J. López Gorgé

A more sensitive modification of the catalase assay with the Clark oxygen electrode: Application to the kinetic study of the pea leaf enzyme

A modification of the method of catalase determination by means of the Clark oxygen electrode is described. The assay is based on measurement of the initial rate at which oxygen is released by catalase in an oxygen-free buffer. Displacement of oxygen was brought about by flushing with nitrogen, and the substrate used was hydrogen peroxide at a 33.5 mm final concentration. The method is rapid and can be used with crude catalase preparations. Its sensitivity is at least 20 times higher than that of previous methods; it has an interval of measurable activity of about 0.01–8.4 μmol of O2/min and, therefore, is applicable to an 840-fold range of catalase concentrations. This modification was applied to the kinetic study of crude extracts of pea leaf catalase. An apparent Km of 0.190 m was calculated.

High-Yield Expression of Pea Thioredoxin m and Assessment of Its Efficiency in Chloroplast Fructose-1,6-Bisphosphatase Activation

A cDNA clone encoding pea (Pisum sativum L.) chloroplast thioredoxin (Trx) m and its transit peptide were isolated from a pea cDNA library. Its deduced amino acid sequence showed 70% homology with spinach (Spinacia oleracea L.) Trx m and 25% homology with Trx f from pea and spinach. After subcloning in the Ndel-BamHI sites of pET-12a, the recombinant supplied 20 mg Trx m/L Escherichia coli culture. This protein had 108 amino acids and was 12,000 D, which is identical to the pea leaf native protein. Unlike pea Trx f, pea Trx m showed a hyperbolic saturation of pea chloroplast fructose-1,6-bisphosphatase (FBPase), with a Trx m/FBPase molar saturation ratio of about 60, compared with 4 for the Trx f/FBPase quotient. Cross-experiments have shown the ability of pea Trx m to activate the spinach chloroplast FBPase, results that are in contrast with those in spinach found by P. Schurmann, K. Maeda, and A. Tsugita ([1981] Eur J Biochem 116: 37–45), who did not find Trx m efficiency in FBPase activation. This higher efficiency of pea Trx m could be related to the presence of four basic residues (arginine-37, lysine-70, arginine-74, and lysine-97) flanking the regulatory cluster; spinach Trx m lacks the positive charge corresponding to lysine-70 of pea Trx m. This has been confirmed by K70E mutagenesis of pea Trx m, which leads to a 50% decrease in FBPase activation.

Fructose-1,6-diphosphatase from spinach leaf chloroplasts: Purification and heterogeneity

Abstract The enzyme fructose- 1,6-diphosphatase (FDPase), involved in the reductive cycle of the pentose phosphate pathway, has been purified from spinach leaves by heating (30 min at 60°), “salting out” with ammonium sulphate (between 30–70% of saturation), filtration through Sephadex G-100 and G-200, fractionation on DEAE-52 cellulose and preparative electrophoresis on polyacrylamide gel. Filtration through DEAE-cellulose led to the isolation of two active fractions (fractions I and II) with very close MWs and isoelectric points. By electrophoresis on acrylamide gel, both fractions gave two active fractions (fractions I a -I b and II a -II b ). The fractions with low electrophoretic migration rate—I b and II b —are stable in acid and neutral pH, have a MW between 90 000 and 110 000 and constitute the native form of the photosynthetic enzyme. The fractions of faster migration rate—I a and II a -originate from the corresponding fractions I b and II b under alkaline conditions, show half the MW of the respective fractions, and behave as subunits of the original dimer form. Measured by electrofocusing, the four active fractions have isoclectric points in the range 4·10–4.30.

IRON DEFICIENCY IN PEA PLANTS EFFECT ON CATALASE, PEROXIDASE, CHLOROPHYLL AND PROTEINS OF LEAVES

SummaryPea plants (Pisum sativum L., var. Lincoln) were grown in nutrient cultures at 4 levels of iron, 0.60 ppm (low), 0.96 ppm (low), 3.0 ppm (normal) and 30 ppm (excess) for 45 days.Leaf extracts were assayed for chlorophyll, proteins, catalase and peroxidase activities. Catalase and chlorophyll were closely related to iron supply. An inverse relationship was observed between peroxidase and catalase activities. Peroxidase was increased both at dificiency and excess iron levels, but was depressed at normal iron supply. The peroxidase/catalase ratio varied with iron supply and showed a minimum value of about 39 at 15 and 30 days growth, at adequate iron supplies.Measurement of catalase activity and the use of peroxidase/catalase ratios appear to be helpful in identifying iron deficiencies in peas.

Cloning, structure and expression of a pea cDNA clone coding for a photosynthetic fructose-1,6-bisphosphatase with some features different from those of the leaf chloroplast enzyme.

A positive clone against pea (Pisum sativum L.) chloroplast fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) antibodies was obtained from a copy DNA (cDNA) library in λgt11. The insert was 1261 nucleotides long, and had an open reading frame of 1143 base pairs with coding capability for the whole FBPase subunit and a fragment of a putative processing peptide. An additional 115 base pairs corresponding to a 3′-untranslated region coding for an mRNA poly(A)+ tail were also found in the clone. The deduced sequence for the FBPase subunit was a 357-amino-acid protein of molecular mass 39253 daltons (Da), showing 82–88% absolute homology with four chloroplastic FBPases sequenced earlier. The 3.1-kilobase (kb)KpnI-SacI fragment of the λgt11 derivative was subcloned between theKpnI-SacI restriction sites of pTZ18R to yield plasmid pAMC100. Lysates ofEscherichia coli (pAMC100) showed FBPase activity; this was purified as a 170-kDa protein which, upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, displayed a 44-kDa band. As occurs with native FBPases, this indicates a homotetrameric structure for the expressed FBPase. When assayed under excess Mg2+ (10 mM), the expressed enzyme had a higher affinity for the substrate than the native pea leaf FBPase; this parameter appears to be substantiated by a tenfold higher specific activity than that of the native enzyme. However, when activated with dithiothreitol plus saturating concentrations of pea thioredoxin (Td) f, both FBPase had similar activities, with a 4:1 Td f-FBPase stoichiometry. In contrast to the native pea chloroplast FBPase, theE. coli-expressed enzyme did not react with the monoclonal antibody GR-PB5. It also had a higher heat sensitivity, with 42% residual activity after heating for 30 min at 60°C, conditions which preserved the native enzyme in a fully active state. These results show the existence of some difference(s) in the conformation of the two FBPases; this could be a consequence of a different expression of the genomic and cDNA clones, or be due to the need for some factor for the correct assembly of the oligomeric structure of the native chloroplast enzyme.

Purification and properties of pea (Pisum sativum L.) thioredoxin f, a plant thioredoxin with unique features in the activation of chloroplast fructose-1,6-bisphosphatase

Thioredoxin (Td) f from pea (Pisum sativum L.) leaves was purified by a simple method, which provided a high yield of homogeneous Td f. Purified Td f had an isoelectric point of 5.4 and a relative molecular mass (Mr) of 12 kilodaltons (kDa) when determined by filtration through Superose 12, but an Mr of 15.8 kDa when determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protein remained fully active for several months when conserved frozen at — 20° C. The pea protein was able to activate fructose1,6-bisphosphatase (FBPase; EC 3.1.3.11), but in contrast to other higher-plant Td f proteins, was not functional in the modulation of NADP+-malate dehydrogenase activity. In spite of the absence of immunological cross-reactions of pea and spinach Td f proteins with the corresponding antibodies, pea Td f activated not only the homologous FBPase, but also the spinach enzyme. The saturation curves for pea FBPase, either with fructose-1,6-bisphosphate in the presence of different concentrations of homologous Td f, or with pea Td f in the presence of excess substrate, showed sigmoid kinetics; this can be explained on the basis of a random distribution of fructose-1,6-bisphosphate, and of the oxidized and reduced forms of the activator, among the four Td f- and substrate-binding sites of this tetrameric enzyme. From the saturation curves of pea and spinach Td f proteins against pea FBPase, a 4:1 stoichiometry was determined for the Td f-enzyme binding. This is in contrast to the 2:1 stoichiometry found for the spinach FBPase. The UV spectrum of pea Td f had a maximum at 277 nm, which shifted to 281 nm after reduction with dithiothreitol (s at 280 nm for 15.8-kDa Mr = 6324 M−1 · cm−1). The fluorescence emission spectrum after 280-nm excitation had a maximum at 334 nm, related to tyrosine residues; after denaturation with guanidine isothiocyanate an additional maximum appeared at 350 nm, which is concerned with tryptophan groups. Neither the native nor the denatured form showed a significant increase in fluorescence after reduction by dithiothreitol, which means that the tyrosine and tryptophan groups in the reduced Td f are similarly exposed. Pea Td f appears to have one cysteine residue more than the three cysteines earlier described for spinach and Scenedesmus Td f proteins.

Cloning and sequencing of a pea cDNA fragment coding for thioredoxin m.

Thioredoxins are low molecular mass proteins (about 12 kD) engaged in different redox processes (Holmgren, 1985; Buchanan, 1992). From a functional point of view two types of thioredoxins coexist in the plant kingdom. Chloroplast thioredoxins are concerned with activation of some organellar enzymes through a light-mediated reduction mechanism (Jacquot, 1984; Cseke and Buchanan, 1986), whereas cytosolic thioredoxins appear to be engaged in other redox functions, such as the synthesis of deoxyribonucleotides from the corresponding ribonucleotides. Two types of thioredoxins have been found in the chloroplast: thioredoxin f is especially competent in Fru-1,6-bisphosphatase activation, whereas thioredoxin m is more effective in the activation of NADP+malate dehydrogenase (Cseke and Buchanan, 1986; Buchanan, 1992). However, in spite of the same cellular location, both chloroplast thioredoxins show a clear phylogenetic divergence. The m form is structurally close to prokaryotic thioredoxins, and the f type is more similar to those from mammals and yeasts (Hartman et al., 1990). AI1 the thioredoxins so far sequenced show the active center Cys-X-ProCys (X = Gly or Ala), which fulfills the redox function through the establishment of a Cys-Cys bridge (Holmgren, 1985). The only chloroplast thioredoxins from higher plants so far sequenced are the f types from spinach (Kamo et al., 1989) and pea (Lepiniec et al., 1992) and the m type from spinach (Wedel et al., 1992). We now describe the isolation of a cDNA clone coding for pea thioredoxin m, as well as the nucleotide sequence and the deduced primary structure of this chloroplast thioredoxin (Table I).

Root Uptake and Partition of Copper, Iron, Manganese, and Zinc in Pinus radiata Seedlings Grown under Different Copper Supplies

Summary In spite of the low Cu needs, a Cu2+ level of 0.03 µM in hydroponic cultures of Pinus radiata seedlings induces a Cu-deficient situation; under these conditions the foliar Cu was 4 mg·kg-1 dry wt after 2.5 months growth, which decreased to 1 mg kg-1 in 5-month-old seedlings. This is shown by a decrease of the growth rate with time, which appears negligible between 7.5 and 9 months in culture. The Cu content of roots, foliar and non-foliar stems, and leaves increases with the micronutrient concentration in the culture solutions, but the response of each organ is inversely related with its distance from the hydroponic medium. In any case P. radiata seedlings showed a very efficient Cu2+ absorption capability of the root, but the nutrient translocation to the aerial organs appears restricted, maybe as a protection mechanism. Until 7.5 months in culture the optimum plant development occurs under excess Cu2+, which shifts to normal Cu2+ levels after 9 months growth; this can be explained as a transient Cu deficiency during the first developmental stage. Mn absorption showed some type of antagonism with that of Cu2+, the needles being the site of the highest Mn concentration in pine. On the contrary, the rate of Fe absorption increases with Cu2+ levels, but the translocation to stems and leaves occurs very slowly. Zn showed a Z-shape partition pattern along root, stems, and needles, with the highest concentrations in root and foliar stem; in addition, Zn absorption appears to be antagonistic to that of Cu.

Binding of photosynthetic fructose-1,6-bisphosphatase to chloroplast membranes

Abstract The binding of stromal fructose-1,6-bisphosphatase (FBPase) activity to thylakoid membranes was investigated in lysates of intact spinach and pea chloroplasts. Binding of spinach FBPase increases with the Mg 2+ concentration of the lysis medium in such a way that 20% of the total enzyme activity becomes associated at 15 mM MgCl 2 . Pea chloroplast FBPase showed a similar interaction, with a 12% linkage to the membranes at 25 MgCl 2 . The apparent K 0.5 -values (Mg 2+ concentration for half-maximum binding) were 1.4 mM (spinach) and 2.2 mM (pea). In the presence of 100 mM KCl or NaCl we have found about 40% of the enzyme activity bound to pea thylakoids; in these cases the apparent K 0.05 -values were 23 mM and 26 mM, respectively. In all cases the double-reciprocal and Hill and Scatchard plots were non-linear, the latter showing a succession of negative and positive cooperativities. Both at 20 mM Mg 2+ or 100 mM K + we found no evidence for any influence of pH on enzyme binding in the range tested (pH 6.5–8.0). From these results we suggest that, under illumination, the negative charges of FBPase and stromal-exposed thylakoid proteins become neutralized, which facilitates the binding of the enzyme to some membranous thioredoxin-like protein. The possible physiological significance of this FBPase-thylakoid membrane interaction is discussed from the point of view of the reductive light-activation of this chloroplast stromal enzyme.

In vitro and in vivo analyses of the mechanism of action of SWEP

Abstract In vitro experiments with intact chloroplasts from hydroponically grown spinach ( Spinacia oleracea L. var. Winter Giant) plants, have shown an I 50 value for SWEP (methyl N -3,4-dichlorophenyl) carbamate) of 0.1 μ M in PS I and II-linked electron transport H 2 0 → NADP + . With thylakoid membranes the I 50 values for PS II-linked Hill reactions H 2 O → [Fe(CN) 6 ] 3− and H 2 O → dichlorophenolindophenol are in the range 0.05-0.1 μ M , whereas the I 50 shifts to 0.45 μ M in short PS II-linked transport chain diphenylcarbazide → dichlorophenolindophenol. Trypsination of PS II-enriched particles produces a negligible increase of the I 50 value in diphenylcarbazide → dichlorophenolindophenol electron transport, a much smaller increase than occurs with diuron- or atrazine-type inhibitors. All these data show SWEP as a strong inhibitor of electron transport in the Q-B region of the PS II-reducing side. However, it appears to have a different binding site than that of urea and triazine herbicides, either on a trypsin resistant or on a non-surface cluster. As a consequence of the NADPH shortage, SWEP brings about a strong inhibition of CO 2 assimilation, with an I 50 of 0.04 μ M , and a lower percentage of trioses-P among the intermediates of the Calvin cycle. In vivo experiments have shown a three to five times higher inhibition of PS II-linked electron transport, when SWEP was supplied through the roots than when it was applied to the leaves. We have found I 50 values of CO 2 assimilation by isolated chloroplasts of foliar disks of 3 and 5 μ M , respectively, when the herbicide was root supplied, as opposed to 10 and 25 μ M after leaf application.