Lectins in Immature Pods of Winged Bean,Psophocarpus tetragonolobus (L.) DC.

Lectin activity in immature pods from 30 strains of winged bean was investigated. Most of the lectin activity occurred in green shells and a small portion in immature seeds. Hemagglutinating activity of green shells was classified into four groups, according to the agglutination of trypsinized human type A, B, and O erythrocytes. Extracts from green shells of four representative strains which showed different hemagglutination patterns gave different elution profiles of lectin activity from ion exchange columns. Four types of lectin activity with different blood group specificities were apparently found in green shells.     

Two Isolectins from Leaves of Winged Bean, Psophocarpus tetragonolobus (L.) DC.

Two isolectins were isolated from leaves of winged bean andcharacterized. They differed from each other in terms of theirimmunological properties, hemagglutinating activities, sugarinhibition patterns, and amino acid compositions. Both lectinswere acidic and one of them (L-I), which was inactive towardtrypsinized human type O erythrocytes, was similar to one ofgreen shell lectins (WGS-1); which resembled basic seed lectinin its immunological properties. The amino-terminal sequenceof L-I was homologous to that of WGS-1. The amino acid compositionof L-I was similar to that of basic seed lectin, but the extentof the homology between amino-terminal sequences was low whenL-I and basic seed lectin were compared. Examination by ELISArevealed that L-I and WGS-1 were distinct from the basic lectinsof seeds and tuberous roots. L-I had a disulfide bridge betweentwo subunits and it exhibited high hemagglutinating activitytoward human type A erythrocytes, as compared to its activitytoward other erythrocytes. By contrast, the properties of asecond acidic lectin from winged bean leaves (L-II) were verysimilar to those of acidic lectins from seeds and tuberous roots,and the similarities extended as far as the immunological properties. (Received January 6, 1994; Accepted August 15, 1994)     

Somatic embryogenesis and plant regeneration from leaf derived callus of winged bean [Psophocarpus tetragonolobus (L.) DC.]

Somatic embryos were obtained from callus cultures derived from leaf explants of the winged bean, Psophocarpus tetragonolobus (L.) DC. Initiation and development of the somatic embryos occurred with a two-step culture method. Callus cultures initiated on MS medium with NAA and BAP, upon transfer to a new medium with IAA and BAP, produced somatic embryos. Maximum embryogenesis of 60% was obtained on induction medium with 0.5 mg/l NAA plus 1.0 mg/l BAP followed by transfer to a secondary medium with 0.1 mg/l IAA and 2.0 mg/l BAP. Optimal embryo germination and plantlet development was achieved on MS medium with 0.2 mg/l BAP plus 0.1 mg/l IBA. The regenerated plants were successfully transferred to glasshouse conditions.Abbreviations MS Murashige and Skoog (1962) medium - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - BAP 6-benzylaminopurine - KN Kinetin     

Structure and regulated expression of Kunitz chymotrypsin inhibitor genes in winged bean [Psophocarpus tetragonolobus (L.) DC.]

We analyzed the structure and the expression of Kunitz chymotrypsin inhibitor (WCI) genes in winged bean. WCI was encoded by a multigene family which comprised at least seven members. From their primary structures, four genes (WCI-2, WCI-3a, WCI-3b, and WCI-x) were expected to be functional ones and the other three (WCI-P1, WCI-P2, and WCI-P3) to be pseudogenes. The nucleotide sequences of the WCI-3a and WCI-3b genes were nearly identical, and they encoded the WCI-3 protein, the major chymotrypsin inhibitor in seeds. The WCI-2 gene also encoded the chymotrypsin inhibitor found in seeds and the WCI-x gene was expected to encode an unidentified chymotrypsin inhibitor. WCI messenger RNA and protein accumulated mainly in developing seeds and tuberous roots, small amounts of WCI mRNA being present in stems and pericarps. In seeds, transient accumulation of WCI mRNA was observed during the seed maturation period. These results suggest that the expression of WCI genes is regulated organ-specifically and developmentally in winged bean.     

Chemical modification studies on a lectin from winged-bean [Psophocarpus tetragonolobus (L.) DC] tubers.

The effect of chemical modification on a D(+)-galactose-specific lectin isolated from winged-bean tubers was investigated to identify the type of amino acid involved in its haemagglutinating activity. Various anhydrides of dicarboxylic acids, such as acetic anhydride, succinic anhydride, maleic anhydride and citraconic anhydride, modified 57-68% of the amino groups of the winged-bean tuber lectin. Treatment with N-acetylimidazole modified only 45% of the total amino groups. Reductive methylation of free amino groups modified 57% of the amino groups. Modification of the amino groups of the lectin by acetic anhydride and succinic anhydride did not lead to any significant change in the haemagglutinating activity (greater than or equal to 75% active). However, citraconylation and maleylation of the lectin led to a significant decrease in the haemagglutinating activity (less than or equal to 20% active). Acetylation and succinylation (3-carboxypropionylation) of the lectin led to a decrease in the pI value of the native lectin from approx. 9.5 to approx. 4.5. Treatment of the lectin with N-bromosuccinimide led to the modification of two and four tryptophan residues per molecule in the absence and in the presence of 8 M-urea respectively. The immunological identity of all the modified lectin preparations showed no gross structural changes except the lectin modified with N-bromosuccinimide in the presence of urea at pH 4.0.     

Winged bean (Psophocarpus tetragonolobus (L.) DC) as a substrate for growth and aflatoxin production by aflatoxigenic strains of Aspergillus spp.

The mold flora of seeds of twelve varieties of winged beans were determined both before and after surface disinfections. When seeds were surface disinfected, molds were detected in 73% of the seeds whereas 81% of the seed that was not disinfected produced molds. Aspergillus spp. were most frequently present while Penicillium spp. occurred in seed of 4 varieties and in less than 4% of the seed. Twelve isolates of A. flavus and A. parasiticus were examined for their ability to produce aflatoxins. Whether aflatoxins were produced and the amount of each varied according to the origin of the isolate and the species of Aspergillus. For example all A. flavus isolates produced at least 2 aflatoxins whereas 4 of the A. parasiticus isolates were nontoxigenic. When ground seeds of winged beans were inoculated with an aflatoxigenic strain of A. parasiticus the level of aflatoxins that occurred varied with the variety. All of the varieties supported greater aflatoxin production than peanuts and 6 of the 12 winged bean varieties gave higher levels of aflatoxins than rice.     

Winged bean (Psophocarpus tetragonolobus (L.) DC) as a substrate for growth and aflatoxin production by aflatoxigenic strains of Aspergillus spp

The mold flora of seeds of twelve varieties of winged beans was determined both before and after surface disinfections. When seeds were surface disinfected, mold fungi were detected in 73% of the seeds whereas 81% of the seed that was not disinfected produced mold fungi. Aspergillus spp. was most frequently present while Penicillium spp. occurred in seed of 4 varieties and in less than 4% of the seed. Twelve isolates oiA. flavus and A. parasiticus were examined for their ability to produce aflatoxins. Whether aflatoxins were produced and the amount of each varied according to the origin of the isolate and the species of Aspergillus. For example all A. parasiticus isolates produced at least 2 aflatoxins whereas 4 of the A. parasiticus isolates were non-toxigenic. When ground seeds of winged beans were inoculated with an aflatoxigenic strain of A. parasiticus the level of aflatoxins that occurred varied with the variety, however, the level of aflatoxin was higher in winged bean than in peanut tissue and 6 of the 12 winged bean varieties contained higher levels of aflatoxins than rice.     

Generality and Diversity of Winged Bean (Psophocarpus tetragonolobus) Protein in Eight Various Lines

Protein components from eight lines of winged bean (Psophocarpus tetragonolobus) seeds which were originally introduced from Papua New Guinea, Indonesia, Nigeria, and Ishigaki, and cultivated in Okinawa and Fukuoka, were investigated. Two major peaks which had sedimentation coefficients, s₂₀, of about 2.5S and about 6.5S (6.0 to 6.6 for the 8 lines), and no larger component were observed in all specimens with more than 90% extraction. Electrophoretic profiles of the “6.5S” component(s) which was separated with Sepharose 6B column chromatography showed a main broad band and a few minor bands which seemed to be essentially similar among the eight lines of winged bean. Thus the “6.5S” protein surely could be regarded as the common storage protein in winged bean seeds. The subunit structure of the “6.5S” component(s) in SDS solution consisted of four major bands. The “2.5S” components were mixtures and combinations of various proteins which were distinctly different from one selection to another.     

Purification and characterization of proteinase inhibitors from winged bean (Psophocarpus tetragonolobus (L.) DC.) seeds

Seven proteinase inhibitors were isolated from winged bean seeds by ion-exchange chromatographies. These inhibitors had molecular weights of around 20,000, included four half-cystine residues, and were Kunitz-type inhibitors. Two (WTI-2 and 3) inhibited bovine trypsin strongly and four (WCI-1, 2, 3, and 4) inhibited bovine alpha-chymotrypsin, but in different ways. One mole of WCI-2 or -3 could inhibit 2 mol of alpha-chymotrypsin. The remaining inhibitor (WTCI-1) could bind both bovine trypsin and alpha-chymotrypsin at the molar ratio of 1:1, but not simultaneously. All four chymotrypsin inhibitors cross-reacted with rabbit anti-WCI-3 serum, while the other inhibitors did not.     

Effect of raw winged bean [Psophocarpus tetragonolobus (L.) DC] tuber lectin on gastrointestinal tract of growing rats

The fraction containing high hemagglutinating activity was prepared from raw winged bean tubers and orally administered to growing rats. The food intake and body weights of these rats decreased as the level of lectin increased and significant lectin activity was detected in the faeces extracted from these rats which is anti-genically similar to the native lectin preparation. Microscopic examination has revealed morphological changes in the intestinal epithelial cells. The binding action of the lectin to the mucosal epithelia of the gastrointestinal tract is indicative of the deleterious effects caused by the winged bean tuber lectin.     

Study of genotypic variation of cultivars of winged bean [Psophocarpus tetragonolobus (Stickm.) DC.] with the help of n-alkane profile

The leaf cuticle is covered by epicuticular wax consisting mainly of straight-chain aliphatic hydrocarbons with a variety of substituted groups. Studies have been concentrated on n-alkanes in epicuticular wax of Winged bean [Psophocarpus tetragonolobus (Stickm.) DC.]. Hydrocarbon constituents especially n-alkane analyses of seven cultivars of Winged bean [Psophocarpus tetragonolobus (Stickm.) DC.] have been undertaken. All the n-alkanes in between C₁₄–C₁₈ and C₂₀–C₃₈ are present in each of the species. Among the species, amount of n-alkanes is maximum in IC112417 and relatively low in EC38825. Scanning electron microscopic views were also taken for epicuticular layers and their hydrocarbons of the leaves of all the genotype species of the plant. Qualitative and quantitative characterization of n-alkanes present in the epicuticular wax extracted from the mature leaves can be used as an effective tool in chemo taxonomical work and also for the study of genotypic variation of the different cultivars.     

Crystallization and preliminary crystallographic data of the major albumin from Psophocarpus tetragonolobus (L.) DC

The major seed albumin from Psophocarpus tetragonolobus (L.) DC, winged bean albumin 1, has been crystallized from ethanol as rhombic needles in a form suitable for high resolution x-ray crystallographic studies. The space group is I4(1)22 with cell parameters a = b = 95.6 A, and c = 86.0 A. Amino acid sequence homology between winged bean albumin 1 and legume 7 S globulins suggests that the former may be related to a structural domain of the 7 S globulins.     

Amino acid sequence of the acidic Kunitz-type trypsin inhibitor from winged-bean seed [Psophocarpus tetragonolobus (L.) DC]

The primary sequence of trypsin inhibitor-2 (WBTI-2) fromPsophocarpus tetragonolobus (L.) DC seeds was determined. This inhibitor consists of a single polypeptide chain of 182 amino acids, including four half-cystine residues, and an N-terminal residue of pyroglutamic acid. The sequence of WBTI-2 showed 57% identity to the basic trypsin inhibitor (WBTI-3) and 50% identity to the chymotrypsin inhibitor (WBCI) of winged bean, and 54% identity to the trypsin inhibitor DE-3 fromErythrina latissima seed. The similarity to the soybean Kunitz trypsin inhibitor (40%) and the other Kunitz-type inhibitors fromAdenanthera pavonina (30%) and wheat (26%) was much lower. Sequence comparisons indicate that thePsophocarpus andErythrina inhibitors are more closely related to each other than to other members of the Kunitz inhibitor family.     

Effects of Temperature and Photoperiod on Flowering in Chickpeas (Cicer arietinum L.)

Factorial combinations of two photoperiods (12 and 15 h), threeday temperatures (20, 25 and 30 °C) and three night temperatures(10, 15 and 20 °C) were imposed on nodulated plants of ninechickpea genotypes (Cicer arietinum L.) grown in pots in growthcabinets. The times to first appearance of open flowers wererecorded. For all genotypes, the rates of progress towards flowering(the reciprocals of the times taken to flower) were linear functionsof mean temperature. There were no interactions between meantemperature and photoperiod but the longer photoperiod increasedthe rate of progress towards flowering. These effects were independentof both radiation integral (the product of irradiance and photoperiod)and the vegetative stature of the plant. Taken in conjunctionwith evidence from work on other long-day species, it is suggestedthat the photo-thermal response of flowering in chickpeas, overthe range of environments normally experienced by the crop,may be described by the equation: 1/f = a+b     

Amino acid sequence of winged bean (Psophocarpus tetragonolobus (L.) DC.) chymotrypsin inhibitor, WCI-3

The complete amino acid sequence of winged bean chymotrypsin inhibitor 3 (WCI-3) was determined by the conventional methods. WCI-3 consisted of 183 amino acid residues, but was heterogeneous in the carboxyl terminal region owing to the loss of one to four carboxyl terminal amino acid residues. The sequence of WCI-3 was highly homologous with those of soybean trypsin inhibitor Tia, winged bean trypsin inhibitor WTI-1, and Erythrina latissima trypsin inhibitor DE-3. One of the reactive site peptide bonds of WCI-3 was identified as Leu(65)-Ser(66), which was located at the same position as those of the other Kunitz-family leguminous proteinase inhibitors.     

Characterization of the acidic lectins from winged bean seed (Psophocarpus tetragonolobus(L.)DC)

The seeds of winged bean, Psophocarpus tetragonolobus(L.)DC, contain two distinct groups of lectins characterized by different erythrocyte hemagglutinating specificities and isoelectric points. Three acidic lectins (I, II, and III) (pI approximately 5.5) were purified to apparent homogeneity by chromatography on Ultrogel AcA44 and SP-Sephadex C-25. These lectins are glycoproteins with relative molecular mass of 54,000. The total carbohydrate content of the acidic lectins was 7% and was comprised of mannose, N-acetylglucosamine, fucose, and xylose in amounts corresponding to 9.2, 4.8, 1.6, and 7.0 mol/54,000 g, respectively. Electrophoresis in dodecyl sulfate, in the presence and absence of 2-mercaptoethanol, gave a single subunit of apparent relative molecular mass 30-32,000, somewhat higher than expected from the native relative molecular mass. On isoelectric focusing in 8 M urea the subunits of the acidic lectins did not show any significant charge heterogeneity as found for the winged bean basic lectins. The acidic lectins have very similar amino acid compositions. They contain essentially no half-cystine, 1-2 methionine residues, and are rich in acidic and hydroxy amino acids. The amino-terminal sequences of lectins II and III were identical while the amino-terminal sequence of lectin I contained five differences in the first 25 residues; the acidic lectins showed extensive sequence homology with the winged bean basic lectins, the other one-chain subunit lectins and the beta subunit of the two-chain subunit legume lectins. The acidic lectins agglutinated trypsinized human (type A, B, AB, and O) erythrocytes but not trypsinized rabbit erythrocytes. They were inhibited by various D-galactose derivatives and D-galactose-containing disaccharides and trisaccharides. N-Acetylgalactosamine was the best inhibitor, and the specificity appears to be directed to beta-D-galactosides. However, compared with winged bean basic lectins and soybean lectin, the winged bean acidic lectins show a low affinity for the inhibitory sugars.     

Sequence and expression of the mRNA encoding the chymotrypsin inhibitor in winged bean (Psophocarpus tetragonolobus (L.) DC.)

The accumulation of the Kunitz-type chymotrypsin inhibitor WCI-3 in winged bean seeds is controlled developmentally. In vitro translation experiments showed that the WCI-3 mRNA was present in 35- and 40-day-old immature seeds after flowering. The size of the in vitro translation product is about 2 000 Da larger than that of the mature WCI-3 protein. The WCI-3 cDNA clones were isolated from a gtll cDNA library of 35-day-old immature seeds by immunoscreening. A nearly full-length cDNA clone was obtained containing an open reading frame of 207 amino acid residues. The deduced sequence of the 183 carboxy terminal amino acids coincides precisely with the amino acid sequence determined for purified WCI-3. The amino terminal extension of 24 residues has the characteristics of a signal peptide. Northern hybridization analysis of total poly(A)⁺ RNA showed that the WCI-3 mRNA is approximately 900 nucleotides long and accumulates in 35- and 40-day-old but not in 30-day-old immature seeds.     

Distribution of Lectins in Tissues, Derived Callus, and Roots of Psophocarpus tetragonolobus (Winged Bean)

The distribution of lectin in parental tissues, roots formed de novo from parental stem tissue, and derived callus cells of Psophocarpus tetragonolobus has been measured by hemagglutinating activity and radioimmunoassay. The antisera used for the radioimmunoassay was raised in rabbits to lectin isolated from seeds by affinity chromatography using insolubilized hog gastric mucin. The distribution of lectin in buffer extracts of the tissues (or cells) and the extracellular medium favors the tissues for in vitro grown roots, regardless of the culture conditions used. The lectin content of the extracellular medium is more significant for callus, regardless of its conditions of culture. The lectin activity of extracts of in vitro grown roots was higher than that of mature roots from whole plants. Differences in relative levels of lectin activity measured by hemagglutination and by radioimmunoassay, and differences in saccharide inhibition of hemagglutination, suggest the presence of multiple lectins in extracts of different tissues.     

四棱豆(Psophocarpus tetragonolobus)酰脲相对丰度与固氮酶活力的关系

四棱豆根瘤固氮酶活力日变化呈双峰曲线,两个峰分别出现在14:30和20:00;其固氮酶活力日变化与叶片酰脲含量变化的相关系数为0.67,且与根、茎、叶的酰脲相对丰度(URA)相关系数分别为0.59,0.61,0.76。在个体发育过程中,根瘤固氮酶活力与叶片酰脲含量以及酰脲相对丰度之间呈极显著相关。贮存在30℃条件下5 h不影响植物材料的酰脲相对丰度。四棱豆叶片酰脲相对丰度可作为估价根瘤固氮酶活力的一个指标。