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At least 685 records · Page 38Linked to original sources
PubMed1996

Novel drought-inducible genes in the highly drought-tolerant cowpea: cloning of cDNAs and analysis of the expression of the corresponding genes.

Ten cDNAs of genes that were induced by dehydration stress were cloned by differential screening from the highly drought-tolerant legume, cowpea (Vigna unguiculata), a major crop in West Africa. The clones were collectively named CPRD (cowpea clones responsive to dehydration). Northern blot analysis revealed that nine of the CPRD genes were induced by dehydration stress, but the timing of induction of mRNA synthesis varied among the CPRD genes. We analyzed the effects of other environmental stresses on the expression of the CPRD8, CPRD14 and CPRD22 genes, and we found that these genes were strongly induced by high-salinity stress but not by cold or heat stress. Drought-stressed cowpea plants accumulated abscisic acid (ABA) to a level that was 160 times higher than that in unstressed plants. The CPRD8 and CPRD22 genes were induced to a significant extent by the application of exogenous ABA but the CPRD14 gene was not. These results indicate the existence of at least two signal-transduction pathways between the detection of water stress and the expression of CPRD genes in cowpea. Sequence analysis of CPRD8 and CPRD22 cDNAs revealed that they encoded putative proteins that were related to old yellow enzyme and group 2 LEA proteins, respectively. The protein encoded by CPRD14 exhibited sequence homology to dihydroflavonol-4-reductase (DFR) and vestitone reductase (VR). Old yellow enzyme, DFR and VR have not been identified as drought-inducible proteins in other plants, whereas LEA genes have been well characterized as drought-inducible genes. The various gene products might function to protect cells from environmental stress.

Abscisic Acid↗
PubMed1997

Accumulation of a glycoprotein that is homologous to a seed storage protein in mung bean hypocotyls at the late stage of tissue elongation.

Physiological changes were examined in the amount of a 50-kDa glycoprotein (gp50) that was recovered in a nuclear fraction from hypocotyls of mung bean (Vigna radiata) seedlings. Immunoblot analysis indicated that the glycoprotein was present in hypocotyls and epicotyls from 4- and 5-day-old seedlings but not in hypocotyls from 2-day-old seedlings. The glycoprotein was not detected in leaves or roots. When we divided hypocotyls of 3-day-old seedlings into elongating region (0 to 1.5 cm below the cotyledon) and the mature region, we found gp50 in the mature region only. The results suggest that the 50-kDa glycoprotein is synthesized de novo and accumulates at the late stage during elongation of cells in the hypocotyl. Furthermore, an antibody specific to gp50 reacted with a major 50-kDa protein in cotyledons, which is known as a storage protein in mung bean cotyledon. Eighteen amino acid residues among 22 amino-terminal residues of gp50 were identical to those of the storage protein from cotyledon. A peptide map of the glycoprotein after digestion with V8 protease was similar to that of the storage protein. Overall, our findings suggest that the glycoprotein recovered in the nuclear fraction is an isoform of the seed storage protein that is expressed only in the mature cells of hypocotyls and epicotyls.

Allergens↗
PubMed1998

An increase in apparent affinity for sucrose of mung bean sucrose synthase is caused by in vitro phosphorylation or directed mutagenesis of Ser11.

A mutational analysis of mung bean (Vigna radiata Wilczek) sucrose synthase was performed by site-directed mutagenesis of the recombinant protein expressed in Escherichia coli, in which two different acidic amino acid residues (Asp or Glu) were introduced at Ser11 (S11D, S11E). Only the wild-type enzyme (Ser11) was phosphorylated in vitro by a Ca(2+)-dependent protein kinase from soybean root nodules, suggesting that this is the specific target residue in mung bean sucrose synthase. The apparent affinity for sucrose was increased in this phosphorylated enzyme and also in the S11D and S11E mutant enzymes, although the affinities for UDP-glucose and fructose were similar in the wild-type, phosphorylated wild-type, and mutant enzymes. These results suggest that a monoanionic (1-) side chain at position 11 mimics the Ser11-P2- residue to bind and cleave sucrose for the synthesis of UDP-glucose. Since the S11E mutant enzyme showed the lowest K(m) (sucrose) and the highest catalytic efficiency of the recombinant proteins, the enzymic properties of this S11E mutant were further characterized. The results showed that replacement of Ser11 with Glu11 modestly protected the sucrose synthesis activity against phenolic glycosides and altered the enzyme nucleotide specificity. We postulate that the introduction of negative charge at Ser11 is possibly involved in the enzymatic perturbation of sucrose synthase.

Amino Acid Sequence↗
PubMed1999

Hypergravity increases the molecular mass of xyloglucans by decreasing xyloglucan-degrading activity in azuki bean epicotyls.

Elongation growth of dark-grown azuki bean (Vigna angularis Ohwi et Ohashi cv. Takara) epicotyls was suppressed by hypergravity at 30 x g and above. Acceleration at 300 x g significantly decreased the mechanical extensibility of cell walls. The amounts of cell wall polysaccharides (pectin, hemicellulose-II and cellulose) per unit length of epicotyls increased under the hypergravity condition. Hypergravity also increased the amounts and the weight-average molecular mass of xyloglucans in the hemicellulose-II fraction, while decreasing the activity of xyloglucan-degrading enzymes extracted from epicotyl cell walls. These results suggest that hypergravity increases the amounts and the molecular mass of xyloglucans by decreasing xyloglucan-degrading activity. Modification of xyloglucan metabolism as well as the thickening of cell walls under hypergravity conditions seems to be involved in making the cell wall mechanically rigid, thereby inhibiting elongation growth of azuki bean epicotyls.

Cell Wall↗
PubMed2000

Changes in the apoplastic pH are involved in regulation of xyloglucan breakdown of azuki bean epicotyls under hypergravity conditions.

Hypergravity inhibited elongation growth of azuki bean (Vigna angularis Ohwi et Ohashi) epicotyls by decreasing the mechanical extensibility of cell walls via the increase in the molecular mass of xyloglucans [Soga et al. (1999) Plant Cell Physiol. 40: 581]. Here, we report that the pH value of the apoplastic fluid in epicotyls increased from 5.8 to 6.6 by hypergravity (300 x g) treatment. When the xyloglucan-degrading enzymes extracted from cell walls of the 1 x g control epicotyls were assayed in buffer at pH 6.6 and 5.8, the activity at pH 6.6 was almost half of that at pH 5.8. In addition, when enzymically active cell wall preparations obtained from 1 x g control epicotyls were autolyzed in buffer at pH 5.8 and 6.6 and then xyloglucans were extracted from the autolyzed cell walls, the molecular mass of xyloglucans incubated at pH 5.8 decreased during the autolysis, while that at pH 6.6 did not change. Thus, the xyloglucans were not depolymerized by autolysis at the pH value (6.6) observed in the hypergravity-treated epicotyls. These findings suggest that in azuki bean epicotyls, hypergravity decreases the activities of xyloglucan-degrading enzymes by increasing the pH in the apoplastic fluid, which may be involved in the processes of the increase in the molecular mass of xyloglucans, leading to the decrease in the cell wall extensibility.

Biomechanical Phenomena↗
PubMed2000

Possible involvement of 65 kda MAP in elongation growth of azuki bean epicotyls.

Although regulation of the dynamics of plant microtubules (MTs) by microtubule-associated proteins (MAPs) has been suggested, the mechanism has not yet been elucidated. As one candidate, a MAP composed of a 65 kDa polypeptide (65 kDa MAP) has been isolated from tobacco cultured cells [Jiang and Sonobe (1993), J. Cell Sci 105: 8911. To investigate the physiological role of the 65 kDa MAP in situ, we analyzed the changes in content and colocalization of this MAP with cortical MTs in relation to elongation growth, using azuki bean epicotyls (Vigna angularis Ohwi et Ohashi). All apical, intermediate, and basal segments prepared from 6 d seedlings showed high growth activity. In 12 d seedlings, growth activity of intermediate and basal segments was low, although that of apical segments was high. The relationship between the growth activity and the orientation of cortical MTs in the epidermal cells was analyzed. Cells could be classified into four types with respect to orientation of cortical MTs: transverse (T), oblique (O), longitudinal (L) to the vertical axis of cells, and random (R). In rapidly growing segments, three types of cells, T, O, L, were observed at similar ratios. In such segments, significant amounts of the 65 kDa MAP were expressed, and it colocalized well with cortical MTs. In segments showing low growth activity, most of the cells showed oblique and longitudinal orientation of cortical MTs. In such segments, the content of the 65 kDa MAP was low. These results suggested involvement of this 65 kDa MAP in regulation of the elongation growth of this epicotyl.

Fabaceae↗
PubMed2001

A new type of endo-xyloglucan transferase devoted to xyloglucan hydrolysis in the cell wall of azuki bean epicotyls.

A new type of xyloglucan-degrading enzyme was isolated from the cell wall of azuki bean (Vigna angularis Ohwi et Ohashi cv. Takara) epicotyls and its characteristics were determined. The enzyme was purified to apparent homogeneity by Concanavalin A (Con A)-Sepharose, cation exchange, and gel filtration columns from a cell wall protein fraction extracted with 1 M sodium chloride. The purified enzyme gave a single protein band of 33 kDa on SDS-PAGE. The enzyme specifically cleaved xyloglucans and showed maximum activity at pH 5.0 when assayed by the iodine-staining method. An increase in reducing power in xyloglucan solution was clearly detected after treatment with the purified enzyme. Xyloglucans with molecular masses of 500 and 25 kDa were gradually hydrolyzed to 5 kDa for 96 h without production of any oligo- or monosaccharide with the purified enzyme. The purified enzyme did not show an endo-type transglycosylation reaction, even in the presence of xyloglucan oligosaccharides. Partial amino acid sequences of the enzyme shared an identity with endo-xyloglucan transferase (EXGT) family, especially with xyloglucan endotransglycosylase (XET) from nasturtium. These results suggest that the enzyme is a new member of EXGT devoted solely to xyloglucan hydrolysis.

Cell Wall↗
PubMed2002

Action of xyloglucan hydrolase within the native cell wall architecture and its effect on cell wall extensibility in azuki bean epicotyls.

Xyloglucan hydrolase (XGH) has recently been purified from the cell wall of azuki bean (Vigna angularis Ohwi et Ohashi) epicotyls as a new type of xyloglucan-degrading enzyme [Tabuchi et al. (2001) Plant Cell Physiol. 42: 154]. In the present study, the effects of XGH on the mechanical properties of the cell wall and on the level and the molecular size of xyloglucans within the native wall architecture were examined in azuki bean epicotyls. When the epidermal tissue strips from the growing regions of azuki bean epicotyls were incubated with XGH, the mechanical extensibility of the cell wall dramatically increased. XGH exogenously applied to cell wall materials (homogenates) or epidermal tissue strips decreased the amount of xyloglucans via the solubilization of the polysaccharides. Also, XGH substantially decreased the molecular mass of xyloglucans in both materials. These results indicate that XGH is capable of hydrolyzing xyloglucans within the native cell wall architecture and thereby increasing the cell wall extensibility in azuki bean epicotyls.

Biomechanical Phenomena↗
PubMed2004

Xyloglucan oligosaccharides cause cell wall loosening by enhancing xyloglucan endotransglucosylase/hydrolase activity in azuki bean epicotyls.

Addition of xyloglucan-derived oligosaccharides shifted the wall-bound xyloglucans to a lower molecular mass distribution and increased the cell wall extensibility of the native epidermal tissue strips isolated from azuki bean (Vigna angularis) epicotyls. To ascertain the mechanism of oligosaccharide function, we examined the action of a xyloglucan endotransglucosylase/hydrolase (XTH) showing both endotransglucosylase and endohydrolase activities, isolated from azuki bean epicotyl cell walls, in the presence of xyloglucan oligosaccharides. The addition of xyloglucan oligosaccharides enhanced the xyloglucan-degrading activity of XTH against isolated xyloglucan substrates. When the methanol-fixed epidermal tissue strips were incubated with XTH, the molecular mass of wall-bound xyloglucans was decreased and the cell wall extensibility increased markedly in the presence of the oligosaccharides. These results suggest that xyloglucan oligosaccharides stimulate the degradation of xyloglucans by enhancing the XTH activity within the cell wall architecture, thereby increasing the cell wall extensibility in azuki bean epicotyls.

Cell Wall↗
PubMed1996

Molecular cloning of two classes of Em-like proteins from the seeds of the leguminous tree Robinia pseudoacacia.

To check for the presence of Em-like proteins in seeds of the leguminous tree Robinia pseudoacacia L. (black locust), a cDNA library constructed from mRNA isolated from developing seeds was screened using radiolabeled cDNA encoding the Em protein from Vigna radiata as a probe. Sequence analysis of the identified cDNA clones revealed two classes of Em proteins in Robinia. The nucleotide sequence data for each class have been submitted to Genbank/EMBL Data library and are available under the accession numbers U40820 and U40821. Northern blot analysis demonstrated that the Robinia Em proteins are translated from mRNAs of approximately 800 nucleotides, which are abundant in the seed but are not expressed in the bark. Southern blot analysis indicated that the Em proteins in Robinia are encoded by at least two genes. The Robinia Em proteins have a high degree of sequence homology with previously characterized Em-like proteins from both monocots and dicots.

Journal Article↗
PubMed1988

Identification of a nodD-dependent locus in the Rhizobium strain NGR234 activated by phenolic factors secreted by soybeans and other legumes.

Transfer of the strain NGR234nodD 1 gene into the narrow host range R. trifolii strain ANU843 on either a 6.7-kb HindIII or 17-kb XhoI fragment broadens the host range of this bacterium to include the tropical legumes Vigna unguiculata, Glycine ussuriensis, Leucaena leucocephala, and siratro (Macroptilium atropurpureum). Contrary to previous data (Bassam et al. 1986), mutagenesis of the 17-kb XhoI fragment with a mini-Mu lac transposon (Mu dII1734) showed that a functional nodD 1 gene was essential for extended host range. Gene expression studies using both Mu dII1734 fusions and a promoter-cloning vector indicated that several loci, including the nodD 1 gene, are constitutively expressed. No evidence was found for regulation of the strain NGR234 nodD 1 gene by its product. Another locus nod-81, was induced only in the presence of exudates from various plant species, including soybean (Glycine max). Whereas the expression of nod-81 was dependent on the presence of a functional nodD 1 gene product, a regulatory nod-box DNA sequence was not detected 5' to this gene by using available oligonucleotide hybridization probes. The nod-81 locus was induced by genistein, daidzein, naringenin, and coumestrol from both cotyledon and root tissue of freshly germinated soybean seedlings. A broad spectrum of commercially available phenolic compounds stimulated induction of the nod-81 locus, including some that antagonize nod gene induction in other Rhizobium species. The nodD 1 gene product from strain NGR234 was shown to determine the spectrum of compounds that induce nod-81 expression.

Base Sequence↗
PubMed2005

GuaB activity is required in Rhizobium tropici during the early stages of nodulation of determinate nodules but is dispensable for the Sinorhizobium meliloti-alfalfa symbiotic interaction.

The guaB mutant strain Rhizobium tropici CIAT8999-10T is defective in symbiosis with common bean, forming nodules that lack rhizobial content. In order to investigate the timing of the guaB requirement during the nodule formation on the host common bean by the strain CIAT899-10.T, we constructed gene fusions in which the guaB gene is expressed under the control of the symbiotic promoters nodA, bacA, and nifH. Our data indicated that the guaB is required from the early stages of nodulation because full recovery of the wild-type phenotype was accomplished by the nodA-guaB fusion. In addition, we have constructed a guaB mutant derived from Sinorhizobium meliloti 1021, and shown that, unlike R. tropici, the guaB S. meliloti mutant is auxotrophic for guanine and induces wild-type nodules on alfalfa and Medicago truncatula. The guaB R. tropici mutant also is defective in its symbiosis with Macroptilium atropurpureum and Vigna unguiculata but normal with Leucaena leucocephala. These results show that the requirement of the rhizobial guaB for symbiosis is found to be associated with host plants that form determinate type of nodules.

Artificial Gene Fusion↗
PubMedDate not supplied

nodSU, two new nod genes of the broad host range Rhizobium strain NGR234 encode host-specific nodulation of the tropical tree Leucaena leucocephala.

Rhizobium species strain NGR234 nodulates at least 35 diverse genera of legumes as well as the nonlegume Parasponia andersonii. Most nodulation genes are located on the 500-kilobase pair symbiotic plasmid, pNGR234a. Previously, three plasmid-borne host range determinants (HsnI, HsnII, and HsnIII) were identified by their ability to extend the nodulation capacity of heterologous rhizobia to include Vigna unguiculata. In this study, we show that HsnII contains two new nod-box linked hsn genes, nodS and nodU.nodS controls nodulation of the tropical tree Leucaena leucocephala, while the nodSU genes regulate nodulation of the pasture legume Desmodium intortum and the grain legume V. unguiculata. Regulation of the nod-box upstream of nodSU by the flavonoid naringenin was shown using a fusion with a promoterless lacZ gene. Determination of the nucleotide sequence of the nodS gene did not reveal homology with any gene in the EMBL library, although Bradyrhizobium japonicum USDA110 contains both nodS and nodU (M. Göttfert, S. Hitz, and H. Hennecke, Molecular Plant-Microbe Interactions 3:308-316, 1990). We suggest that broad host range in NGR234 is controlled in part by a nodD gene which interacts with a wide range of flavonoids, and in part by host-specific nod genes such as nodS.

Amino Acid Sequence↗
PubMed1997

Rhizobia modulate root-hair-specific expression of extensin genes.

Three cDNAs (ext3, ext127, and ext26), originally isolated by differential screening from a root-hair cDNA library of Vigna unguiculata, were found to encode extensin-like cell wall proteins. Transcripts homologous to these cDNAs were only detected in root hairs where mRNA levels decreased 1 day after inoculation with rhizobia. This coincided with the onset of root-hair deformation, the first morphological step in the Rhizobium-legume interaction. Decreases in transcript levels following inoculation with wild-type Rhizobium sp. NGR234 were more pronounced than with NGR delta nodABC, a mutant deficient in Nod-factor production. Inoculation with a rhizobial strain carrying a mutation in a gene encoding a transcriptional activator for nod genes (NGR delta nodD1) did not repress mRNA levels, indicating that a second nodulation signal may be present that is nodD dependent. Application of purified NodNGR factors only affected transcript levels of ext3. The genomic locus of the gene homologous to ext26 (Ext26G) was cloned. In the 5' flanking region, several potential TATA boxes and CAP signals were identified. Part of the promoter region shares homology with the Pisum sativum seed lectin promoter and the Nicotiana tabacum nitrate reductase promoter region. Nonetheless, the function of these homologous regions in gene regulation is unknown.

Amino Acid Sequence↗
PubMed1997

Induction of chalcone synthase expression by rhizobia and nod factors in root hairs and roots.

Chalcone synthase (CHS) of Vigna unguiculata is encoded by a gene family that is abundantly transcribed in leaves and nodules. Inoculation with Rhizobium sp. NGR234, which nodulates V. unguiculata, or with NGR delta nodABC, a mutant deficient in Nod factor production, induced rapid accumulation of CHS mRNAs in roots and root hairs. As both Nod+ and Nod- bacteria provoke responses, induction of CHS gene expression may involve symbiotic or defense responses. Four days after inoculation with the wild-type Rhizobium sp., the transcript levels increased in roots but decreased in root hairs. Use of a region unique to the 5' end of a specific CHS gene (VuCHS1) showed that increases of transcript levels in root hairs 24 h after inoculation with both rhizobia were specific to this gene. Transcripts of this gene in roots were only detectable 4 days after treatment with NGR234. It is possible therefore that accumulation of VuCHS1 follows the infection pathway of rhizobia entering legume roots. Purified Nod factors induced accumulation of transcripts, showing that they might be part of the signal transduction pathway leading to CHS expression.

Acyltransferases↗
PubMed1999

The nolL gene from Rhizobium etli determines nodulation efficiency by mediating the acetylation of the fucosyl residue in the nodulation factor.

The nodulation factors (Nod factors) of Rhizobium etli and R. loti carry a 4-O-acetyl-L-fucosyl group at the reducing end. It has been claimed, based on sequence analysis, that NolL from R. loti participates in the 4-O-acetylation of the fucosyl residue of the Nod factors, as an acetyl-transferase (D. B. Scott, C. A. Young, J. M. Collins-Emerson, E. A. Terzaghi, E. S. Rockman, P. A. Lewis, and C. E. Pankhurst. Mol. Plant-Microbe Interact. 9:187-197, 1996). Further support for this hypothesis was obtained by studying the production of Nod factors in an R. etli nolL::Km mutant. Chromatographic and mass spectrometry analysis of the Nod factors produced by this strain showed that they lack the acetyl-fucosyl substituent, having a fucosyl group instead. Acetyl-fucosylation was restored upon complementation with a wild-type nolL gene. These results indicate that the nolL gene determines 4-O-acetylation of the fucosyl residue in Nod factors. Analysis of the predicted NolL polypeptide suggests a transmembranal location and that it belongs to the family of integral membrane transacylases (J. M. Slauch, A. A. Lee, M. J. Mahan, and J. J. Mekalanos. J. Bacteriol. 178:5904-5909, 1996). NolL from R. loti was also proposed to function as a transporter; our results show that NolL does not determine a differential secretion of Nod factors from the cell. We also performed plant assays that indicate that acetylation of the fucose conditions efficient nodulation by R. etli of some Phaseolus vulgaris cultivars, as well as of an alternate host (Vigna umbellata).

Acetylation↗
PubMed1999

Rhizobium sp. strain NGR234 and R. fredii USDA257 share exceptionally broad, nested host ranges.

Genetically, Rhizobium sp. strain NGR234 and R. fredii USDA257 are closely related. Small differences in their nodulation genes result in NGR234 secreting larger amounts of more diverse lipo-oligosaccharidic Nod factors than USDA257. What effects these differences have on nodulation were analyzed by inoculating 452 species of legumes, representing all three subfamilies of the Leguminosae, as well as the nonlegume Parasponia andersonii, with both strains. The two bacteria nodulated P. andersonii, induced ineffective outgrowths on Delonix regia, and nodulated Chamaecrista fasciculata, a member of the only nodulating genus of the Caesalpinieae tested. Both strains nodulated a range of mimosoid legumes, especially the Australian species of Acacia, and the tribe Ingeae. Highest compatibilities were found with the papilionoid tribes Phaseoleae and Desmodieae. On Vigna spp. (Phaseoleae), both bacteria formed more effective symbioses than rhizobia of the "cowpea" (V. unguiculata) miscellany. USDA257 nodulated an exact subset (79 genera) of the NGR234 hosts (112 genera). If only one of the bacteria formed effective, nitrogen-fixing nodules it was usually NGR234. The only exceptions were with Apios americana, Glycine max, and G. soja. Few correlations can be drawn between Nod-factor substituents and the ability to nodulate specific legumes. Relationships between the ability to nodulate and the origin of the host were not apparent. As both P. andersonii and NGR234 originate from Indonesia/Malaysia/Papua New Guinea, and NGR234's preferred hosts (Desmodiinae/Phaseoleae) are largely Asian, we suggest that broad host range originated in Southeast Asia and spread outward.

Carbohydrate Sequence↗
PubMed2004

Flavonoids, NodD1, NodD2, and nod-box NB15 modulate expression of the y4wEFG locus that is required for indole-3-acetic acid synthesis in Rhizobium sp. strain NGR234.

Flavonoids secreted by host plants activate, in conjunction with the transcriptional activator NodD, nod gene expression of rhizobia resulting in the synthesis of Nod factors, which trigger nodule organogenesis. Interestingly, addition of inducing flavonoids also stimulates the production of the phytohormone indole-3-acetic acid (IAA) in several rhizobia. Here, the molecular basis of IAA synthesis in Rhizobium sp. NGR234 was investigated. Mass spectrometric analysis of culture supernatants indicated that NGR234 is capable of synthesizing IAA via three different pathways. The production of IAA is increased strongly by exposure of NGR234 to daidzein in a NodD1-, NodD2-, and SyrM2-dependent manner. This suggests that the y4wEFG locus that is downstream of nod-box NB15 encodes proteins involved in IAA synthesis. Knockout mutations in y4wE and y4wF abolished flavonoid-inducible IAA synthesis and a functional y4wF was required for constitutive IAA production. The promoter activity of NB15 and IAA production both were enhanced by introduction of a multicopy plasmid carrying nodD2 into NGR234. Surprisingly, the y4wE mutant still nodulated Vigna unguiculata and Tephrosia vogelii, although the nodules contained less IAA and IAA conjugates than those formed by the wild-type bacterium.

Bacterial Proteins↗