Paolo Pupillo

Istituto delle Scienze Neurologiche, Ospedale Bellaria | IRCCS Bologna · Pharmacy and Biotechnology

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...

Populations of Pulmonaria on Apennine highlands in the province of Bologna were investigated. Except for a few isolated sites with P. officinalis, most populations are referable to P. apennina on the basis of leaf shape, indumentum and chromosome number (2n = 22). Pulmonaria apennina occurs in two morphs: i) a green morph with few or no leaf spots,...

The hybrid Anemonoides nemorosa × A.ranunculoides is recorded for the first time in Italy at the southern periphery of Bologna (N Italy, Emilia-Romagna). Its status is supported by both morphological features and chromosome number (2n = 31). For this taxon, a new nomenclatural combination is proposed.

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...

Relationships between age, time of emergence, and leaf traits of individuals were investigated in a population of Erythronium dens-canis L. in a hilly woodland area named Farneto-C, near Bologna, Italy. In 2015, 591 individuals were counted, 19 of which were flowering (FLO), 442 were mature non-flowering (MNF) and 130 were juveniles (JUV). FLO emer...

Relationships between age, time of emergence, and leaf traits of individuals were investigated in a population of Erythroniumdens-canis L. in a hilly woodland area named Farneto-C , near Bologna, Italy. In 2015, 591 individuals were counted, 19 of which were flowering (FLO), 442 were mature non-flowering (MNF) and 130 were juveniles (JUV). FLO emer...

Populations of Pulmonaria with unique features and associated with shady and dry conditions were found in high beech woods on the west side of Mt. Marzola and in other dolomitic sites of Trentino (southern Alps). Morpho-statistical and karyological analyses suggest that these populations represent a new subspecies of P. officinalis, Pulmonaria offi...

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 variegati...

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 conten...

BAM1 is a plastid-targeted β-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 res...

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), a...

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 s...

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 t...

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...

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...

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 assim...

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) w...

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 whic...

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...

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 formati...

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 stoichiometri...

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 t...

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 monodeh...

NAD(P)H:(quinone-acceptor) oxidoreductase [NAD(P)H-QR], a plant, cytosolic protein, was purified from cultured sugarbeet cells by a combination of ammonium sulfate fractionation, FPLC Superdex 200 gel filtration, Q-Sepharose anion-exchange chromatography, and a final Blue Sepharose CL-6B affinity chromatography with an NADPH gradient. The subunit m...

The benzothiadiazole derivative acibenzolar-S-methyl (BTH; Bion, Syngenta), a well known chemical inducer of systemic acquired resistance (SAR), was tested for its ability to protect pear (Pyrus communis cv. Abate Fetel) from fire blight following inoculation with Erwinia amylovora. Two-year-old pear plants sprayed with BTH 10 days before inoculati...

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 s...

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 cofac...

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...

Here, we report the first crystal structure of a photosynthetic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) complexed with NADP. The enzyme, purified from spinach chloroplasts, is constituted of a single type of subunit (A) arranged in homotetramers. It shows non-regulated NADP-dependent and NAD-dependent activities, with a preference for NADP...

Cytochromeb 561 (cytb 561) is a trans-membrane cytochrome probably ubiquitous in plant cells. In vitro, it is readily reduced by ascorbate or by juglonol, which in plasma membrane (PM) preparations from plant tissues is efficiently produced by a PM-associated NAD(P)H∶quinone reductase activity. In bean hypocotyl PM, juglonol-reduced cytb 561 was no...

Plant cytochrome b5 reductases (b5R) are assumed to be part of an ER-associated redox chain that oxidizes NADH to provide electrons via cytochrome b5 (cyt b5) to ER-associated fatty acyl desaturase and related hydroxylases, as in mammalian cells. Here we report on cDNA cloning of a novel maize b5R, NFR II, strongly related to a previously cloned cD...

The high potential, ascorbate-reducible b-type cytochrome of plant plasma membranes, named cytochrome b-561, has been purified to homogeneity from etiolated bean hypocotyls. The pure protein migrated in denaturing electrophoresis as a broad band of approximately 55 kDa, and was found to be glycosylated. Optical redox titrations of partially purifie...

In higher plants, NAD(P)H:quinone reductase (NQR) is the only flavoreductase known to reduce quinone substrates directly to hydroquinones by a two-electron reaction mechanism. This enzymatic activity is believed to protect aerobic organisms from the oxidative action of semiquinones. For this reason plant NQR has recently been suggested to be relate...

A novel NADH-dependent, soluble flavoreductase of 60 kDa, active toward ferric chelates and quinones, has been purified from maize seedlings. Two closely related isoforms were separated. The two isoforms are similar in several biochemical features, with the exception of the apparent molecular mass of their subunits (29 and 31 kDa, respectively). Th...

We previously purified an NADH-dependent Fe3+-chelate reductase (NFR) from maize roots with biochemical features of a cytochrome-b5 reductase (b5R) [Sparla, Bagnaresi, Scagliarini and Trost (1997) FEBS Lett. 414, 571-575]. We have now cloned a maize root cDNA that, on the basis of sequence information, calculated parameters and functional assay, co...

We previously purified an NADH-dependent Fe3+-chelate reductase (NFR) from maize roots with biochemical features of a cytochrome-b(5) reductase (b(5)R) [Sparla, Bagnaresi, Scagliarini and Trost (1997) FEBS Lett. 414, 571-575]. We have now cloned a maize root cDNA that, on the basis of sequence information, calculated parameters and functional assay...

Isoforms of NAD(P)-dependent glyceraldehyde-3-phosphate dehydrogenases (EC 1.2.1.13) have been separated from spinach chloroplast extracts by FPLC-anion exchange chromatography in phosphate buffer and purified to homogeneity. Peak I from Q-Sepharose corresponds to a tetramer of A-subunits of 36 kDa showing a constant ratio of NADPH- to NADH-activit...

The NAD(P)H:quinone oxidoreductase activity of tobacco leaves is catalyzed by a soluble flavoprotein [NAD(P)H-QR] and membrane-bound forms of the same enzyme. In particular, the activity associated with the plasma membrane cannot be released by hypoosmotic and salt washing of the vesicles, suggesting a specific binding. The products of the plasma-m...

The plasma membrane (PM) of higher plants contains a major ascorbate-reducible, high-potentialb-type cytochrome, named cytochromeb 561 (cytb 561). In this paper a rapid purification protocol for the cytb 561 of bean hypocotyls PM is described. An almost 200-fold increase of cytb 561 specific concentration was achieved with respect to the PM fractio...

The plasma membrane (PM) redox studies started in the 1920s by measuring the reduction rate of impermeable redox dyes (e.g.. indigodisulfonate) by normal and tumor cells (Crane et al., 1985, 1990 and references therein). In the 1960s it was shown that: (i) erythrocytes reduce the impermeable ferricyanide with concomitant release of protons (Dormand...

Quinone oxidoreductase activities dependent on pyridine nucleotides are associated with the plasma membrane (PM) in zucchini (Cucurbita pepo L.) hypocotyls. In the presence of NADPH, lipophilic ubiquinone homologs with up to three isoprenoid units were reduced by intact PM vesicles with a Km of 2 to 7 [mu]M. Affinities for both NADPH and NADH were...

The NADH-dependent Fe(3+)-chelate reductase (NFCHR) of tomato (Lycopersicon esculentum L.) roots, a strategy I species, was investigated. The Fe(3+)-citrate reductase (FeCitR) assay was strongly inhibited by p-hydroxymercuribenzoic acid (PHMB); moreover, the inhibitor was found to be more specific to the FeCitR assay than to the Fe(3+)-EDTA reducta...

NAD(P)H:(quinone-acceptor) oxidoreductase [NAD(P)H-QR], a plant cytosolic protein, was purified from cultured sugarbeet cells by a combination of ammonium sulfate fractionation, FPLC Superdex 200 gel filtration, Q-Sepharose anion-exchange chromatography, and a final Blue Sepharose CL-6B affinity chromatography with an NADPH gradient. The subunit mo...

Iron-deficient maize seedlings exhibit a starvation syndrome characterized by an increase in different parameters such as root fresh weight (+ 30%), protein (+ 25%) and plasma membrane-associated NADH Fe³⁺ −EDTA reductase (NFR; +45%). NFR activity was found associated with 9 000g (20 min) and 110 000 g (1 h) sediments, purified plasma membrane and...

Cell suspension cultures of Beta vulgaris L., treated with calcium chelators or untreated, were used to characterize pyndine nucleotide-dependent diaphorases of microsomes. The microsomal activity of NADH-dependent duroquinone reductase from cultures treated with 10 mM Na2EGTA for 24 h increased by a factor of 1.8 with respect to control microsomes...

D-Glyceraldehyde 3-phosphate: NADP+ oxidoreductase, nonphosphorylating (GNR; EC 1.2.1.9) purified from spinach leaves was investigated by initial velocity analysis. The hyperbolic saturation curves became nonhyperbolic when NADP+ was varied at elevated D-glyceraldehyde 3-phosphate (G3P) concentrations (sigmoidicity) or when G3P was varied at low NA...

Light modulation of chloroplast glyceraldehyde 3-phosphate dehydrogenase (NAD(P)-GAPDH; EC 1.2.1.13) has been investigated. Complete activation of NADPH-dependent activity is achieved at 25 W.m–2 photosynthetically active radiation in spinach (Spinacia oleracea L.) and 100 W.m–2 in maize (Zea mays L.) leaves. Light activation is stronger in spinach...

Spinach (Spinacia oleracea L.) chloroplast NAD(P)-dependent glyceraldehyde 3-phosphate dehydrogenase (NAD(P)-GAPDH; EC 1.2.1.13) was purified. The association state of the protein was monitored by fast protein liquid chromatography-Superose 12 gel filtration. Protein chromatographed in the presence of NADP+ and dithiothreitol consisted of highly NA...

The steady state kinetics of glyceraldehyde 3-phosphate:NADP(+) oxidoreductase (GNR) (EC 1.2.1.9) have been investigated. The enzyme exhibits hyperbolic behavior over a wide range of substrate concentrations. Double-reciprocal plots are nearly parallel or distantly convergent with limiting K(m) values of 2 to 5 micromolar for NADP(+) and 20 to 40 m...

The intracellular distribution of NADPH- and NADH-dependent duroquinone reductase (NAD (P)H-DQR) from etiolated zucchini hypocotyls (Cucurbita pepo L.) was investigated. About 80% of this enzyme is in the supernatant fraction and is probably cytosolic. Particulate NAD (P)H-DQR was largely (42%) found in association with the plasma membrane and was...

Scagliarini, S., Pupillo, P. and Valenti, V. 1988. Isoforms of NADP-dependent malic enzyme in tissues of the greening maize leaf.—J. exp. Bot. 39: 1109–1119. The compartmentation of the isoforms of NADP-dependent malic enzyme (E.C. 1.1.1.40) has been studied in cell-free extracts and in enzymatically-isolated protoplasts of mesophyll tissue (MT) an...

An NAD(P)H dehydrogenase stimulated by quinone (P Pupillo, V Valenti, L de Luca, R Hertel 1986 Plant Physiol 80: 384-389) was solubilized from washed microsomes of zucchini squash hypocotyls (Cucurbita pepo L.) by use of 1% Triton X-100. The solubilized enzyme remained in solution in aqueous buffer and could be purified by a combination of Sepharos...

The leaves of maize seedlings contain two principal isozymes of fructose 1,6-bisphosphate aldolase (E.C. 4.1.2.13), one chloroplastic and one cytosolic (Gasperini and Pupillo, 1982). Mesophyll protoplasts were separated from bundle sheath (BS) strands of both light-grown and dark-grown maize leaves. Aldolase isozymes were separated from extracts of...

Mesophyll protoplasts of one-month-old maize leaves were separated enzymatically from bundle sheath strands, and purified by centrifugation through a Percoll layer. The protoplasts and BS strands were essentially pure as judged by microscopy, chl a/b ratios, and levels of enzyme markers (PEP carboxylase and NADP-malic enzyme). Chioroplasts were obt...

Some properties of microsomal electron transfer chains, dependent for oxidase activity on addition of NADH or NADPH, duroquinone, and oxygen (L. De Luca et al., 1984, Plant Sci Lett 36: 93-98) are described. Activity is characterized by negatively cooperative kinetics toward reduced pyridine nucleotides, with limiting K(m) of 10 to 50 micromolar at...

The distribution of glucose 6-phosphate dehydrogenase and D-glyceraldehyde 3-phosphate: NADP reductase activities in maize leaf blades was investigated by differential tissue fractionation. The reliability of this technique was verified on the basis of distribution of several marker enzymes (NADP-malate dehydrogenase and phosphoenolpyruvate carboxy...

In microsomes from Cucurbita hypocotyls a duroquinone (DQ) stimulated NADH oxidase was found which is strongly activated by addition of 0.01–0.1% Triton X-100. After density gradient centrifugation and polyethyleneglycol (PEG) fractionation this enzyme occurs in membranes carrying plasma membrane markers. Another NADH oxidase localized at the endop...

Two different forms of glucose 6-phosphate dehydrogenase (EC 1.1.1.49) have been purified from etiolated and green leaves, respectively, of 6-day maize (Zea mays L. cv Fronica) seedlings. The procedure includes an ammonium sulfate step, an ion exchange chromatography, and a second gel filtration in Sephadex G-200 in the presence of NADP(+) to take...

Aldolase from maize leaves (Zea mays L., cv. Fronica) has been partially purified to specific activities of 200–300 nmol min−1 mg−1 protein. Etiolated leaves contain two isozymes characterized by pI 4.3 and 5.8, respectively, in a 2:1 to 3:1 ratio. The cytosolic pI 5.8 activity declines during greening, concomitant with the relative increase of pI...

NAD-dependent malic enzyme (EC 1.1.1.39) was obtained from isolated mitochondria of cauliflower buds (Brassica oleracea L., var. botrytis). The NAD-linked activity is accompanied by a minor NADP-linked activity. Some contaminant NADP-malic enzyme from the supernatant and the plasma membrane is usually present in crude mitochondrial preparations. NA...

A definite membrane fraction from Cucurbita hypocotyls, maize coleoptiles, and other plant tissues contains a NADP-dependent malic enzyme activity, up to 10% of overall tissue activity, and probably other soluble proteins. This "malic enzyme particle" is identified as plasmalemma on the basis of sedimentation behavior, density distribution in sucro...

Etiolated maize leaves (Zea mays L.) contain a major isozyme of NADP-dependent malic enzyme (L-malate dehydrogenase, decarboxylating, EC 1.1.1.40) having an isoelectric point of 5.280.03, a Km (L-malate) 0.3–0.6 mM at pH 7.45; a broad pH optimum around pH 6.9 under the conditions of assay; a molecular weight of 280,000 (sometimes accompanied by a m...

A procedure is described for the purification of three glyceraldehyde phosphate dehydrogenases from a batch of beet leaves. Glyceraldehyde 3-phosphate:NADP+ reductase, nonphosphorylating (EC 1.2.1.9) has been purified over 1500-fold. The Mr of this enzyme is 190,000 and its subunits have an Mr of 53,000, suggesting a tetramer as the active form. It...

Protein-lipopolysaccharide (pr-LPS) complexes were purified from a virulent strain of Pseudomonas tabaci (NCPPB 1427) and two incompatible strains (P. aptata NCPPB 2664 and P. lachrymans NCPPB 1436). They all contained two major polypeptides with mol. wts of 20 000 and 40 000. Slide agglutination tests with O- or pr-LPS-antisera produced against an...

Incorporation of 14C-L-leucine into young spruce seedlings (Picea abies), a process innibited by NaF, FCCP and azide, is less sensitive to low cyanide concentrations than for larch (Laris decidua) seedlings, in keeping with previous observations on biosynthetic capacity.

A protein-lipopolysaccharide (pr-LPS) complex has been selectively detached from washed living cells of Erwinia chrysanthemi NCPPB 454 by treatment with disodium ethylene diamine tetraacetate (EDTA). The material was partially purified by ultrafiltration, then chemically characterized, and its purity was checked by spectrophotometry and gel chromat...

Exocellular pectate lyases produced by three strains of Erwinia chrysanthemi in polypectate broth or in infiltrated Dieffenbachia leaves have been separated by isoelectric focusing. The proteins can be classified into three main groups on the basis of their immunological and enzymological properties and isoelectric points: “alkaline” (pI 9·7 to 9·0...

The ligand-dependent dissociation of spinach chloroplast glyceraldehyde-phosphate dehydrogenase (Mr 600000) to protomers of Mr about 145000, previously shown by us in 1973, has been further characterized by the technique of velocity sedimentation in sucrose gradients. The process exhibits cooperativity and is accompanied by an increase of the appar...

A procedure for the purification of NADP-linked glyceraldehyde-3-phosphate dehydrogenase from autotrophically grown Euglena gracilis is described. The NADP activity has been purified 103-fold over crude extracts with a 30-fold purification of the NAD activity. A second NAD-dependent activity is eliminated by salting out. The specific activity obser...

Spinach chloroplast glyceraldehyde phosphate dehydrogenase (d-glyceraldehyde-3-phosphate: NADP oxidoreductase, phosphorylating; EC 1.2.1.13) is an equilibrium mixture of aggregates of a basic protomer (Mr about 145,000) and is active with both NADP and NAD. The enzyme is primarily “tetrameric” (Mr about 600,000), although minor amounts of smaller a...

Crude extracts of species of several groups of green plants were examined to determine the specificity of the glyceraldehyde-3-phosphate dehydrogenases. The results are consistent with the view that in all green plants both the NAD- and the NADP-dependent activities of chloroplasts are catalyzed by one enzyme which is different from the NAD-linked...