Studies on the distribution of 14C-malformin A in major fractions of Phaseolus vulgaris L.

The distribution pattern of ¹⁴C-malformin in major fractionsof Phaseolus vulgaris L. seedlings shifted during water treatmentin the absence of malformin. From these shifts, and by comparisonof the ¹⁴C distribution patterns at the base and top of theseedlings, it was concluded that some ¹⁴C-malformin enters thecell and proceeds to the cell wall via intermediate compounds.As a working hypothesis it was suggested that in roots ¹⁴C-malforminfirst appears in a soluble "small molecules" fraction, bindsto a soluble protein fraction, and proceeds via die wall lipidfraction to the wall itself. Direct binding of some ¹⁴C-malforminto the wall fraction was not precluded. In leaves, the pathwayof ¹⁴C-malformin to the cell wall was similar in some respectsto that in roots. ¹ Present address: American Cyanamid, P.O. Box 400, Princeton,New Jersey 08540, U.S.A. (Received January 21, 1976; )     

Isolation and characterization of the subunits of Phaseolus vulgaris alpha-amylase inhibitor.

An alpha-amylase inhibitor (PHA-I) of the white kidney bean (Phaseolus vulgaris) was found to be composed of two kinds of subunits and they were isolated on a size-exclusion column by HPLC under denaturing conditions. The alpha-subunit was free from tryptophan and cysteine and the beta-subunit contained no methionine or cysteine. There was no marked resemblance in tryptic peptide map between these subunit polypeptides. The alpha-subunit contained 28% by weight of carbohydrate, mainly made up of high mannose-type oligosacharides, whereas the sugar moiety of the beta-subunit amounted to 7% by weight and seemed to be predominantly composed of xylomannose-type oligosaccharides. By SDS-PAGE following deglycosylation, the molecular weights of the polypeptides of alpha- and beta-subunits were shown to be 7,800 and 14,000, respectively. These values were consistent with molecular sizes obtained for alpha- and beta-subunits by gel permeation HPLC in 6 M guanidine hydrochloride. The molecular weight of the native PHA-I, 28,800, obtained by gel permeation HPLC under non-denaturing conditions, suggested a heterodimeric structure for PHA-I.     

Growth and cell wall composition of glucose-limited bean cells (Phaseolus vulgaris L.)

Glucose-limited bean cells (Phaseolus vulgaris L.) were grown in a modified bacterial fermentor at a constant pH of 4.8. The cultures were kept in steady state at different specific growth rates varying from 0.00216 h^–1 to 0.0106 h^–1. Culture conditions are described that are needed to start a continuous culture. First, it was essential to use log-phase cells as starting material. Second, it was important to increase the dilution rate gradually, otherwise cells in the culture aggregated. Cells grown at the highest dilution rate employed contained twice as much protein per gram dry weight as cells grown at the lowest dilution rate. The composition of the cell walls also varied with the dilution rate in contrast to their relatively constant composition when grown in batch culture.     

Isolation and Identification of the Precursor of Ethane in Phaseolus vulgaris L

Ethane production by homogenates of Phaseolus vulgaris L. cv. Harvester was studied. The precursor of ethane was identified as linolenic acid. The liberation of ethane was optimum at pH 4.2 and was highest from homogenates of leaves and apical buds. When roots were homogenized in linolenic acid solution, ethane and ethylene production were stimulated. In corn root homogenates, ethylene biosynthesis was stimulated nearly 8-fold by linolenic acid. The enzyme responsible for ethane production from oat root homogenates was soluble and had a high molecular weight.     

Partitioning of 14C-photoassimllates within seeds of Phaseolus coccineus (Lam.) and Phaseolus coccineus x Phaseolus vulgaris L.

The objective of this study was to determine partitioning within seeds of ¹⁴C-photoassimilates at three stages of seed development in two Phaseolus crosses — P. coccineus Lam. selfed, and P. coccineus x P. vulgaris L. Abortion of the interspecific embryos occurred when the seed reached 10 mm seed length. When expressed as sink strength (% dpm) or sink activity (% dmp/d.wt.) there were no differences in partitioning of ¹⁴C-photoassimilates when whole seeds were analyzed. If the seed was divided into seed coat, liquid endosperm, and embryo, the sink activity of the interspecific embryo was higher than that of the embryo in the selfed seed. Therefore, abortion of these interspecific Phaselus embryos appeared not to be caused by a lack of photoassimilates.Assistant Professor, Professors, respectively.Contribution from the Agr. Expt. Station, University of Minnesota, St. Paul, MN 55108. Paper No. 13,548, Scientific Journal Series. This research was supported in part by the Science and Education Administration of the United States Department of Agriculture under Grant 59-2271-9-2-020-0 and in part by a grant from the Minnesota Soybean Research and Promotion Council.     

Apical Dominance in Phaseolus vulgaris L.

Although determinations of the ABA content of lateral buds ofPhaseolus vulgaris revealed no difference between decapitatedand intact control plants in the first 12 h following decapitation,a relative decrease in the ABA content of lateral buds of decapitatedplants was detectable 24 h following decapitation. Shoot decapitationwas also observed to result in a decrease in the ABA contentof stem tissue. The application of IAA to the stem of decapitatedplants prevented these changes and increased the ABA contentof stem tissue relative to that of intact plants. The levelsof IAA and ABA were also determined in the stem tissue fromthe nodes of intact bean plants. The possible interdependenceof these two plant hormones was further investigated by a studyof [2_–14ClABA metabolism. The results are discussed inrelation to the possible role of these hormones in apical dominance. Key words: Apical dominance, Abscisic acid, Indole-3-acetic acid     

Modification of cell wall polysaccharides during cell elongation in Phaseolus vulgaris hypocotyls

The effect of gibberellic acid (GA) and naphthylacetic acid (NAA) on hypocotyl elongation and cell wall polysaccharides was studied using Phaseolus vulgaris seedlings grown in light condition. The hypocotyl was demarcated into two segments — one near the root was called lower and the one near the cotyledon was called upper. The upper segment showed a typical sigmoidal growth curve while lower segment did not show any growth at all. GA promoted the growth of upper segment while NAA showed clear inhibition in both the segments. Xyloglucan content showed a clear inverse correlation with growth. Pectic polysaccharides did not show a clear trend, though showed an initial inverse correlation with growth. It is concluded that degradation of low and high molecular weight xyloglucans are involved in cell wall loosening which in turn may be responsible for the elongation growth of Phaseolus hypocotyls in light.     

Purification and characterization of a thermostable beta-galactosidase from kidney beans (Phaseolus vulgaris L.) cv. PDR14

Using five different steps, beta-Galactosidase has been purified from kidney beans to apparent electrophoretic homogeneity with approximately 90-fold purification with a specific activity of 281 units mg-1 protein. A single band was observed in native PAGE. Activity staining of the native gel with 5-bromo 4-chloro 3-indoxyl beta-D-galactopyranoside (X-Gal) at pH 4.0 also produced a single band. Analytical gel filtration in Superdex G-75 revealed the molecular mass of the native protein to be approximately 75 kD. 10% SDS-PAGE under reducing conditions showed two subunits of molecular masses, 45 and 30 kD, respectively. Hence, beta-galactosidase from kidney beans is a heterodimer. A typical protein profile with lambda max at 280 nm was observed and A280/A260 ratio was 1.52. The N-terminal sequence of the 45 kD band showed 86% sequence homology with an Arabidopsis thaliana and 85% with Lycopersicon esculentum putative beta-galactosidase sequences. The Electrospray Mass Spectrometric analysis of this band also revealed a peptide fragment that had 90% sequence homology with an Arabidopsis thaliana putative beta-galactosidase sequence. The N-terminal sequencing of the 30 kD band as well as mass spectrometric analysis both by MALDI-TOF and ES MS revealed certain sequences that matched with phytohemagglutinin of kidney beans. The optimum pH of the enzyme was 4.0 and it hydrolysed o- and p-nitrophenyl beta-D galactopyranoside with a Km value of 0.63 mmol/L and 0.74 mmol/L, respectively. The energy of activation calculated from the Arrhenius equation was 14.8 kcal/mol enzyme site. The enzyme was found to be comparatively thermostable showing maximum activity at 67 degrees C. Thermal denaturation of the enzyme at 65 degrees C obeys single exponential decay with first order-rate constant 0.105 min-1. Galactose, a hydrolytic product of this enzyme was a competitive inhibitor with a Ki of 2.7 mmol/L.     

Cytokinin metabolism in Phaseolus vulgaris L.

The major cytokinins in stems of decapitated, disbudded bean plants have been identified by enzymic degradation, Sephadex LH20 and reversed phase high performance liquid chromatography, and by combined gas chromatography-mass spectrometry as 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-9--D-ribofuranosylpurine (zeatin riboside), 6-(4-hydroxy-3-methylbutylamino)-9--D-ribofuranosylpurine (dihydrozeatin riboside), and the 5-phosphates of these compounds (zeatin ribotide and dihydrozeatin ribotide). Minor cytokinins in this tissue were tentatively identified as dihydrozeatin-O--D-glucoside and zeatin ribotide-O--D-glucoside. [8-¹⁴C-]Dihydrozeatin appeared to be rapidly metabolized to dihydrozeatin ribotide when supplied to segments of stems from decapitated plants. These results are discussed in relation to the metabolism and distribution of cytokinins in the whole plant.Abbreviations TEAB triethyl ammonium bicarbonate - UV ultra-violet - GCMS gas chromatography-mass spectrometry - HPLC high performance liquid chromatography - TMS trimethyl silyl     

Isolation, characterization and cloning of a cDNA encoding a new antifungal defensin from Phaseolus vulgaris L. seeds

The PvD1 defensin was purified from Phaseolus vulgaris (cv. Pérola) seeds, basically as described by Terras et al. [Terras FRG, Schoofs HME, De Bolle MFC, Van Leuven F, Ress SB, Vanderleyden J, Cammue BPA, Broekaer TWF. Analysis of two novel classes of plant antifungal proteins from radish (Raphanus sativus L.) seeds. J Biol Chem 1992;267(22):15301–9], with some modifications. A DEAE-Sepharose, equilibrated with 20 mM Tris–HCl, pH 8.0, was initially utilized for the separation of peptides after ammonium sulfate fractionation. The basic fraction (the non-retained peak) obtained showed the presence of one unique band in SDS–Tricine gel electrophoresis with a molecular mass of approximately 6 kDa. The purification of this peptide was confirmed after a reverse-phase chromatography in a C2/C18 column by HPLC, where once again only one peak was observed and denominated H1. H1 was submitted to N-terminal sequencing and the comparative analysis in databanks revealed high similarity with sequences of different defensins isolated from other plants species. The N-terminal sequence of the mature defensin isolated was used to produce a degenerated primer. This primer allowed the amplification of the defensin cDNA by RT-PCR from mRNA of P. vulgaris seeds. The sequence analysis of the cloned cDNA, named PVD1, demonstrated 314 bp encoding a polypeptide of 47 amino acids. The deduced peptide presented high similarity with plant defensins of Vigna unguiculata (93%), Cicer arietinum (95%) and Pachyrhizus erosus (87%). PvD1 inhibited the growth of the yeasts, Candida albicans, Candida parapsilosis, Candida tropicalis, Candida guilliermondii, Kluyveromyces marxiannus and Saccharomyces cerevisiae. PvD1 also presented an inhibitory activity against the growth of phytopathogenic fungi including Fusarium oxysporum, Fusarium solani, Fusarium lateritium and Rizoctonia solani.     

Isozymes of Glutamine Synthetase in Phaseolus vulgaris L. and Phaseolus lunatus L. Root Nodules

The glutamine synthetase (GS) isozymes in the plant fraction of nodule extracts from 62 cultivars of Phaseolus vulgaris L. and one cultivar of Phaseolus lunatus L. were analyzed by polyacrylamide gel electrophoresis. All P. vulgaris nodule extracts displayed two GS activity bands: a nodule-specific band (GS_n1) and a band (GS_n2) similar to the single band (GS_r) present in root extracts. In nodule extracts of P. lunatus, the GS_n1 band was detected, but the GS_n2 band was barely detectable. In contrast to P. vulgaris, the GS_n2 band and the GS_r band of P. lunatus appeared to be different. The electrophoretic mobility of the GS_n1 band in P. vulgaris was governed by both the plant cultivar and the development stage of the nodule. In nodule extracts of P. vulgaris and P. lunatus, the zone of GS_n1 activity coincided with six to nine distinct protein bands as revealed after treatment of gels, which had previously been stained for GS activity, with Coomassie blue. All these protein bands were shown to consist of polypeptides of identical molecular weight (approximately 47,000 daltons) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our results indicate that P. vulgaris continuously generates isozymes of GS_n1 of increasing electrophoretic mobility during the course of nodule development.     

Tissue Cultures of Phaseolus vulgaris L.

Abstract

Callus production and plant regeneration from different explants of Phaseolus vulgaris L. cv. Giza are reported. Calli cultures were induced from leaf, hypocotyl, embryo and root explants. Rapid growth of callus was achieved by leaf explants cultured on MS salts, B5 vitamins and supplemented with 2,4— dichlorophenoxyacetic acid (2, 4—D)+0.5 mg/l kinetin (kin). Addition of casein hydrolysate at 2 g/l to maintenance medium enhanced callus growth and hindered the early appearance of necrotic parts. This report also provides a detailed method for production of multiple shoots directly from the wounded edges of immature cotyledon explant via organogenesis on 1 mg/l benzyladenine (BA) or indirectly on 0.5 mg/l naphthaleneacetic acid (NAA)+2 mg/l BA. The regeneration of bean plants through the two ways described here (direct or indirect) may be of use in genetic improvement of bean.     

Biochemical characterization of profilin from seeds of Phaseolus vulgaris L

The isoform composition of the 14.4 kDa profilin polypeptide was analyzed in seeds, leaves, flowers, roots and root-nodules from Phaseolus vulgaris L. Isoforms of pIs approximately 4.4-5 were present in all the tissues analyzed. The biochemical features of the protein present in seed tissue were determined. Seed profilin bound to Phenyl-Sepharose under low salt conditions which suggested a hydrophobic interaction; however, it was not associated with microsomal membranes nor it partitioned as a hydrophobic protein in Triton X-114. Fractions eluting from poly-L-proline or Phenyl-Sepharose columns contained well detectable amounts of profilin but no actin, suggesting that most of the protein was not present as profilactin in the seed. However, seed profilin appeared to be in some kind of complex since several molecular weight species were observed on native gels. In addition, profilin was found preferentially in the embryo axis and light microscopic immunolocalization showed a cytoplasmic distribution in this tissue.     

Isolation and characterization of lectins from kidney beans (Phaseolus vulgaris)

The bioactive properties of lectins obtained from raw and canned red kidney bean (Phaseolus vulgaris) were studied to determine the changes in their bioactivity during the canning process. Phytohaemagglutinin (PHA) was extracted using Affi-gel Blue gel and thyroglobulin-Sepharose and had a molecular weight of 32 kDa. Both the raw and the canned kidney beans possessed the ability to agglutinate red blood cells and inhibit α-glucosidase. The activity found in the canned beans was similar to that from the in the raw kidney beans. However, the amount of lectin that could be extracted from thyroglobulin-Sepharose was much less in the canned samples than in the raw kidney bean samples. The extracted lectin from the raw kidney beans was also subjected to a heating and cooling treatment using a differential scanning calorimeter. The lectin had a nonset denaturation temperature of 77.76 °C and it did not renature upon cooling. In this study, we demonstrated that extracts from raw red kidney bean and canned red kidney bean contain bioactive compounds capable of inhibiting HIV-1 RT in vitro.     

Ureide Metabolism in Non-nodulated Phaseolus vulgaris L.

The distribution of ureide-N was studied throughout vegetativeand reproductive growth of non-nodulated Phaseolus vulgarisL. (bushbean) grown in nitrate nutrient solution. Largest increasesin ureide-N per plant were correlated with flowering and earlypod formation and with seed filling. Highest amounts of ureidesper organ were measured in stems and axillary trifoliates. Highestconcentrations (µmol ureide-N g^–1 fr. wt.) weremeasured in young developing organs and stems. Seeds did notaccumulate ureides until the ureide content of pods had reacheda maximum. Results obtained using the inhibitor of xanthine oxidase, allopurinol,are consistent with the origin of ureides via purine degradationbut alternative pathways cannot be discounted. Leaves and stems were shown to have the ability to degrade allantoatevia an enzymic process.     

Comparative Water Relations of Phaseolus vulgaris L. and Phaseolus acutifolius Gray

Leaf area expansion, dry weight, and water relations of Phaseolus vulgaris L. and P. acutifolius Gray were compared during a drying cycle in the greenhouse to understand the characteristics which contribute to the superior drought tolerance of P. acutifolius. Stomates of P. acutifolius closed at a much higher water potential than those of P. vulgaris, delaying dehydration of leaf tissue. P. acutifolius had a more deeply penetrating root system, which also contributes to its drought tolerance. Root-shoot ratios did not differ between the two species either under well watered or water stressed conditions. Leaf osmotic potential was also similar in the two species, with no apparent osmotic adjustment during water stress. These results indicate that P. acutifolius postpones dehydration and suggest that sensitive stomates and a deeply penetrating root system are characteristics which, if incorporated into cultivated beans, might increase their drought tolerance.     

Late Transplanting and the Yield of Phaseolus vulgaris L.

The prediction that very high seed yields of dry beans (Phaseolusvulgaris L.) would be produced by the delayed transplantingof large plants has been tested in a factorial experiment withfour dates of transplanting and eight plant populations. Therewere significant differences in yield between transplantingdates and between population densities, and there was a significantdate-density interaction. At low plant densities (up to about30 plants m^–2) the three transplanted treatments yieldedless than the hand-sown controls, and late transplanting yieldedless than early. At the highest density the situation was reversed;all three transplanted treatments out-yielded the controls andlate transplanting tended to out-yield plants transplanted early.The biggest yield was 340 g seed m^–2 from a transplantedcrop grown at 35 plants m^–2. The data on yield fitted a modified rectangular hyperbola ofthe form where y is yield per unit area, p is the number of plants perunit area, t is the number of days between sowing and transplanting,and B_o, n, m, and p are arbitrary parameters. This equationaccounted for 91 per cent of the variation in yield with t andp. It is suggested that late transplanting had adverse effects,due to transplanting ‘shock’ and which were mostmarked at low plant densities; and beneficial effects, ascribableto an effect on plant ‘plasticity’, which were mostmarked at high plant densities. Possible physiological mechanismsof these effects are discussed. Phaseolus vulgaris, yield, density, transplanting     

Transport of 14C-Assimilates in Seedlings of Phaseolus vulgaris L. in Relation to Vascular Anatomy

Patterns of ¹⁴C-photosynthate translocation in bean (Phaseolusvulgaris L.) seedlings have been investigated in relation tovascular-bundle continuity between exporting and importing organsby use of radioassay and tissue-clearing techniques. Assimilatefrom the primary leaves reaches the first trifoliate leaf byan indirect route. There is no direct vascular connection betweenthe primary leaves and distal tissues. Bundles of the primaryleaf petiole connect with an anastomosis at the node, and allbundles which originate from this structure descend the stem.Assimilate from primary leaves moves down the stem, and is thentransferred to an ascending pathway, the bundles of which traversethe anastomosis at the second node. The lateral leaflets ofthe first trifoliate leaf are served differentially by primaryleaves with respect to assimilate supply. Differences are relatedto position, and may be accounted for by differences in vascularcontinuity.