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PubMed2003

Molecular and biochemical characterization of VR-EILs encoding mung bean ETHYLENE INSENSITIVE3-LIKE proteins.

ETHYLENE INSENSITIVE3 (EIN3) is a transcription factor involved in the ethylene signal transduction pathway in Arabidopsis. Two full-length cDNA clones, pVR-EIL1 and pVR-EIL2, encoding EIN3-LIKE proteins were isolated by reverse transcriptase-polymerase chain reaction and by screening the cDNA library of mung bean (Vigna radiata) hypocotyls. VR-EIL1 and VR-EIL2 share 70% identity and display varying degrees of sequence conservation (39%-65%) with previously isolated EIN3 homologs from Arabidopsis, tobacco (Nicotiana tabacum) and tomato (Lycopersicon esculentum) plants. Gel retardation assay revealed that both VR-EILs were able to interact specifically with optimal binding sequence-1, the recently identified optimal binding sequence for tobacco TEIL, with the binding of VR-EIL2 being more efficient than that of VR-EIL1. Transient expression analysis using a VR-EIL::smGFP fusion gene in onion (Allium cepa) epidermal cells indicated that the VR-EIL proteins were effectively targeted to the nucleus. The fusion protein of VR-EIL2 with GAL4 DNA-binding domain strongly activated transcription of a reporter gene in yeast cells, and an essential domain for transcription-stimulating activity was localized to the amino-terminal acidic region that consists of 50 amino acid residues. In contrast with what has been previously found in EIN3- and TEIL-overexpressing Arabidopsis plants, transgenic tobacco seedlings expressing the VR-EIL genes under the control of cauliflower mosaic virus 35S promoter did not exhibit a constitutive triple response. Instead, they displayed a markedly enhanced proliferation of root hairs, one of the typical ethylene response phenotypes, and increased sensitivity to exogenous ethylene. In addition, the pathogenesis-related (PR) genes encoding beta-1,3-glucanase, osmotin, and PR1 were constitutively expressed in 35S::VR-EIL lines without added ethylene, and were hyperinduced in response to ethylene treatment. These results indicate that VR-EILs are functional in tobacco cells, thereby effectively transactivating the GCC-box-containing PR genes and enhancing sensitivity to ethylene. The possible physiological role of VR-EILs is discussed in the light of the suggestion that they are active components of the ethylene-signaling pathway and their heterologous expressions constitutively turn on a subset of ethylene responses in tobacco plants.

Active Transport, Cell Nucleus↗
PubMed2003

Functional characterization and expression of a cytosolic iron-superoxide dismutase from cowpea root nodules.

An iron-superoxide dismutase (FeSOD) with an unusual subcellular localization, VuFeSOD, has been purified from cowpea (Vigna unguiculata) nodules and leaves. The enzyme has two identical subunits of 27 kD that are not covalently bound. Comparison of its N-terminal sequence (NVAGINLL) with the cDNA-derived amino acid sequence showed that VuFeSOD is synthesized as a precursor with seven additional amino acids. The mature protein was overexpressed in Escherichia coli, and the recombinant enzyme was used to generate a polyclonal monospecific antibody. Phylogenetic and immunological data demonstrate that there are at least two types of FeSODs in plants. An enzyme homologous to VuFeSOD is present in soybean (Glycine max) and common bean (Phaseolus vulgaris) nodules but not in alfalfa (Medicago sativa) and pea (Pisum sativum) nodules. The latter two species also contain FeSODs in the leaves and nodules, but the enzymes are presumably localized to the chloroplasts and plastids. In contrast, immunoblots of the soluble nodule fraction and immunoelectron microscopy of cryo-processed nodule sections demonstrate that VuFeSOD is localized to the cytosol. Immunoblot analysis showed that the content of VuFeSOD protein increases in senescent nodules with active leghemoglobin degradation, suggesting a direct or indirect (free radical-mediated) role of the released Fe in enzyme induction. Therefore, contrary to the widely held view, FeSODs in plants are not restricted to the chloroplasts and may become an important defensive mechanism against the oxidative stress associated with senescence.

Amino Acid Sequence↗
PubMed2003

Effect of manganese toxicity on the proteome of the leaf apoplast in cowpea.

Excess manganese (Mn) supply causes formation of visible brown depositions in the cell walls of leaves of cowpea (Vigna unguiculata), which consist of oxidized Mn and oxidized phenols. Because oxidation of Mn and phenolic compounds in the leaf apoplast was proposed to be catalyzed by apoplastic peroxidases (PODs), induction of these enzymes by Mn excess was investigated. POD activity increased upon prolonged Mn treatment in the leaf tissue. Simultaneously, a significant increase in the concentration of soluble apoplastic proteins in "apoplastic washing fluid" was observed. The identity of the released proteins was systematically characterized by analysis of the apoplast proteome using two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry. Some of the identified proteins exhibit sequence identity to acidic PODs from other plants. Several other proteins show homologies to pathogenesis-related proteins, e.g. glucanase, chitinase, and thaumatin-like proteins. Because pathogenesis-related-like proteins are known to be induced by various other abiotic and biotic stresses, a specific physiological role of these proteins in response to excess Mn supply remains to be established. The specific role of apoplastic PODs in the response of plants to Mn stress is discussed.

Amino Acid Sequence↗
PubMed2004

Evidence that sucrose loaded into the phloem of a poplar leaf is used directly by sucrose synthase associated with various beta-glucan synthases in the stem.

Sucrose (Suc) synthase (SuSy) is believed to function in channeling UDP-Glc from Suc to various beta-glucan synthases. We produced transgenic poplars (Populus alba) overexpressing a mutant form (S11E) of mung bean (Vigna radiata) SuSy, which appeared in part in the microsomal membranes of the stems. Expression of SuSy in these membranes enhanced the incorporation of radioactive Suc into cellulose, together with the metabolic recycling of fructose (Fru), when dual-labeled Suc was fed directly into the phloem of the leaf. This overexpression also enhanced the direct incorporation of the glucosyl moiety of Suc into the glucan backbone of xyloglucan and increased recycling of Fru, although the Fru recycling system for cellulose synthesis at the plasma membrane might differ from that for xyloglucan synthesis in the Golgi network. These findings suggest that some of the Suc loaded into the phloem of a poplar leaf is used directly by SuSys associated with xyloglucan and cellulose synthases in the stem. This may be a key function of SuSy because the high-energy bond between the Glc and Fru moieties of Suc is conserved and used for polysaccharide syntheses in this sink tissue.

Base Sequence↗
PubMed2004

Use of the fluorescent timer DsRED-E5 as reporter to monitor dynamics of gene activity in plants.

Fluorescent proteins, such as green fluorescent protein and red fluorescent protein (DsRED), have become frequently used reporters in plant biology. However, their potential to monitor dynamic gene regulation is limited by their high stability. The recently made DsRED-E5 variant overcame this problem. DsRED-E5 changes its emission spectrum over time from green to red in a concentration independent manner. Therefore, the green to red fluorescence ratio indicates the age of the protein and can be used as a fluorescent timer to monitor dynamics of gene expression. Here, we analyzed the potential of DsRED-E5 as reporter in plant cells. We showed that in cowpea (Vigna unguiculata) mesophyll protoplasts, DsRED-E5 changes its fluorescence in a way similar to animal cells. Moreover, the timing of this shift is suitable to study developmental processes in plants. To test whether DsRed-E5 can be used to monitor gene regulation in plant organs, we placed DsRED-E5 under the control of promoters that are either up- or down-regulated (MtACT4 and LeEXT1 promoters) or constitutively expressed (MtACT2 promoter) during root hair development in Medicago truncatula. Analysis of the fluorescence ratios clearly provided more accurate insight into the timing of promoter activity.

DNA Primers↗
PubMed1993

Covalent and Noncovalent Dimers of the Cyanide-Resistant Alternative Oxidase Protein in Higher Plant Mitochondria and Their Relationship to Enzyme Activity.

Evidence for a mixed population of covalently and noncovalently associated dimers of the cyanide-resistant alternative oxidase protein in plant mitochondria is presented. High molecular mass (oxidized) species of the alternative oxidase protein, having masses predicted for homodimers, appeared on immunoblots when the sulfhydryl reductant, dithiothreitol (DTT), was omitted from sodium dodecyl sulfate-polyacrylamide gel sample buffer. These oxidized species were observed in mitochondria from soybean (Glycine max [L.] Merr. cv Ransom), Sauromatum guttatum Schott, and mung bean (Vigna radiata [L.] R. Wilcz). Reduced species of the alternative oxidase were also present in the same mitochondrial samples. The reduced and oxidized species in isolated soybean cotyledon mitochondria could be interconverted by incubation with the sulfhydryl reagents DTT and azodicarboxylic acid bis(dimethylamide) (diamide). Treatment with chemical cross-linkers resulted in cross-linking of the reduced species, indicating a noncovalent dimeric association among the reduced alternative oxidase molecules. Alternative pathway activity of soybean mitochondria increased following reduction of the alternative oxidase protein with DTT and decreased following oxidation with diamide, indicating that electron flow through the alternative pathway is sensitive to the sulfhydryl/disulfide redox poise. In mitochondria from S. guttatum floral appendix tissue, the proportion of the reduced species increased as development progressed through thermogenesis.

Journal Article↗
PubMed2005

The strigolactone germination stimulants of the plant-parasitic Striga and Orobanche spp. are derived from the carotenoid pathway.

The seeds of parasitic plants of the genera Striga and Orobanche will only germinate after induction by a chemical signal exuded from the roots of their host. Up to now, several of these germination stimulants have been isolated and identified in the root exudates of a series of host plants of both Orobanche and Striga spp. In most cases, the compounds were shown to be isoprenoid and belong to one chemical class, collectively called the strigolactones, and suggested by many authors to be sesquiterpene lactones. However, this classification was never proven; hence, the biosynthetic pathways of the germination stimulants are unknown. We have used carotenoid mutants of maize (Zea mays) and inhibitors of isoprenoid pathways on maize, cowpea (Vigna unguiculata), and sorghum (Sorghum bicolor) and assessed the effects on the root exudate-induced germination of Striga hermonthica and Orobanche crenata. Here, we show that for these three host and two parasitic plant species, the strigolactone germination stimulants are derived from the carotenoid pathway. Furthermore, we hypothesize how the germination stimulants are formed. We also discuss this finding as an explanation for some phenomena that have been observed for the host-parasitic plant interaction, such as the effect of mycorrhiza on S. hermonthica infestation.

Abscisic Acid↗
PubMed2006

The role of hydrogen peroxide-producing and hydrogen peroxide-consuming peroxidases in the leaf apoplast of cowpea in manganese tolerance.

The apoplast is considered the leaf compartment decisive for manganese (Mn) toxicity and tolerance in cowpea (Vigna unguiculata). Particularly apoplastic peroxidases (PODs) were proposed to be key enzymes in Mn toxicity-induced processes. The presented work focuses on the characterization of the role of hydrogen peroxide (H2O2)-producing (NADH peroxidase) and H2O2-consuming peroxidase (guaiacol POD) in the apoplastic washing fluid (AWF) of leaves for early stages of Mn toxicity and genotypic differences in Mn tolerance of cowpea. Leaf AWF of the Mn-sensitive cultivar (cv) TVu 91 but not of the Mn-tolerant cv 1987 showed an increase of guaiacol-POD and NADH-peroxidase activities at elevated AWF Mn concentrations. two-dimensional resolutions of AWF proteins revealed that cv TVu 91 expressed more and additional proteins at high Mn treatment, whereas Mn-tolerant cv TVu 1987 remained nearly unaffected. In both cultivars, NADH-peroxidase activity and accompanied H2O2 formation rate in vitro were significantly affected by Mn2+, p-coumaric acid, and metabolites occurring in the AWF. The total phenol concentration in the AWF was indicative of advanced stages of Mn toxicity but was rather unrelated to early stages of Mn toxicity and genotypic differences in Mn tolerance. The NADH oxidation by AWF PODs was significantly delayed or enhanced in the presence of the protein-free AWF from cv TVu 1987 or cv TVu 91, respectively. High-performance liquid chromatography analysis of AWF indicates the presence of phenols in cv TVu 1987 not observed in cv TVu 91. We conclude from our studies that the H2O2-producing NADH peroxidase and its modulation by stimulating or inhibiting phenolic compounds in the leaf apoplast play a major role for Mn toxicity and Mn tolerance in cowpea.

Fabaceae↗
PubMed2006

Identification of a mung bean arabinofuranosyltransferase that transfers arabinofuranosyl residues onto (1, 5)-linked alpha-L-arabino-oligosaccharides.

Arabinofuranosyltransferase activity was identified in Golgi membranes obtained from mung bean (Vigna radiata) hypocotyls. The enzyme transfers the arabinofuranosyl (Araf) residue from UDP-beta-L-arabinofuranose to exogenous (1, 5)-linked alpha-L-arabino-oligosaccharides labeled at their reducing ends with 2-aminobenzamide. The transferred residue was shown, using 1H-nuclear magnetic resonance spectroscopy and alpha-L-arabinofuranosidase treatment, to be alpha-L-Araf and to be linked to O-5 of the nonreducing terminal Araf residue of the acceptor oligosaccharide. The enzyme was nonprocessive because only a single Araf residue was added to the acceptor molecule. Arabino-oligosaccharides with a degree of polymerization between 3 and 8 were acceptor substrates. The 2-aminobenzamide-labeled arabino-tetra- and pentasaccharides were the most effective acceptor substrates analyzed. The enzyme has a pH optimum between 6.5 and 7.0 and its activity is stimulated by Mn2+ and Co2+ ions. The apparent Km and Vmax values of the arabinofuranosyltransferase for UDP-arabinofuranose are 243 microm and 243 pmol min(-1) mg protein(-1), respectively. The highest enzyme activity was detected in the elongating regions of mung bean hypocotyls. The data show that UDP-arabinofuranose is the donor molecule for the generation of arabino-oligosaccharides composed of Araf residues.

Arabinose↗
PubMed2006

Localization of green fluorescent protein fusions with the seven Arabidopsis vacuolar sorting receptors to prevacuolar compartments in tobacco BY-2 cells.

We have previously demonstrated that vacuolar sorting receptor (VSR) proteins are concentrated on prevacuolar compartments (PVCs) in plant cells. PVCs in tobacco (Nicotiana tabacum) BY-2 cells are multivesicular bodies (MVBs) as defined by VSR proteins and the BP-80 reporter, where the transmembrane domain (TMD) and cytoplasmic tail (CT) sequences of BP-80 are sufficient and specific for correct targeting of the reporter to PVCs. The genome of Arabidopsis (Arabidopsis thaliana) contains seven VSR proteins, but little is known about their individual subcellular localization and function. Here, we study the subcellular localization of the seven Arabidopsis VSR proteins (AtVSR1-7) based on the previously proven hypothesis that the TMD and CT sequences correctly target individual VSR to its final destination in transgenic tobacco BY-2 cells. Toward this goal, we have generated seven chimeric constructs containing signal peptide (sp) linked to green fluorescent protein (GFP) and TMD/CT sequences (sp-GFP-TMD/CT) of the seven individual AtVSR. Transgenic tobacco BY-2 cell lines expressing these seven sp-GFP-TMD-CT fusions all exhibited typical punctate signals colocalizing with VSR proteins by confocal immunofluorescence. In addition, wortmannin caused the GFP-marked prevacuolar organelles to form small vacuoles, and VSR antibodies labeled these enlarged MVBs in transgenic BY-2 cells. Wortmannin also caused VSR-marked PVCs to vacuolate in other cell types, including Arabidopsis, rice (Oryza sativa), pea (Pisum sativum), and mung bean (Vigna radiata). Therefore, the seven AtVSRs are localized to MVBs in tobacco BY-2 cells, and wortmannin-induced vacuolation of PVCs is a general response in plants.

Amino Acid Sequence↗
PubMed2006

Dynamic response of prevacuolar compartments to brefeldin a in plant cells.

Little is known about the dynamics and molecular components of plant prevacuolar compartments (PVCs) in the secretory pathway. Using transgenic tobacco (Nicotiana tabacum) Bright-Yellow-2 (BY-2) cells expressing membrane-anchored yellow fluorescent protein (YFP) reporters marking Golgi or PVCs, we have recently demonstrated that PVCs are mobile multivesicular bodies defined by vacuolar sorting receptor proteins. Here, we demonstrate that Golgi and PVCs have different sensitivity in response to brefeldin A (BFA) treatment in living tobacco BY-2 cells. BFA at low concentrations (5-10 microg mL(-1)) induced YFP-marked Golgi stacks to form both endoplasmic reticulum-Golgi hybrid structures and BFA-induced aggregates, but had little effect on YFP-marked PVCs in transgenic BY-2 cells at both confocal and immunogold electron microscopy levels. However, BFA at high concentrations (50-100 microg mL(-1)) caused both YFP-marked Golgi stacks and PVCs to form aggregates in a dose- and time-dependent manner. Normal Golgi or PVC signals can be recovered upon removal of BFA from the culture media. Confocal immunofluorescence and immunogold electron microscopy studies with specific organelle markers further demonstrate that the PVC aggregates are distinct, but physically associated, with Golgi aggregates in BFA-treated cells and that PVCs might lose their internal vesicle structures at high BFA concentration. In addition, vacuolar sorting receptor-marked PVCs in root-tip cells of tobacco, pea (Pisum sativum), mung bean (Vigna radiata), and Arabidopsis (Arabidopsis thaliana) upon BFA treatment are also induced to form similar aggregates. Thus, we have demonstrated that the effects of BFA are not limited to endoplasmic reticulum and Golgi, but extend to PVC in the endomembrane system, which might provide a quick tool for distinguishing Golgi from PVC for its identification and characterization, as well as a possible new tool in studying PVC-mediated protein traffic in plant cells.

Brefeldin A↗
PubMed1994

Characterization and Localization of a Phenoloxidase in Mung Bean Hypocotyl Cell Walls.

The occurrence of proteins able to oxidize polyphenols even in the absence of H2O2 was recently reported in mung bean (Vigna radiata L.) hypocotyl cell wall extracts (R. Goldberg, A. Chabanet, A.M. Catesson [1993] In K.G. Welinder, S.K. Rasmussen, C. Penel, H. Greppin, eds, Plant Peroxidases: Biochemistry and Physiology, pp. 296-300). Therefore, the possible presence of a laccase in the extracts was investigated using immunocytological and biochemical approaches. An enzyme catalyzing phenol oxidation in the presence of molecular O2 was extracted and purified from the cell walls. This 38-kD cationic protein, like o-diphenoloxidases, was unable to oxidize p-diphenols or p-diamines. However, it crossreacted with an anti-laccase antiserum and, like laccases, its activity was inhibited by N-cetyl-N,N,N-trimethylammonium bromide but not by ferulic acid salts. Immunolabeling data showed that the 38-kD oxidase was absent from all cellulosic cell walls. It was localized only in lignifying and lignified cell walls. This restricted localization suggests that this laccase-like phenoloxidase could participate in the lignification process but not in the primary wall stiffening, which develops in the epidermal and cortical tissues along the mung bean hypocotyl.

Journal Article↗
PubMed1994

Immobilized and Free Apoplastic Pectinmethylesterases in Mung Bean Hypocotyl.

The nature and the action pattern of apoplastic pectinmethylesterase (PME) isoforms were investigated in mung bean [Vigna radiata (L.) Wilzeck] hypocotyls. Successive extractions of neutral and alkaline PME isoforms present in hypocotyl native cell walls (referred to as PE1, PE2, PE3, PE4, with increasingly basic isoelectric points) revealed that solubilization of PE1, PE2, and PE4 did not induce any significant decrease in the cell-wall-bound PME activity. The in vitro de-esterification occurring when isolated cell walls were incubated with pectin resulted, then, from the activity of PE3. In addition, pH control of PME activity was shown to be much stronger for enzymes bound to cell walls, in their native state or reintroduced after solubilization, than for enzymes in solution. Mature cell walls showed much more activity than young cell walls, and were relatively enriched in two acidic PME isoforms missing in young cell walls. One acidic PME was also detected in the extracellular fluid. The acidic and neutral isoforms that could be easily transferred from their binding sites to their substrate might be those involved in the demethylation process developing along the mung bean hypocotyl.

Journal Article↗
PubMed1994

Biosynthesis of Cardiolipin in Plant Mitochondria.

The properties of cardiolipin synthase were investigated in mitochondria and submitochondrial fractions from etiolated mung bean (Vigna radiata L.) seedlings. Direct evidence is presented that the enzyme utilizes CDP-diacylglycerol in addition to phosphatidylglycerol for the synthesis of cardiolipin. Cardiolipin synthase had an alkaline pH optimum of about 9 and required divalent cations for activity. Maximal activity was obtained in the presence of 16 mM MnCl2. The apparent Km values for CDP-diacylglycerol and phosphatidylglycerol were 0.8 and 50 [mu]M, respectively. Cardiolipin synthase was localized predominantly in the inner membrane of mung bean mitochondria and displayed a substrate species specificity. Highest activities were measured with the dioleoyl species of both CDP-diacylglycerol and phosphatidylglycerol, and somewhat lower activities were measured with mixed species of the two substrates containing a palmitoyl and an oleoyl group. On the other hand, the cardiolipin synthase hardly used the dipalmitoyl species and strongly discriminated against CDP-dipalmitoylglycerol from a mixture with CDP-dioleoylglycerol.

Journal Article↗
PubMed1995

Magnesium Adenosine 5[prime]-Triphosphate-Energized Transport of Glutathione-S-Conjugates by Plant Vacuolar Membrane Vesicles.

By characterization of the uptake of glutathione-S-conjugates, principally dinitrophenyl-S-glutathione (DNP-GS), by vacuolar membrane vesicles, we demonstrate that a subset of energy-dependent transport processes in plants are not H+-coupled but instead are directly energized by MgATP. The most salient features of this transport pathway are: (a) its specific, obligate requirement for MgATP as energy source; (b) the necessity for hydrolysis of the [gamma]-phosphate of MgATP for uptake; (c) the insensitivity of uptake to uncouplers of the transtonoplast H+ gradient (carbonylcyanide 4-trifluoromethoxyphenylhydrazone, gramicidin-D, and NH4Cl); (d) its pronounced sensitivity to vanadate and partial inhibition by vinblastine and verapamil; (e) the lack of chemical modification of DNP-GS either during or after transport; (f) the capacity of S-conjugates of chloroacetanilide herbicides, such as metolachlor-GS, but not free herbicide, to inhibit uptake; and (g) the ability of vacuolar membrane vesicles purified from a broad range of plant species, including Arabidopsis, Beta, Vigna, and Zea, to mediate MgATP-dependent, H+-electrochemical potential difference-independent DNP-GS uptake. On the basis of these findings it is proposed that the transport of DNP-GS across the vacuolar membrane of plant cells is catalyzed by a glutathione-conjugate transporter that directly employs MgATP rather than the energy contained in the transtonoplast H+-electrochemical potential difference to drive uptake. The broad distribution of the vacuolar DNP-GS transporter and its inhibition by metolachlor-GS are consistent with the notion that it plays a general role in the vacuolar sequestration of glutathione-conjugable cytotoxic agents.

Journal Article↗
PubMed1995

Characterization and Physiological Function of Class I Low-Molecular-Mass, Heat-Shock Protein Complex in Soybean.

Examination of an ammonium sulfate-enriched fraction (70-100% saturation) of heat-shock proteins (HSPs) by nondenaturing polyacrylamide gel electrophoresis revealed the presence of a high molecular mass complex (280 kD) in soybean (Glycine max) seedlings. This complex cross-reacted with antibodies raised against soybean class I low-molecular-mass (LMW) HSPs. Dissociation of the complex by denaturing polyacrylamide gel electrophoresis showed the complex to contain at least 15 polypeptides of the 15-to 18-kD class I LMW HSPs that could be detected by staining, radiolabeling, and western blotting. A similar LMW-HSP complex was observed in mung bean (Vigna radiata L.; 295 kD), in pea (Pisum sativum L.; 270 kD), and in rice (Oryza sativa L.; 310 kD). The complex was stable under high salt conditions (250 mM KCI), and the integrity was not affected by 1% Nonidet P-40 and 3 [mu]g/ML RNase treatment. The size of the isolated HSP complex in vitro was conserved to 55[deg]C; however, starting at 37.5[deg]C, it changed to higher molecular forms in the presence of soluble proteins. The isolated HSP complex was able to protect up to 75% of the soluble proteins from heat denaturation in vitro.

Journal Article↗
PubMed1995

1-Chloro-2,4-Dinitrobenzene-Elicited Increase in Vacuolar Glutathione-S-Conjugate Transport Activity.

Unlike most other characterized organic solute transport in plants, uptake of the model compound S-(2,4-dinitrophenyl)glutathione (DNP-GS) and related glutathione-S-conjugated by vacuolar membranes is directly energized by MgATP. Here we show that exogenous application of the DNP-GS precursor 1-chloro-2,4-dinitrobenzene (CDNB) to seedlings of Vigna radiata (mung bean) increases the capacity of vacuolar membrane vesicles isolated from hypocotyls for MgATP-dependent DNP-GS transport in vitro. Our findings are 4-fold: (a) Pretreatment of seedlings with CDNB causes a progressive increase in MgATP-dependent DNP-GS uptake by vacuolar membrane vesicles, whereas the same range of CDNB concentrations causes only marginal stimulation when the compound benoxacor [4-(dichloroacetyl)-3,4-dihydro-3-methyl-2H-1,4-benzoxazine] is included in the pretreatment solution. (b) Increased DNP-GS uptake is accompanied by a proportionate and selective increase in Vmax(DNP-GS) but not in Km(DNP-GS) or Km(MgATP). (c) CDNB-enhanced DNP-GS uptake is not accompanied by a change in the density profile or sidedness of the vacuolar membrane fraction. (d) Basal and CDNB-enhanced DNP-GS uptake are indistinguishable in terms of their inhibitor profiles. On the basis of these findings, it is inferred that pretreatment with CDNB increases the amount or recruitment of functional transporter into the vacuolar membrane and that agents such as benoxacor antagonize the effects otherwise seen with CDNB in this sytem.

Journal Article↗
PubMed1995

Morphological and Molecular Characteristics of Host-Conditioned Ineffective Root Nodules in Cowpea.

In cowpea (Vigna unguiculata [L.] Walp.) a recessive allele, designated cpi, elicits the formation of non-N2-fixing nodules with all bacterial isolates tested. Comparisons of mutant and wild-type nodules demonstrated that the ineffective nodules were anatomically similar to the wild type and contained both infection threads and bacteroids. Ineffective nodules were smaller, however, largely because of the reduced size of the infected cells. Additionally, the number of bacteroids was reduced and senescence occurred prematurely in the infected cells. Grafting studies demonstrated that the defect in nodule development was controlled by the root rather than the shoot. Northern analysis of four nodulin genes indicated that in the ineffective nodules transcript levels of the early nodulin VuENOD2 were initially reduced but were equivalent to wild-type nodules by 21 d. In contrast, transcript levels of the early nodulin VuB were initially similar in both genotypes but as the nodules matured the mRNA levels declined more slowly in the ineffective nodules. The late nodulins leghemoglobin and uricase were expressed in the ineffective nodules but at greatly reduced levels. Thus, the cpi-conditioned defect in nodulation is associated with impaired bacteroid maturation and maintenance, altered nodulin expression, and accelerated senescence.

Journal Article↗