysteines substituted by serines (C18S, C274S, C285S) still showed strong redox regulation. Fully active GapB was a tetramer of B-subunits, and, when incubated with NAD, it associated to a high molecular weight oligomer showing low NADPH-dependent activity. The C-terminal GapB mutants (C358S, C349S, C349S/C358S) were active tetramers unable to aggregate to higher oligomers in the presence of NAD, whereas other mutants (C18S, C274S, C285S) again behaved like GapB. We conclude that a regulatory disulfide, between Cys-349 and Cys-358 of the C-terminal extension of GapB, does form in the presence of oxidized thioredoxin. This covalent modification is required for the NAD-dependent association into higher oligomers and inhibition of the NADPH-activity. By leading to GAPDH autoinhibition, thioredoxin and NAD may thus concur to the dark inactivation of the enzyme in vivo.