Phaseolus vulgaris — Recalcitrant potential

Since the ability to genetically engineer plants was established, researchers have modified a great number of plant species to satisfy agricultural, horticultural, industrial, medicinal or veterinary requirements. Almost thirty years after the first approaches to the genetic modification of pulse crops, it is possible to transform many grain legumes. However, one of the most important species for human nutrition, Phaseolus vulgaris, still lacks some practical tools for genomic research, such as routine genetic transformation. Its recalcitrance towards in vitro regeneration and rooting significantly hampers the possibilities of improvement of the common bean that suffers from many biotic and abiotic constraints. Thus, an efficient and reproducible system for regeneration of a whole plant is desired. Although noticeable progress has been made, the rate of recovery of transgenic lines is still low. Here, the current status of tissue culture and recent progress in transformation methodology are presented. Some major challenges and obstacles are discussed and some examples of their solutions are presented.     

Analysis of rhizobial strains nodulating Phaseolus vulgaris from Hispaniola Island,a geographic bridge between Meso and South America and the first historical link with Europe

Hispaniola Island was the first stopover in the travels of Columbus between America and Spain, and played a crucial role in the exchange of Phaseolus vulgaris seeds and their endosymbionts. The analysis of recA and atpD genes from strains nodulating this legume in coastal and inner regions of Hispaniola Island showed that they were almost identical to those of the American strains CIAT 652, Ch24-10 and CNPAF512, which were initially named as Rhizobium etli and have been recently reclassified into Rhizobium phaseoli after the analysis of their genomes. Therefore, the species R. phaseoli is more abundant in America than previously thought, and since the proposal of the American origin of R. etli was based on the analysis of several strains that are currently known to be R. phaseoli, it can be concluded that both species have an American origin coevolving with their host in its distribution centres. The analysis of the symbiovar phaseoli nodC gene alleles carried by different species isolated in American and European countries suggested a Mesoamerican origin of the α allele and an Andean origin of the γ allele, which is supported by the dominance of this latter allele in Europe where mostly Andean cultivars of common beans have been traditionally cultivated.     

Protective role of Mangifera indica, Cucumis melo and Citrullus vulgaris peel extracts in chemically induced hypothyroidism

An investigation was made to evaluate the pharmacological importance of fruit peel extracts of Mangifera indica (MI), Citrullus vulgaris (CV) and Cucumis melo (CM) with respect to the possible regulation of tissue lipid peroxidation (LPO), thyroid dysfunctions, lipid and glucose metabolism. Pre-standardized doses (200 mg/kg of MI and 100 mg/kg both of CV and CM), based on the maximum inhibition in hepatic LPO, were administered to Wistar albino male rats for 10 consecutive days and the changes in tissue (heart, liver and kidney) LPO and in the concentrations of serum triiodothyronine (T₃), thyroxin (T₄), insulin, glucose, α-amylase and different lipids were examined. Administration of three test peel extracts significantly increased both the thyroid hormones (T₃ and T₄) with a concomitant decrease in tissue LPO, suggesting their thyroid stimulatory and antiperoxidative role. This thyroid stimulatory nature was also exhibited in propylthiouracil (PTU) induced hypothyroid animals. However, only minor influence was observed in serum lipid profile in which CM reduced the concentrations of total cholesterol and low-density lipoprotein-cholesterol (LDL-C), while CV decreased triglycerides and very low-density lipoprotein-cholesterol (VLDL-C). When the combined effects of either two (MI + CV) or three (MI + CV + CM) peel extracts were evaluated in euthyroid animals, serum T₃ concentration was increased in response to MI + CV and MI + CV + CM treatments, while T₄ level was elevated by the combinations of first two peels only. Interestingly, both the categories of combinations increased T₄ levels, but not T₃ in PTU treated hypothyroid animals. Moreover, a parallel increase in hepatic and renal LPO was observed in these animals, suggesting their unsafe nature in combination. In conclusion the three test peel extracts appear to be stimulatory to thyroid functions and inhibitory to tissue LPO but only when treated individually.     

Arabinogalactan from Phaseolus atropurpureus leaves

Water-soluble polysaccharide material comprising d-galactose (53·0%), l-arabinose (33·2%) and d-glucuronic acid (13·8%) has been isolated from the leaves of Phaseolus atropurpureus. Acid hydrolysis, periodate oxidation and methylation have indicated a highly branched structure. The principal interglycosidic linkages have been tentatively identified as 1,3- and 1,6-linked d-galactopyranose and 1,3-linked l-arabinofuranose residues. In synthesising polysaccharide with these structural features, P. atropurpureus differs from other legumes such as soybean, lucerne and Centrosema.     

Expression and interaction of the CBLs and CIPKs from immature seeds of kidney bean (Phaseolus vulgaris L.)

Protein phosphorylation plays a key regulatory role in a variety of cellular processes. To better understand the function of protein phosphorylation in seed maturation, a PCR-based cloning method was employed and five cDNA clones (pvcipk1-5) for protein kinases were isolated from a cDNA library prepared from immature seeds of kidney bean (Phaseolus vulgaris L.). The deduced amino acid sequences showed that the five protein kinases (PvCIPK1-5) are members of the sucrose non-fermenting 1-related protein kinase type 3 (SnRK3) family, which interacts with calcineurin B-like proteins (CBLs). Two cDNA clones (pvcbl1 and 2) for CBLs were further isolated from the cDNA library. The predicted primary sequences of the proteins (PvCBL1 and 2) displayed significant identity (more than 90%) with those of other plant CBLs. Semi-quantitative RT-PCR analysis showed that the isolated genes, except pvcbl1, are expressed in leaves and early maturing seeds, whereas pvcbl1 is constitutively expressed during seed development. Yeast two-hybrid assay indicated that among the five PvCIPKs, only PvCIPK1 interacts with both PvCBL1 and PvCBL2. These results suggest that calcium-dependent protein phosphorylation-signaling via CBL-CIPK complexes occurs during seed development.     

Biosynthesis of coumestrol in Phaseolus aureus

Feeding experiments with 4′,7-dihydroxyisoflavone-[4-¹⁴C] (daidzein), 2′,4′,7-trihydroxyisoflavone-[T] and (±)-4′,7-dihydroxyisoflavanone-[T] (dihydrodaidzein) in suspension cultures of mung bean (Phaseolus aureus Roxb.) roots have shown that daidzein is a better precursor of the coumestan coumestrol than is the trihydroxyisoflavone and that dihydrodaidzein can also be converted very efficiently. The results provide further evidence for the intermediacy of a pterocarp-6a-en in coumestrol biosynthesis, and also indicate the possible existence of a 'metabolic grid' of isoflavones and isoflavanones in P. aureus.     

Mussel beds in deeper water provide an unusual situation for competitive interactions between the seastars Leptasterias polaris and Asterias vulgaris

The common seastars Leptasterias polaris and Asterias vulgaris show competitive interactions in shallow subtidal communities in the northern Gulf of St. Lawrence, particularly during summer when aggregations of the two seastars forage on mussel beds at 1-2 m in depth. We examined interactions between the two seastars in a different situation, in a mussel bed at 6 m in depth (a rare situation in this region). In the deeper mussel bed, seastars were three times more abundant than in the shallower beds, and the mussels were larger. The deeper bed disappeared rapidly due to the intense predation. Although decreased prey abundance should have favored interference interactions, we did not detect either partitioning of mussels by size or avoidance of A. vulgaris by L. polaris as previously reported when mussels are in short supply in shallower water. The lack of an avoidance behavior by L. polaris, together with the higher proportion of L. polaris than A. vulgaris that were feeding, suggests that in this situation, the dominance of A. vulgaris (observed in shallower water) is attenuated, or that L. polaris may dominate.     

Effects of substrate on interactions between juvenile sea scallops (Placopecten magellanicus Gmelin) and predatory sea stars (Asterias vulgaris Verrill) and rock crabs (Cancer irroratus Say)

We investigated the effect of substrate (glass bottom, sand, granule, pebble) on predation of juvenile sea scallops (Placopecten magellanicus) by sea stars (Asterias vulgaris) and rock crabs (Cancer irroratus) at two prey sizes (11-15 mm and 24-28 mm shell height), and two prey densities (10 and 30 scallops per aquarium) in laboratory experiments. Specifically, we quantified predation rate and underlying behaviours (proportion of time a predator spent searching for and handling prey, encounter rate between predators and prey, and various outcomes of encounters). We detected a significant gradual effect of particle size of natural substrates on sea star predation: specifically, predation rate on and encounter rate with small scallops tended to decrease with increasing particle size (being highest for sand, intermediate for granule, and lowest for pebble). Substrate type did not significantly affect predation rates or behaviours of sea stars preying on large scallops or of rock crabs preying on either scallop size classes. Other factors, such as prey size and density, were important in the scallop-sea star and scallop-rock crab systems. For example, predation rate by sea stars and crabs and certain sea star behaviours (e.g. probability of consuming scallops upon capture) were significantly higher with small scallops than with large scallops. As well, in interactions between small scallops and sea stars, predation rate and encounter rate increased with prey density, and the proportion of time sea stars spent searching was higher at low prey density than high prey density. Thus, substrate type may be a minor factor determining predation risk of seeded scallops during enhancement operations; prey size and prey density may play a more important role. However, substrate type still needs to be considered when choosing a site for scallop enhancement, as it may affect other scallop behaviours (such as movement).     

Quantification of abnormal repetitive behaviour in captive European starlings (Sturnus vulgaris)

Stereotypies are repetitive, unvarying and goalless behaviour patterns that are often considered indicative of poor welfare in captive animals. Quantifying stereotypies can be difficult, particularly during the early stages of their development when behaviour is still flexible. We compared two methods for objectively quantifying the development of route-tracing stereotypies in caged starlings. We used Markov chains and T-pattern analysis (implemented by the software package, Theme) to identify patterns in the sequence of locations a bird occupied within its cage. Pattern metrics produced by both methods correlated with the frequency of established measures of stereotypic behaviour and abnormal behaviour patterns counted from video recordings, suggesting that both methods could be useful for identifying stereotypic individuals and quantifying stereotypic behaviour. We discuss the relative benefits and disadvantages of the two approaches.     

Effect of trimethyllead chloride on slowly activating (SV) channels in red beet (Beta vulgaris L.) taproots

The patch-clamp technique was used to examine the effect of trimethyllead chloride (Met₃PbCl) on SV channel activity in red beet (Beta vulgaris L.) taproot vacuoles. It was found that in the control bath the macroscopic currents showed the typical slow activation and a strong outward rectification of the steady-state currents. An addition of Met₃PbCl to the bath solution blocked, in a concentration-dependent manner, SV currents in red beet vacuoles. The time constant τ increased several times in the presence of 100 μM trimethyllead chloride at all voltages tested. When single channel properties were analyzed, only little channel activity could be recorded in the presence of 100 μM Met₃PbCl. Trimethyllead chloride decreased significantly (by about one order of magnitude) the open probability of single channels. The recordings of single channel activity obtained in the presence and absence of Met₃PbCl showed that organolead only slightly (by ca. 10%) decreased the unitary conductance of single channels. It was also found that Met₃PbCl diminished significantly the number of SV channel openings, whereas it did not change the opening times of the channels. Taken together, these results suggest that Met₃PbCl binding site is located outside the channel selectivity filter.     

Fixed-bed decolorization of Reactive Blue 172 by Proteus vulgaris NCIM-2027 immobilized on Luffa cylindrica sponge

Proteus vulgaris NCIM-2027 cells immobilized on Luffa cylindrica (Loofa) completely decolorized C.I. Reactive Blue 172 at 37 °C and pH 8.0 under 5-h static incubation with high total organic carbon (TOC) and chemical oxygen demand (COD) reduction. The repeated-batch decolorization experiments also indicate good reusability of the immobilized biocatalyst. Some oxidoreductive enzymes were shown to be involved in the decolorization and degradation process. Loofa immobilized cells were also able to decolorize a mixture of reactive dyes in batch mode (in terms of ADMI value) with significant reduction in TOC and COD. Loofa immobilized cells were also used for continuous decolorization of individual and mixture of reactive dyes in a fixed bed bioreactor.     

Metallothioneins and trace elements in digestive gland,gills, kidney and gonads of Octopus vulgaris

Metallothionein-like proteins (MT) and V, Cr, Co, Ni, Zn, Cu, As and Cd were determined in digestive gland, gills, kidney and gonads of Octopus vulgaris, from the Portuguese coast. To our knowledge these are the first data on MT in octopus. High concentrations (µg g^− 1, dry mass) of Zn (48050) and Cd (555) were found in digestive gland, and MT reached levels one order of magnitude above the ones registered in wild bivalves. Significantly higher levels of MT in digestive gland and gills of specimens from A and B were in line with elevated Cd concentrations. Principal component analyses (PCA) point to MT-Cd and MT-Cr associations in digestive gland and gills. Despite the high levels of Zn in specimens from B, association with Zn was not obtained. Due to the affinity of MT to various elements, it should not be excluded the possibility of Cd replacing Zn in Zn-MT. Kidney presented higher levels of Cd, Co, Ni and As than gills and gonads, and in the case of As surpassing the levels in digestive gland, but PCA showed no relation with MT. Likewise the MT levels in gonads had no correspondence to the metal concentration variation.     

Inhibition of estrogen signaling through depletion of estrogen receptor alpha by ursolic acid and betulinic acid from Prunella vulgaris var. lilacina

Extracts of Prunella vulgaris have been shown to exert antiestrogenic effects. To identify the compounds responsible for these actions, we isolated the constituents of P. vulgaris and tested their individual antiestrogenic effects. Rosmarinic acid, caffeic acid, ursolic acid (UA), oleanolic acid, hyperoside, rutin and betulinic acid (BA) were isolated from the flower stalks of P. vulgaris var. lilacina Nakai (Labiatae). Among these constituents, UA and BA showed significant antiestrogenic effects, measured as a decrease in the mRNA level of GREB1, an estrogen-responsive protein; the effects of BA were stronger than those of UA. UA and BA were capable of suppressing estrogen response element (ERE)-dependent luciferase activity and expression of estrogen-responsive genes in response to exposure to estradiol, further supporting the suppressive role of these compounds in estrogen-induced signaling. However, neither UA nor BA was capable of suppressing estrogen signaling in cells ectopically overexpressing estrogen receptor α (ERα). Furthermore, both mRNA and protein levels of ERα were reduced by treatment with UA or BA, suggesting that UA and BA inhibit estrogen signaling by suppressing the expression of ERα. Interestingly, both compounds enhanced prostate-specific antigen promoter activity. Collectively, these findings demonstrate that UA and BA are responsible for the antiestrogenic effects of P. vulgaris and suggest their potential use as therapeutic agents against estrogen-dependent tumors.     

Evidence for genetic differentiation of Octopus vulgaris (Mollusca, Cephalopoda) fishery populations from the southern coast of Brazil as revealed by microsatellites

Octopus vulgaris is a cephalopod species in several oceans and commonly caught by artisanal and industrial fisheries. In Brazil, O. vulgaris populations are mainly distributed along the southern coast and have been subjected to intensive fishing during recent years. Despite the importance of this marine resource, no genetic study has been carried out to examine genetic differences among populations along the coast of Brazil. In this study, 343 individuals collected by commercial vessels were genotyped at six microsatellite loci to investigate the genetic differences in O. vulgaris populations along the southern coast of Brazil. Genetic structure and levels of differentiation among sampling sites were estimated via a genotype assignment test and F-statistics. Our results indicate that the O. vulgaris stock consists of four genetic populations with an overall significant analogous F_ST (Φ_CT = 0.10710, P < 0.05) value. The genetic diversity was high with an observed heterozygosity of Ho = 0.987. The negative values of F_IS found for most of the loci examined suggested a possible bottleneck process. These findings are important for further steps toward more sustainable octopus fisheries, so that this marine resource can be preserved for long-term utilization.     

Calcium and oxalate content of the leaves of Phaseolus vulgaris at different calcium supply in relation to calcium oxalate crystal formation

Soluble and insoluble oxalate and insoluble calcium were measured in the leaves of Phaseolus vulgaris. The plants were grown in nutrient solutions with two different concentrations of calcium. Two developmental stages of the leaves were studied. Although the content of insoluble calcium differs widely according to leaf age and growth conditions, the percentage bound in crystals is nearly the same in all cases. In the growing leaves, concentrations of total oxalate are independent of calcium supply, thus, showing that the known rise in numbers of crystals, and of cells containing them, is not induced via oxalate biosynthesis. Fully expanded leaves contain more oxalate when grown in a nutrient solution with higher calcium concentration. Amounts of oxalate in percent of dry weight are similar to those given in the literature for other legume leaves.