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.     

The influence of aluminium availability on phosphate uptake in Phaseolus vulgaris L. and Phaseolus lunatus L.

Aluminium toxicity is one of the major limiting factors of crop productivity on acid soils. High levels of available aluminium in soil may induce phosphorus deficiency in plants. This study investigates the influence of Aluminium (Al) on the phosphate (P_i) uptake of two Phaseolus species, Phaseolus vulgaris L. var. Red Kidney and Phaseolus lunatus L. The two bean species were treated first with solutions of Al at different concentrations (0, 25, 50 and 100 μM, pH 4.50) and second with solutions of P_i (150 μM) at pH 4.50. The higher the Al concentration the higher the Al concentration sorbed but P. vulgaris L var. Red Kidney adsorbed significantly more Al than P. lunatus L. Both species released organic acids: P. vulgaris L var. Red Kidney released fumaric acid and P. lunatus L. fumaric and oxalic acids which could have hindered further Al uptake.The two bean species showed a sigmoid P_i uptake trend but with two different mechanisms. P. vulgaris L var. Red Kidney showed a starting point of 3 h whereas P. lunatus L. adsorbed P_i immediately within the first minutes. In addition, P. vulgaris L var. Red Kidney presented significantly higher P_i uptake (higher uptake rate ‘k’ and higher maximum adsorption ‘a’ of the kinetic uptake model). The Al treatments did not significantly influence P_i uptake. Results suggest that P. lunatus L. might adopt an external Al detoxification mechanism by the release of oxalic acid. P. vulgaris L var. Red Kidney on the other hand seemed to adopt an internal detoxification mechanism even if the Al sorbed is poorly translocated into the shoots. More detailed studies will be necessary to better define Al tolerance and/or resistance of Phaseolus spp.     

Phytochrome Chromophore Biosynthesis : Both 5-Aminolevulinic Acid and Biliverdin Overcome Inhibition by Gabaculine in Etiolated Avena sativa L. Seedlings

Etiolated Avena sativa L. seedlings grown in the presence of gabaculine (5-amino-1,3-cyclohexadienylcarboxylic acid) contained reduced levels of phytochrome as shown by spectrophotometric and immunochemical assays. Photochromic phytochrome levels in gabaculine-grown plants were estimated to be 20% of control plants, while immunoblot analysis showed that the phytochrome protein moiety was present at approximately 50% of control levels. Gabaculine-grown seedlings administered either 5-aminolevulinic acid or biliverdin exhibited a rapid increase of spectrophotometrically detectable phytochrome. Phytochrome concentrations estimated immunochemically did not similarly increase throughout treatment with either compound. Similar experiments with 5-amino[4-¹⁴C] levulinic acid showed radiolabeling of phytochrome with kinetics that paralleled the spectrally detected increase. These results are consistent with (a) the intermediacy of both 5-aminolevulinic acid and biliverdin in the biosynthetic pathway of the phytochrome chromophore and (b) the lack of coordinate regulation of chromophore and apoprotein synthesis in Avena seedlings.     

Mating system of wild Phaseolus lunatus L. and its relationship to population size

Using isozyme variation in a naturally pollinated seed family for 10 wild Phaseolus lunatus L. (Lima bean) populations, ranging in sizes from 10 to 60 reproductive individuals, we estimated levels of outcrossing (t) and parental inbreeding coefficient (F). We also examined the relationship between outcrossing rate and population size. Average estimates of the single-locus outcrossing rate (ts) ranged from 0.024 to 0.246 (mean=0.091+/-0.065). Estimates of the multilocus outcrossing rate (tm) ranged from 0.027 to 0.268, and averaged 0.096+/-0.071. Inbreeding coefficients based on genotypic frequencies of maternal plants were positive and significantly greater than zero (F=0.504), suggesting an excess of homozygotes in all the populations studied. There was indirect evidence of nonrandom mating for outcrosses and this was mainly attributed to self-fertilisation since the averaged difference between tm and ts, which provides a measure of biparental inbreeding, represents only 1% of the autogamy rate. No significant correlation was observed between outcrossing rate and population size. Estimates of t showed significant heterogeneity among populations and factors explaining this tendency are suggested.     

In vivo endoproteolytically cleaved phaseolin is stable and accumulates in developing Phaseolus lunatus L. seeds

Phaseolin is the most abundant storage protein of bean seeds. To modify its amino-acidic composition by protein engineering, for the improvement of its nutritional value, regions which could be modified without detrimental effects on structural features of the protein must be identified. Data presented here, on the characterisation of the major storage protein of lima bean (Phaseolus lunatus L.) seeds, a phaseolin-like glycoprotein, provide good indications on one of such region. Phaseolus lunatus phaseolin consists of four major oligomers containing two subunit classes. Polypeptides of one class show a molecular mass ranging from 38.5 kDa to 32 kDa, while the molecular mass of polypeptides belonging to the other class ranges from 27 kDa to 21 kDa. The subunits originate from the cleavage of precursor forms, with molecular masses of 58 kDa and 54 kDa, which are still present — in residual amounts — in the mature protein. Comparison of their N-terminal sequences with those of the subunits demonstrate that cleavage occurs in a region of the molecule that instead remains uncleaved in phaseolins of the other species. Since this region can accommodate such a drastic modification, we suggest it could be a good candidate for in vitro manipulation.     

Chlorophyll mutations induced by gamma radiation in Phaseolus vulgaris L]

In a study of chlorophyll mutants of Phaseolus vulgaris L. through Co60 gamma radiation, five types of mutants, classified as albino, cream, yellow, yellow-green and light green were obtained; all were lethal; their segregation was always proportionally lower than the Mendelian. Gamma radiation-induced mutations in black beans do not depart significantly from those obtained elsewhere in barley and wheat.     

Molecular diversity of native bradyrhizobia isolated from lima bean (Phaseolus lunatus L.) in Peru

The diversity of a collection of 21 bradyrhizobial isolates from Lima bean (Phaseolus lunatus L.) was assayed by molecular methods. Moderately high to high genetic diversity was revealed by multilocus enzyme electrophoresis (MLEE) analysis of seven enzyme loci and genomic fingerprints with ERIC and BOX primers. Two groups with differences in growth rate were found among the isolates and their differentiation as two divergent bradyrhizobial lineages was supported by PCR-RFLP of the rpoB gene and sequence analysis of the 16S rDNA and dnaK genes. Isolates with slow growth (SG) were identified as Bradyrhizobium yuanmingense, while extra-slow growing isolates (ESG) constitute a new lineage different from all described Bradyrhizobium species. Three distinct symbiotic genotypes were detected among Lima bean bradyrhizobia by PCR-RFLP and sequence analysis of the nifH and nodB genes. One genotype was found in the ESG lineage and two in B. yuanmingense. Another symbiotic genotype was detected in B. yuamingense isolated from Lespedeza plants. The identified bradyrhizobial lineages constitute sympatric species effectively nodulating Lima bean on the coast of Peru.     

Pattern of enzymes involved in cyanogenesis and HCN metabolism in cell cultures of Phaseolus lunatus L. varieties

Callus and cell suspension cultures were established from root and shoot tips of aseptically-grown seedlings of highly cyanogenic Phaseolus lunatus L. varieties. The content of cyanogenic glucosides in the explanted seedling sections decreased during storage, the derived callus cells were free of cyanogenic glycosides. In spite of the non-existence of cyanogenic glucosides, the cyanogen degrading linamarase, the cyanide detoxifying enzyme -cyanoalanine synthase and also hydroxynitrile lyase were still present in suspension cultures. The linamarase activity equalled the total -glucosidase activity, of which up to 80% was found in the culture medium. In contrast the -cyanoalanine synthase and the hydroxy nitrile lyase were entirely localized in the cell biomass.~Botanical Institute, Technical University Braunschweig     

The Linamarin beta-Glucosidase in Costa Rican Wild Lima Beans (Phaseolus lunatus L.) Is Apoplastic

Analysis of mesophyll protoplasts and cell wall extracts of leaf discs of Costa Rican wild lima bean (Phaseolus lunatus L.) shows that the linamarase activity is confined to the apoplast. Its substrate linamarin, together with the related enzyme hydroxynitrile lyase, is found inside the cells. This compartmentation prevents cyanogenesis from occurring in intact tissue, and suggests that linamarin has to be protected during any translocation across the linamarase rich apoplast.     

Lectin and lectin-related proteins in Lima bean (Phaseolus lunatus L.) seeds: biochemical and evolutionary studies

Lectin-related polypeptides are a class of defence proteins found in seeds of Phaseolus species. In Lima bean (P. lunatus), these proteins and their genes have been well characterized in the Andean morphotype, which represents one of the two gene pools of this species. To study the molecular evolution of the lectin family in Lima bean we characterized the polypeptides belonging to this multigene family and cloned the genes belonging to the Mesoamerican gene pool. The latter gene pool contains components similar to those of the Andean pool, namely: an amylase inhibitor-like (AIL), an arcelin-like (ARL) lectin and the less abundant Lima bean lectin (LBL). These proteins originate from an ancestor gene of the lectin type which duplicated to yield the lectin gene and the progenitor of ARL and AIL. In this species. ARL represents an evolutionary intermediate form that precedes AIL. Phylogenetic analysis supports an Andean origin for Lima bean. The molecular evolutionary studies were extended to the genes of common bean and demonstrated that true lectin genes and the ancestor of lectin-related genes are the result of a duplication event that occurred before speciation. Lima and common bean followed different evolutionary pathways and in the latter species a second duplication event occurred that gave rise, in Mesoamerican wild genotypes, to arcelin genes.     

Increased Bean (Phaseolus vulgaris L.) Nodulation Competitiveness of Genetically Modified Rhizobium Strains

Rhizobium leguminosarum bv. phaseoli strain collections harbor heterogeneous groups of bacteria in which two main types of strains may be distinguished, differing both in the symbiotic plasmid and in the chromosome. We have analyzed under laboratory conditions the competitive abilities of the different types of Rhizobium strains capable of nodulating Phaseolus vulgaris L. bean. R. leguminosarum bv. phaseoli type I strains (characterized by nif gene reiterations and a narrow host range) are more competitive than type II strains (that have a broad host range), and both types are more competitive than the promiscuous rhizobia isolated from other tropical legumes able to nodulate beans. Type I strains become even more competitive by the transfer of a non-Sym, 225-kilobase plasmid from type II strain CFN299. This plasmid has been previously shown to enhance the nodulation and nitrogen fixation capabilities of Agrobacterium tumefaciens transconjugants carrying the Sym plasmid of strain CFN299. Other type I R. leguminosarum bv. phaseoli transconjugants carrying two symbiotic plasmids (type I and type II) have been constructed. These strains have a diminished competitive ability. The increase of competitiveness obtained in some transconjugants seems to be a transient property.     

Cyanogenesis of Wild Lima Bean (Phaseolus lunatus L.) Is an Efficient Direct Defence in Nature

In natural systems plants face a plethora of antagonists and thus have evolved multiple defence strategies. Lima bean (Phaseolus lunatus L.) is a model plant for studies of inducible indirect anti-herbivore defences including the production of volatile organic compounds (VOCs) and extrafloral nectar (EFN). In contrast, studies on direct chemical defence mechanisms as crucial components of lima beans'' defence syndrome under natural conditions are nonexistent. In this study, we focus on the cyanogenic potential (HCNp; concentration of cyanogenic glycosides) as a crucial parameter determining lima beans'' cyanogenesis, i.e. the release of toxic hydrogen cyanide from preformed precursors. Quantitative variability of cyanogenesis in a natural population of wild lima bean in Mexico was significantly correlated with missing leaf area. Since existing correlations do not by necessity mean causal associations, the function of cyanogenesis as efficient plant defence was subsequently analysed in feeding trials. We used natural chrysomelid herbivores and clonal lima beans with known cyanogenic features produced from field-grown mother plants. We show that in addition to extensively investigated indirect defences, cyanogenesis has to be considered as an important direct defensive trait affecting lima beans'' overall defence in nature. Our results indicate the general importance of analysing ‘multiple defence syndromes’ rather than single defence mechanisms in future functional analyses of plant defences.     

Direct trade-off between cyanogenesis and resistance to a fungal pathogen in lima bean (Phaseolus lunatus L.)

1.  Plants are simultaneously attacked by multiple herbivores and pathogens. While some plant defences act synergistically, others trade-off against each other. Such trade-offs among resistances to herbivores and pathogens are usually explained by the costs of resistance, i.e. resource limitations compromising a plant's overall defence.
2.  Here, we demonstrate that trade-offs can also result from direct negative interactions among defensive traits. We studied cyanogenesis (release of HCN) of lima bean (Fabaceae: Phaseolus lunatus ) and effects of this efficient anti-herbivore defence on resistance to a fungal pathogen (Melanconiaceae: Colletotrichum gloeosporioides ).
3.  Leaf tissue destruction by fungal growth was significantly higher on high cyanogenic (HC) lima bean accessions than on low cyanogenic (LC) plants. The susceptibility of HC accessions to the fungal pathogen was strongly correlated to reduced activity of resistance-associated polyphenol oxidases (PPOs) in leaves of these plants. LC accessions, in contrast, showed high PPO activity, which was correlated with distinct resistance to C. gloeosporioides .
4.  Experimentally applied, gaseous HCN reduced PPO activity and significantly increased the size of lesions caused by C. gloeosporioides in LC leaves.
5.  Field observations of a wild lima bean population in Mexico revealed a higher infection rate of HC compared to LC plant individuals. The types of lesions observed on the different cyanogenic plants in nature were similar to those observed on HC and LC plants in the laboratory.
6.   Synthesis. We suggest that cyanogenesis of lima bean directly trades off with plant defence against fungal pathogens and that the causal mechanism is the inhibition of PPOs by HCN. Our findings provide a functional explanation for the observed phenomenon of the low resistance of HC lima beans in nature.     

Responses of Phaseolus lunatus callus tissues to cytokinin-active ureidopurines

Three ureidopurine derivatives were tested for their ability to promote the cytokinin-dependent growth of callus tissues derived from three genotypes of Phaseolus lunatus (cv. Jackson Wonder, cv. Kingston and P.I. 260415). In all three test systems, the ureidopurines exhibited the same relative cytokinin activities (6-phenylureidopurine > 6-p-tolylureidopurine > 6-cyclohexylureidopurine) and were less active than the aminopurine derivatives, N⁶-benzyl-adenine and N⁶-cyclohexylmethyladenine. Callus tissues of Jackson Wonder, which become cytokinin-autonomous if grown on cytokinin-active urea derivatives, retained cytokinin-dependence on media containing optimal concentrations of ureidopurines. In this respect, the biological activity of the ureidopurines was similar to that of the aminopurine derivatives and differed from that of cytokinin-active urea derivatives tested in this system.     

Cerium Relieves the Inhibition of Chlorophyll Biosynthesis of Maize Caused by Magnesium Deficiency

Lanthanoids (Ln) were demonstrated to improve chlorophyll formation and the growth of plants. But the mechanism of the fact that Ln promotes chlorophyll biosynthesis of plants is poorly understood. The main aim of the study was to determine Ln effects in chlorophyll formation of maize under magnesium (Mg) deficiency. Maize plants were cultivated in Hoagland’s solution. They were subjected to Mg deficiency and to cerium administered in Mg-deficient Hoagland’s media, and then the contents of various chlorophyll precursors and gen expressions of the key enzymes of chlorophyll biosynthesis were examined. The decrease of chlorophyll contents in maize leaves caused by Mg deficiency suggested an inhibition of chlorophyll synthesis that was inhibited by a reduction of the precursors as measured by analyzing the contents of δ-aminolevulinic acid, porphobilinogen, uroporphyrinogen III, Mg–protoporphyrin IX, and protochlorophyll, as well as the expression levels of magnesium chelatase, magnesium-protoporphyrin IX methyltransferase, and chlorophyll synthase; Mg deficiency significantly inhibited the transformation from coproporphyrinogen III or protoporphyrin IX to chlorophyll. However, cerium addition significantly relieved the inhibition of chlorophyll biosynthesis in maize caused by Mg deficiency and increased chlorophyll content and promoted a series of transformations from δ-aminolevulinic acid to chlorophyll and maize growth under Mg deficiency. It implied that cerium might partly substitute for the role of Mg.     

Effect of seed size and plant growth on nodulation and nodule development in lima bean (Phaseolus lunatus L.)

Lima bean (Phaseolus lunatus L.) cultivars vary widely in their growth habit and seed size. Preliminary experiments indicated that a large-seeded pole cultivar (King of the Garden) formed many more nodules than a small-seeded bush cultivar (Henderson). The relative importance of seed size and shoot mass in determining nodule number and mass was assessed in five lima bean cultivars differing in seed size and growth habit. Between cultivars, significant positive correlations between initial seed mass, plant weight and nodule number and mass were observed during the first four weeks after planting. Comparisons within cultivars indicated a strong correlation between nodule mass and shoot dry weight. The influence of plant morphology on nodule formation and mass was secondary to the effects of seed and shoot mass. As plants matured, the increase in nodule mass paralleled the increase in plant mass, while nodule number was relatively stable after day 18. These results suggest that the highly regulated process of nodule formation was under the influence of seed derived factors, while the continued accumulation of nodule tissue was related to shoot growth.     

Contrasting rDNA evolution in lima bean (Phaseolus lunatus L.) and common bean (P. vulgaris L., Fabaceae)

Phaseolus vulgaris has two 5S rDNA sites in chromosomes 6 and 10 and from two up to nine 45S rDNA sites depending on the accession. The presence of three 45S rDNA sites, in chromosomes 6, 9 and 10, is considered the ancestral state for the species. For P. lunatus, only one 5S and one 45S rDNA sites in distinct chromosomes were known. In order to investigate the homeologies among these rDNA-bearing chromosomes and the stability of the rDNA sites in P. lunatus, rDNA and P. vulgaris chromosome-specific probes were hybridized in situ to P. lunatus. The chromosomes bearing the 5S and the 45S rDNA of P. lunatus are homeologous to chromosomes 10 and 6 of P. vulgaris, respectively. In contrast to the common bean, no variation in the number of rDNA loci was detected, except for a duplication of the 5S rDNA in the same chromosome in a small group of cultivars. These results suggest that the 5S rDNA site in chromosome 10 and the 45S rDNA site in chromosome 6 represent the ancestral loci in the genus. The 5S rDNA site in chromosome 10 of P. vulgaris is located in the long arm, while in P. lunatus it is present in the short arm, suggesting the occurrence of a transposition or a pericentric inversion after separation of both lineages.