Analysis of the Proteome of Common Bean (Phaseolus vulgaris L.) Roots after Inoculation with Rhizobium etli

Proteomics techniques were used to identify the underlying mechanism of the early stage of symbiosis between the common bean (Phaseolus vulgaris L.) and bacteria. Proteins from roots of common beans inoculated with bacteria were separated using two-dimensional polyacrylamide gel electrophoresis and identified using mass spectrometry. From 483 protein spots, 29 plant and 3 bacterial proteins involved in the early stage of symbiosis were identified. Of the 29 plant proteins, the expression of 19 was upregulated and the expression of 10 was downregulated. Upregulated proteins included those involved in protein destination/storage, energy production, and protein synthesis; whereas the downregulated proteins included those involved in metabolism. Many upregulated proteins involved in protein destination/storage were chaperonins and proteasome subunits. These results suggest that defense mechanisms associated with induction of chaperonins and protein degradation regulated by proteasomes occur during the early stage of symbiosis between the common bean and bacteria.     

Common Bean (Phaseolus vulgaris L.) Mutants Defective in Root Nodule Formation: II GENETIC ANALYSIS

Genetic analysis of crosses between two induced, ineffectivelynodulating mutants of common bean, NOD238 and NOD109, revealedthat their mutated nodulation phenotype is under the controlof the same locus in both mutants. The two mutants also resultedallelic for poor pod fertility, the other trait common to themutants. F₁ plants from crosses with their wild types nodulatedeffectively and had wild type pod fertility. Ineffective nodulationand poor pod fertility traits co-segregated in the F₂generationin which plants with the mutant nodulation and pod fertilityphenotypes represented 12.5% of the total population. Analysisin F₃confirmed that these plants were homozygous for both mutatedcharacters. The results indicated both mutant traits studiedare determined by a single recessive allele, named sym-2, whoseinheritance is negatively affected by its pleiotropic effecton pod fertility determining a deficit of ineffectively nodulatingcombinations. In an allelism test with the non-nodulating mutantof common bean NOD125 it was found that ineffective nodulationis controlled at a different locus and that the two loci arenot linked. Key words: Phaseolus valgaris, nitrogen fixation, nodulation mutants, genetics     

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.     

Nodulation of Pole Bean (Phaseolus vulgaris L.) by Rhizobium Species of Two Cross-Inoculation Groups

Physiology and morphology of pole bean (Phaseolus vulgaris L. cv Kentucky Wonder) root nodules induced by two Rhizobium species of different cross-inoculation groups have been compared. Root nodules induced by Rhizobium sp. 127E15, which is a strain of the cowpea group Rhizobium, were pinkish, had irregular shapes, and were only partially effective. Their peak acetylene reduction activity was 4.36 μmol of C₂H₄ formed per g of fresh nodules per h at 30 days after inoculation. The effective nodules induced by Rhizobium phaseoli 127K14, which is a strain of the bean group Rhizobium, were dark red, spherical, and showed peak acetylene reduction activity of 15.95 μmol of C₂H₄ formed per g of fresh nodules per h at 15 days after inoculation. The partial effectiveness of 127E15-induced nodules was associated with fewer infected cells, a delay in the increase of bacteroid population within the host cells, abundance of cytoplasmic vesicles in the host cells, more bacteroids within a membrane envelope (peribacteroid membrane), and the inability of bacteroids to completely fill up the host cytoplasmic space. The 127K14-induced nodules were fully mature, with host cells filled with bacteroids by 12 days after inoculation. In contrast, the 127E15-induced nodules did not reach a similar developmental stage even 30 days after inoculation.     

Common Bean (Phaseolus vulgaris L.) Mutants Defective in Root Nodule Formation: I. PHYSIOLOGICAL CHARACTERIZATION

Two ineffectively nodulating, allelic mutants of the commonbean (Phaseolus vulgaris L.) lines RIZ30 and RIZ36 were studied.In both mutants the nodulation phenotype was characterized bythe formation of tumour-like swellings when inoculated withbean Rhizobium strains. Late formation of pink nodules was observedin mutant plants grown in pots with soil or in hydroponic conditionswith perlite. In the absence of mineral nitrogen, mutant growthand nodulation were poor; a low peak of acetylene reductionactivity being detected 45 d after inoculation. Mutant growthin the presence of mineral nitrogen was similar to that of thewild type. The mutants were also characterized by poor pod fertility(seed per pod). The nodulation and pod fertility phenotypesare specifically controlled at the root and shoot levels, respectively,as assessed with grafting experiments. Mutant shoots graftedon wild type, nitrogen-fixing roots produced a number of seedssimilar to the wild type despite the expression of the poorpod fertility phenotype. Nitrogen and seed yields of wild typeshoots, grafted on mutant, ineffectively nodulating roots wereseverely reduced when compared with control wild type plantsalthough nitrogen accumulation during vegetative phase was similar. Key words: Phaseolus valgaris, nitrogen fixation, nodulation mutants, grafting, nitrogen assimilation     

普通菜豆种质资源表型鉴定及多样性分析

对646份普通菜豆核心种质在贵州毕节进行了表型鉴定,结果表明,普通菜豆具有丰富的形态性状多样性。总变异数为367,平均遗传丰富度为8.34,变异范围2~31;遗传多样性指数为0.63,变异范围为0.02~0.91。通过多变量的主成分分析,前3个主成分的贡献率较大,分别为17.73%、15.35%和11.33%。依据表型鉴定数据信息,将供试种质聚类并划分为4组。Ⅰ组的遗传多样性较高,主要为直立有限的大粒资源,大多资源属于安第斯基因库;Ⅱ组的遗传多样性最低,为直立无限生长习性的小粒资源,属于中美基因库;Ⅲ组的遗传多样性最高,主要以蔓生无限为主,包括小部分直立无限和匍匐无限的资源;Ⅳ组的遗传多样性较低,为蔓生无限生长习性、株高最高、分枝数最少的资源。筛选到大粒、多荚、长荚、宽荚等具有特异性状的种质资源35份。     

Effect of Salt Stress on Carbon Metabolism and Bacteroid Respiration in Root Nodules of Common Bean (Phaseolus vulgaris L.)

Abstract: In the present work, we examined the effect of salinity on growth, N fixation and carbon metabolism in the nodule cytosol and bacteroids of Phaseolus vulgaris, and measured the O₂ consumption by bacteroids incubated with or without the addition of exogenous respiratory substrates. The aim was to ascertain whether the compounds that accumulate under salt stress can increase bacteroid respiration and whether this capacity changes in response to salinity in root nodules of Phaseolus vulgaris. The plants were grown in a controlled environment chamber, and 50, 100 mM or no NaCl (control) was added to the nutrient solution. Two harvests were made, at the vegetative growth period and at the beginning of the reproductive period. The enzyme activities in the nodule cytosol were reduced by the salt treatments, while in the bacteroid cytosol the enzyme activities increased at high salt concentrations at the first harvest and for ADH in all treatments. The data presented here confirm that succinate and malate are the preferred substrates for bacteroid respiration in common bean, but these bacteroids may also utilize glucose, either in control or under saline conditions. The addition of proline or lactate to the incubation medium significantly raised oxygen consumption in the bacteroids isolated from plants treated with salt.     

Genetic Analysis of Turkey Common Bean (Phaseolus vulgaris L.) Genotypes by Simple Sequence Repeats Markers

Russian Journal of Genetics - Common bean (Phaseolus vulgaris L.), besides being an agricultural product that can be consumed as fresh vegetable, is a significant legume widely being planted in...     

Semienclosed Tube Cultures of Bean Plants (Phaseolus vulgaris L.) for Enumeration of Rhizobium phaseoli by the Most-Probable-Number Technique

The semienclosed tube culture technique of Gibson was modified to permit growth of common bean (Phaseolus vulgaris L.) roots in humid air, enabling enumeration of the homologous (nodule forming) symbiont, Rhizobium phaseoli, by the most-probable-number plant infection method. A bean genotype with improved nodulation characteristics was used as the plant host. This method of enumeration was accurate when tubes were scored 3 weeks after inoculation with several R. phaseoli strains diluted from aqueous suspensions, peat-based inoculants, or soil. A comparison of population sizes obtained by most-probable-number tube cultures and plate counts indicated that 1 to 3 viable cells of R. phaseoli were a sufficient inoculant to induce nodule formation.     

Thigmotropism in Organs of the Bean Plant (Phaseolus vulgaris L.)

When each vegetative aerial organ of the young bean plant isunilaterally mechanically perturbed by gentle rubbing, it exhibitspositive thigmotropism. The internodes undergo only moderatethigmotropism, but leaves and petioles bend much more, especiallywhen they are rubbed on their abaxial surfaces. When the abaxialsurface of one petiole is rubbed, it bends down, but the oppositepetiole bends upward at the stem-petiole pulvinus. These observationsare interpreted in terms of the vining habit of bean plants. Bean, Phaseolus vulgaris, thigmotropism, mechanical perturbation     

普通菜豆种质资源遗传多样性研究进展

普通菜豆于15世纪从美洲直接引入中国,作为重要的食用豆类作物之一,在我国的许多地区广泛种植。本文对普通菜豆的起源、驯化、传播以及遗传多样性研究等方面的进展进行综述,并根据普通菜豆育种和生产中存在的问题及今后的研究方向提出一些建议,旨在为普通菜豆的种质收集保存以及合理利用提供参考。     

On Vessel Member Differentiation in the Bean (Phaseolus vulgaris L.)

Certain ultrastructural features of vessel member differentiationwere examined in the primary xylem of petiole of bean (Phaseolusvulgaris L.). The cells used had helical secondary wall thickeningsand simple perforation plates. The primary cell wall increasesin thickness before the helices of secondary wall develop. Ina common wall between two vessel members of different ages,theprimary thickening occurs first in the older cell so thatfor a time the middle lamella is located closer to the youngercell rather than medianly. Apparently the helix is depositedafter the primary wall of a given cell reaches maximum thickness.The perforation of the end wall is preceded by primary thickeningof the part of the wall that is later removed. The marginalregion remains relatively thin and becomes covered with a rimof secondary wall. Vesicles with fibrous content appear nearthe surface within the end wall shortly before the perforationoccurs. A highly vacuolated protoplast with a much enlargednucleus and numerous organdIes is present during cell wall differentiation.After that process is completed, the protoplast disintegratesand the primary wall bearing the helix is hydrolysed where itis exposed to the cell lumen and, under certain conditions,also under the secondary wall.     

Ontogenetic Changes in Root Nodule Subpopulations of Common Bean (Phaseolus vulgaris L.): II. PROTEIN CONTENT AND CARBOHYDRATE POOLS

Root nodules of common bean (Phaseolus vulgaris L. GN 1140)show different ontogenetic patterns of nitrogen fixation indifferent parts of the root system. The objective of the presentstudy was to search for nodule physiological factors which couldexplain these differences. Plants were grown to maturity undercontrolled environment conditions. Root nodules were regularlyharvested from two zones of the root system, and assayed forsoluble cytosolic content and carbohydrate status. Nodule concentrationsof soluble carbohydrates, glucose, fructose or sucrose, didnot change significantly during the pod-filling decline in nitrogenaseactivity. Nodule concentrations of sucrose appeared more relatedto nodule ontogeny than to the ontogenetic stage of the shoot.Total soluble protein and leghaemoglobin concentrations werehighly correlated to nitrogenase activity. In the top zone ofthe root system proteolytic capacity increased during pod-filling,indicating a role in nodule senescence. Proteolytic capacityalso increased in the mid zone, but to a lesser extent, andwithout correlation to nitrogenase activity. The two nodulesubpopulations showed different nitrogenase activity in relationto their leghaemoglobin and carbohydrate status. We suggestthat physiological differences within the root nodule subpopulations,possibly in combination with the distance from the shoot, makesthe top zone more susceptible to a systemic increase in proteolyticcapacity during rapid pod-filling. Key words: Nitrogen fixation, protein, carbohydrate, ontogeny, Phaseolus vulgaris     

BAC-end Sequence Analysis and a Draft Physical Map of the Common Bean (Phaseolus vulgaris L.) Genome

Common bean (Phaseolus vulgaris L.) is a legume that is an important source of dietary protein in developing countries throughout the world. Utilizing the G19833 BAC library for P. vulgaris from Clemson University, 89,017 BAC-end sequences were generated giving 62,588,675 base pairs of genomic sequence covering approximately 9.54% of the genome. Analysis of these sequences in combination with 1,404 shotgun sequences from the cultivar Bat7 revealed that approximately 49.2% of the genome contains repetitive sequence and 29.3% is genic. Compared to other legume BAC-end sequencing projects, it appears that P. vulgaris has higher predicted levels of repetitive sequence, but this may be due to a more intense identification strategy combining both similarity-based matches as well as de novo identification of repeats. In addition, fingerprints for 41,717 BACs were obtained and assembled into a draft physical map consisting of 1,183 clone contigs and 6,385 singletons with ~9x coverage of the genome.     

Genome-Wide Association Studies of Anthracnose and Angular Leaf Spot Resistance in Common Bean (Phaseolus vulgaris L.)

The common bean (Phaseolus vulgaris L.) is the world’s most important legume for human consumption. Anthracnose (ANT; Colletotrichum lindemuthianum) and angular leaf spot (ALS; Pseudocercospora griseola) are complex diseases that cause major yield losses in common bean. Depending on the cultivar and environmental conditions, anthracnose and angular leaf spot infections can reduce crop yield drastically. This study aimed to estimate linkage disequilibrium levels and identify quantitative resistance loci (QRL) controlling resistance to both ANT and ALS diseases of 180 accessions of common bean using genome-wide association analysis. A randomized complete block design with four replicates was performed for the ANT and ALS experiments, with four plants per genotype in each replicate. Association mapping analyses were performed for ANT and ALS using a mixed linear model approach implemented in TASSEL. A total of 17 and 11 significant statistically associations involving SSRs were detected for ANT and ALS resistance loci, respectively. Using SNPs, 21 and 17 significant statistically associations were obtained for ANT and angular ALS, respectively, providing more associations with this marker. The SSR-IAC167 and PvM95 markers, both located on chromosome Pv03, and the SNP scaffold00021_89379, were associated with both diseases. The other markers were distributed across the entire common bean genome, with chromosomes Pv03 and Pv08 showing the greatest number of loci associated with ANT resistance. The chromosome Pv04 was the most saturated one, with six markers associated with ALS resistance. The telomeric region of this chromosome showed four markers located between approximately 2.5 Mb and 4.4 Mb. Our results demonstrate the great potential of genome-wide association studies to identify QRLs related to ANT and ALS in common bean. The results indicate a quantitative and complex inheritance pattern for both diseases in common bean. Our findings will contribute to more effective screening of elite germplasm to find resistance alleles for marker-assisted selection in breeding programs.     

Cell-Wall Proteins Induced by Water Deficit in Bean (Phaseolus vulgaris L.) Seedlings

In the last few years, much attention has been given to the role of proteins that accumulate during water deficit. In this work, we analyzed the electrophoretic patterns of basic protein extracts, enriched for a number of cell-wall proteins, from bean (Phaseolus vulgaris L.) seedlings and 21-d-old plants subjected to water deficit. Three major basic proteins accumulated in bean seedlings exposed to low water potentials, with apparent molecular masses of 36, 33, and 22 kD, which we refer to as p36, p33, and p22, respectively. Leaves and roots of 21-d-old plants grown under low-water-availability conditions accumulated only p36 and p33 proteins. In 21-d-old plants subjected to a fast rate of water loss, both p33 and p36 accumulated to approximately the same levels, whereas if the plants were subjected to a gradual loss of water, p33 accumulated to higher levels. Both p36 and p33 were glycosylated and were found in the cell-wall fraction. In contrast, p22 was not glycosylated and was found in the soluble fraction. The accumulation of these proteins was also induced by abscisic acid (0.1-1.0 mM) treatment but not by wounding or by jasmonate treatment.     

Ontogenetic Changes in Root Nodule Subpopulations of Common Bean (Phaseolus vulgaris L.): I. NITROGENASE ACTIVITY AND RESPIRATION DURING POD-FILLING

Nitrogenase activity is commonly measured on a whole plant basis,or only on parts of the root system. In the present paper, activityin different root nodule subpopulations was followed throughoutreproductive growth, in order to characterize the pod-fillingdecline in nitrogenase activity. Inoculated common bean plantswere grown to maturity under controlled environment conditions.Nitrogenase activity (H₂ evolution in air) and nodule respiration(CO₂ evolution) were measured in three separate zones of theroot system with a non-destructive, open flow, gas-exchangesystem. Nitrogenase activity in the top zone drastically declinedat the initiation of pod-filling, whereas nitrogenase activityin the mid zone was stable during the same period. Hence, thepod-filling decline was limited to a certain nodule subpopulationand not of a systemic type. Nodule respiration showed a similar,but less pronounced pattern. The sharp decline in nitrogenaseactivity was not paralleled in nitrogenase specific activity.Nitrogenase activity is not likely to be limited by the availabilityof oxygen or carbohydrates at the onset of pod-filling becausespecific nodule respiration did not change significantly atthis time. In the top zone, nitrogenase specific activity declinedgradually throughout the measurements, whereas in the mid-partof the root system specific activity peaked and gradually declined2-4 weeks later. The dissimilarities between specific and totalnitrogenase activity were explained by differences in nodulegrowth rates. The data suggest that the oldest nodule populationloses activity at the onset of pod-filling. At the same time,nodules grow and nitrogenase activity increases in younger distalparts of the root system. Estimating total nitrogen fixationin this symbiosis by partial sampling of nodulated root systemsis likely to be very misleading. Key words: Nitrogen fixation, respiration, pod-filling decline, Phaseolus vulgaris, ontogeny