Separation and biological properties of Phaseolus vulgaris isolectins

Two isolectins (L₄E₀-PHA and L₀E₄-PHA) from red kidney beans (Phaseolus vulgaris) were isolated by affinity chromatography on immobilized thyroglobulin and by chromatography on hydroxyapatite. The L₄E₀-PHA siolectin was not retarded on the affinity column and was eluted from the hydroxyapatite column with a 0.1 M Phosphate buffer. This isolectin had no erythroagglutinating activity but had high lymphoagglutinating and lymphocyte stimulating activities. The L₀E₄-PHA isolectin was adsorbed on the affinity column and was eluted from the hydroxyapatite column with a 0.25 M Phosphate buffer. This isolectin has a high erythroagglutinating activity, a very low lymphoagglutinating activity and no lymphocyte stimulating activity. These two isolectins are shown however to be closely related with respect to their oligomeric structure and reactivity towards anti-PHA antibodies. The lack of mitogenic activity of the L₀E₄-PHA isolectin suggests that in the other isolectins, the E monomer is not responsible for their mitogenic activity and that the membrane glycoproteins, which contain the E monomer — specific oligosaccharide, are not involved in the process inducing mitosis.     

Binding of hydrophobic ligands to plant lectins: Titration with arylaminonaphthalenesulfonates

Binding of the Hydrophobic ligands 1,8-anilinonaphthalenesulfonic acid (ANS) and 2,6-toluidinylnaphthalenesulfonic acid (TNS) to a variety of plant lectins was studied by lectin-induced alteration of the fluorescence spectra of the two ligands. With one exception, all legume lectins examined bound ANS, with affinity constants ranging from 10³ to 10⁴ M^?1. Similar ANS binding was noted for some nonlegume lectins. Titration of the five isolectins from Phaseolus vulgaris with ANS indicated positive cooperative binding of ANS to the two isolectins E₄ and E₃L₁. Titrations with TNS revealed high-affinity sites for this ligand in a number of lectins. Addition of haptenic sugars did not inhibit binding of ANS, suggesting that the hydrophobic binding sites of lectins are independent of the carbohydrate binding sites.     

A unique cytoplasmic male sterility (CMS) determinant is present in three Phaseolus species characterized by different mitochondrial genomes

Previous results have shown that cytoplasmic male sterility (CMS) in lines from Phaseolus coccineus and Phaseolus vulgaris contain the same CMS-specific sequence, raising the question of whether this sequence rearrangement arose before divergence of the two species or afterward with subsequent transfer by introgression. Hybridization patterns of total DNA from eight P. vulgaris lines with cytoplasm from P. coccineus and three P. vulgaris lines were examined in order to analyze the mitochondrial DNA (mtDNA) diversity within each species and to determine differences between CMS lines derived from the two species. Three restriction enzymes and 17 heterologous mtDNA sequences were used. The analysis of the different hybridization patterns revealed a considerable diversity in mtDNA organization particularly within P. coccineus. We obtained distinctive hybridization patterns for the five CMS lines tested. The resulting classification showed that mitochondrial genomes from P. coccineus CMS lines group with those of fertile P. coccineus but not with CMS lines from P. vulgaris. The groupings concur with the taxonomic classification of these lines. The results support the hypothesis of a single ancient origin of the CMS determinant and exclude the transfer of cytoplasm by introgression from P. vulgaris to P. coccineus and P. coccineus ssp polyanthus.     

Biochemical evidence bearing on the domestication of<Emphasis Type="Italic">Phaseolus</Emphasis> (Fabaceae) beans

The genusPhaseolus (Fabaceae) consists of some 50 species, all of which are distributed in the Americas. Four of these contain cultigens.P. vulgaris (common bean),P. lunatus (lima bean),P. acutifolius (tepary bean),P. coccineus subsp.coccineus (runner bean); andP. coccineus subsp.polyanthus (no English vernacular name). Biochemical markers—phaseolin seed storage protein and isozymes—have provided new evidence on the organization of the first three species. Domestication has possibly caused a strong reduction in genetic diversity inP. vulgaris andP. acutifolius. BothP. vulgaris andP. lunatus cultivars result from at least two independent domestications, in Mesoamerica and in the Andes. These two species consist of two gene pools, each of which includes wild ancestors and their respective cultivated descendants. Our findings suggest the need for additional emphasis on genetic conservation of wild ancestors and their use in breeding programs and for a comparison of inter-gene pool vs. intra-gene pool crosses in breeding programs.     

Analyses of Phaseolus vulgaris L. and P. coccineus Lam. hybrids by RFLP: preferential transmission of P. vulgaris alleles

Summary Restriction fragment length polymorphism (RFLP) was determined among P. vulgaris genotypes and Phaseolus species using 19 probes. The incidence of polymorphism was high (70–86%) between species, but relatively low (22–26%) between genotypes of P. vulgaris. Suitable probes were identified for the analysis of P. vulgaris and P. coccineus hybrids. The segregation pattern in F₂ populations was Mendelian for two probes (LHB and VEE20) and non-Mendelian for GS-g, CHS, and CHI. Statistical analyses indicated gametic selection with preferential transmission of the P. vulgaris alleles, which may account for the selective recovery of P. vulgaris progeny types observed earlier. The available hybrids of P. vulgaris and P. coccineus and the high degree of interspecific RFLP will facilitate the construction of a linkage map for Phaseolus.     

Genetic structure and mating systems in wild and cultivated populations of Phaseolus coccineus and P. vulgaris (Fabaceae)

We present a study of the genetic variation, genetic structure, and outcrossing rate of two wild populations of Phaseolus coccineus ssp. formosus (both from Central Mexico), two cultivated populations of P. coccineus ssp. coccineus (one from Central Mexico, the other from Chiapas), and one cultivated population of P. vulgaris (from Central Mexico), using seven electrophorectic loci. All the populations of P. coccineus showed high and similar levels of genetic variation, without differences among wild and cultivated populations (H ranged between 0.187 to 0.271). In contrast, P. vulgaris has low levels of genetic variation (H = 0.041). The three P. coccineus populations from Central Mexico were genetically similar, while the cultivated P. coccineus from Chiapas and the cultivated P. vulgaris from Central Mexico were very different. In all the loci and all the populations for both species, the fixation indices (F) were positive and differed significantly from random mating expectation in 59% of the cases for P. coccineus. The highest F was the estimated for P. vulgaris. For P. coccineus, the multilocus outcrossing rate estimates ranged from 0.592 to 0.698, and these did not differ significantly among populations. For P. coccineus, we also estimated the outcrossing rate for each of 60 different families, showing great variation, with a majority of families with a t between 0.8 and 1, but a substantial fraction of the families with a t as low as 0.12–0.20. The domestication process has neither eroded the levels of genetic variation of the cultivated populations of P. coccineus nor changed its mating system.     

RELATIONS D‘ACANTHOSCELIDES OBTECTUS AVEC DIFFÉRENTES ESPÈCES DE PHASEOLUS: INFLUENCE SUR LA FÉCONDITÉ ET POSSIBILITÉS DE DÉVELOPPEMENT LARVAIRE

Résumé La présence des gousses et graines de six espèces de Phaseolus déclenche une ponte importante chez A. obtectus et stimule significativement la production ovarienne. Cependant, les meilleures performances ne sont pas observées avec P. vulgaris, hôte habituel, mais avec P. coccineus.Pour toutes ces espèces le tégument de la graine représente une barrière importante pour la pénétration des larves. Chez certaines espèces sauvages, et dans les conditions expérimentales, il permet une protection totale de la graine. Cette protection semble diminuer avec la domestication chez P. vulgaris.Lorsque les graines sont perforées artificiellement, les larves (une par graine) pénètrent sans difficulté, quelle que soit l'espèce. Les graines de P. coccineus et de P. vulgaris permettent le développement complet de la majorité des larves, tandis que dans celles de P. metcalfei et de P. acutifolius ne peut se développer qu'une faible partie de la population larvaire. Certains Phaseolinae peuvent donc stimuler la ponte sans permettre le développement des larves.Les femelles de la F₁ provenant de P. coccineus ont des performances reproductrices supérieures à celles des femelles provenant de P. vulgaris (variétés sauvage ou cultivée). P. coccineus pourrait être considéré comme une des plantes-hôtes d'origine.

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Summary The presence of pods and seeds of six Phaseolus species induces significant egg-laying in A. obtectus and strongly stimulates ovarian production. However, the best performances are not observed with P. vulgaris, the common host, but with P. coccineus.For all six species, the seed testa is an important barrier against larval penetration. For some wild species, it can give total protection of the seed, but this protection is reduced with domestication in P. vulgaris.For all species, when the seeds are artificially perforated, larvae (one per seed) penetrate easily. Most of the larvae can complete their development in seeds of P. coccineus and P. vulgaris, but only a small proportion of the larval population can develop in seeds of P. metcalfei and P. acutifolius. Thus certain Phaseolinae seeds stimulate egg-laying without allowing larval growth.Females of the F₁ reared in P. coccineus have superior reproductive capacities than those reared in P. vulgaris. P. coccineus could be considered as one of the original host-plants of A. obtectus.

     

Embryo development in reciprocal crosses of Phaseolus vulgaris L. and P. coccineus Lam.

Summary Embryo development was examined in reciprocal crosses of Phaseolus vulgaris cv. Great Northern and P. coccineus cv. Scarlet Runner. The formation of abnormal (shrunken and underdeveloped) embryos constituted the primary crossing barrier between the two species when P. coccineus was the female parent. Plants of P. coccineus X P. vulgaris were obtained by embryo culture. Although the P. vulgaris X P. coccineus cross resulted in normal seed development, the fertility of the resulting hybrids was much lower (27%) than that of the reciprocal hybrids (81%). Three classes of F₂ embryos, normal, shrunken, and underdeveloped were formed on reciprocal F₁s and the frequencies did not differ between reciprocal populations. Thus, the interactions between embryo and endosperm and/or maternal parent rather than cytoplasmic-nuclear effects seem to be important in the determination of the extent of embryo growth. The examination of pollen fertility of F₂ plants and the development of F₂ and F₃ embryos suggests that the formation of abnormal embryos and reduced male fertility are independent events. The P. vulgaris — P. coccineus crosses may be useful in studying the possible involvement of interspecific differences in hormonal metabolism in the development of hybrid embryos.     

Nodulation and Nitrogen Fixation Efficacy of Rhizobium fredii with Phaseolus vulgaris Genotypes

Phaseolus plant introduction (PI) genotypes (consisting of 684 P. vulgaris, 26 P. acutifolius, 39 P. lunatus, and 5 P. coccineus accessions) were evaluated for their ability to form effective symbioses with strains of six slow-growing (Bradyrhizobium) and four fast-growing (Rhizobium fredii) soybean rhizobia. Of the 684 P. vulgaris genotypes examined, three PIs were found to form effective nitrogen-fixing symbioses with the R. fredii strains. While none of the Bradyrhizobium strains nodulated any of the genotypes tested, some produced large numbers of undifferentiated root proliferations (hypertrophies). A symbiotic plasmid-cured R. fredii strain failed to nodulate the P. vulgaris PIs and cultivars, suggesting that P. vulgaris host range genes are Sym plasmid borne in the fast-growing soybean rhizobia.     

Metabolism of C-zeatin in phaseolus embryos : occurrence of o-xylosyldihydrozeatin and its ribonucleoside

The metabolism of trans-[8-¹⁴C]zeatin was examined in embryos of Phaseolus acutifolius A. Gray P.I. 321637 and Phaseolus coccineus Lam. cvs Scarlet Runner and Desiree. In both species zeatin was converted to ribosylzeatin, ribosylzeatin 5′-monophosphate, O-glucosyl-9-ribosylzeatin and the recently discovered O-xylosyl derivatives of zeatin and ribosylzeatin (Turner, JE, DWS Mok, MC Mok, G Shaw 1987 Proc Natl Acad Sci USA. In press). Two new metabolites, identified by enzyme degradation and gas chromatography-mass spectrography analyses as O-xylosyldihydrozeatin and its ribonucleoside, were recovered from P. coccineus embryos. From this and previous studies it may be concluded that the potential to form O-xylosyl derivatives of zeatin is present only in embryos of three Phaseolus species (P. vulgaris L., P. coccineus, and P. acutifolius), but not in P. lunatus L., while the reduction of the side chain is most prominent in P. coccineus.     

Genetic diversity and structure of a worldwide collection of Phaseolus coccineus L

Phaseolus coccineus L. is closely related to P. vulgaris and is the third most important cultivated Phaseolus species. Little is known about the patterns of its diversity. In this work, a representative collection of its worldwide diversity was initially developed. The collection includes 28 wild forms (WFs) and 52 landraces (LRs) from Mesoamerica (the crop domestication area), and 148 LRs from Europe (where the crop was introduced in the sixteenth century). The collection was studied by using 12 SSR molecular markers that were developed for the P. vulgaris genome. They were proved to be effective and reliable in P. coccineus in this work. Fourteen LRs of P. dumosus (previously identified as a subspecies of P. coccineus) were also studied. The genetic diversity, population structure and phylogenetic relationships were investigated. The results indicate that: (a) the European and Mesoamerican gene pools are clearly differentiated, (b) a certain reduction of diversity occurred with introduction into Europe, and (c) the Mesoamerican LRs (P. dumosus included) and WFs are closely related and are connected by a high gene flow. Inferences on the domestication process of P. coccineus are also presented. This study provides a picture of the genetic diversity distribution and outcomes with introduction into the Old World, which was not available before. It also underlines that the genetic diversity of both WFs and LRs is an important source for Phaseolus spp. breeding programs and deserves to be preserved in situ and ex situ.     

Separation and partial characterization of isolectins with different subunit compositions from Datura stramonium seeds

The lectin from Datura stramonium seeds was separated into three individual isolectins by hydrophobic-interaction chromatography on phenyl-Sepharose. Two of these isolectins are homodimers made up of two A- or two B-subunits, whereas the third is a heterodimer composed of one A- and one B-subunit. Analysis of the homodimeric AA- and BB-isolectins revealed that the A- and B-subunits have similar but not identical M_r values (32 000 and 28 000, respectively), amino acid and carbohydrate compositions. The A-subunit has a higher affinity for N-acetyl-D-glucosamine oligomers than the B-subunit, whereas the latter is more specific for the carbohydrate determinants of some animal glycoproteins such as fetuin, asialofetuin and ovomucoid.     

Gibberellins in Suspensors of Phaseolus coccineus L. Seeds

Analysis of extracts from 6300 (1.2 grams fresh weight) Phaseolus coccineus suspensors by combined gas chromatography-mass spectrometry has demonstrated the presence of five C₁₉-gibberellins, GA₁, GA₄, GA₅, GA₆, GA₈, and one C₂₀-GA, GA₄₄. The major GAs present were GA₁ and GA₈. Data are discussed in relation to previous results obtained in P. coccineus seed as well as in the embryo-suspensor system.     

Use of chloroplast DNA polymorphisms for the phylogenetic study of seven Phaseolus taxa including P. vulgaris and P. coccineus

The genetic variability of seven Phaseolus taxa has been evaluated on the basis of molecular data and the results have used to clarify the phyletic relationships between several taxa of the P. coccineus L. complex. Chloroplast DNA (cpDNA) from 33 populations was digested with six restriction endonucleases, revealing some polymorphisms that made it possible to divide most of the taxa into two main groups: the subspecies of P. coccineus on the one hand, and P. vulgaris L., P. polyanthus Greenman and P. costaricensis (Freytag and Debouck) on the other hand. P. polyanthus is closer to P. vulgaris than the other taxa of the second group and should be considered as a separate species. The position of the wild species P. costaricensis is intermediate between P. coccineus and P. polyanthus. P. glabellus shows sufficient polymorphisms at the cpDNA level to be recognized as a separate species, as previously suggested from total seed-protein electrophoretic studies. These results favour the hypothesis of a common phylogeny for P. vulgaris, P. polyanthus, P. costaricensis and P. coccineus from a single wild ancestor. Although cpDNA is generally known to be uniform at the intraspecific level, some additional polymorphisms were also detected within P. vulgaris, P. polyanthus and P. coccineus. Further studies are required to understand the significance of the latter.     

Investigations into the inter-relationships ofPhaseolus vulgaris L. andP. Coccineus LAM

The crossability ofP. coccineus (self-incompatible) andP. vulgaris (self-compatible) was much higher whenvulgaris was used as the female parent. Reciprocal difference in crossability was due to the retarded development of embryo and endosperm tissue during the immediate post-fertilization period in the crossP. coccineus ×P. vulgaris . Backcrossing the reciprocal F₁ hybrids to the maternal species was more successful than to the paternal species.Two varieties ofP. vulgaris differed significantly in their ability to hybridize with six genotypes ofP. coccineus; this difference was shown to be genetical.The F₁ hybrids were readily distinguishable from the parent species at the seedling stage and could be classified into two distinct morphological types; one group showed and abnormal wrinkling of the leaves. The frequency of the abnormal progeny in the F₁ generation could be satisfactorily explained on the basis of either allelic or genic interaction. The importance of this genetic system in the divergence of the two species is discussed.     

Chloroplast DNA as an evolutionary marker in thePhaseolus vulgaris complex

We have analyzed the changes occurring in the chloroplast DNA (cpDNA) of taxa belonging to thePhaseolus vulgaris complex to help clarify relationships among species of this complex. Two restriction maps for 11 restriction enzymes comprising the whole chloroplast genome from a wildP. vulgaris and a wildP. coccineus accession were constructed. These maps allowed us to compare a total of 330 restriction sites between the two genomes in order to identify polymorphisms, assess the type of mutations detected, and identify regions of high variability. A region, located in the large single-copy region near the borders with the inverted repeats, accounted for a large portion of the variation. Most of the mutations detected were due to restriction sites gains or losses. Variable and conserved regions were then evaluated in 30 accessions belonging to taxa of theP. vulgaris complex. Phylogenetic analyses were made using parsimony methods. Conclusions obtained from such analyses were the following: (1) there was high cpDNA variability withinP. coccineus but not inP. vulgaris. (2)P. coccineus subsp.glabellus showed a very distinct cpDNA type that strongly suggests that it actually belongs to a different but as yet undetermined section of the genus. Our cpDNA observations are supported by distinctive morphological traits and reproductive biology of this taxon. (3) InP. coccineus subsp.darwinianus (also classified asP. polyanthus), the cpDNA lineage was in disagreement with data obtained from nuclear markers and suggested a reticulated origin by hybridization betweenP. coccineus as the male parent and an ancestralP. polyanthus type, closely allied toP. vulgaris, as the seed parent. This initial cross was presumably followed by repeated backcrossing toP. coccineus. Our cpDNA studies illustrate the importance of molecular markers in elucidating phylogenetic relationships. They also indicate that accurate phylogenies will require analyses of both nuclear and cytoplasmic genomes.     

Cytokinin autonomy in tissue cultures of phaseolus: a genotype-specific and heritable trait

Intra- and interspecific differences in cytokinin requirement were detected in callus cultures of Phaseolus vulgaris L. and P. lunatus L. Of the ten genotypes of P. vulgaris tested in the present study, one required cytokinin for callus growth, six exhibited some to moderate growth on cytokinin-free medium, and the remaining three grew uniformly in the absence of cytokinin. In contrast, six of the P. lunatus genotypes were strictly cytokinin-dependent, while four genotypes displayed irregular amount of callus growth on cytokinin-free medium. The genotype-specific behavior of Phaseolus callus tissues was independent of the tissue of origin and the time in culture. The inheritance of the cytokinin requirement of Phaseolus tissue cultures was studied in hybrid tissues resulting from crosses between a strictly cytokinin-dependent genotype (P.I. 200960) and two independent genotypes (cv. G 50 and P.I. 286303) of P. vulgaris. Fresh weights of hybrid tissues on cytokinin-free medium were intermediate between and significantly different from the parental tissues. No differences were found between reciprocal hybrids. These results suggest that cytokinin autonomy in tissue cultures of P. vulgaris is a genetic trait under nuclear control. Both parental and intermediate phenotypes were recovered in the F₂ progeny. The frequency distribution of cytokinin-dependent progeny in F₂ and backcross populations indicates that the cytokinin requirement of P. vulgaris callus tissue may be regulated by one set of alleles.     

Distribution of the protein “phaseolin“ in some representatives ofViciaceae

Using immunochemical methods the authors investigated the evolutionary taxonomic distribution of the reserve seed protein “phaseolin” in cultivars ofPhaseolus vulgaris, in a series of species ofPhaseolus, and in representatives of some additional genera ofViciaceae. “Phaseolin” is typical of the seed ofPhaseolus vulgaris L.: it was detected in all 658 investigated cultivars — and also in species related toPhaseolus vulgaris L.(Ph. vulgaris L. ssp.aborigineus Burk.,Ph. polyanthus Green,Ph. dumosus Macf.,Ph. coccineus L., and in an undescribed species from the group ofPh. vulgaris L. -Ph. coccineus L.). A protein immunochemically somewhat similar to ?phaseolin“ occurs inPh. acufifolius A. Gray. In all other taxa“phaseolin” is absent.     

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.     

Identification of anti-lung cancer extract from Chlorella vulgaris C-C by antioxidant property using supercritical carbon dioxide extraction

In this study, supercritical carbon dioxide extraction (SC-CO₂) technology was developed to extract the active components (such as antioxidants) from a novel microalga, Chlorella vulgaris C-C, due to its superior advantages over conventional solvent or ultrasonic extraction methods. Using SC-CO₂ and ultrasonic extractions, the polyphenol contents of C. vulgaris C-C were 13.40 and 0.46 (mg_{gallic acid}/g lyophilized extract), respectively. The flavonoid content obtained from SC-CO₂ (3.18 mg_quercetin/g lyophilized extract) was also significantly higher than from ultrasonic extraction (0.86 mg_quercetin/g lyophilized extract). Investigation of C. vulgaris C-C extract from SC-CO₂ indicates strong antioxidant activities in radical scavenging, ferric reducing power and metal chelating abilities. In cell proliferation assay, the extract of C. vulgaris C-C inhibits human lung cancer H1299, A549, and H1437 cells in a dose-dependent manner. In addition, the treatment with extracts of C. vulgaris C-C effectively reduced the migration of tumor cells, suggesting the potential of using the C. vulgaris C-C extract to inhibit lung cancer metastasis.