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PubMed1998

nodD2 of Rhizobium sp. NGR234 is involved in the repression of the nodABC operon.

Transcriptional regulators of the lysR family largely control the expression of bacterial symbiotic genes. Rhizobium sp. NGR234 contains at least four members of this family: two resemble nodD, while two others are more closely related to syrM. Part of the extremely broad host range of NGR234 can be attributed to nodD1, although the second gene shares a high degree of DNA sequence homology with nodD2 of R. fredii USDA191. A nodD2 mutant of NGR234 was constructed by insertional mutagenesis. This mutant (NGR omega nodD2) was deficient in nitrogen fixation on Vigna unguiculata and induced pseudonodules on Tephrosia vogelii. Several other host plants were tested, but no correlation could be drawn between the phenotype and nodule morphology. Moreover, nodD2 has a negative effect on the production of Nod factors: mutation of this gene results in a fivefold increase in Nod factor production. Surprisingly, while the structure of Nod factors from free-living cultures of NGR omega nodD2 remained unchanged, those from V. unguiculata nodules induced by the same strain are non-fucosylated and have a lower degree of oligomerization. In other words, developmental regulation of Nod factor production is also abolished in this mutant. Competitive RNA hybridizations, gene fusions and mobility shift assays confirmed that nodD2 downregulates expression of the nodABC operon.

Artificial Gene Fusion↗
PubMed1995

Correlation between the induction of a gene for delta 1-pyrroline-5-carboxylate synthetase and the accumulation of proline in Arabidopsis thaliana under osmotic stress.

The isolation and characterization is reported of a cDNA for delta 1-pyrroline-5-carboxylate (P5C) synthetase (cAtP5CS), an enzyme involved in the biosynthesis of proline, from a cDNA library prepared from a dehydrated rosette plant of Arabidopsis thaliana. Southern blot analysis suggested that only one copy of the corresponding gene (AtP5CS) is present in A. thaliana. The deduced amino acid sequence of the P5CS protein (AtP5CS) from A. thaliana exhibited 74% homology to that of the P5CS from Vigna aconitifolia. Northern blot analysis revealed that the gene for P5CS was induced by dehydration, high salt and treatment with ABA, while it was not induced by heat or cold treatment. Moreover, the simultaneous accumulation of proline was observed as a result of the former treatments in A. thaliana. A cDNA for P5C reductase (cAtP5CR) was also isolated from A. thaliana and Northern blot analysis was performed. The AtP5CR gene was not induced to a significant extent by dehydration or high-salt stress. These observations suggest that the AtP5CS gene plays a principal role in the biosynthesis of proline in A. thaliana under osmotic stress.

1-Pyrroline-5-Carboxylate Dehydrogenase↗
PubMed1999

PR-1 protein inhibits the differentiation of rust infection hyphae in leaves of acquired resistant broad bean.

Treatment of broad bean leaves with salicylic acid (SA) or 2, 6-dichloro-isonicotinic acid (DCINA) induces resistance against the rust fungus Uromyces fabae resulting in reduced rust pustule density. Light-microscopy studies showed that in induced resistant plants the rust fungus is inhibited immediately after penetration through the stomatal pore. The differentiation of infection structures growing within the intercellular space of the leaf, i.e. infection hyphae and haustorial mother cells, is inhibited. Furthermore, low-temperature scanning electron microscopy studies of freeze fractures revealed protrusions at the tips of infection hyphae growing in induced resistant broad bean leaves. Treatment of in vitro-differentiating rust infection structures with intercellular fluids (IFs) from induced resistant plants confirmed that the fungus is sensitive towards an apoplastic anti-fungal activity only after having formed appressoria. Other legume rusts such as U. vignae and U. appendiculatus were likewise inhibited in the presence of IF from SA-treated broad bean leaves. Heterologous antibodies were used to study changes in the extracellular pathogenesis-related (PR) protein pattern after resistance induction. Western blots indicated that chitinases and beta-1,3-glucanases were present in both induced and control plants. In contrast, PR-1 proteins were newly synthesized in response to SA or DCINA application. The major induced PR-1 protein was purified and exhibited strong differentiation-inhibiting activity towards U. fabae infection structures. We conclude that the inhibition of rust infection hyphae in acquired resistant broad bean plants is mainly due to the anti-fungal activity of this induced basic PR-1 protein.

Antifungal Agents↗
PubMed1999

Asparaginyl endopeptidase (VmPE-1) and autocatalytic processing synergistically activate the vacuolar cysteine proteinase (SH-EP).

A vacuolar cysteine proteinase, designated SH-EP, is synthesized in cotyledons of germinated Vigna mungo seeds and is responsible for degradation of the seed proteins accumulated in protein bodies (protein storage vacuoles). SH-EP belongs to the papain proteinase family and has a large N-terminal prosegment consisting of 104 amino acid residues and a C-terminal prosegment of 10 amino acid residues. It has been suggested that an asparaginyl endopeptidase, V. mungo processing enzyme 1 (VmPE-1), is involved in the N-terminal post-translational processing of SH-EP. The recombinant proform of SH-EP (rSH-EP) was produced in Escherichia coli cells, purified to homogeneity and refolded by stepwise dialysis. 31P-NMR analysis of intact germinated cotyledons revealed that the vacuolar pH of cotyledonary cells changes from 6.04 to 5.47 during seed germination and early seedling growth. rSH-EP was converted in vitro to the mature form through autocatalytic processing at a pH mimicking the vacuolar pH at the mid and late stages of seed germination, but not at the pH of the early stage. VmPE-1 accelerated the rate of processing of rSH-EP in vitro at the pH equivalent to the vacuolar pH at the early and mid stages of germination. In addition, the cleavage sites of the in vitro processed intermediates and the mature form of SH-EP were identical to those of SH-EP purified from germinated cotyledons of V. mungo. We propose that the asparaginyl endopeptidase (VmPE-1)-mediated processing mainly functions in the activation of proSH-EP at the early stage of seed germination, and both VmPE-1-mediated and autocatalytic processings function synergistically in the activation of proSH-EP in cotyledons at the mid and late stages.

Amino Acid Sequence↗
PubMed2005

Specificity patterns indicate that auxin exporters and receptors are the same proteins.

A study of transport and action of synthetic auxin analogues can help to identify transporters and receptors of this plant hormone. Both aspects--transportability and action on growth--were tested with 2-naphthoxyacetic acid (2-NOA) and compared across several plant species. 2-NOA stimulates elongation effectively at low concentrations in petioles of the gymnosperm Ginkgo biloba L., in hypocotyls or internodes of the dicot legumes, mung bean (Vigna mungo L.) and pea (Pisum sativum L.), in cotyledons of onion (Allium cepa L.) and in leaf bases of chive (Allium schoenoprasum L.), the latter two of the monocot order Asparagales. In contrast, elongation of coleoptile segments of maize (Zea mays L.) is poorly responsive to 2-NOA. Significant auxin-like transport of 2-NOA was observed in segments of mung bean hypocotyls, pea internodes, and chive leaf bases, but not in segments of the grass coleoptiles. Thus, for the two assays, elongation and polar transportability, the same difference in ligand specificity was observed between the grass and all other species assayed. This finding supports the hypothesis that a common protein mediates auxin efflux as well as auxin action on elongation.

Allium↗
PubMed2003

Bioremediation of crude oil polluted soil by the white rot fungus, Pleurotus tuberregium (Fr.) Sing.

Bioremediation has become an attractive alternative to physicochemical methods of remediation of polluted sites. White rot fungi (WRF) are increasingly being investigated and used in bioremediation, because of their ability to degrade an extremely diverse range of very persistent or toxic environmental pollutants. The white rot fungus, Pleurotus tuberregium, was examined for its ability to ameliorate crude oil polluted soil. This was inferred from the ability of the polluted soil to support seed germination and seedling growth in Vigna unguiculata, at 0, 7 and 14 days post treatment. Results obtained from the present study showed that bioremediation of soil contaminated with crude oil was possible, especially when the fungus had been allowed to establish and fully colonize the substrate mixed with the soil. There were significant improvements in % germination, plant height and root elongation values of test plants, when seeds were planted 14 days post soil treatment. At 1 to 5% crude oil pollution, % germination values were comparable with the values in control plants in the 14 days treatment, and significantly higher than values obtained in the day 0 treatment. Also, at the highest level of crude oil pollution (15%), there was about 25% improvement in % germination value over the 0 day treatment. This trend of improvement in values was also observed for plant height, root elongation and biomass accumulation as well as decreased total hydrocarbon content.

Biodegradation, Environmental↗
PubMed2005

Mechanoreceptors rather than sedimentable amyloplasts perceive the gravity signal in hypergravity-induced inhibition of root growth in azuki bean.

Elongation of primary roots of azuki bean (Vigna angularis Ohwi et Ohashi) was suppressed under hypergravity conditions produced by centrifugation, such that the growth rate decreased in proportion to the logarithm of the magnitude of the gravity. The removal of the root cap did not influence the hypergravity-induced inhibition of root growth, although it completely inhibited the gravitropic root curvature. Lanthanum and gadolinium, blockers of mechanoreceptors, nullified the growth-inhibitory effect of hypergravity. These results suggest that the gravity signal for the hypergravity-induced inhibition of root growth is perceived independently from that of gravitropism, which involves amyloplasts as statoliths. Horizontal and basipetal hypergravity suppressed root growth as did acropetal hypergravity, all of which were nullified by the presence of lanthanum or gadolinium. These findings suggest that mechanoreceptors on the plasma membrane perceive the gravity signal independently of the direction of the stimuli and roots may utilise it to regulate their growth rate.

Centrifugation↗
PubMed2006

Fragments of ATP synthase mediate plant perception of insect attack.

Plants can perceive a wide range of biotic attackers and respond with targeted induced defenses. Specificity in plant non-self-recognition occurs either directly by perception of pest-derived elicitors or indirectly through resistance protein recognition of host targets that are inappropriately proteolyzed. Indirect plant perception can occur during interactions with pathogens, yet evidence for analogous events mediating the detection of insect herbivores remains elusive. Here we report indirect perception of herbivory in cowpea (Vigna unguiculata) plants attacked by fall armyworm (Spodoptera frugiperda) larvae. We isolated and identified a disulfide-bridged peptide (+ICDINGVCVDA-), termed inceptin, from S. frugiperda larval oral secretions that promotes cowpea ethylene production at 1 fmol leaf(-1) and triggers increases in the defense-related phytohormones salicylic acid and jasmonic acid. Inceptins are proteolytic fragments of chloroplastic ATP synthase gamma-subunit regulatory regions that mediate plant perception of herbivory through the induction of volatile, phenylpropanoid, and protease inhibitor defenses. Only S. frugiperda larvae that previously ingested chloroplastic ATP synthase gamma-subunit proteins and produced inceptins significantly induced cowpea defenses after herbivory. Digestive fragments of an ancient and essential plant enzyme, inceptin functions as a potent indirect signal initiating specific plant responses to insect attack.

Amino Acid Sequence↗
PubMed1976

Hydrogen evolution: A major factor affecting the efficiency of nitrogen fixation in nodulated symbionts.

Nitrogenase-dependent hydrogen evolution from detached legume nodules and from reaction mixtures containing cell-free nitrogenase has been well established, but the overall effect of hydrogen evolution on the efficiency of nitrogen fixation in vivo has not been critically assessed. This paper describes a survey which revealed that hydrogen evolution is a general phenomenon associated with nitrogen fixation by many nodulated nitrogen-fixing symbionts. An evaluation of the magnitude of energy loss in terms of the efficiency of electron transfer to nitrogen, via nitrogenase, in excised nodules suggested that hydrogen production may severely reduce nitrogen fixation in many legumes where photosynthate supply is a factor limiting fixation. With most symbionts, including soybeans, only 40-60% of the electron flow to nitrogenase was transferred to nitrogen. The remainder was lost through hydrogen evolution. In situ measurements of hydrogen evolution and acetylene reduction by nodulated soybeans confirmed the results obtained with excised nodules. In an atmosphere of air, a major portion of the total electron flux available for the reduction of atmospheric nitrogen by either excised nodules or intact nodulated plants was utilized in the production of hydrogen gas. Some non-leguminous symbionts, such as Alnus rubra, and a few legumes (i.e., Vigna sinensis) apparently have evolved mechanisms of minimizing net hydrogen production, thus increasing their efficiency of electron transfer to nitrogen. Our results indicate that the extent of hydrogen evolution during nitrogen reduction is a major factor affecting the efficiency of nitrogen fixation by many agronomically important legumes.

Journal Article↗
PubMed1979

Further examination of presumptive Rhizobium trifolii mutants that nodulate Glycine max.

Two recent reports described the isolation of derivatives of a Rhizobium trifolii strain that had gained the ability to nodulate Glycine max and Vigna radiata and that had demonstrated altered patterns of carbon source utilization, free-living nitrogen fixation, and hydrogen uptake. More extensive characterization of these strains now supports the conclusion that these strains are R. japonicum and are not derived from the putative parent R. trifolii.

Journal Article↗
PubMed1984

Formation of cyanide from carbon 1 of 1-aminocyclopropane-1-carboxylic acid during its conversion to ethylene.

It has been shown that 1-aminocyclopropane-1-carboxylic acid (ACC) is the immediate precursor of ethylene, which is derived from C-2 and C-3 of ACC. When [1-(14)C]ACC was administered to etiolated mungbean (Vigna radiata) hypocotyls, approximately 16% of the ACC was converted to ethylene and about 10% of the radioactivity was converted to [(14)C]asparagine in 7 hr. In etiolated epicotyls of common vetch (Vicia sativa), after 7 hr about 14% of the ACC was converted to ethylene and 16% of the radioactivity was converted to beta-cyanoalanine plus gamma-glutamyl-beta-cyanoalanine. Itis known that in most plants cyanide is metabolized to asparagine via the intermediate beta-cyanoalanine, whereas in a fewplants such as V. sativa, beta-cyanoalanine is converted to the conjugate gamma-glutamyl-beta-cyanoalanine. We confirmed that [(14)C]cyanide was metabolized into [(14)C]asparagine in mungbean and into [(14)C]cyanoalanine plus its conjugate in V. sativa. Moreover, after feeding plant tissue with [1-(14)C]ACC, [(14)C]asparagine isolated from mungbean and beta-[(14)C]cyanoalanine from V. sativa were labeled in the C-4 position, as would be expected if these two compounds were derived from [(14)C]cyanide. When the conversion of ACC to ethylene in V. sativa tissue was inhibited by high temperature (41 degrees C), the conversion of [1-(14)C]ACC to beta-[(14)C]cyanoalanine and gamma-glutamyl-beta-[(14)C]cyanoalanine was similarly inhibited. When [carboxyl-(14)C]ACC was administered to mungbean and V. sativa, (14)CO(2) was recovered in an amount equivalent to the amount of ethylene produced. These data indicate that in the conversion of ACC to ethylene the carboxyl group yields CO(2), and C-1 is released as HCN.

Journal Article↗
PubMed1985

Conservation of nodulation genes between Rhizobium meliloti and a slow-growing Rhizobium strain that nodulates a nonlegume host.

Parasponia, a woody member of the elm family, is the only nonlegume genus whose members are known to form an effective nitrogen-fixing symbiosis with a Rhizobium species. The bacterial strain RP501 is a slow-growing strain of Rhizobium isolated from Parasponia nodules. Strain RP501 also nodulates the legumes siratro (Macroptilium atropurpureum) and cowpea (Vigna unguiculata). Using a cosmid clone bank of RP501 DNA, we isolated a 13.4-kilobase (kb) EcoRI fragment that complemented insertion and point mutations in three contiguous nodulation genes (nodABC) of Rhizobium meliloti, the endosymbiont of alfalfa (Medicago sativa). The complemented R. meliloti nod mutants induced effective nitrogen-fixing nodules on alfalfa seedlings but not on siratro, cowpeas, or Parasponia. The cloned RP501 nodulation locus hybridized to DNA fragments carrying the R. meliloti nodABC genes. A 3-kb cluster of Tn5 insertion mutations on the RP501 13.4-kb EcoRI fragment prevented complementation of R. meliloti nodABC mutations.

Journal Article↗
PubMed1987

K regulates bacteroid-associated functions of Bradyrhizobium.

Cowpea Bradyrhizobium 32H1 cells, when grown under 0.2% O(2), synthesize nitrogenase, as well as a methylammonium (ammonium) transport system and an electrogenic K(+)/H(+) antiporter. This effect was seen in growth medium containing 8-12 mM K(+) but not with 50 muM K(+). Addition of K(+) to cells growing under low O(2) tensions in low-K(+) medium led to various phenotypic properties associated with bacteroids, including the ability to reduce acetylene, induction of an ammonium transport carrier and the K(+)/H(+) antiporter, and increased synthesis of two heme-biosynthetic enzymes, delta-aminolevulinate synthase and delta-aminolevulinate dehydratase. K(+) addition caused the repression of glutamine synthetase and of capsular polysaccharide synthesis, functions related to the free-living state. A similar pattern of regulation was observed in Bradyrhizobium japonicum. In addition, K(+)-mediated depression in Bradyrhizobium 32H1 was inhibited by exudate of Vigna unguiculata, its host plant. We conclude that K(+) ions, in addition to low O(2) tension, are needed for the expression of several bacteroid-related functions in bradyrhizobia and thus are a major controlling influence in bacteroid development.

Journal Article↗
PubMed1987

Rhizobium symbiotic genes required for nodulation of legume and nonlegume hosts.

Parasponia, a woody member of the elm family, is the only nonlegume genus whose members are known to form an effective nitrogen-fixing symbiosis with Bradyrhizobium or Rhizobium species. The Bradyrhizobium strain Rp501, isolated from Parasponia nodules, also nodulates the legumes siratro (Macroptilium atropurpureum) and cowpea (Vigna unguiculata). To test whether some of the same genes are involved in the early stages of legume and nonlegume nodulation, we generated transposon Tn5 insertions in the region of three evolutionarily conserved genes (nodA, nodB, and nodC) required for legume nodulation in several Rhizobium and Bradyrhizobium species. Assays of these mutant Rp501 strains on legume hosts and Parasponia seedlings established that nodABC are required for nodulation of legume and nonlegume hosts, indicating that nonlegumes and legumes can respond to the same bacterial signal(s). In addition, a strain carrying a Tn5 insertion adjacent to the nodABC genes vigorously nodulated Rp501 legume hosts but was incapable of nodulating Parasponia, possibly identifying a nonlegume-specific nodulation function.

Journal Article↗
PubMed1990

Genomic RNA of an insect virus directs synthesis of infectious virions in plants.

Newly synthesized virions of flock house virus (FHV), an insect nodavirus, were detected in plant cells inoculated with FHV RNA. FHV was found in whole plants of barley (Hordeum vulgare), cowpea (Vigna sinensis), chenopodium (Chenopodium hybridum), tobacco (Nicotiana tabacum), and Nicotiana benthamiana and in protoplasts derived from barley leaves. Virions produced in plants contained newly synthesized RNA as well as newly synthesized capsid protein. These results show that the intracellular environment in these plants is suitable for synthesis of a virus normally indigenous only to insects. Such synthesis involves, minimally, translation of viral RNA, RNA replication, and virion assembly. Inoculation of barley protoplasts with FHV virions resulted in synthesis of small amounts of progeny virions, suggesting that FHV virions are capable of releasing their RNA in plant cells. In N. benthamiana, virions resulting from inoculation with RNA were detected not only in inoculated leaves but also in other leaves of inoculated plants, suggesting that virions could move in this plant species. Such movement probably occurs by a passive transport through the vascular system rather than by an active transport involving mechanisms that have evolved for plant viruses.

Animals↗
PubMed1991

Localization of xanthine dehydrogenase in cowpea root nodules: implications for the interaction between cellular compartments during ureide biogenesis.

Immunocytochemistry was used to assess the location of xanthine dehydrogenase (EC 1.1.1.204) in the infected region of nodules of cowpea (Vigna unguiculata [L.] Walpers cv. Queen Anne Blackeye). Polyclonal antibodies raised against purified cowpea xanthine dehydrogenase were used to localize this enzyme at the electron microscopic level. Sparse nonspecific labeling was observed after treatment of nodule sections with preimmune serum. Although immune serum cross-reacted with the ground cytoplasm of both infected and uninfected cells, significantly more labeling was observed in the uninfected cells. No labeling above background was observed in peroxisomes, mitochondria, proplastids, endoplasmic reticulum, cytoplasmic or peribacteroid membranes, peribacteroid spaces, or bacteroids. The enzyme is soluble and not present in any organelle or membrane. The greater concentration of xanthine dehydrogenase in the uninfected cells suggests that xanthine or a precursor to xanthine, rather than uric acid, is the intermediate that moves from infected to uninfected cells during ureide biogenesis.

Journal Article↗
PubMed1993

Purification, characterization, and cDNA cloning of an NADPH-cytochrome P450 reductase from mung bean.

We report here the isolation and deduced amino acid sequence of the flavoprotein, NADPH-cytochrome P450 (cytochrome c) reductase (EC 1.6.2.4), associated with the microsomal fraction of etiolated mung bean seedlings (Vigna radiata var. Berken). An 1150-fold purification of the plant reductase was achieved, and SDS/PAGE showed a predominant protein band with an apparent molecular mass of approximately 82 kDa. The purified plant NADPH-P450 reductase gave a positive reaction as a glycoprotein, exhibited a typical flavoprotein visible absorbance spectrum, and contained almost equimolar quantities of FAD and FMN per mole of enzyme. Specific antibodies revealed the presence of unique epitopes distinguishing the plant and mammalian flavoproteins as demonstrated by Western blot analyses and inhibition studies. Peptide fragments from the purified plant NADPH-P450 reductase were sequenced, and degenerate primers were used in PCR amplification reactions. Overlapping cDNA clones were sequenced, and the deduced amino acid sequence of the mung bean NADPH-P450 reductase was compared with equivalent enzymes from mammalian species. Although common flavin and NADPH-binding sites are recognizable, there is only approximately 38% amino acid sequence identity. Surprisingly, the purified mung bean NADPH-P450 reductase can substitute for purified rat NADPH-P450 reductase in the reconstitution of the mammalian P450-catalyzed 17 alpha-hydroxylation of pregnenolone or progesterone.

Amino Acid Sequence↗
PubMed1995

A mechanical strain-induced 1-aminocyclopropane-1-carboxylic acid synthase gene.

Ethylene production is observed in all higher plants, where it is involved in numerous aspects of growth, development, and senescence. 1-Aminocyclopropane-1-carboxylic acid synthase (ACC synthase; S-adenosyl-L-methionine methylthioadenosine-lyase, EC 4.4.1.14) is the key regulatory enzyme in the ethylene biosynthetic pathway. We are reporting an ACC synthase gene in Vigna radiata (mung bean) that is inducible by mechanical strain. The ACC synthase cDNA AIM-1 was induced by mechanical strain within 10 min, reaching a maximum at 30 min, showing a dramatic reduction after 60 min, and showing no detectable message by 3 hr. The kinetics of induction for AIM-1 was similar to a mechanical strain-induced calmodulin (MBCaM-1) in V. radiata, whereas the kinetics of its decline from maximum was different. When plants were subjected to calcium-deficient conditions, supplemental calcium, calcium chelators, calcium storage releasers, calcium ionophore, or calmodulin antagonists, there was no effect on AIM-1, indicating that the mechanical strain-induced AIM-1 expression is a calcium-independent process. Induction of MBCaM-1 in all cases behaved in the same way as AIM-1, suggesting that they share similar mechanically activated cis- and/or trans-acting elements in their promoter.

Calcium↗