Five different copy DNA clones coding for the same leghemoglobin were isolated from a winged-bean... more Five different copy DNA clones coding for the same leghemoglobin were isolated from a winged-bean (Psophocarpus tetragonolobus L.) nodule library. Although identical in sequence, they each possess a different side of polyadenylation located 93-128 nucleotides downstream of two overlapping AAUAAA putative signal sequences. By analysis of the untranslated 3' ends, a potential mRNA secondary structure can be predicted which could explain the observed polyadenylation heterogeneity. The structure is a size-variable hairpin, creating a net topological distance of 25-27 nucleotides between the canonical signal sequence and the different polyadenylation sites observed. We suggest that this type of variable secondary structure could be one among other causes that determines the apparent flexibility of plant polyadenylation. It could also confer particular properties to the mRNA in relation to stability, translation efficiency and-or nuclear export.
Higher plants possess a large set of the classical guaiacol peroxidases (class III peroxidases, E... more Higher plants possess a large set of the classical guaiacol peroxidases (class III peroxidases, E.C. 1.11.1.7). These enzymes have been implicated in a wide array of physiological processes such as H 2 O 2 detoxification, auxin catabolism and lignin biosynthesis and stress response (wounding, pathogen attack, etc.). During the last 10 years, molecular cloning has allowed the isolation and characterization of several genes encoding peroxidases in plants. The achievement of the large scale Arabidopsis genome sequencing, combined with the DNA complementary to RNA (cDNA) expressed sequence tags projects, provided the opportunity to draw up the first comprehensive list of peroxidases in a plant. By screening the available databases, we have identified 73 peroxidase genes throughout the Arabidopsis genome. The evolution of the peroxidase multigene family has been investigated by analyzing the gene structure (intron/exon) in correlation with the phylogenetic relationships between the isoperoxidases. An evolutionary pattern of extensive gene duplications can be inferred and is discussed. Using a cDNA array procedure, the expression pattern of 23 peroxidases was established in the different organs of the plant. All the tested peroxidases were expressed at various levels in roots, while several were also detected in stems, leaves and flowers. The specific functions of these genes remain to be determined. q
Nodule senescence is one aspect of nitrogen fixation that is important to study from the perspect... more Nodule senescence is one aspect of nitrogen fixation that is important to study from the perspective of improving the host-bacteroid interaction. In winged bean nodules, a 21-kilodalton protein is specifically expressed when senescence begins. Using subcellular fractionation, we observed that this plant protein interacts with the bacteroids. Microsequencing of the protein allowed us to obtain a specific oligonucleotide that was used to isolate the corresponding nodule cDNA. Sequence analysis of this cDNA revealed that the 21-kilodalton protein has all of the features of a legume Kunitz protease inhibitor. Subsequent analysis confirmed that this nodulin is indeed a protease inhibitor. lmmunocytochemical study showed that the protease inhibitor is exclusively localized i11 infected senescent cells of the nodule, particularly in disorganized bacteroids, the peribacteroid membrane, vacuole membranes, and in the vacuole fluid. The specific expression of a protease inhibitor at senescence may be of particular interest if the targeted proteolytic activity is important for the symbiotic relationship. This point is discussed in relation to the known nodule proteases.
The cell wall polymer lignin is believed to be condensed by specific cell wall-localized oxidored... more The cell wall polymer lignin is believed to be condensed by specific cell wall-localized oxidoreductases. In many plants species, including poplar, the peroxidase-directed oxidation of the lignin analogue syringaldazine (SYR) has been localized to cells that undergo secondary wall formation, a process that includes lignification. As a first step to analyse the corresponding peroxidases. we have isolated previously two anionic isoenzymes (PXP 3-4 and PXP 5) from poplar xylem (Populus trichocarpa), which use SYR as a substrate. Here, we demonstrate that these enzymes are responsible for the visualized SYR oxidation in the developing xylem. The cDNA that corresponds to PXP 3-4 was isolated and the deduced protein was found closely related to the other SYR-oxidizing peroxidase PXP 5 (ca. 98% of identity). PXP 3-4 was expressed in a baculovirus expression system yielding high levels of active peroxidase (3 mg/l medium). The heterologously produced protein showed characteristics similar t...
Nodule senescence is one aspect of nitrogen fixation that is important to study from the perspect... more Nodule senescence is one aspect of nitrogen fixation that is important to study from the perspective of improving the host-bacteroid interaction. In winged bean nodules, a 21-kilodalton protein is specifically expressed when senescence begins. Using subcellular fractionation, we observed that this plant protein interacts with the bacteroids. Microsequencing of the protein allowed us to obtain a specific oligonucleotide that was used to isolate the corresponding nodule cDNA. Sequence analysis of this cDNA revealed that the 21-kilodalton protein has all of the features of a legume Kunitz protease inhibitor. Subsequent analysis confirmed that this nodulin is indeed a protease inhibitor. lmmunocytochemical study showed that the protease inhibitor is exclusively localized i11 infected senescent cells of the nodule, particularly in disorganized bacteroids, the peribacteroid membrane, vacuole membranes, and in the vacuole fluid. The specific expression of a protease inhibitor at senescence may be of particular interest if the targeted proteolytic activity is important for the symbiotic relationship. This point is discussed in relation to the known nodule proteases.
Solutions were obtained from the cell wall free space of red light-grown cucumber (Cucumis sativu... more Solutions were obtained from the cell wall free space of red light-grown cucumber (Cucumis sativus L.) hypocotyl sections by a low-speed centrifugation technique. The centrifugate contained NAD and peroxidase but no detectable cytoplasmic contamination, as indicated by the absence of the activity of glucose-6phosphate dehydrogenase from the cell wall solution. Peroxidase activity centrifuged from the cell wall of red light-grown cucumber hypocotyl section could be resolved into at least three cathodic isoforms and two anodic isoforms by isoelectric focusing. Treatment of red light-grown cucumber seedlings with a 10-minute pulse of high-intensity blue light increased the level of cell wall peroxidase by about 60% and caused a qualitative change in the anodic isoforms of this enzyme. The increase in peroxidase activity was detectable within 25 minutes after the start of the blue light pulse, was maximal at 35 minutes, and declined to control levels by 45 minutes of irradiation. The inhibitory effect of blue light on hypocotyl elongation was more rapid than the effect of blue light on total wall peroxidase activity, leading to the conclusion that growth and peroxidase activity are not causally related.
NAD kinase activity has been found in a soluble, cytoplasmic fraction and in the chloroplasts pre... more NAD kinase activity has been found in a soluble, cytoplasmic fraction and in the chloroplasts prepared from green spinach leaves. A small amount of both the cytoplasmic and the chloroplastic NAD kinase activities was retained on a calmodulin-Sepharose affinity column. The cytoplasmic NAD kinase eluted from the affinity column was found to be enhanced by calmodulin in a Ca2+-dependent manner. The chloroplastic enzyme which is located exclusively in the stroma and not in the envelope and thylakoid fractions was not affected by Ca 2+ and calmodulin. The stromal fraction of purified chloroplasts contained only a negligible amount of calmodulin, most probably due to cytoplasmic contamination. Based on these data, two different mechanisms for the light-dependent modulation of spinach NAD kinase activity are suggested. 0 7 2 1 -7 7 1 4 / 8 2 / 0 0 0 1 / 0 1 1 9 / $ 01.00
1981. Light dependency of the cytokinin-induced bud initiation in protonemata of the moss Funaria... more 1981. Light dependency of the cytokinin-induced bud initiation in protonemata of the moss Funaria hygrometrica. -Physiol. Plant, 53: 13-18.
ABSTRACT The activity of NAD+ and NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase isozym... more ABSTRACT The activity of NAD+ and NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase isozymes (EC 1.2.1.12 and EC 1.2.1.13, respectively) were measured in spinach (Spinacia oleracea L. cv. Nobel) leaves grown under different photoperiodic treatments in order to discriminate between the early events of floral induction and processes related to acclimation. Glycolysis-linked isozyme activities were increased not only during floral induction and acclimation, but also during acclimation alone, suggesting that the changes in cytosolic activities were most probably associated with acclimation. In contrast, the chloroplast-linked isozyme activities only increased during flower induction and appeared to be specifically associated with the initiation of the flowering process. The relative activity changes in the chloroplast and cytosol compartments may thus be supposed to be among the first signs of translation of the photoperiodic signal into cytosolic and cellular metabolic adaptation, whereby the leaf moves rapidly into a new metabolic state.
The Arabidopsis genome encodes many secretory guaiacol peroxidases (class III plant peroxidases, ... more The Arabidopsis genome encodes many secretory guaiacol peroxidases (class III plant peroxidases, EC 1.11.1.7). These higher plant enzymes are found either in the vacuole or in the apoplast, where several functions have been attributed to them. Their localisation within the cell wall matrix is most likely important for their activity. In the present work, a gel consisting of polygalacturonate chains cross-linked by Ca 2 + and embedded in polyacrylamide was used to separate proteins from Arabidopsis leaves having an affinity for the Ca 2 + -mediated conformation of pectin. This chromatographic technique selected a small number of cationic isoperoxidases able to bind to Ca 2 + -pectate but not to Ca 2 + -alginate, a polyuronate gel similar to Ca 2 + -pectate. This result suggested that some of the Arabidopsis peroxidases have an affinity for pectin in vivo. Such a property could allow them to be properly distributed within the cell wall network. In addition, eleven cDNAs encoding an Arabidopsis peroxidase were expressed in the baculovirus-insect cell system. The capacity of the resulting recombinant peroxidases to bind Ca 2 + -pectate and Ca 2 + -alginate was also assessed. It appeared that 3 of them exhibited a Ca 2 + -pectate binding activity that was resistant to the action of NaCl. The binding of these recombinant peroxidases to Ca 2 + -alginate was much weaker than to Ca 2 + -pectate, confirming the specificity of the interaction with the pectic structure.
An apoplastic isoperoxidase from zucchini (APRX) was shown to bind strongly to polygalacturonic a... more An apoplastic isoperoxidase from zucchini (APRX) was shown to bind strongly to polygalacturonic acid in their Ca 2 ؉induced conformation. By homology modeling, we were able to identify a motif of four clustered arginines (positions 117, 262, 268, and 271) that could be responsible for this binding. To verify the role of these arginine residues in the binding process, we prepared three mutants of APRX (M1, R117S; M2, R262Q/R268S; and M3, R262Q/R268S/R271Q). APRX and the three mutants were expressed as recombinant glycoproteins by the baculovirus-insect cell system. This procedure yielded four active enzymes with similar molecular masses that were tested for their ability to bind Ca 2 ؉pectate. Recombinant wild-type APRX exhibited an affinity for the pectic structure comparable to that of the native plant isoperoxidase. The mutations impaired binding depending on the number of arginine residues that were replaced. M1 and M2 showed intermediate affinities, whereas M3 did not bind at all. This was demonstrated using an in vitro binding test and on cell walls of hypocotyl cross-sections. It can be concluded that APRX bears a Ca 2 ؉ -pectate binding site formed by four clustered arginines. This site could ensure that APRX is properly positioned in cell walls, using unesterified domains of pectins as a scaffold.
Five different copy DNA clones coding for the same leghemoglobin were isolated from a winged-bean... more Five different copy DNA clones coding for the same leghemoglobin were isolated from a winged-bean (Psophocarpus tetragonolobus L.) nodule library. Although identical in sequence, they each possess a different side of polyadenylation located 93-128 nucleotides downstream of two overlapping AAUAAA putative signal sequences. By analysis of the untranslated 3' ends, a potential mRNA secondary structure can be predicted which could explain the observed polyadenylation heterogeneity. The structure is a size-variable hairpin, creating a net topological distance of 25-27 nucleotides between the canonical signal sequence and the different polyadenylation sites observed. We suggest that this type of variable secondary structure could be one among other causes that determines the apparent flexibility of plant polyadenylation. It could also confer particular properties to the mRNA in relation to stability, translation efficiency and-or nuclear export.
Higher plants possess a large set of the classical guaiacol peroxidases (class III peroxidases, E... more Higher plants possess a large set of the classical guaiacol peroxidases (class III peroxidases, E.C. 1.11.1.7). These enzymes have been implicated in a wide array of physiological processes such as H 2 O 2 detoxification, auxin catabolism and lignin biosynthesis and stress response (wounding, pathogen attack, etc.). During the last 10 years, molecular cloning has allowed the isolation and characterization of several genes encoding peroxidases in plants. The achievement of the large scale Arabidopsis genome sequencing, combined with the DNA complementary to RNA (cDNA) expressed sequence tags projects, provided the opportunity to draw up the first comprehensive list of peroxidases in a plant. By screening the available databases, we have identified 73 peroxidase genes throughout the Arabidopsis genome. The evolution of the peroxidase multigene family has been investigated by analyzing the gene structure (intron/exon) in correlation with the phylogenetic relationships between the isoperoxidases. An evolutionary pattern of extensive gene duplications can be inferred and is discussed. Using a cDNA array procedure, the expression pattern of 23 peroxidases was established in the different organs of the plant. All the tested peroxidases were expressed at various levels in roots, while several were also detected in stems, leaves and flowers. The specific functions of these genes remain to be determined. q
Nodule senescence is one aspect of nitrogen fixation that is important to study from the perspect... more Nodule senescence is one aspect of nitrogen fixation that is important to study from the perspective of improving the host-bacteroid interaction. In winged bean nodules, a 21-kilodalton protein is specifically expressed when senescence begins. Using subcellular fractionation, we observed that this plant protein interacts with the bacteroids. Microsequencing of the protein allowed us to obtain a specific oligonucleotide that was used to isolate the corresponding nodule cDNA. Sequence analysis of this cDNA revealed that the 21-kilodalton protein has all of the features of a legume Kunitz protease inhibitor. Subsequent analysis confirmed that this nodulin is indeed a protease inhibitor. lmmunocytochemical study showed that the protease inhibitor is exclusively localized i11 infected senescent cells of the nodule, particularly in disorganized bacteroids, the peribacteroid membrane, vacuole membranes, and in the vacuole fluid. The specific expression of a protease inhibitor at senescence may be of particular interest if the targeted proteolytic activity is important for the symbiotic relationship. This point is discussed in relation to the known nodule proteases.
The cell wall polymer lignin is believed to be condensed by specific cell wall-localized oxidored... more The cell wall polymer lignin is believed to be condensed by specific cell wall-localized oxidoreductases. In many plants species, including poplar, the peroxidase-directed oxidation of the lignin analogue syringaldazine (SYR) has been localized to cells that undergo secondary wall formation, a process that includes lignification. As a first step to analyse the corresponding peroxidases. we have isolated previously two anionic isoenzymes (PXP 3-4 and PXP 5) from poplar xylem (Populus trichocarpa), which use SYR as a substrate. Here, we demonstrate that these enzymes are responsible for the visualized SYR oxidation in the developing xylem. The cDNA that corresponds to PXP 3-4 was isolated and the deduced protein was found closely related to the other SYR-oxidizing peroxidase PXP 5 (ca. 98% of identity). PXP 3-4 was expressed in a baculovirus expression system yielding high levels of active peroxidase (3 mg/l medium). The heterologously produced protein showed characteristics similar t...
Nodule senescence is one aspect of nitrogen fixation that is important to study from the perspect... more Nodule senescence is one aspect of nitrogen fixation that is important to study from the perspective of improving the host-bacteroid interaction. In winged bean nodules, a 21-kilodalton protein is specifically expressed when senescence begins. Using subcellular fractionation, we observed that this plant protein interacts with the bacteroids. Microsequencing of the protein allowed us to obtain a specific oligonucleotide that was used to isolate the corresponding nodule cDNA. Sequence analysis of this cDNA revealed that the 21-kilodalton protein has all of the features of a legume Kunitz protease inhibitor. Subsequent analysis confirmed that this nodulin is indeed a protease inhibitor. lmmunocytochemical study showed that the protease inhibitor is exclusively localized i11 infected senescent cells of the nodule, particularly in disorganized bacteroids, the peribacteroid membrane, vacuole membranes, and in the vacuole fluid. The specific expression of a protease inhibitor at senescence may be of particular interest if the targeted proteolytic activity is important for the symbiotic relationship. This point is discussed in relation to the known nodule proteases.
Solutions were obtained from the cell wall free space of red light-grown cucumber (Cucumis sativu... more Solutions were obtained from the cell wall free space of red light-grown cucumber (Cucumis sativus L.) hypocotyl sections by a low-speed centrifugation technique. The centrifugate contained NAD and peroxidase but no detectable cytoplasmic contamination, as indicated by the absence of the activity of glucose-6phosphate dehydrogenase from the cell wall solution. Peroxidase activity centrifuged from the cell wall of red light-grown cucumber hypocotyl section could be resolved into at least three cathodic isoforms and two anodic isoforms by isoelectric focusing. Treatment of red light-grown cucumber seedlings with a 10-minute pulse of high-intensity blue light increased the level of cell wall peroxidase by about 60% and caused a qualitative change in the anodic isoforms of this enzyme. The increase in peroxidase activity was detectable within 25 minutes after the start of the blue light pulse, was maximal at 35 minutes, and declined to control levels by 45 minutes of irradiation. The inhibitory effect of blue light on hypocotyl elongation was more rapid than the effect of blue light on total wall peroxidase activity, leading to the conclusion that growth and peroxidase activity are not causally related.
NAD kinase activity has been found in a soluble, cytoplasmic fraction and in the chloroplasts pre... more NAD kinase activity has been found in a soluble, cytoplasmic fraction and in the chloroplasts prepared from green spinach leaves. A small amount of both the cytoplasmic and the chloroplastic NAD kinase activities was retained on a calmodulin-Sepharose affinity column. The cytoplasmic NAD kinase eluted from the affinity column was found to be enhanced by calmodulin in a Ca2+-dependent manner. The chloroplastic enzyme which is located exclusively in the stroma and not in the envelope and thylakoid fractions was not affected by Ca 2+ and calmodulin. The stromal fraction of purified chloroplasts contained only a negligible amount of calmodulin, most probably due to cytoplasmic contamination. Based on these data, two different mechanisms for the light-dependent modulation of spinach NAD kinase activity are suggested. 0 7 2 1 -7 7 1 4 / 8 2 / 0 0 0 1 / 0 1 1 9 / $ 01.00
1981. Light dependency of the cytokinin-induced bud initiation in protonemata of the moss Funaria... more 1981. Light dependency of the cytokinin-induced bud initiation in protonemata of the moss Funaria hygrometrica. -Physiol. Plant, 53: 13-18.
ABSTRACT The activity of NAD+ and NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase isozym... more ABSTRACT The activity of NAD+ and NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase isozymes (EC 1.2.1.12 and EC 1.2.1.13, respectively) were measured in spinach (Spinacia oleracea L. cv. Nobel) leaves grown under different photoperiodic treatments in order to discriminate between the early events of floral induction and processes related to acclimation. Glycolysis-linked isozyme activities were increased not only during floral induction and acclimation, but also during acclimation alone, suggesting that the changes in cytosolic activities were most probably associated with acclimation. In contrast, the chloroplast-linked isozyme activities only increased during flower induction and appeared to be specifically associated with the initiation of the flowering process. The relative activity changes in the chloroplast and cytosol compartments may thus be supposed to be among the first signs of translation of the photoperiodic signal into cytosolic and cellular metabolic adaptation, whereby the leaf moves rapidly into a new metabolic state.
The Arabidopsis genome encodes many secretory guaiacol peroxidases (class III plant peroxidases, ... more The Arabidopsis genome encodes many secretory guaiacol peroxidases (class III plant peroxidases, EC 1.11.1.7). These higher plant enzymes are found either in the vacuole or in the apoplast, where several functions have been attributed to them. Their localisation within the cell wall matrix is most likely important for their activity. In the present work, a gel consisting of polygalacturonate chains cross-linked by Ca 2 + and embedded in polyacrylamide was used to separate proteins from Arabidopsis leaves having an affinity for the Ca 2 + -mediated conformation of pectin. This chromatographic technique selected a small number of cationic isoperoxidases able to bind to Ca 2 + -pectate but not to Ca 2 + -alginate, a polyuronate gel similar to Ca 2 + -pectate. This result suggested that some of the Arabidopsis peroxidases have an affinity for pectin in vivo. Such a property could allow them to be properly distributed within the cell wall network. In addition, eleven cDNAs encoding an Arabidopsis peroxidase were expressed in the baculovirus-insect cell system. The capacity of the resulting recombinant peroxidases to bind Ca 2 + -pectate and Ca 2 + -alginate was also assessed. It appeared that 3 of them exhibited a Ca 2 + -pectate binding activity that was resistant to the action of NaCl. The binding of these recombinant peroxidases to Ca 2 + -alginate was much weaker than to Ca 2 + -pectate, confirming the specificity of the interaction with the pectic structure.
An apoplastic isoperoxidase from zucchini (APRX) was shown to bind strongly to polygalacturonic a... more An apoplastic isoperoxidase from zucchini (APRX) was shown to bind strongly to polygalacturonic acid in their Ca 2 ؉induced conformation. By homology modeling, we were able to identify a motif of four clustered arginines (positions 117, 262, 268, and 271) that could be responsible for this binding. To verify the role of these arginine residues in the binding process, we prepared three mutants of APRX (M1, R117S; M2, R262Q/R268S; and M3, R262Q/R268S/R271Q). APRX and the three mutants were expressed as recombinant glycoproteins by the baculovirus-insect cell system. This procedure yielded four active enzymes with similar molecular masses that were tested for their ability to bind Ca 2 ؉pectate. Recombinant wild-type APRX exhibited an affinity for the pectic structure comparable to that of the native plant isoperoxidase. The mutations impaired binding depending on the number of arginine residues that were replaced. M1 and M2 showed intermediate affinities, whereas M3 did not bind at all. This was demonstrated using an in vitro binding test and on cell walls of hypocotyl cross-sections. It can be concluded that APRX bears a Ca 2 ؉ -pectate binding site formed by four clustered arginines. This site could ensure that APRX is properly positioned in cell walls, using unesterified domains of pectins as a scaffold.
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