Tepary Research Articles

The Effect of Feeding Tepary Bean (Phaseolus acutifolius) Proteinase Inhibitors on the Growth and Pancreas of Young Mice

The effect of tepary proteinase inhibitor (TI) isolated from unheated tepary bean on the growth, nitrogen digestion and pancreatic hypertrophy of mice was assessed. The mice were fed a whole protein based diet containing either 0, 100, 200, 400 or 600 mg TI per 100 g of diet. Growth performance of mice fed whole egg or 100 mg TI/100 g diet was superior to that of mice fed 200, 400 and 600 TI mg/100 g diet. The growth inhibition of mice fed trypsin inhibitor above 200 mg TI/100 g diet was accompanied by a lower apparent digestibility of protein and lower food conversion efficiency. The pancreas weight relative to body weight of animals fed TI was significantly higher than that of animals fed whole egg. The results of th e present study also indicate that TI is not responsible for the death of mice and rats fed raw tepary bean flour.

Paraheliotropism in two Phaseolus species: combined effects of photon flux density and pulvinus temperature, and consequences for leaf gas exchange

Phaseolus species exhibit paraheliotropic (light-avoiding) leaf movements in response to environmental stimuli. We investigated the response of two ecologically contrasting species, Phaseolus vulgaris (mesic) and Phaseolus acutifolius (xeric), to various combinations of photosynthetic photon flux density (PFD) and pulvinus temperature to compare the control and consequences, in terms of photosynthetic gas exchange, of paraheliotropism under identical conditions. Leaflet angles were measured in a cuvette that exposed the leaf to controlled PFD and pulvinus temperature. A gas exchange system was used to determine carbon assimilation, stomatal conductance and water use efficiency (WUE) in response to environmental factors and leaf movements. The effect of tilting leaves was compared with an equivalent reduction of PFD for horizontal leaves. Both species showed significant (but different) responses to PFD and temperature, with increased light avoidance at high levels of both factors. P. acutifolius exhibited greater light avoidance under equivalent conditions. The gas exchange consequences of changing leaf angles were similar but not identical to those of an equivalent modification of radiation level. The photosynthetic cost of the leaf movements was negligible, but there was a substantial increase in WUE, especially for P. acutifolius. Thus, the benefits of paraheliotropism are achieved at virtually no cost to the plant.

A major QTL for common bacterial blight resistance derives from the common bean great northern landrace cultivar Montana No.5

Knowledge of the evolutionary origin and sources of pest resistance genes will facilitate gene deployment and development of crop cultivars with durable resistance. Our objective was to determine the source of common bacterial blight (CBB) resistance in the common bean Great Northern Nebraska #1 (GN#1) and GN#1 Selection 27 (GN#1 Sel 27). Several great northern cultivars including GN#1, GN#1 Sel 27, and Montana No.5 (the female parent of the common x tepary bean interspecific population from which GN #1 and GN # 1 Sel 27 were derived) and known susceptible checks were evaluated for CBB reaction in field and greenhouse environments. These genotypes and CBB resistant and susceptible tepary bean including Tepary #4, the male parent and presumed contributor of CBB resistance toGN#1 and GN#1 Sel 27, were assayed for presence or absence of three SCAR markers tightly linked with independent QTLs conditioning CBB resistance. The parents and F2 of Montana No. 5/GN #1 Sel 27 and Montana No.5/Othello(CBB susceptible) were screened for CBB reaction and SCAR markers. CBB resistance in Montana No.5 was comparable to that of GN#1 and GN#1 Sel27. The SAP6 SCAR marker present in GN#1 and GN#1 Sel 27 was also present in Montana No.5, and it co-segregated (R 2 =35%) with the CBB resistance in the Montana No.5/Othello F2 population. Although a few CBB resistant and susceptible transgressive segregants were found in the F2 of MontanaNo.5/GN #1 Sel 27 and later confirmed by F3 progeny tests, SAP6 SCAR marker was present in all progenies. None of the tepary bean specific CBB resistance-linked SCAR markers were present in GN#1, GN#1 Sel 27, or Montana No.5. A cluster analysis of 169 polymorphic PCR-based markers across three common bean and Tepary #4 indicated that GN#1, GN#1 Sel 27, and Montana No.5 were closely related, and not related at all with Tepary #4.Thus, these results clearly indicate Montana No.5, not Tepary #4, as the source of CBB resistance in GN#1 and GN#1 Sel 27.

Purification and acute toxicity of a lectin extracted from tepary bean ( Phaseolus acutifolius)

Lectins are proteins or glycoproteins known for their ability to agglutinate cells, especially erythrocytes. Several lectins are toxic to mammalian cells both in vitro and in vivo, inhibit growth when incorporated into the diet and are toxic when injected into animals. On the other hand, the use of lectins has been suggested as an alternative in cancer treatment. The objective of the present study was to evaluate the acute toxicity of a lectin extracted and purified from tepary bean ( Phaseolus acutifolius, G-400-34) on CD-1 mice. Fetuin affinity chromatography, electrophoretic pattern and Western-immunoblot techniques were used to purify and partially characterize the lectin. This tepary bean line presented high levels of lectin activity. Western blot revealed that the sera reacted with the known PHA subunits in the 34–40 kD range. Four major lectin and lectin-related glycopolypeptides were identified. The purified lectin from tepary bean was a tetrameric one that ran at about 115–120 kD. The LD 50 (ip) was 1100 mg/kg body weight for males and 1120 mg/kg body weight for females. The body weight of experimental animals decreased in a dose-dependent manner. The main target organs affected were small intestine, spleen and thymus. The lectin isolated from tepary bean showed affinity towards fetuin, with high agglutination values and low acute toxicity.

An optimized Agrobacterium -mediated transformation procedure for Phaseolus acutifolius A. Gray

To improve Agrobacterium tumefaciens-mediated transformation of Phaseolus acutifolius, we examined the effect of different factors on T-DNA transfer by measuring transient expression levels of an intron-containing β-glucuronidase gene. Improved transformation frequencies were obtained with an A. tumefaciens strain carrying nopaline-type virulence genes and when calli were infected with Agrobacterium cells in the early-log growth phase. Optimized co-cultivation was performed at 22°C under a 16/8-h (day/night) photoperiod in an acidic medium (pH 5.5) in the presence of 200 µM acetosyringone. By combining the best treatments, an efficient and reproducible transformation procedure was established for the P. acutifolius genotype NI576. Southern and immunoblot analyses confirmed the stable integration and expression of the transgenes in the primary transgenic plants and their progeny.

Light strongly promotes gene transfer from Agrobacterium tumefaciens to plant cells.

Light conditions during Agrobacterium-based plant transformation, the most routinely used method in plant genetic engineering, differ widely and, to our knowledge, have not been studied systematically in relation to transformation efficiency. Here, light effects were examined in two already optimized transformation procedures: coculture of Agrobacterium tumefaciens with callus from two genotypes of the crop plant Phaseolus acutifolius (tepary bean) and coculture of root segments from two ecotypes of Arabidopsis thaliana. Except for the light conditions during coculture, all steps followed established procedures. Coculture was done either under continuous darkness, under a commonly used photoperiod of 16 h light/8 h darkness or under continuous light. beta-glucuronidase (GUS) production due to the transient expression of an intron-containing uidA gene in the binary vector was used to evaluate T-DNA transfer. In all situations, uidA expression correlated highly and positively with the light period used during coculture; it was inhibited severely by darkness and enhanced more under continuous light than under a 16 h light/8 h dark photoperiod. The promotive effect of light was observed with Agrobacterium strains harboring either a nopaline-, an octopine- or an agropine/succinamopine-type non-oncogenic helper Ti plasmid. The observed positive effect of light has obvious implications for developing and improving transient and stable transformation protocols, specifically those involving dark coculture conditions.

Subcellular Localization of a Novel Transcription Factor in Watered and Drought Sressed Phaseolus acutifolius

Subcellular Localization of a Novel Transcription Factor in Watered and Drought Sressed <i>Phaseolus acutifolius</i>

Uptake of cesium-137 and strontium-90 from contaminated soil by three plant species; application to phytoremediation.

A field test was conducted to determine the ability of three plant species to extract 137Cs and 90Sr from contaminated soil. Redroot pigweed (Amaranthus retroflexus L.), Indian mustard [Brassica juncea (L.) Czern.], and tepary bean (Phaseolus acutifolius A. Gray) were planted in a series of spatially randomized cells in soil that was contaminated in the 1950s and 1960s. We examined the potential for phytoextraction of 90Sr and 137Cs by these three species. Concentration ratios (CR) for 137Cs for redroot pigweed, Indian mustard, and tepary bean were 2.58, 0.46, and 0.17, respectively. For 90Sr they were substantially higher: 6.5, 8.2, and 15.2, respectively. The greatest accumulation of both radionuclides was obtained with redroot pigweed, even though its CR for 90Sr was the lowest, because of its relatively large biomass. There was a linear relationship between the 137Cs concentration in plants and its concentration in soil only for redroot pigweed. Uptake of 90Sr exhibits no relationship to 90Sr concentrations in the soil. Estimates of time required for removal of 50% of the two contaminants, assuming two crops of redroot pigweed per year, are 7 yr for 90Sr and 18 yr for 137Cs.

Planting Date and Genotype Effects on Tepary Bean Productivity

Our objective was to evaluate production potential of eight tepary bean (Phaseolus acutifolius A. Gray) genotypes and three planting dates. Significant variation (P &lt; 0.05) existed among eight genotypes and three planting dates in 1997 and 1998. The genotype ×planting date interaction was nonsignificant (P &gt; 0.05) for seed yield and harvest index. Seed yields of eight genotypes, when averaged over three planting dates and 2 years, varied from 1618 to 1988 with a mean of 1816 kg·ha-1, indicating that tepary bean is adapted to Virginia's agro-climatic conditions. The harvest index (ratio between seed and total plant weight, expressed as percentage) ranged from 38% to 47%. Seed weight varied from 12.6 to 18.8 g with a mean of 14.5 g. Genotypes with tan-colored seeds had significantly larger seed than those with black or white seeds. Planting dates significantly affected seed yield, seed weight, and harvest index. The highest seed yield (2239 kg·ha-1) and harvest index were obtained from the late May plantings.

Thermal inactivation of tepary bean ( Phaseolus acutifolius), soybean and lima bean protease inhibitors: effect of acidic and basic pH

Thermal stability of the protease inhibitor of tepary bean ( Phaseolus acutifolius) was studied by comparing the antitryptic activities of a crude extract and the purified inhibitor (TBPI) to those of three purified protease inhibitors, soybean trypsin inhibitor (Kunitz; KSTI), soybean trypsin and chymotrypsin inhibitor (BBI) and lima bean trypsin inhibitor (LBTI). Three purified inhibitors, TBPI, BBI and LBTI, were found to be heat-stable when subjected to boiling (100 °C) at either neutral or acidic pH, or to autoclaving at neutral pH. The tepary bean crude extract and KSTI were found to be heat labile when boiled or autoclaved at neutral pH. At alkaline pH, all the purified protease inhibitors were heat labile, losing antitryptic activity rapidly upon heating to 100 °C. These data suggest that the trypsin inhibitor of TBPI is heat-stable, and similar to the Bowman–Birk family of inhibitors, and can be rapidly inactivated by heat under alkaline conditions.

The effects of a protease inhibitor fraction from tepary bean ( Phaseolus acutifolius) on in vitro cell proliferation and cell adhesion of transformed cells

Some protease inhibitors (PI), such as the soybean Bowman–Birk protease inhibitor (SBBI), have been described as anticarcinogenic agents. Although PI are ubiquitous compounds in living organisms, the anticarcinogenic potential of PIs other than SBBI remain poorly explored. We evaluated the antiproliferative effect of a protein fraction from tepary bean ( Phaseolus acutifolius) seeds with protease inhibitor activity (TPIF), on normal and on malignant cells. TPIF was obtained after precipitation with ammonium sulfate and gel filtration, and its bioactivity was assayed in vitro on HeLa cells, normal 3T3 fibroblasts and 3T3/v-mos transformed fibroblasts. TPIF showed antiproliferative and cytotoxic effects on 3T3/v-mos transformed fibroblasts in a dose_dependent way. On the contrary, TPIF was only cytostatic for normal 3T3 cells at the highest doses assayed, and had no effect on epithelial HeLa cells proliferation. Sublethal TPIF doses also stimulated cell adhesion of poorly adherent 3T3/v-mos cell line.

Improvement of drought adapted tepary bean ( Phaseolusacutifolius A. Gray var. latifolius) yield through biological nitrogen fixation in semi-arid SE-Kenya

Tepary bean (TB), a drought adapted bean variety has recently assumed prominence among resource poor farmers in semi-arid SE-Kenya. However, its productivity is still low because of inadequate inputs. Greenhouse experiments were initially carried out to determine the number of rhizobia specific to TB in the soils of SE-Kenya, nodulation and nitrogen fixation of the same. The most probable number (MPN) of Rhizobia cells capable of nodulating TB were 1.0×10 2 cells per gram of soil in SE-Kenya. Subsequent field experiments were conducted at Kiboko KARI sub-station over two rainy seasons, i.e. long rains (LR) 1999 (March–June) and short rains (SR) 1999/2000 (October–January). The main objective was to screen various Rhizobia strains effectivity in nitrogen fixation with TB. The field trials showed that from the five Rhizobium strains tested only R3254 significantly increased pod dry weight, seed dry weight and seed yield/ha. Top dressed fertilizer N had no significant impact on growth and seed formation of tepary bean. Total nitrogen (N) concentration in the plant tissues was significantly enhanced in the course of the vegetative growth in R3254 treatment. This is an indication that Rhizobium strain R3254 can be used in the improvement of TB production for the resource poor farmers of semi-arid SE-Kenya.

Dehydration-avoidance responses of tepary bean lines differing in drought resistance

Summary Four tepary bean (Phaseolus acutifolius) lines were studied for the responses of the dry matter accumulation and partitioning, the growth characteristics of the root and the shoot genotypes, and the potential seed yield to drought. Comparatively, two lines were high (NE # 8A and NE # 19) and two lines (NE # 5 and NE # 7) were low in the seed yield under well-watered conditions. The two high yielding lines remained higher in the seed yield than the two low yielding lines under drought conditions. NE # 19 is characterized by a deep root penetration and a balanced root : shoot growth pattern, along with the greatest mass of root in the deepest soil profile. Its leaves exhibited sensitive stomates in response to drought stress. Except for having less depth of its root penetration, NE # 8A was very similar to NE # 19. NE # 8A showed less reduction than NE # 19 in the potential seed yield as affected by the water deficit. It appeared to use less soil water, suggesting operating sensitive stomates. However, the RWC of NE # 8A leaves showed more reduction than NE # 19 in response to the drought. NE # 19 could cope with more severe water stress due to its deeply penetrating root along with the sensitive stomates of the leaf genotype.

A quick method for assessing chloroplastic starch granules by flow cytometry

Summary A crude but rapid method for assessing chloroplastic starch granules by flow cytometry is introduced. It yields information on the average number of starch granules in chloroplasts within minutes. Two species of beans, Phaseolus vulgaris and Phaseolus acutifolius, were used to develop the tech nique. This new technique cannot replace the conventional enzymatic method when absolute quantification of starch (in glucose equivalents) is required. However, if a quick check of relative starch content is sufficient when addressing issues such as stress and photosynthetic regulation through large-scale screening of plants, the new technique may be the method of choice.

Salinity and Boron Effects on Growth and Yield of Tepary and Kidney Beans

Crops tolerant to salt and boron have an advantage when grown in soils, near power plants, that are contaminated by saline cooling tower water or by waste products such as coal fly ash containing high levels of B. In addition, tolerant crops may have higher yields in arid and semi arid regions where salt and B problems coexist. This greenhouse study was conducted in order to compare the response of tepary bean (Phaseolus acutifolius A. Gray) and kidney bean (Phaseolus vulgaris L.) to combinations of high levels of solution B and chloride salts. The plants of both species were grown in 10-L plastic buckets containing silica sand and were irrigated with half-strength Hoagland's No. 1 nutrient solution to which boric acid and a mixture of NaCl and CaCl2·2H2O on a 1:1 equivalent ratio were added. Boron treatments consisted of 5, 20, and 25 mg·L-1 and the target electrical conductivity (EC) levels were 1, 4, 8, 12, and 15 dS·m-1. The maximum growth and yield for tepary bean occurred at 5 mg·L-1 B and 4 dS·m-1. These values of B and salinity were 6.5 and 4 times higher than those threshold values reported for kidney bean. Furthermore, tepary bean produced profitable yield (80% of control) in the 5 mg·L-1 B, 8 dS·m-1 treatment and it even produced beans at B concentration as high as 20 mg·L-1 B, four times higher than that observed for kidney bean.

Purification and characterization of two α-amylase inhibitors from seeds of tepary bean ( Phaseolus acutifolius A. Gray)

Two proteinaceous α-amylase inhibitors termed αAI-Pa1 and αAI-Pa2 were purified from seeds of a cultivated tepary bean ( Phaseolus acutifolius A. Gray, cv. PI311897). The two inhibitors differed in their specificity towards α-amylases of insect pests such as bruchids, although neither showed any inhibitory activity against α-amylases of mammalian, bacterial or fungal origin. αAI-Pa2 resembles two common bean inhibitors, αAI-1 and αAI-2, in several characteristics such as N-terminal amino acid sequences and oligomeric structure being composed of α and β subunits. In contrast αAI-Pa1 is composed of a single glycopolypeptide with a molecular mass of 35 kDa, and its N-terminal amino acid sequence resembled that of seed lectins in tepary bean and common bean. The information on the two tepary bean α-amylase inhibitors may be useful not only for providing insight into critical structure for the specificity towards different α-amylase enzymes but also for enhancing insect resistance in crops.

Digestive amylase from the larger grain borer, Prostephanus truncatus Horn

A combination of ion-exchange chromatography, preparative electrophoresis and gel filtration chromatography allowed a 1209-fold purification of one of the two major digestive α-amylases from larvae of the larger grain borer, Prostephanus truncatus Horn. The purified enzyme showed a molecular mass of 60.2 kDa, an isoelectric point of 4.7 and an optimal pH for activity of 6.0. The enzyme was heat labile and it was recognized by proteinaceous inhibitors from amaranth seeds ( Amaranthus hypochondriacus), whereas extracts from maize ( Zea mays) and tepary bean ( Phaseolus acutifolius) produced very low inhibition. When the enzyme was measured at different stages of development, maximal activity was found in the second instar larvae. Activity drastically decreased to a very low level during the pupae stage and increased again at the adult stage. A zymogram of the different developmental stages showed two main bands of α-amylase activity, which almost disappeared at the pupae stage to increase again during the adult stage, revealing a new, smaller band. This new band may be required for a better adaptation of the adult insect to its new environment.

IN VITRO REGENERATION AND AGROBACTERIUM-MEDIATED TRANSFORMATION OF PHASEOLUS VULGARIS L. (COMMON BEAN) AND P. ACUTIFOLIUS A. GRAY (TEPARY BEAN)

Dry Phaseolus beans are an important staple Food and immature bean pods are popular as a vegetable. Phaseolus seeds are deficient in the sulfur-containing amino acids and the most important Phaseolus species, the common bean (P. vulgaris) is susceptible to many pests and diseases, Genetic modification of the crop could contribute to solving some of these problems. However, the recalcitrance of Phaseolus towards in vitro regeneration is a major bottleneck in transgenic plant production, It has been reported that, in an attempt to avoid the use of in vitro regeneration, transgenic plants have been produced through particle bombardment of seedling meristems of P, vulgaris. This approach relies on screening rather than selection for transformed tissues and is therefore inefficient. We anticipate that a regeneration procedure that involves a callus phase is more suitable for obtaining transgenic plants because selection for transformed cells can be more readily achieved in callus than in organized meristems, Regeneration from callus in P. vulgaris is hard to attain but it has been reported for two genotypes that were constructed from a P, vulgaris x P, acutifolius (tepary bean) hybrid, We found that the same mode of regeneration can be obtained in a wide range of P, acutifolius genotypes. The regeneration system enabled us to produce transgenic plants of a wild genotype of P. acutifolius with Agrobacterium. P, acutifolius can now be used as a 'bridge species' to introgress transgenes into the common bean. Optimized cocultivation and regeneration procedures are now applied to cultivated tepary and common bean genotypes in order to obtain transgenic plants with Agrobacterium.

Effect of Atmospheric CO2 Enrichment on Root Growth and Carbohydrate Allocation of Phaseolus spp.

A glasshouse experiment was conducted with plants of Phaseolus grown in liquid culture. Root growth parameters (biomass, diameter, length, growth rate, zone of cell division), root rheological components (wall extensibility, water potential yield threshold, water potential), shoot growth, carbon allocation, and abscisic acid (ABA) concentration were measured in Phaseolus acutifolius A. Gray at ambient (550 µmol mol-1) and elevated (700 µmol mol-1) atmospheric CO2 concentrations. For contrast, measurements of above- and belowground growth were conducted on Phaseolus vulgaris L. in the same treatments. Under nonlimiting conditions of water and nutrients, elevated CO2 increased root and shoot growth of P. acutifolius but not P. vulgaris. While root mass was increased by nearly 60% in P. acutifolius, there was no effect of atmospheric CO2 on any of the rheological components measured. In contrast, starch and ABA accumulated in roots of P. acutifolius. The concentration of starch in roots of P. acutifolius increased by 10-fold, while root concentrations of ABA doubled. From the data it is concluded that CO2 enrichment is favorable for root growth in some species in that more carbon is allocated to belowground growth. In addition, ABA may play a role in growth responses and/or allocation of photosynthates at elevated CO2 in P. acutifolius.

The arcelin-5 gene of Phaseolus vulgaris directs high seed-specific expression in transgenic Phaseolus acutifolius and Arabidopsis plants.

The regulatory sequences of many genes encoding seed storage proteins have been used to drive seed-specific expression of a variety of proteins in transgenic plants. Because the levels at which these transgene-derived proteins accumulate are generally quite low, we investigated the utility of the arcelin-5 regulatory sequences in obtaining high seed-specific expression in transgenic plants. Arcelin-5 is an abundant seed protein found in some wild common bean (Phaseolus vulgaris L.) genotypes. Seeds of Arabidopsis and Tepary bean (Phaseolus acutifolius A. Gray) plants transformed with arcelin-5 gene constructs synthesized arcelin-5 to levels of 15% and 25% of the total protein content, respectively. To our knowledge, such high expression levels directed by a transgene have not been reported before. The transgenic plants also showed low plant-to-plant variation in arcelin expression. Complex transgene integration patterns, which often result in gene silencing effects, were not associated with reduced arcelin-5 expression. High transgene expression was the result of high mRNA steady-state levels and was restricted to seeds. This indicates that all requirements for high seed-specific expression are cis elements present in the cloned genomic arcelin-5 sequence and trans-acting factors that are available in Arabidopsis and Phaseolus spp., and thus probably in most dicotyledonous plants.