Proline metabolism in response to highest nitrogen dosages in green bean plants (Phaseolus vulgaris L. cv. Strike)

The objective of the present work was to determine what impact extremely high nitrogen dosages would have on proline metabolism in order to use this amino acid as a bioindicator of N status of green bean plants (Phaseolus vulgaris L. cv. Strike). In this effort, we identified the most favourable pathway of proline synthesis under our experimental conditions. The N was applied to the nutrient solution in the form of NH₄NO₃ at 5.4 mmol/L (N1, optimal level), 11.6 mmol/L (N2), 17.4 mmol/L (N3), and 23.2 mmol/L (N4). Our results indicate that the application of high N dosages inPhaseolus is characterized by the accumulation of NO₃⁻, NH₄⁺ and proline in root and foliar organs. However, although the enzymes in charge of proline biosynthesis, ornithine-δ-aminotransferase (OAT, EC 2.6.1.13) and Δ¹-pyrroline-5-carboxylate synthetase (P5CS, EC 2.7.2.11/1.2.2.41) vary in behaviour depending on the N status, in our experiment, this amino acid appears to be synthesized mainly by the enzyme ornithine-δ-aminotransferase. This suggests predominance of the ornithine pathway over the glutamine pathway. Finally, under our experimental conditions, proline can be defined as a good indicator of N excess of green bean plants.     

Phenolic and Oxidative Metabolism as Bioindicators of Nitrogen Deficiency in French Bean Plants (Phaseolus vulgaris L. cv. Strike)

Abstract: The aim of the present work was to determine the effect of abiotic stress, such as nitrogen (N) deficiency, on phenol and oxidative metabolism. In addition, we analyzed whether the response of the two metabolic processes is a good bioindicator of N deficiency in French bean plants. The N was applied to the nutrient solution in the form of NH₄NO₃ at 1.35 mM (N1), 2.7 mM (N2) and 5.4 mM (N3), this latter dosage being considered optimal. The results indicated that application of 1.35 and 2.70 mM of N can be defined as suboptimal or deficient, as it depressed foliar biomass of the French bean plants in our experiment. In addition, abiotic stress from the application of these N dosages stimulated the enzymes PPO, POD and CAT, and inhibited PAL and SOD activities, resulting in the lowest foliar accumulation of phenolic compounds and H₂O₂.     

Macronutrient content in leaves of bean plants (Phaseolus vulgaris,L.) grown with differents rates of N/S as fertilizers

Summary The effect of increasing rates of nitrogen (N) and sulphur (S) as fertilizers on the yield, leaf area and N, P, S, Ca, Mg, NO₃ ⁻ and SO₄ ⁼ content in leaves of bean (Phaseolus vulgaris, L.) were studied in a hydroponic culture experiment under greenhouse conditions. Bean plants responded significantly to all treatments with differents N/S ratios. When plants grew with high N/S ratios, the leaf content of N, Ca and NO₃ ⁻ increased while the content of K, P and SO₄ ⁼ decreased. However, optimal yield and leaf area were not obtained. Optimal leaf and fruit dry matter was obtained at N/S ratio value of 1.41. When lower N/S rates were used, optimal leaf and fruit dry matter was only observed when the leaf N/S ratio was between 15 and 16. At high sulphate levels in the nutrient solution there is no interaction with nitrate which is easily observed, resulting in an increase in yield. An interaction between nitrate and sulphate in the nutrient solution was found at a N/S ratio of 0.81 which produced in leaves a synergic effect between P-K, an antagonistic effect between N-P and N-K and a lower yield. This research was supported by Fundacion ‘Ramon Areces’.     

Proline metabolism and NAD kinase activity in greenbean plants subjected to cold-shock

The aim of the present work was to examine the relationship between proline metabolism and NAD kinase activity in greenbeans submitted to cold-shock. For this, 15-day-old greenbean plants were subjected to a temperature of 4 degrees C (cold shock) for 180 min. Our results indicate that the plants showed foliar accumulation of proline, with the enzymes ornithine-delta-aminotransferase (OAT) and proline dehydrogenase (PDH) appearing as determinant in this accumulation under cold-shock. Also, we found a close relationship between the Ca(2+)-CaM-dependent NAD kinase activity and proline metabolism, suggesting that the adaptive responses or acclimation of plants to cold stress are preceded by increased [Ca(2+)](cyt).     

Possible role played by R1 protein in starch accumulation in bean (Phaseolus vulgaris) seedlings under phosphate deficiency

The effect of phosphate (Pi) deficiency on starch accumulation was studied in bean (Phaseolus vulgaris). After 3 weeks of Pi deprivation total Pi concentration in root and shoot was reduced by 68% and 42%, respectively; however, only shoot growth was affected. In leaves, Pi deprivation induced glucose, fructose and starch accumulation. Pi deficiency did not affect starch synthesis, but it reduced its mobilization during the dark period. At the same time, starch produced by Pi deficient plants have fewer Pi bound and was also less susceptible to beta-amylase hydrolysis. R1 protein is the protein responsible of phosphorylating C3 and C6 glucosyl residues of the polyglucan, increasing the hydration capacity and the interaction with amylolytic enzymes. Pi deprivation did not change the amount of R1 protein detected in total extracts but decreased its association with starch granules.     

Rapid changes in oxidative metabolism as a consequence of elicitor treatment of suspension-cultured cells of French bean (Phaseolus vulgaris L.)

Stressed plant cells often show increased oxygen uptake which can manifest itself in the transient production of active oxygen species, the oxidative burst. There is a lack of information on the redox status of cells during the early stages of biotic stress. In this paper we measure oxygen uptake and the levels of redox intermediates NAD/NADH and ATP and show the transient induction of the marker enzyme for redox stress, alcohol dehydrogenase. Rapid changes in the redox potential of elicitor-treated suspension cultures of French bean cells indicate that, paradoxically, during the period of maximum oxygen uptake the levels of ATP and the NADH/NAD ratio fall in a way that indicates the occurrence of stress in oxidative metabolism. This period coincides with the maximum production of active oxygen species particularly H₂O₂. The cells recover and start producing ATP immediately upon the cessation of H₂O₂ production. This indicates that the increased O₂ uptake is primarily incorporated into active O₂ species. A second consequence of these changes is probably a transient compromising of the respiratory status of the cells as indicated in expression of alcohol dehydrogenase. Elicitor-induced bean ADH was purified to homogeneity and the M_r 40 000 polypeptide was subjected to amino acid sequencing. 15% of the whole protein was sequenced from three peptides and was found to have nearly 100% sequence similarity to the amino acid sequence for pea ADH1 (PSADH1). The cDNA coding for the pea enzyme was used to demonstrate the transient induction of ADH mRNA in elicitor-treated bean cells. Enzyme activity levels also increased transiently subsequently. Increased oxygen uptake has previously been thought to be associated with provision of energy for the changes in biosynthesis that occur rapidly after perception of the stress signal. However the present work shows that this rapid increase in oxygen uptake as a consequence of elicitor action is not wholly associated with respiration.     

Molecular cloning of the cDNA encoding a stress-inducible protein phosphatase 1 (PP1) catalytic subunit from French bean (Phaseolus vulgaris L.)

A cDNA showing high sequence similarity (>70%) to plant protein phosphatase 1 catalytic subunit variants from other species has been isolated from a cDNA library derived from mRNAs expressed in elicitor-treated suspension-cultured cells. The clone appears to be a near full-length 1431 bp with a 172 bp 5-untranslated region and a 317 bp 3-untranslated region. The open reading frame, determined by sequence similarity, codes for a protein with predicted M _r of 35552. Alternatively an ATG situated to the 5 end of the putative start site would increase the protein size by 6 amino acids.The mRNA for Pvpp1 was shown to be rapidly induced by elicitor treatment of suspension-cultured cells of French bean. The cloned cDNA represents one of the few examples of a gene product that is probably involved in dephosphorylation events arising after the initial responses to biotic stress.Abbreviations PAL phenylalanine ammonia-lyase - PP1 protein phosphatase 1 - Pvpp1 Phaseolus vulgaris protein phosphatase 1     

Bean lectins IV: genetic variation in the non-denatured structure of lectins from different Phaseolus vulgaris L. cultivars

Summary Variation in the native conformation of bean lectins was examined using electrophoresis of non-denatured total protein extracts and purified albumin and globulin lectin. The observed variation was related to the genetic variation reported previously for lectin polypeptide composition as revealed by two-dimensional isoelectricfocusing-sodium dodecyl sulfate polyacrylamide gel electrophoresis (IEF-SDS/PAGE). When eleven cultivars with different IEF-SDS/PAGE lectin polypeptide compositions were compared, eight had unique non-denatured lectin patterns and three had identical patterns. For some cultivars differences in non-denatured lectin patterns were observed between the purified albumin and globulin lectin preparations.     

Implications of calcium nutrition on the response of Phaseolus vulgaris L. to salinity

The effect of Ca²⁺ level in the growth medium on the response of germination and early seedling growth of Phaseolus vulgaris to NaCl salinity was investigated. When NaCl concentration was increased germination and early seedling growth was decreased. The addition of Ca²⁺ to the media increased both germination percentage and seedling growth. Chloride concentrations were not affected by the level of Ca²⁺. Potassium and Ca²⁺ concentrations and transport from roots to shoots were decreased by NaCl, but were restored by increasing Ca²⁺ in the medium. The opposite was true for Na⁺. Leakage of NO₃ ^- and H₂PO₄ ^- was increased by salinity and reduced by high Ca²⁺ in the medium. The results are discussed in terms of the beneficial effects of calcium for plant growth under saline conditions.     

Selection for improved nitrogen fixation inGlycine max (L.) Merr. andPhaseolus vulgaris L.

Summary While symbiotic nitrogen (N₂) fixation byG. max andP. vulgaris reduces their need for combined N, N₂ fixation under field conditions is rarely maximized. This paper reviews constraints to N₂ fixation in these species, then examines the genetic variability recorded for traits affecting N₂ fixation and the further work needed in this area. It considers emerging programs for the improvement of N₂ fixation inG. max andP. vulgaris and pays particular attention to methodological considerations.Scientific Journal Series, Minnesota Agri. Exp. Station. No 14190.     

Nitrate uptake by bean (Phaseolus vulgaris L.) roots under phosphate deficiency

Bean (Phaseolus vulgaris L.) plants were cultured for 19 d on complete or on phosphate deficient culture media. Low inorganic phosphate concentration in the roots decreased ATP level and nitrate uptake rate. The mechanisms which may control nitrate uptake rate during phosphate deficiency were examined. Plasma membrane enriched fractions from phosphate sufficient and phosphate deficient plants were isolated and compared. The decrease in total phospholipid content was observed in plasma membranes from phosphate deficient roots, but phospholipid composition was similar. No changes in ATPase and proton pumping activities measured in isolated plasma membrane of phosphate sufficient and phosphate deficient bean roots were noted. The electron microscope observations carried out on cortical meristematic cells of the roots showed that active ATPases were found in plasma membrane of both phosphate sufficient and phosphate deficient plants. The decrease in inorganic phosphate concentration in roots led to increased nitrate accumulation in roots, accompanied by a corresponding alterations in NO₃ distribution between shoots and roots. Nitrate reductase activity in roots of phosphate deficient plants estimated in vivo and in vitro was reduced to 50–60% of the control. The increased NO₃ concentration in root tissue may be explained by decreased NR activity and lower transport of nitrate from roots to shoots. Therefore, the reduction of nitrate uptake during phosphate starvation is mainly a consequence of nitrate accumulation in the roots.     

Alleviation of Salt Stress in Common Bean (Phaseolus vulgaris) by Exogenous Abscisic Acid Supply

In this work the effect of abscisic acid (ABA) and 100 mM NaCl on common bean (Phaseolus vulgaris var. Coco) growth, nitrogenase activity, and nodule metabolism was studied. Experiments were carried out in a controlled environmental chamber and plants, at the vegetative growth stage (16 days old), were treated with ABA (1 μM and 10 μM) and 48 h later were exposed to saline treatment. Results revealed that plant dry weight, nodule dry weight, nitrogen fixation (acetylene reduction activity and ureides content), and most enzymes of ammonium and ureides metabolism were affected by both ABA and NaCl. The addition of 1 μM ABA to the nutrient solution before the exposure to salt stress reduced the negative effect of NaCl. Based on our results, we suggest that ABA application improves the response of Phaseolus vulgaris symbiosis under saline stress conditions, including the nitrogen fixation process and enzymes of ammonium assimilation and purine catabolism.     

Nuclear and chloroplast microsatellite diversity in Phaseolus vulgaris L. from Sardinia (Italy)

Studies of the level and the structure of the genetic diversity of local varieties of Phaseolus vulgaris are of fundamental importance, both for the management of genetic resources and to improve our understanding of the pathways of dissemination and the evolution of this species in Europe. We have here characterized 73 local bean populations from Sardinia (Italy) using seed traits and molecular markers (phaseolins, nuSSRs and cpSSRs). American landraces and commercial varieties were also included for comparison. We see that: (a) the Sardinian material is distinct from the commercial varieties considered; (b) the variation in the seed traits is high and it mostly occurs among populations (95%); (c) compared to the American sample and the commercial varieties, the Sardinian collection has a low level of diversity; (d) the majority (>95%) of the Sardinian individuals belong to the Andean gene pool; (e) the Sardinian material shows a strong genetic structure, both for cpSSRs and nuSSRs; (f) the nuSSRs and cpSSRs concur in differentiating between gene pools, but a lack of congruence between nuclear and chloroplast has been observed within gene pools; and (g) there are three putative hybrids between the Andean and Mesoamerican gene pools. Despite the relatively low level of diversity, which is probably due to a strong founder effect, the Sardinian landraces are worth being conserved and studied further because of their distinctiveness and because hybridization within and between the gene pools could generate variation that will be useful for breeding. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cytokinin metabolism in Phaseolus vulgaris L.

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

Yield and biological nitrogen fixation of common bean (Phaseolus vulgaris L.) in Peru

Two field experiments were performed to evaluate the nitrogen fixation potential of twenty common bean cultivars and breeding lines during summer and winter seasons of 1986 and 1988, respectively. The ¹⁵N isotope dilution method was used to quantify N₂ fixation. The cultivars and breeding lines were variable in terms of their N₂ fixation. The cv. Caballero was very efficient, with more than 50% N derived from the atmosphere and 60–80 kg N ha^–1 fixed in both seasons. Other cultivars were less efficient, since the poorest ones derived less than 30% of their nitrogen from the atmosphere and fixed less than 20 kg N ha^–1. After additional testing the best cultivars may be used directly by the farmers for cultivation. The experiments have provided information about which genotypes may be used to breed for enhanced fixation in common bean.     

Stable transformation of Phaseolus vulgaris via electric-discharge mediated particle acceleration

Summary Transgenic Phaseolus vulgaris or common bean has been produced using electric-discharge particle acceleration. The method uses particle acceleration to introduce DNA into bean seed meristems. Multiple shoots are then generated and screened to recover transgenic plants at a rate of 0.03% germline transformed plants/shoot. We have been able to recover transgenic plants using both GUS and herbicide screening to introduce the gus, bar, and bean golden mosaic virus coat protein genes into the navy bean cultivar, Seafarer. The transgenic plants have been characterized over 5 generations of self-fertilization with no loss of introduced genes or expression. In addition, several families have been crossed with non-transgenic parents and these plants also show expected inheritance patterns. The introduced bar gene has been shown to confer strong resistance in transgenic beans to basta herbicide application in the greenhouse.Abbreviations BGMV bean golden mosaic virus - PAT phosphinothricin acetyltransferase     

Five primary trisomics from translocation heterozygote progenies in common bean,Phaseolus vulgaris L.

Summary Twelve distinct phenotypic groups of plants were isolated from nondisjunction progenies of 11 translocation heterozygote stocks. All the plants in these phenotypic groups originated in the light weight seed class. Five of the 12 phenotypic groups of plants have been verified as primary trisomics. They are all phenotypically distinguishable from each other and from disomics. One of the five primary trisomic groups, puckered leaf, was directly recovered as a primary trisomic from the original translocation heterozygote progenies. Three of the five trisomics — weak stem, dark green leaf, and convex leaf — originated first as tertiary trisomics. The related primary trisomics were isolated later from progenies of selfed tertiary trisomics. The fifth group, chlorotic leaf, originated at a low frequency among the progenies of three other trisomics: puckered leaf, convex leaf, and dark green leaf. The chlorotic leaf did not set seed under field conditions. The remaining four groups — puckered leaf, dark green leaf, convex leaf, and weak stem — are fertile, though sensitive to high temperature conditions. The transmission rate of the extra chromosome on selfing ranges from 28% to 41%. Physical identification of the extra chromosome has not been achieved for any of the five trisomic groups. Two trisomic groups, dark green leaf and convex leaf, have produced tetrasomics at low frequency. The phenotypes of these two tetrasomics are similar to the corresponding trisomics but more exaggerated.Fla. Agr. Expt. Stn. Journal Series No. 7137     

Nodule-specific glutamine synthetase is expressed before the onset of nitrogen fixation in Phaseolus vulgaris L.

Glutamine synthetase expression was studied in developing root-nodules of common bean with regard to the time-course of specific activity, antigen accumulation, polypeptide composition and in vitro translation products. This analysis shows that the nodule-specific GS polypeptide (GS-gamma) is detected prior to the nitrogenase acetylene-reducing activity, and that its accumulation together with that of the GS-alpha and GS-beta polypeptides vary with nodule age. GS-gamma is present in ineffective nodules, although in a lower ratio to GS-beta than in wild-type nodules. Comparisons of in vitro translated and in vivo synthesized GS polypeptides suggest no post-translational modifications. The possible factors and mechanisms involved in the regulation of expression of GS polypeptides are discussed.     

Inheritance of resistance to potato y viruses in Phaseolus vulgaris L

Summary Resistance to watermelon mosaic virus-2 in Phaseolus vulgaris L. is conferred by two distinct dominant alleles at independent loci. Based on segregation data one locus is designated Wmv, the other, Hsw. The dominant allele Wmv from cv. Great Northern 1140 prevents systemic spread of the virus but viral replication occurs in inoculated tissue. In contrast, Hsw confers both local and systemic resistance to WMV-2 below 30C. At higher temperatures, plants that carry this allele in the absence of modifying or epistatic factors develop systemic veinal necrosis upon inoculation with the virus that results in rapid death. Patho-type specificity has not been demonstrated for either allele; both factors confer resistance to every isolate tested. A temperature-sensitive shift in epistasis is apparent between dominant alleles at these loci. Because Hsw is very tightly linked if not identical to the following genes for hypersensitivity to potyviruses I, (bean common mosaic virus), Bcm, (blackeye cowpea mosaic virus), Cam, (cowpea aphid-borne mosaic virus) and Hss (soybean mosaic virus), parental, reciprocal dihybrid F₁ populations, and selected F₃ families were inoculated with each of these viruses and held at 35 C. F₁ populations developed vascular necrosis completely or primarily limited to inoculated tissue, while F₃ families from WMV-2-susceptible segregates were uniformly susceptible to these viruses. The relationship between Hsw, Wmv and other genes for potyvirus resistance suggest patterns in the evolution of resistance and viral pathogenicity. Characterization of the resistance spectrum associated with each factor provides an additional criterion to distinguish genes for plant virus resistance.