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Allelic variation of a dehydrin gene cosegregates with chilling tolerance during seedling emergence.

Dehydrins (DHNs, LEA D-11) are plant proteins present during environmental stresses associated with dehydration or low temperatures and during seed maturation. Functions of DHNs have not yet been defined. Earlier, we hypothesized that a approximately 35-kDa DHN and membrane properties that reduce electrolyte leakage from seeds confer chilling tolerance during seedling emergence of cowpea (Vigna unguiculata L. Walp.) in an additive and independent manner. Evidence for this hypothesis was not rigorous because it was based on correlations of presence/absence of the DHN and slow electrolyte leakage with chilling tolerance in closely related cowpea lines that have some other genetic differences. Here, we provide more compelling genetic evidence for involvement of the DHN in chilling tolerance of cowpea. We developed near-isogenic lines by backcrossing. We isolated and determined the sequence of a cDNA corresponding to the approximately 35-kDa DHN and used gene-specific oligonucleotides derived from it to test the genetic linkage between the DHN presence/absence trait and the DHN structural gene. We tested for association between the DHN presence/absence trait and both low-temperature seed emergence and electrolyte leakage. We show that allelic differences in the Dhn structural gene map to the same position as the DHN protein presence/absence trait and that the presence of the approximately 35-kDa DHN is indeed associated with chilling tolerance during seedling emergence, independent of electrolyte leakage effects. Two types of allelic variation in the Dhn gene were identified in the protein-coding region, deletion of one Phi-segment from the DHN-negative lines and two single amino acid substitutions.

Alleles↗

Investigation of the action patterns of pectinmethylesterase isoforms through kinetic analyses and NMR spectroscopy. Implications In cell wall expansion.

Well characterized pectin samples were incubated with cell wall-bound and -solubilized pure isoforms of pectinmethylesterase from mung bean hypocotyls (Vigna radiata). Both enzyme activity and average product structure were determined at intervals along the deesterification pathway at pH 5.6 and 7.6. The latter analyses were performed by 13C NMR spectroscopy, and the degree of esterification was probed by both 13C NMR and potentiometric measurements. A dichotomy was observed in the behavior of the alpha and gamma isoforms when compared with that of the beta isoenzyme. Ideal blockwise deesterification mechanisms reproduced the experimental average structures (methylester distribution) throughout the course of the reaction. In the case of the alpha and gamma isoforms, a single chain mechanism associated with a free carboxyl group at the second nearest neighbor position could be postulated at pH 5.6, whereas some multiple attack character was required to reproduce the data at pH 7.6. Several mechanisms that differed from the preceding ones were compatible with the data for the beta isoform at the two pH values. Both the nature of the polysaccharides produced in these reactions and the role of pectinmethylesterase in the cell wall-stiffening process along the growth gradient are discussed.

Carboxylic Ester Hydrolases↗

Posttranslational removal of the carboxyl-terminal KDEL of the cysteine protease SH-EP occurs prior to maturation of the enzyme.

SH-EP is a cysteine protease from germinating mung bean (Vigna mungo) that possesses a carboxyl-terminal endoplasmic reticulum (ER) retention sequence, KDEL. In order to examine the function of the ER retention sequence, we expressed a full-length cDNA of SH-EP and a minus-KDEL control in insect Sf-9 cells using the baculovirus system. Our observations on the synthesis, processing, and trafficking of SH-EP in Sf-9 cells suggest that the KDEL ER-retention sequence is posttranslationally removed either while the protein is still in the ER or immediately after its exit from the ER, resulting in the accumulation of proSH-EP minus its KDEL signal. It is this intermediate form that appears to progress through the endomembrane system and is subsequently processed to form mature active SH-EP. The removal of an ER retention may regulate protein delivery to a functional site and present an alternative role for ER retention sequences in addition to their well established role in maintaining the protein composition of the ER lumen.

Animals↗

Control of Mung bean pectinmethylesterase isoform activities. Influence of pH and carboxyl group distribution along the pectic chains.

Well-characterized pectin samples with a wide range of degrees of esterification (39-74%) were incubated with the solubilized pure alpha and gamma isoforms of pectinmethylesterase, from mung bean hypocotyl (Vigna radiata). Enzyme activity was determined at regular intervals along the deesterification pathway at pH 5.6 and pH 7.6. It has been demonstrated that the distribution of the carboxyl units along the pectin backbone controls the activity of the cell wall pectinmethylesterases to a much greater extent than the methylation degree, with a random distribution leading to the strongest activity. Polygalacturonic acid was shown to be a competitive inhibitor of the alpha isoform activity at pH 5.6 and to inhibit the gamma isoform activity at both pH 5.6 and pH 7.6. Under these conditions, the drop in enzyme activity was shown to be correlated to the formation of deesterified blocks of 19 +/- 1 galacturonic acid residues through simulations of the enzymatic digestion according to the mechanisms established previously (Catoire, L., Pierron, M., Morvan, C., Herve du Penhoat, C., and Goldberg, R. (1998) J. Biol. Chem. 273, 33150-33156). However, even in the absence of inhibition by the reaction product, activity dropped to negligible levels long before the substrate had been totally deesterified. Comparison of alpha and gamma isoform cDNAs suggests that the N-terminal region of catalytic domains might explain their subtle differences in activity revealed in this study. The role of pectinmethylesterase in the cell wall stiffening process along the growth gradient is discussed.

Amino Acid Sequence↗

Identification of a membrane-associated cysteine protease with possible dual roles in the endoplasmic reticulum and protein storage vacuole.

SH-EP is a vacuolar cysteine proteinase from germinated seeds of Vigna mungo. The enzyme has a C-terminal propeptide of 1 kDa that contains an endoplasmic reticulum (ER) retention signal, KDEL. The KDEL-tail has been suggested to function to store SH-EP as a transient zymogen in the lumen of the ER, and the C-terminal propeptide was thought to be removed within the ER or immediately after exit from the ER. In the present study, a protease that may be involved in the post-translational processing of the C-terminal propeptide of SH-EP was isolated from the microsomes of cotyledons of V. muno seedlings. cDNA sequence for the protease indicated that the enzyme is a member of the papain superfamily. Immunocytochemistry and subcellular fractionation of cotyledon cells suggested that the protease was localized in both the ER and protein storage vacuoles as enzymatically active mature form. In addition, protein fractionations of the cotyledonary microsome and Sf9 cells expressing the recombinant protease indicated that the enzyme associates with the microsomal membrane on the luminal side. The protease was named membrane-associated cysteine protease, MCP. The possibility that a papain-type enzyme, MCP, exists as mature enzyme in both ER and protein storage vacuoles will be discussed.

Amino Acid Sequence↗

High affinity association of myo-inositol trisphosphates with phytase and its effect upon the catalytic potential of the enzyme.

A neutral phytase from germinating mung bean (Vigna radiata) seeds dephosphorylates myo-inositol hexakisphosphate sequentially to myo-inositol. The enzyme also binds with higher affinity to myo-inositol trisphosphates (1,4,5), (2,4,5), and (1,3,4) isomers without catalysis. The high affinity complex elicits Ca(2+) mobilization in vitro from microsomes/vacuoles via the formation of a ternary complex with the receptor for Ins(1,4,5)P(3). As a sequel to our previous report, we have carried out a detailed characterization of the two sites and examined the mutual interactions between them. Presaturation of the high affinity site leads to an increase in the affinity of the enzyme for phytic acid and its rate of dephosphorylation as well. From the products of limited tryptic cleavage of phytase, two peptides, each with one activity, have been isolated. The larger peptide ( approximately 66 kDa) contains the catalytic site, and the smaller peptide ( approximately 5 kDa) has the high affinity myo-inositol trisphosphate-binding site. The interaction between the dual activities of phytase has been observed also at the level of the two peptides. A sequence homology search using N-terminal 12 amino acid residues of the 5-kDa fragment has revealed significant homology with the Homer class of proteins implicated in signaling pathways involving metabotropic glutamate receptor and myo-inositol 1,4,5-trisphosphate receptor. These results indicate a second role of phytase in Ca(2+) mobilization during germination of mung been seed via a salvage pathway that involves allosteric activation by myo-inositol trisphosphate.

6-Phytase↗

In vitro fusion of plant Golgi membranes can be influenced by divalent cations.

The fusogenic activity of plant Golgi membranes was studied in a cell-free system by assaying lipid mixing and content leakages of fluorescence probes. Golgi membranes from mung bean (Vigna radiata L.) hypocotyl cells fused to liposomes in the absence of any cytosolic proteins and nucleotides. It was demonstrated that the fusion was mediated by integral membrane protein(s), and was influenced by divalent cations (mm). Mg(2+), Ca(2+), and Mn(2+) ions enhanced the lipid mixing by reducing repulsive forces between membranes. In the content leakage assay, Mg(2+) ions also showed a stimulative effect. However, other divalent cations were inhibitory. It is suggested that the fusion system of Golgi membranes comprises at least two components: one that mediates the formation of fusion intermediates prior to pore opening, and one that mediates the subsequent processes. The latter must be sensitive to divalent cations at millimolar concentrations. The fusion of Golgi and biological membranes was induced by divalent cations. We speculated about the biological role of the fusion system studied here.

Calcium↗

Chitinolytic and cellulolytic Pseudomonas sp. antagonistic to fungal pathogens enhances nodulation by Mesorhizobium sp. Cicer in chickpea.

Pseudomonas strains isolated from the rhizosphere of chickpea (Cicer arietinum L.) and green gram (Vigna radiata L.) were screened for the production of chitinases and cellulases. Five Pseudomonas strains were found to produce appreciable amounts of both enzymes in culture-free supernatants and showed growth inhibition of the two fungi Pythium aphanidermatum (Oomycete) and Rhizoctonia solani (Basidiomycete) in plates on potato dextrose agar medium. The fungal growth inhibition was not correlated with cell wall-degrading enzyme activity, which suggested that other antifungal compounds produced by these rhizobacteria were also involved in antagonism. Coinoculation of the Pseudomonas strains with the Mesorhizobium sp. Cicer strain Ca 181 resulted in a significant increase in nodule biomass when grown under sterilized chillum jar conditions. The results suggest that hydrolytic enzymes produced by Pseudomonas sp. contribute to suppression of plant diseases by inhibiting growth of phytopathogenic fungi and also promote nodulation of legumes by rhizobia.

Antibiosis↗

Bionomics and polymorphism in Callosobruchus subinnotatus (Coleoptera: Bruchidae).

Callosobruchus subinnotatus (Pic) is the major insect pest of stored bambara groundnuts, Vigna subterranea (L.) Verdcourt, in sub-Saharan West Africa, but little is currently known about its biology or how it may be controlled. A series of laboratory studies was performed to investigate the bionomics of and differences between two apparently different morphs of adult of each sex of this species, here termed 'active' and 'normal'. Major differences in their morphology, physiology and behaviour were identified and are described in detail for the first time. They provide clear evidence of the existence of an adult polymorphism among populations of this species, which is comparable in certain respects to that previously described for C. maculatus (Fabricius) and C. chinensis Linnaeus. Adults can be separated into the correct morph based on characteristic differences in elytral and pygidial colour and pattern. 'Normal' adults are characterized by having high fecundity, short adult life and are relatively sedentary while 'active' adults exhibit reproductive diapause (suspension of reproductive activity), are long lived, and show (at least in females) increased dispersal tendencies. These characteristics suggest adaptation of the 'active' and 'normal' morphs respectively to the different environments of field and seed stores, and the significance of the polymorphism in the life history of C. subinnotatus is discussed in this context. The design of any effective control regime for this bruchid needs to take account of and could potentially be based upon the existence of polymorphism in C. subinnotatus.

Animals↗

The relationship between rumen bacterial growth, intake of dry matter, digestible organic matter and volatile fatty acid production in buffalo (Bos bubalis) calves.

1. The production rates of bacteria in the rumen of buffalo (Bos bubalis) calves were estimated using an isotope-dilution technique. A series of fifteen experiments was done with animals given green maize and nine experiments with animals given cowpea (Vigna unguiculata). 2. The turnover time ranged from 205 to 567 min in the group given green maize and from 330 to 648 min in animals offered cowpea. The production rates of bacteria were (mean +/- SE; g/d) 145.77 +/- 7.240 and 237.09 +/- 11.847 in animals given green maize and cowpea respectively. 3. There was a significant correlation between bacterial production rates and dry matter intake, digestible organic matter and total volatile fatty acids formed in the rumen. 4. Regression equations obtained for the two foodstuffs were different suggesting that the bacterial growth rate may vary depending upon the quantity and quality of foodstuff digested and possibly the ratio nitrogen:energy of the foodstuff.

Animal Feed↗

A survey of the nutritional and haemagglutination properties of legume seeds generally available in the UK.

Eighty-five samples from fifteen different legume seed lines generally available in the UK were examined by measurements of their net protein utilization by rats and by haemagglutination tests with erythrocytes from a number of different animal species. From these results the seeds were classified into four broad groups. Group a seeds from most varieties of kidney (Phaseolus vulgaris), runner (Phaseolus coccineus) and tepary (Phaseolus acutifolius) beans showed high reactivity with all cell types and were also highly toxic. Group b, which contained seeds from lima or butter beans (Phaseolus lunatus) and winged bean (Psophocarpus tetragonolobus), agglutinated only human and pronase-treated rat erythrocytes. These seeds did not support proper growth of the rats although the animals survived the 10 d experimental period. Group c consisted of seeds from lentils (Lens culinaris), peas (Pisum sativum), chick-peas (Cicer arietinum), blackeyed peas (Vigna sinensis), pigeon peas (Cajanus cajan), mung beans (Phaseolus aureus), field or broad beans (Vicia faba) and aduki beans (Phaseolus angularis). These generally had low reactivity with all cells and were non-toxic. Group d, represented by soya (Glycine max) and pinto (Phaseolus vulgaris) beans, generally had low reactivity with all cells but caused growth depression at certain dietary concentrations. This growth depression was probably mainly due to antinutritional factors other than lectins. Lectins from group a seeds showed many structural and immunological similarities. However the subunit composition of the lectin from the tepary bean samples was different from that of the other bean lectins in this or any other groups.

Animals↗

Dietary modification of potential vitamin K supply from enteric bacterial menaquinones in rats.

Rats given a low-fibre diet based on boiled white rice developed symptoms of severe vitamin K deficiency within 23 d. Inclusion of autoclaved black-eye beans (Vigna unguiculata) in the diet prevented the bleeding syndrome. To test the hypothesis that deficiency resulted from low phylloquinone intake exacerbated by inadequate production of menaquinones by the enteric bacteria, a follow-up experiment was carried out in which groups of rats were given an all-rice diet, a rice + beans diet or a stock diet. Rats on the all-rice diet had significantly lower faecal concentrations of the main menaquinone-producing bacterial species (Bacteroides fragilis and Bacteroides vulgatus) than animals on either of the other two diets. This coupled with the much lower faecal output on this diet suggests that total menaquinone production was low for the all-rice diet. The alterations in faecal flora were associated with several significant changes in caecal metabolism. Rats given the stock diet had much shorter caecal transit times and a considerably greater proportion of butyric acid in volatile fatty acid end-products than did rats on either of the other two diets.

Animals↗

Comparative effects on blood lipids and faecal steroids of five legume species incorporated into a semi-purified, hypercholesterolaemic rat diet.

The cholesterolaemic effects in rats of a diet (VS) containing Bambara groundnuts (Vigna subterranea), a popular legume eaten in Nigeria, were compared with diets PV, PS, LC and PL, containing baked beans (Phaseolus vulgaris), marrowfat peas (Pisum sativum), lentils (Lens culinaris Medik.) and butter beans (Phaseolus lunatus) respectively. Sixty Sprague-Dawley rats were fed on hypercholesterolaemic semi-purified diets supplemented with 10 g cholesterol and 5 g cholic acid/kg and formulated to provide 40% of energy from fat, as in a typical Western-type human diet. Legumes were substituted for 330 g/kg of the semi-purified diet on a dry-matter basis, which was modified to maintain the same contribution of energy sources as the control diet C3. Another ten rats were fed on control diet C2, which was similar to diet C3 but with no added cholesterol. The rats were fed for 8 weeks and plasma cholesterol levels were measured at weeks 4 and 8. The diets incorporating the five different legume species produced very different cholesterolaemic effects. Diets PV and PL were more potent at lowering raised plasma cholesterol levels than diets PS and LC. Inclusion of the Bambara groundnut into the semi-purified diet resulted in an exaggeration of hypercholesterolaemia. Differences in cholesterol-lowering capacity of the various legume diets in this experiment could not be related to concentrations of faecal bile acids or neutral sterols. However, there was evidence that the inclusion of legumes in the diets reduced the faecal excretion of secondary bile acids.

Analysis of Variance↗

New legume sources as therapeutic agents.

This review evaluates the potential health benefits of three legume sources that rarely appear in Western diets and are often overlooked as functional foods. Fenugreek (Trigonella foenum graecum) and isolated fenugreek fractions have been shown to act as hypoglycaemic and hypocholesterolaemic agents in both animal and human studies. The unique dietary fibre composition and high saponin content in fenugreek appears to be responsible for these therapeutic properties. Faba beans (Vicia faba) have lipid-lowering effects and may also be a good source of antioxidants and chemopreventive factors. Mung beans (Phaseolus aureus, Vigna radiatus) are thought to be beneficial as an antidiabetic, low glycaemic index food, rich in antioxidants. Evidence suggests that these three novel sources of legumes may provide health benefits when included in the daily diet.

Animals↗

Characterization of a lectin from cowpeas.

A lectin without blood group specificity was isolated from cowpeas (Vigna unguiculata (L.) Walp.). This lectin was prepared from a crude saline extract by acid precipitation and Sephadex gel filtration. Purification steps generally resulted in increased agglutination activity. Homogeneity was demonstrated by polyacrylamide gel electrophoresis, and ultracentrifugation. The native lectin had a molecular weight of approximately 55,000 as shown by gel filtration, polyacrylamide electrophoresis, and analytical ultracentrifugation. Amino acid analysis showed a very low sulfur amino acid content and relatively high content of aspartic and glutamic acids. Total neutral sugar analysis indicated less than 1% of saccharides. The lectin interacted with several saccharides.

Animals↗

Proximate composition, phytic acid, polyphenols and digestibility (in vitro) of four brown cowpea varieties.

Four cultivars of brown cowpea (Vigna unguiculata. L. Walp) were analysed for their proximate composition, phytic acid, polyphenols and protein and starch digestibility (in vitro). Crude protein values ranged from 20.07 to 24.60%. Ether extract and crude fiber contents varied from 1.77 to 1.96% and 4.27 to 4.95%, respectively. All the four cowpea varieties differed significantly for their starch content which ranged from 46.84 to 53.63%. Antinutrients, phytic acid and polyphenols, showed significant variations among the varieties. In vitro digestibility of protein and starch of grains had a narrow variation. The variety CS-46 containing minimum amount of antinutritional factors had the highest protein digestibility.

Fabaceae↗

Nutritional quality of microwave-cooked and pressure-cooked legumes.

Eight whole legumes, namely Bengal gram (Cicer arietinum), broad beans (Vicia faba), Cowpea (Vigna catjang), field beans (Dolichos lablab), green gram (Phaseolus aureus Roxb), horse gram (Dolichos biflorus), lentils (Lens esculenta) and French beans (Phaseolus vulgaris), were cooked under pressure or in a microwave oven and were analysed for nutrient composition. Raw legumes served as control. The range of nutrients analysed in 100 g cooked samples were as follows: moisture, 62.8-69.7 g; protein, 14.7-24.3 g; fat, 0.9-5.9 g; ash, 1.7-4.6 g; iron, 3.3-8.6 mg; calcium, 50-209 mg; phosphorus, 249-429 mg; and thiamin, 0.14-0.32 mg. Cooking methods did not affect the nutrient composition of legumes. However, thiamine decreased in cooked samples. Cooking altered the dietary fibre content of some legumes. The mean in vitro protein digestibility of pressure-cooked and microwaved samples was 79.8% and 74.7%, respectively. The in vitro starch and protein digestibility of pressure-cooked samples were higher.

Cooking↗

Distribution and utilization of 15N in cowpeas injected into the stem under influence of water deficit.

Investigations were carried out on Vigna unguiculata L. Walp. to estimate the distribution and utilization of 15N in different organs after stem injection during vegetative, flowering and pod filling stage. During flowering effects of water deficit were also examined. In well watered plants, within 4 days after injection, 65% of 15N accumulated in leaves. This was drastically reduced to 42% by water deficit. 15N accumulation in stems increased under water deficit. The translocation of 15N from the stem base to roots were not altered by water deficit. During pod filling 62% of recovered 15N in the plants had accumulated in seeds, 24% in leaves and 11% in stems within 4 days, whereas the uptake of nitrogen in pod walls and roots remained low (2%). These results demonstrate that the method of injecting very small quantities (1 mg/plant) of 15N into the stem base allows an exact and detailed quantitative assessment of N translocation/distribution with regard to different organs, different growth stages and different treatments.

Fabaceae↗