Isolation of xyloglucans from etiolated Glycine max and Vigna sesquipedalis hypocotyls

Xyloglucans isolated from cell walls of etiolated Glycine maxand Vigna sesquipedalis hypocotyls were subjected to fragmentationanalysis with cellulase for structural comparison with thosederived from Phaseolus aureus hypocotyls. The xyloglucans fromG. max and V. sesquipedalis had glucose, xylose, galactose andfucose in the approximate molar ratio of 10:6:4:1 and 10:7:3:1,respectively. However, the results of cellulase fragmentationanalysis of xyloglucans from the three species suggested thatthe basic structure of the xyloglucans in the cell walls ofthese bean-hypocotyls is almost the same; the structure is basedon two repeating oligosaccharide units, one of which consistsof glucose and xylose and the other of glucose, xylose, galactoseand fucose. ¹ Present address: Toppan Printing Co., Ltd., Okaji, Sendai980, Japan. (Received February 3, 1977; )     

Effects of defoliation on growth and yield in groundnut (Arachis hypogea), cowpeas (Vigna unguiculata), soyabean (Glycine max) and green gram (Vigna aurens)

Defoliation reduced the dry weight of stems, pods, grains and size of individual grains in all four of the legume crops studied and the dry weight of flowering inflorescence stalk in the case of cowpeas and green gram only. The adverse effect of defoliation was more pronounced when defoliation was complete than when half of the number of leaves were removed. The greatest reduction in grain yield occurred when the plants were defoliated during the early podding stage, the percentage reduction being 59.7, 79.0, 86.4 and 95.3 in groundnut, cowpeas, soyabeans and green gram respectively when completely defoliated at this stage and 43.3, 14.0, 42.4 and 46.1 respectively when only half defoliated. The results show that assimilates produced by the leaves during the early stages of growth are used in the growth of stems and leaves, but the assimilates produced during the reproductive stage are used mainly for the growth of the pods. In groundnut, pod number and grain weight were positively correlated with stem weight. It appears that defoliation reduced pod number by depressing the growth of stems and this in turn reduced the number of flowering nodes. The reasons for the differences between the crops in their response to the defoliation treatments and the practical implications of the findings in relation to pest and disease control and plucking of leaves for human consumption are discussed.     

Effects of aluminium on tap-root elongation of soybean (Glycine max), cowpea (Vigna unguiculata) and green gram (Vigna radiata) grown in the presence of organic acids

The role of fulvic, malic, and oxalic acids in alleviating the toxic effects of aluminium (Al) on tap-root elongation of soybean cv. Fitzroy, cowpea cv. Vita 4, and green gram cv. Berken was studied. Treatments consisted of a factorial combination of four Al concentrations (0, 12.5, 25 and 50 µM as Al(NO₃)₃·9H₂O) and two concentrations either of malic or oxalic acid (0, 50 µM) or fulvic acid (0, 65 mg L^-1 of organic carbon). The free monomeric Al in solution was determined using a pyrocatechol violet procedure which distinguishes between monomeric and organically complexed Al. Fulvic acid completely alleviated the toxic effect of Al at all concentrations on soybean and cowpea and at concentrations <25 µM on green gram. The non-toxic Al-fulvate complex remained in solution. Both malic and oxalic acid, at the concentrations tested, failed to alleviate Al toxicity on any species; a much higher proportion of the added Al remained in monomeric form in the presence of these acids.     

Root growth of soybean (Glycine max L. Merr.) and cowpea (Vigna unguiculata Walp.) on a hydromorphic toposequence in Western Nigeria

Summary The effects of variations in edaphic and hydrologic factors over a short distance on the root growth of soybean cv. Hernon 237 and cowpea cv. IT 82 E-60 were studied in a hydromorphic toposequence. During the growing season the water table (WT) fluctuated from 0.43 to 0.94 m (high), 0.60 to 1.12 m (medium) and 0.72 to 1.51 m (low), respectively in 1983 and from 0.47 to 0.84 m (high), 0.63 to 1.13 m (medium) and 0.83 to 1 20m (low). respectively, in 1984. Poor soil aeration did not limit growth, even for high WT.Root penetration into the deeper soil was prevented at the low and medium water table sites by the presence of a naturally occurring compacted gravel layer at the 0.30–0.40 m depth. The absence of this layer at the high water table site resulted in root growth and proliferation of soybean roots even within the capillary fringe zone immediately above the water table. Cowpea roots, however, were not observed in this saturated soil zone. Cowpea roots penetrated deeper in high than in medium and low WT. Evapotranspiration (Et) and E_t/E_o values of both crops were significantly greater at the high than at medium or low water table.     

De Novo Purine Synthesis in Nitrogen-Fixing Nodules of Cowpea (Vigna unguiculata [L.] Walp.) and Soybean (Glycine max [L.] Merr.)

Partially purified, cell-free extracts from nodules of cowpea (Vigna unguiculata L. Walp. cv. Caloona) and soybean (Glycine max L. Merr. cv. Bragg) showed high rates of de novo purine nucleotide and purine base synthesis. Activity increased with rates of nitrogen fixation and ureide export during development of cowpea plants; maximum rates (equivalent to 1.2 micromoles N₂ per hour per gram fresh nodule) being similar to those of maximum nitrogen fixation (1-2 micromoles N₂ per hour per gram fresh nodule). Extracts from actively fixing nodules of a symbiosis not producing ureides, Lupinus albus L. cv. Ultra, showed rates of de novo purine synthesis 0.1% to 0.5% those of cowpea and soybean. Most (70-90%) of the activity was associated with the particulate components of the nodule, but up to 50% was released from this fraction by osmotic shock. The accumulated end products with particulate fractions were inosine monophosphate and aminoimidazole carboxamide ribonucleotide. Further metabolism to purine bases and ureides was restricted to the soluble fraction of the nodule extract. High rates of inosine monophosphate synthesis were supported by glutamine as amide donor, lower rates (10-20%) by ammonia, and negligible rates with asparagine as substrate.     

Scab disease of cowpea (Vigna unguiculata) caused by Sphaceloma a species of the fungus

Scab disease of cowpea (Vigna unguiculuta) was shown to be caused by Sphaceloma sp. It affects all above ground parts of the plant. The first symptom of the disease, appearing within 3 to 6 days of inoculation, is puckering of the lamina. Spots on mature leaves are white with or without brown margins. Typical scab lesions on petiole, stem, peduncle and pod are white turning dark brown when chlamydospores are formed and are oblong-elongate except for pod lesions that are ovoid. The most destructive phase is scab of the flowering axis which causes flower and, or, pod abortion or completely prevents flower formation. Inoculation of asparagus pea (V. sesquipedalis) with a cowpea isolate of Sphaceloma sp. produced symptoms similar to those on cowpea. Inoculated hyacinth bean (Lablab niger) produced atypical mild lesions. The following legumes were not affected when artificially inoculated with the fungus: black gram (Phaseolus mungo), green gram (P, aureus), French bean (P. vulgaris), Lima bean (P. lunatus), groundnut (Arachis hypogaea), and soyabean (Glycine max). The characteristics of the fungus on potato dextrose agar are described.     

Plant regeneration by organogenesis in Glycine max

A procedure for the regeneration of fertile plants by organogenesis from tissue cultures of soybeans, Glycine max is described. Seeds were germinated on reduced inorganic salt MS medium containing 5M BA. Cotyledonary nodes were excised and cultured on the same medium. Presence of BA in the medium during seed germination and culture of nodal explants was required for multiple shoot and shoot-bud formation. Histological analyses established the de novo nature of shoot regeneration. Separate reduction of the concentration of inorganic salts or substitution of sucrose for fructose during culture had minimal effects on the regeneration response. Conversely, if the BA was reduced, the inhibition response could not be overcome by increased salt concentration or altered carbon source.Abbreviations BA benzyladenine - IAA indoleacetic acid - SAS secondary axillary shoots - MS Murashige and Skoog (1962) medium     

Isolation and metabolism of Vigna unguiculata root nodule protoplasts

Axenic cultures of bacteroid-containing protoplasts were isolated from root nodules of Vigna unguiculata L. Walp. Dimensions of the protoplasts were 35 to 135 m long x 35 to 95 m wide. Yields were about 30 to 50 mg dry weight per gram fresh weight of nodules. About 5x10⁸ protoplasts packed into 1 ml of basal medium under the influence of gravity. When incubated in hypertonic, nitrogen-free media, freshly isolated protoplasts began to reduce acetylene to ethylene after a lag period of 24 to 48 h. Various additions to the basal medium showed that the system possessed functional glycolytic and tricarboxylic acid pathways. Endogenous application of various intermediary metabolites stimulated both acetylene reduction and respiration, though not often equally. As acetylene reduction, but not respiration, was inhibitable by both asparagine and glutamine, the system appears suitable for the study of mechanisms controlling symbiotic nitrogen fixation.Abbreviations BSA bovine serum albumine - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - PEP phospho(enol)pyruvate - UMKL 76 University of Malaga, Kuala Lumpur, Rhizobium, No. 76 - TCAC tricarboxylic acid cycle     

Development and polymorphism of Vigna unguiculata ssp. unguiculata microsatellite markers used for phylogenetic analysis in asparagus bean (Vigna unguiculata ssp. sesquipedialis (L.) Verdc.)

Asparagus bean (V. unguiculata ssp. sesquipedialis), a specific form of cowpea (V. unguiculata L. Walp.), is cultivated as a vegetable crop throughout eastern and southern Asia for its tender long pods. Little is known about the genetic relationship between asparagus bean and the broader species, particularly the dominant ssp. unguiculata. We report here the development and transferability of simple sequence repeat (SSR) markers, over 40% of which are EST-derived, from ssp. unguiculata to asparagus bean and the use of a subset of the polymorphic markers to assess the genetic diversity of asparagus bean cultivars from diverse geographic origins across China. A total of 410 EST derived SSR (eSSR) markers and 600 SSR markers derived from cowpea genespace sequences (GSS) were developed, with a cross-subspecies transferability of 100% and 98.5%, respectively. In a recombinant inbred line population of asparagus bean, a 1:1 segregation was observed for most loci. Principal coordinate analysis (PCA) and phylogenetic clustering based on 62 alleles detected by 14 polymorphic SSR markers distinguished ssp. unguiculata and sesquipedialis into separate groups. Improved asparagus bean cultivars in China generally have a narrow genetic basis compared with landraces varieties. This suggests that asparagus bean breeding programs need to consider utilizing landrace germplasm to enhance genetic variability and ensure long-term gains from selection and reduce genetic vulnerability to pathogen/pest epidemics. Because of their transferability across subspecies, the SSR markers described in this study could be effectively employed in cross-subspecies trait introgression breeding from ssp. unguiculata to sesquipedialis.     

Cotyledonary mRNA Species and Germinability of Immature Vigna unguiculata Seeds

We have defined four stages in the development of cowpea seeds:I(9–11 days after flowering), II (13–15), HI (17–19)and IV (22–24). Poly A⁺ RNA fractions were prepared fromcotyledons of developing (stages I–IV) and germinating(0, 12, 24 and 48 h after imbibition) seeds. Poly A⁺ RNAs fromstages I–III exhibited high translation activities witha maximum at stage II, and the activity was markedly reducedat stage IV. In cotyledons of germinating seeds, the translationactivity was low until 12 h after the onset of imbibition butrose thereafter. Analysis of in vitro translation products withSDS-polyacrylamide gel electrophoresis and fluorography showedthat the abundant mRNA population underwent a distinct changebetween stages II and III of seed development. Since the mRNApopulation at stage III was very similar to that of stage IV(mature seeds), it appears that, as far as mRNA species areconcerned, the prerequisites for germination are fully availablein the developing seeds by stage III. This assumption was supportedby the fact that immature seeds at stage III exhibited highgermination rates and normal axial growth and produced -amylaseat levels similar to those produced by mature seeds. Severalpolypeptides which have been regarded as translation productsof stored mRNA (poly A⁺ RNA from dry seeds) were detected atearlier stages of germination. (Received September 29, 1988; Accepted January 25, 1989)     

Nodulins and nodulin genes of Glycine max

Summary Nodulins are organ-specific plant proteins induced during symbiotic nitrogen fixation. Nodulins play both metabolic and structural roles within infected and uninfected nodule cells. In soybean, several nodulin genes, coding for abundant nodulins, have been identified and isolated. Structural analysis of some of these genes has revealed their possible mode of regulation and the subcellar location of the protein product. Studies of ineffective symbiosis based on cultivar-strain genotype differences suggested that both partners influence the expression of nodulin genes. Concomitant with nodule organogenesis, the Rhizobium undergoes substantial differentiation leading to the accumulation of nodule-specific bacterial proteins, bacteroidins. The major structural alteration occuring in the infected cell is the formation of a membrane enclosing the bacteroid (peribacteroid membrane). A number of nodulins are specifically targetted to this membrane during endosymbiosis. The induction of nodulins and bacteroidins leads to the formation of an effective nodule. Nodulin genes can be induced in vitro by factors derived from nodules suggesting that trans-activators may be involved in derepression of the host genes necessary for Rhizobium-legume symbiosis.     

Molecular Cloning and Characterization of Stored mRNA in Cotyledons of Vigna unguiculata

From a gt1O cDNA library constructed from the total polyadenylatedRNA (poly A⁺ RNA) of mature cowpea cotyledons, we selected recombinantphages that hybridized with a cDNA probe complementary to polyA⁺ RNA from cotyledons collected 24 h after the onset of imbibition(cDNA-B) but that did not hybridize with a probe from cotyledonsat developmental stage II (13 to 15 days after flowering; cDNA-A).cDNA inserts of two of these phages were subcloned in pUC18plasmids and the resultant plasmids were designated pSAS5 andpSAS1O, respectively. We also selected two recombinant phagesthat hybridized with cDNA-A but not with cDNA-B, and one ofthem was subcloned in pUC18 to generate pSASC1. We then characterizedthe pSASlO cDNA insert as the cDNA of a putative stored mRNAof cowpea seeds and the pSASC1 insert as a reference cDNA. TheRNA-blot hybridization after gel electrophoresis, with pSAS10as probe, indicated that an essentially full-length cDNA hadbeen cloned, and the hybrid-select translation of pSAS10 mRNAgave a 10-kDa product. pSASC1 mRNA was shown to code for a 46-kDapolypeptide, which corresponds in size to one of the major storageproteins of the cowpea. pSAS10 mRNA was detectable only in cotyledonsat developmental stage III (17 to 19 days after flowering) orlater, and the level of the mRNA began to decline when seedsgerminated. The mRNA was also present in cowpea embryonic axes.mRNA hybridizable to pSAS10 cDNA was found in extracts of someother plants. (Received July 17, 1989; Accepted October 17, 1989)     

Cloning and Characterization of Trypsin Inhibitor Gene from Vigna unguiculata

A trypsin inhibitor, member of Bowman-Birk family, was isolated and purified from cowpea (Vigna unguiculata]). Polyclonal antibodies were raised against cowpea trypsin inhibitor (CpTI) protein in rabbits. The gene for CpTI was amplified by polymerase chain reaction and cloned in a bacterial expression vector pVCATFR18416. The expression of CpTI protein in BL21 (DE3) strain of Escherichia coli was confirmed by western blot studies. The CpTI gene was also sequenced and found to exhibit 100% homology with already published sequence of CpTI gene.     

NaCl胁迫下野生和栽培大豆幼苗体内离子的再转运

采用NaCl根际处理和叶面饲喂^22Na方法,研究了野生大豆(Glycine soja)——耐盐的BB52、盐敏感的N23232和栽培大豆(Glycine max)——较耐盐的Lee68幼苗在盐胁迫及解除过程中对Na^ 、Cl^-的吸收和再转运。结果表明,在NaCl根际处理12h过程中,BB52和Lee68幼苗根对Na^ 、Cl^-吸收和向茎、叶的运输逐渐增加,10h时趋于稳定,Na^ 、Cl^-含量高低顺序是根＞茎＞叶。但N23232的Na^ 、Cl^-含量则是茎＞根＞叶。在用NaCl对根处理10h后再解除NaCl处理的0～36h内,BB52吸收的Na^ 、Cl^-较多地留于根部或转运至根茎过渡区,叶中较少。N23232吸收的Na^ 较多地转运至茎部,而Cl^-含量在幼苗各部分无差异。叶片饲喂^22Na 10h后,BB52吸收^22Na较N23232多,并较多地向根部运输。从离子再转运角度讨论了BB52的耐盐性。     

Functional associations between the metabolome and manganese tolerance in Vigna unguiculata

Genotypic- and silicon (Si)-mediated differences in manganese (Mn) tolerance of cowpea (Vigna unguiculata) arise from a combination of symplastic and apoplastic traits. A detailed metabolomic inspection could help to identify functional associations between genotype- and Si-mediated Mn tolerance and metabolism. Two cowpea genotypes differing in Mn tolerance (TVu 91, Mn sensitive; TVu 1987, Mn tolerant) were subjected to differential Mn and Si treatments. Gas chromatography-mass spectrometry (GC-MS)-based metabolite profiling of leaf material was performed. Detailed evaluation of the response of metabolites was combined with gene expression and physiological analyses. After 2 d of 50 μM Mn supply TVu 91 expressed toxicity symptoms first in the form of brown spots on the second oldest trifoliate leaves. Silicon treatment suppressed symptom development in TVu 91. Despite higher concentrations of Mn in leaves of TVu 1987 compared with TVu 91, the tolerant genotype did not show symptoms. From sample cluster formation as identified by independent component analysis (ICA) of metabolite profiles it is concluded that genotypic differences accounted for the highest impact on variation in metabolite pools, followed by Mn and Si treatments in one of two experiments. Analysis of individual metabolites corroborated a comparable minor role for Mn and Si treatments in the modulation of individual metabolites. Mapping individual metabolites differing significantly between genotypes onto biosynthetic pathways and gene expression studies on the corresponding pathways suggest that genotypic Mn tolerance is a consequence of differences (i) in the apoplastic binding capacity; (ii) in the capability to maintain a high antioxidative state; and (iii) in the activity of shikimate and phenylpropanoid metabolism.     

Elicitation of Necrosis in Vigna unguiculata Walp. by Homogeneous Aspergillus niger Endo-Polygalacturonase and by alpha-d-Galacturonate Oligomers

Endo-polygalacturonase (PG) was purified from a commercial preparation of Aspergillus niger pectinase by means of carboxymethylcellulose chromatography, preparative isoelectric focusing, and gel permeation through Sephadex G-50. The enzyme was electrophoretically homogeneous and consisted of a single polypeptide chain with a molecular weight of 33,500. The enzyme exhibited a specific activity significantly higher than those of purified polygalacturonases from phytopathogenic fungi. Galacturonate oligomers with a degree of polymerization higher than four appeared quickly as products of the enzymic hydrolysis of Napolygalacturonate. The oligomers were later degraded to di- and monogalacturonate. The homogeneous enzyme and growing mycelium of Aspergillus niger separately elicited a necrotic response in cowpea (Vigna unguiculata Walp.) pods. Heat-inactivated PG and PG inactivated with specific antibodies did not elicit necrosis, suggesting that the catalytic activity of the enzyme is necessary for its function as an elicitor. The PG-released oligosaccharides from Vigna cell wall and the galacturonides with a degree of polymerization greater than four separately elicited necrosis, whereas di- and monogalacturonate did not.     

Proteinase inhibitors from Vigna unguiculata subsp. cylindrica: I. Occurrence of thiol proteinase inhibitors in plants and purification from Vigna unguiculata subsp. cylindrica

Inhibitors of the thiol proteinase, papain (EC 3.4.22.2), were shown to be present in 11 species of 10 genera of plants. The inhibitor activity was nondialyzable, and precipitated by ammonium sulfate. Tissue cultures from a number of plant genera consisting of rapidly dividing cells contained latent papain inhibitor that could be activated upon heating. Four isoinhibitors of plant thiol proteinases from seeds of the legume Vigna unguiculata subsp. cyclindrica were purified to apparent homogeneity by acrylamide gel electrophoresis with or without sodium dodecyl sulfate. The inhibitors were present in very small amounts compared to the trypsin inhibitors and the degree of purification of the homogeneous isoinhibitors on the assumption that all were present initially in equal amounts was 15,000- to 60,000-fold. The isoinhibitors did not inhibit pepsin, bromelain, and the serine proteinases, trypsin, chymotrypsin, and subtilisin. They were specific for papain, chymopapain, and ficin but their inhibition of the proteinase, esterase, and amidase activities of the three enzymes differed.     

Nitrogen fixation by soybeans (Glycine max L.) in Zambia

Soybeans (Glycine max L.) are being introduced as a cash crop to small scale farmers in Zambia for rotation in their farming systems. The objectives of this study were to compare and select the most approriate non-fixing reference crop for estimating N₂ fixation by soybeans and assess yields and N₂ fixation of soybeans in Zambia. Nitrogen isotope dilution techniques using¹⁵N-labelled organic or inorganic materials were utilized. Two nonnodulating soybean cultivars, Clark RJ1 and N77 or in their absence Pearl millet (Panicum glaucum L.) were judged to be appropriate reference crops. A local soybean fixing cultivar (Glycine max L. cv. Magoye) rated highest among three cultivars tested for its ability to support symbiotic N₂ fixation byB. japonicum under the experimental conditions. Values of percent N derived from atomosphere for this cultivar were in the order of 65 to 70%.deceased.Contribution no R531 of the Saskatchewan Institute of Pedology. Present address (REK): Esso Chemical Canada, P.O. Box 3010, Lethbridge, Alberta Canada T1J 4A9.