Growth and nitrogen assimilation in nodules in response to nitrate levels in Vicia faba under salt stress

This study analyses the effects of salt on the effective symbiosisof faba bean (Vicia faba L. var. minor cv. Alborea) and salt-tolerantRhizobium leguminosarum biovar. viciae strain GRA19 grown withtwo KNO₃ levels (2 and 8 mM). The addition of 8 mM KNO₃ to thegrowth medium increases plant tolerance to salinity even witha concentration of 100 mM NaCl. This KNO₃ level in control plantsreduced the N₂ fixation. For 2 and 8 mM KNO₃ the plants treatedwith NaCl reduced N₂ fixation to identical values. The activityof the enzymes mediating ammonium assimilation in nodules (GS,NADH-GOGAT and NADH-GDH) was decreased by high KNO₃ levels.The results show that NADH-GOGAT activity was more markedlyinhibited than was GS activity by salinity, therefore NADH-GOGATlimits the ammonium assimilation by nodules in V. faba undersalt stress. The total proline content in the nodule was notrelated to salt tolerance and thus does not serve as a salttoleranceindex for V. faba. Key words: Glutamate synthase, glutamine synthetase, N₂ fixation, nitrate, salinity     

Influence of host genotypes on growth,symbiotic performance and nitrogen assimilation in faba bean (Vicia faba L.) under salt stress

Fifteen genotypes of faba bean (Vicia faba L.) were inoculated with salt-tolerant Rhizobium leguminosarum biovar. viciae strain GRA 19 in solution culture with 0 (control) and 75 mM NaCl added immediately after transplanting. Genotypes varied in their tolerance of high levels of NaCl. Physiological parameters (dry weight of shoot and root, number and dry weight of nodules) were not affected by salinity in lines VF46, VF64 and VF112. Faba bean line VF60 was sensitive to salt stress. Host tolearance appeared to be a major requisite for nodulation and N₂ fixation under salt stress. Tolerant line VF112 sustained nitrogen fixation under saline conditions. Activity of the ammonium assimilation enzymes glutamine synthetase and glutamate synthase, and soluble protein content, were reduced by salinity in all genotypes tested. Evidence presented here suggests a need to select faba bean genotypes that are tolerant to salt stress.Abbreviations ARA acetylene reduction activity - NADH-GOGAT NADH-dependent glutamate synthase - GS glutamine synthetase     

The Effects of Exogenous Amino Acid on Acetylene Reduction Activity of Vicia faba L. cv. Fiord

The feed back control mechanism proposed to explain the inhibitionof N₂ fixation by N was investigated using Vicia faba cv. Fiord.Plants were grown under controlled conditions without mineralN in coarse river sand. Asparagine was supplied to plants activelyfixing N₂ by absorption through cut roots and via a wick ordirect injection into the stem just above the bottom leaf. Responsesin N₂ fixation were measured by acetylene reduction (AR). Feedingplants with [¹⁴C]-labelled asparagine showed that the amidewas taken up when exogenously applied. Asparagine (10 mM) suppliedby the above procedures resulted in a 50-70% inhibition of ARby 48 h. Glutamine produced a similar effect. The cut root methodallowed higher levels of these amides to be supplied but theinhibition observed with 10 mM asparagine was only increasedslightly with higher levels of the amide. The antibiotic Securopenprevented bacterial contamination of root solutions of asparagineand glutamine and had no effect on nodule activity. It is concludedthat accumulation of asparagine of glutamine or the resultantincrease in the pool of soluble N in the plant cause a feedbackeffect on the activity of nitrogenase.Copyright 1993, 1999 AcademicPress Vicia faba, faba bean, asparagine, inhibition of N₂ fixation     

Bacteroid oxalate oxidase and soluble oxalate in nodules of faba beans (Vicia faba L.) submitted to water restricted conditions: possible involvement in nitrogen fixation

The inhibitory effect exerted by water stress on acetylene reductionactivity (ARA) by nodulated roots of faba beans (Vicia fabaL.) was correlated with a 40% decline in the organic acid poolof nodule cytosol. Oxalate concentration was lowered (–55%)whereas a stimulation of the bacteroid oxalate oxidase concomitantlyoccurred. This enzyme was characterized by an optimal activityat pH 8 but, as in higher plants, exhibited a K_m for oxalateof 1.4 mM and an inhibition by substrate excess. Oxalate providedto bacteroid incubations supported C₂H₂ reduction up to 2.5mM whereas higher concentrations were strongly inhibitory. Incontrast, purified symbiosomes incubated with oxyleghaemoglobinreduced C₂H₂ in the presence of oxalate concentrations up to10 mM. The peribacteroid membrane (PBM), in controlling theoxalate flux to the bacteroids avoided the substrate inhibitionwhich would limit its efficiency. Thus, oxalate present in highconcentration in faba bean nodules could play a role as complementarysubstrate for bacteroids slowing down the nitrogen fixationdecline induced by water restricted conditions. Key words: Faba bean, water stress, oxalate, acetylene reduction, bacteroid     

Use of the Expolinear Growth Model to Analyse the Growth of Faba bean, Peas and Lentils at Three Densities: Fitting the Model

The expolinear equation for crop growth (Goudriaan and MonteithAnnalsof Botany66: 695–701, 1990) was fitted to measurementsof above ground dry weight made on two cultivars of each ofthree species, faba bean (Vicia fabaL.), peas (Pisum sativumL.)and lentils (Lens culinarsMedic.), each grown at three densitiesat the University of Reading, UK in 1992 and 1993. The expolinearequation fitted the data well but required frequent samplingto obtain good estimates of the parameters. The equation hasthree parameters,R_mthe maximum relative growth rate,C_ma maximumcrop growth rate, andt_bthe time at which the crop effectivelyreaches a linear phase of growth.R_mdid not differ between densities,cultivars or species but differed between years.C_mincreasedwith increased density and was lower for lentils than for fababeans or peas.t_bdecreased with increased density for faba beanbut not for the other species. Incorporating an extinction coefficientfor solar radiation and the maximum fraction of radiation interceptedenabled reasonably accurate time courses of leaf area indexto be derived, as suggested by Goudriaan (1994. In: MontiethJL, Scott RK, Unsworth MH, eds.Resource capture by crops. Nottingham:Nottingham University Press, 99–110).Copyright 1998 Annalsof Botany Company Expolinear equation, grain legumes, crop growth rate, crop density, relative growth rate, growth modelling, faba bean,Vicia fabaL., peas,Pisum sativumL., lentils,Lens culinarsMedic.     

The Effect of the Accumulation of Carbohydrate and Organic Nitrogen on Nitrogen Fixation (Acetylene Reduction) of Faba Bean cv. Fiord

Plants of faba bean cv. Fiord were grown under controlled conditions,without mineral N, in coarse river sand. Twenty-five days aftersowing when plants had at least eight fully opened leaves andwere nodulated and actively fixing N₂, half were topped andkept debudded for 21 d. Changes in dry weight, N₂ fixation (acetylenereduction activity), soluble carbohydrate, starch, soluble Nand total N in plants were monitored over the period. Both debudded and control plants grew and accumulated dry matter.Debudding resulted in a significant increase in the concentrationof soluble carbohydrate, starch and soluble N. but had onlya small effect on the total N concentration. A strong positivelinear relation between total plant weight and N content ofboth control and debudded plants showed that even under conditionsof excess supply of carbohydrate, faba beans have little capacityto store N. Soluble N accumulated in debudded plants presumablybecause less N was needed for the formation of new tissues thanin control plants. AR continued to increase throughout the experimentin control plants but declined in debudded plants from 6 to13 d after debudding and remained low until the end of the experiment.The decline was associated with an increase in available carbohydrateand in soluble N. The results of this experiment are consistentwith a feed back control of N₂ fixation by the soluble poolof N.Copyright 1994, 1999 Academic Press Vicia faba, faba bean, debudding, soluble N, inhibition of N₂ fixation     

The Distribution of Nitrate Assimilation Between Root and Shoot in Vicia faba L.

Nitrate assimilation was examined in two cultivars (Banner Winterand Herz Freya) of Vicia faba L. supplied with a range of nitrateconcentrations. The distribution between root and shoot wasassessed. The cultivars showed responses to increased applied nitrateconcentration. Total plant dry weight and carbon content remainedconstant while shoot: root dry weight ratio, total plant nitrogen,total plant leaf area and specific leaf area (SLA) all increased.The proportion of total plant nitrate and nitrate reductase(NR) activity found in the shoot of both cultivars increasedwith applied nitrate concentrations as did NO₃^–: Kjeldahl-Nratios of xylem sap. The cultivars differed in that a greaterproportion of total plant NR activity occurred in the shootof cv. Herz Freya at all applied nitrate concentrations, andits xylem sap NO₃^–: Kjeldahl-N ratio and SLA were consistentlygreater. It is concluded that the distribution of nitrate assimilationbetween root and shoot of V. faba varies both with cultivarand with external nitrate concentration. Vicia faba L., field bean, nitrate assimilation, nitrate reductase, xylem sap analysis     

Nodulation and Nitrogen Fixation of Faba Bean (Vicia faba L.) as Affected by Removal of the Cotyledons and Nitrate Supply

Cotyledons of faba bean (^{Vicia faba} L. cv. Fiord) were removedto determine whether an apparent delay in nodulation of thiscultivar could be attributed to an inhibitor from these organs.Cotyledons were left intact or excised from seedling plants14 and 18 d after sowing and plants grown with or without 2·5mm NO₃^–. Seedling growth was depressed when cotyledons were removed onday 14 but not when removed on day 18. Removal of the cotyledonsat day 14 reduced nodule number and nodule weight in the absenceof NO₃^–, but in the presence of NO₂^–, nodule numberwas unaffected and only nodule weight was reduced. Cotyledonremoval at day 18 increased both nodule number and nodule weightwith +NO₃^– but not with –NO₂^–. Acetylene reduction(AR) was markedly depressed by NO₃^–. Cotyledon removalat day 14 decreased AR but removal at day 18 resulted in anincrease in AR. We suggest from these results that faba beancotyledons have an inhibitory effect on nodule activity andon nodulation and this interacts with NO₃^–. This can beexplained through a ‘feed-back’ regulation of N₂fixation by soluble N in the seedling. Vicia faba, faba bean, nodule number, nodulation, nodule activity, acetylene reduction, N₂ fixation, cotyledon removal, nitrate     

Carbon and Nitrogen Assimilation by Vicia faba L. at Low Temperature: the Importance of Concentration and Form of Applied-N

Low temperature (6 C) growth was examined in two cultivarsof Vicia faba L. supplied with 4 and 20 mol m^–3 N as nitrateor urea. Both cultivars showed similar growth responses to increasedapplied-N concentration regardless of N-form. Total leaf areaincreased, as did root, stem and leaf dry weight, total carboncontent and total nitrogen content. In contrast to findingsat higher growth temperatures, 20 mol m^–3 urea-N gavesubstantially greater growth (all parameters measured) than20 mol m^–3 nitrate-N. The increased carbon content per plant associated with increasedapplied nitrate or urea concentration, or with urea in comparisonto nitrate, was due to a greater leaf area per plant for CO₂uptake and not an increased CO₂, uptake per unit area, carbon,chlorophyll or dry weight, all of which either remained constantor decreased. Nitrate reductase activity was substantial inplants given nitrate but negligible in plants given urea. Neitherfree nitrate nor free urea contributed greatly to nitrogen levelsin plant tissues. It is concluded that there is no evidence for a restrictionin nitrate reduction at 6 C, and it is likely that urea givesgreater growth than nitrate because of greater rates of uptake. Vicia faba, broad bean, low temperature growth, carbon assimilation, nitrogen assimilation     

Phenotypic and genetic diversity of Moroccan rhizobia isolated from Vicia faba and study of genes that are likely to be involved in their osmotolerance

Rhizobia are symbiotic nitrogen-fixing bacteria in root nodules of legumes. In Morocco, faba bean (Vicia faba L.), which is the main legume crop cultivated in the country, is often grown in marginal soils of arid and semi-arid regions. This study examines the phenotypic diversity of rhizobia nodulating V. faba isolated from different regions in Morocco for tolerance to some abiotic stresses. A total of 106 rhizobia strains isolated from nodules were identified at the species level by analysing 16S rDNA. Additionally, for selected strains recA, otsA, kup and nodA fragments were sequenced. 102 isolates are likely to belong to Rhizobium leguminosarum or R. laguerreae and 4 isolates to Ensifer meliloti. All strains tolerating salt concentrations of 428 or 342 mM NaCl as well as 127 or 99 mM Na2SO4 were highly resistant to alkaline conditions (pH 10) and high temperature (44 °C). Three strains: RhOF4 and RhOF53 (both are salt-tolerant) and RhOF6 (salt-sensitive) were selected to compare the influence of different levels of salt stress induced by NaCl on growth and on trehalose and potassium accumulation. We find a direct correlation between the trehalose contents of the rhizobial strains and their osmotolerance.     

Effects of NaCl on growth and nitrogen fixation and assimilation of inoculated and KNO3 fertilized Vicia faba L. and Pisum sativum L. plants

Faba bean (Vicia faba L. var. minor cv. Alborea) and pea (Pisum sativum L. cv. Lincoln) plants, inoculated with Rhizobium leguminosarum biovar. viciae strain GRA19, were treated with salt (100 mM NaCl) and/or nitrate (8 mM KNO₃) to test whether plants grown with inorganic-nitrogen are more tolerant to salinity than plants entirely reliant upon fixed nitrogen. According to the growth inhibition recorded, pea plants dependent on dinitrogen fixation proved more tolerant to salt stress than those N-fertilized, in contrast to results obtained for faba bean plants. This study therefore confirms that plants dependent on nitrogen fixation are not always more sensitive to salinity than are N-fertilized plants. Nitrate addition did not reduce the specific nitrogenase activity in pea, but did in faba bean. However, nodulation was inhibited in both legumes. The specific nitrogenase activity was more affected by salt treatment in N-fertilized plants for both legumes. The activity of the enzymes mediating ammonium assimilation in nodules (GS, NADH-GOGAT) was inhibited by salt stress both in N-fixing and in N-fertilized pea and faba bean plants.     

Transport of Nitrate and Calcium into Legume Root Nodules

Nitrate transport into nodulated plants of soybean (Glycinemax), cowpea (Vigna unguiculata) and faba bean (Vicia faba)was investigated. Nitrate entering the root system of soybeandid not pass out of the vascular system into nodular tissuesin detectable quantities. On the other hand, nitrate could passfrom soil through the outer surface of nodules but did not penetratethe infected tissue. Similarly, nitrate was restricted to corticaltissues of cowpea and faba bean. Thus, nitrate cannot inhibitnitrogen fixation as a result of reduction to nitrite by nitratereductase within the bacteroid zone. These results are, however,consistent with an effect of nitrate on an oxygen diffusionresistance located in the nodule cortex. Unlike nitrate, measurable quantities of ⁴⁵calcium were transportedvia the xylem into infected and cortical tissues of soybeannodules: it also passed from the soil into the free space ofthe nodule cortex. Key words: Nitrate, legume nodules, calcium     

Effects of NaCl on Growth, Nitrogen Incorporation and Chemical Composition of Inoculated and NH4NO3 Fertilized Vicia faba (L. ) Plants

Vicia faba cv. Maris Bead was grown either on fixed nitrogenor on ammonium nitrate. After 4 weeks growth, nutrient solutionswere supplemented with 50, 75 and 100 mol m^–3 NaCl for15 d. Five harvests were made at weekly intervals, beginningat 4 weeks. Effects of salinity were directly related to dose,plant growth (fresh and dry weight) being depressed in bothN-fixing and N-fertilized plants. The number of nodules perplant and the proportion of those formed which developed intothe active nitrogen fixing state were depressed by salt stress.Increased size of nodules in salt-stressed plants only partlycompensated for the lower specific nitrogenase activity. Theeffects of salinity on plant nitrogen content were more pronouncedon N-flxing than on N-fertilized plants. The former took upmore Na⁺ and Cl^– than the latter: the implications ofthis and of ionic imbalance are discussed. Key words: Vicia faba, Growth, Salt stress, Nodulation     

Effects of salt stress on nitrogenase activity and growth of four legumes

The effects of salt stress on nitrogenase (N2-ase) activity, growth and nitrogen content ofVicia faba (L.),Medicago sativa (L.) Merrill,Glycine max andVigna sinensis (L.) were investigated. Four levels of salinity were applied and salt treatments were imposed on inoculated and N-fertilized plants.M. sativa tolerated mild levels of salinity but higher salt concentrations depressed N₂-ase activity of this species. The other three legumes were considerably affected by salt treatments, and N₂-ase activity was significantly reduced by salinity. Vicia faba, carrying elongate nodules, could restore a partial N₂-ase activity upon recovery from salt stress whereasG. max andV. sinesis, both with spherical nodules, could not regain significant activity when salinity was removed. Salt stress retarded growth of both inoculated and N-fertilized plants. The nitrogen content of both treatments was also affected by salinity and the effect was more severe for inoculated than N-fertilized plants.     

The Development of Primary Root Nodules on Vicia faba L. Grown at Two Temperatures

The development of primary root nodules on the field bean (Viciafaba L.) grown at 10 and 18 °C was examined. The sequenceof anatomical changes observed was the same in both temperatureregimes. Nodules developed much more slowly at 10 °C andthe nodules were much larger when corresponding anatomical changesoccurred. Primary root nodules eventually ceased growth in bothtemperature regimes but ultimate nodule volume was nearly twiceas great as 10 °C as at 18 °C. The larger size did notcompensate for the lower specific nitrogenase activity of cold-grownnodules: nitrogen fixation (acetylene reduction) rates on awhole plant basis were much lower at 10 °C than at 18 °C.There was no difference in the total number of primary rootnodules in the two temperature regimes but their distributionwas biased towards the upper part of the root and the epicotylat 18 °C. Vicia faba L., field bean, nodulation, nitrogen fixation, temperature     

A Mutation in Vicia faba Results in Ineffective Nodules with Impaired Bacteroid Differentiation and Reduced Synthesis of Late Nodulins

Although numerous reports have documented the effect of bacterially-inducedineffectiveness on root nodule structure, function, and plantgene expression, few studies have detailed the effect of theplant genome on similar parameters. In this report effective(N₂-fixing) broadbean {Vicia faba L.) and plant-controlled ineffective(non-N₂-fixing) broadbean recessive for the sym-1 gene werecompared for nodule structure, developmental expression of noduleenzyme activities, enzyme proteins, and mRNAs involved in Nassimilation, leghemoglobin (Lb) synthesis, and acetylene reductionactivity (ARA). During development of effective wild-type nodules,glutamine synthetase (GS), aspartate aminotransferase (AAT),phosphoenolpyruvate carboxylase (PEPC) and NADH-glutamate synthase(GOGAT) activities and enzyme proteins increased coincidentwith nodule ARA. The increases in GS, AAT, and PEPC were associatedwith increased synthesis of mRNAs for these proteins. Synthesisof Lb polypeptides and mRNAs during development of effectivenodules was similar to that of GS, AAT, and PEPC. By contrast,ineffective sym-1 nodules displayed little or no ARA and hadneither the increases in enzyme activities nor enzyme proteinsand mRNAs as seen for effective nodules. The effect of the sym-1gene appeared to occur late in nodule development at eitherthe stage of bacterial release from infection threads or differentiationof bacteria into bacteroids. High in vitro enzyme activities,enzyme polypeptides, and mRNA levels in parental effective noduleswere dependent upon a signal associated with effective bacteroidsthat was lacking in sym-1 nodules. Nodule organogenesis didnot appear to be a signal for the induction of GS, PEPC, AAT,and Lb expression in sym-1 nodules. Key words: Vicia faba, mutation, sym-1 gene, nodules     

Carbon Dioxide Fixation by Guard Cell Protoplasts of Commelina communis

Biochemical studies of epidermal tissue may not reflect metabolismof the guard cells which represent less than 5% of the tissuevolume. Pure samples of guard cell protoplasts of Commelinacommunis were therefore used to investigate CO₂ fixation ratesand ¹⁴C-labelling patterns of metabolites in the light and thedark. Qualitatively, results were similar in most respects tothose obtained in a previous study (Schnabl, 1980) for guardcell protoplasts of Vicia faba. CO₂ fixation rates by guardcell protoplasts of C. communis were the same in the light andthe dark but about 50 times lower than the values Schnabl obtainedfor V.faba. The ¹⁴C-labelling pattern of metabolites in C. communiswas also similar in the light and the dark: over 60% of thetotal fixed was in malate with only 1% in sugar phosphates.Label was also detected in starch, aspartate, glutamate andcitrate but not in glycollate as previously recorded in V. fabaguard cell protoplasts. The results confirm the view that the reductive pentose phosphatepathway does not occur in guard cells of C. communis. Key words: CO₂ fixation, Guard cell protoplasts, Stomata     

Ammonia assimilation by rhizobium cultures and bacteroids.

The enzymes involved in the assimilation of ammonia by free-living cultures of Rhizobium spp. are glutamine synthetase (EC. 6.o.I.2), glutamate synthase (L-glutamine:2-oxoglutarate amino transferase) and glutamate dehydrogenase (ED I.4.I.4). Under conditions of ammonia or nitrate limitation in a chemostat the assimilation of ammonia by cultures of R. leguminosarum, R. trifolii and R. japonicum proceeded via glutamine synthetase and glutamate synthase. Under glucose limitation and with an excess of inorganic nitrogen, ammonia was assimilated via glutamate dehydrogenase, neither glutamine synthetase nor glutamate synthase activities being detected in extracts. The coenzyme specificity of glutamate synthase varied according to species, being linked to NADP for the fast-growing R. leguminosarum, R. melitoti, R. phaseoli and R. trifolii but to NAD for the slow-growing R. japonicum and R. lupini. Glutamine synthetase, glutamate synthase and glutamate dehydrogenase activities were assayed in sonicated bacteroid preparations and in the nodule supernatants of Glycine max, Vicia faba, Pisum sativum, Lupinus luteus, Medicago sativa, Phaseolus coccineus and P. vulgaris nodules. All bacteroid preparations, except those from M. sativa and P. coccineus, contained glutamate synthase but substantial activities were found only in Glycine max and Lupinus luteus. The glutamine synthetase activities of bacteroids were low, although high activities were found in all the nodule supernatants. Glutamate dehydrogenase activity was present in all bacteroid samples examined. There was no evidence for the operation of the glutamine synthetase/glutamate synthase system in ammonia assimilation in root nodules, suggesting that ammonia produced by nitrogen fixation in the bacteroid is assimilated by enzymes of the plant system.     

Studies on Glutamate Synthase from the Leaves of Higher Plants

Ferredoxin-dependent glutamate synthase has been isolated andpurified 170-fold from field bean (Vicia faba cv. Minor) leaves.The enzyme has a molecular weight of 145 000 and does not appearto contain non-haem iron. The K^m values of the enzyme for glutamine,2-oxoglutarate, and ferredoxin are 330, 150, and 2 µMrespectively. The specificity of the enzyme is extremely highand no substitutes for glutamine have been found. Azaserineis a potent inhibitor of glutamate synthase. Chloroplasts from four species of plants have been shown tocarry out a light-dependent glutamate synthesis from glutamine.This reaction is also highly specific for glutamine and purifiedasparagine cannot substitute for glutamine.     

A Comparison of the Rate of Maintenance Respiration in Some Crop Legumes and Tobacco Determined by Three Methods

Carbon exchange was measured on whole plants of field bean,lucerne, chick pea, kidney bean, pea and tobacco. The maintenance respiration rate was measured in three ways:(i) by allowing the CO₂ efflux to decay in prolonged darknessto an asymptotic value which was then taken to be the maintenancevalue (the dark decay method); (ii) by plotting the dark CO₂efflux as a function of the net CO₂ uptake over a range of irradiancesand taking maintenance as the dark CO₂ efflux when the net CO₂uptake was zero (the dynamic method); and (iii) by plottingthe total CO₂ uptake as a function of the growth rate and takingmaintenance respiration as the CO₂ efflux when the growth ratewas zero (the zero growth rate method). The range of valuesfor the maintenance coefficient over all species was from 1.6to 2.1 per cent of the dry weight per day, 1.8 to 2.1 per centand 2.7 to 2.9 per cent as determined by these three methodsrespectively. There was a linear relationship, common to allspecies, between the maintenance respiration rate (dark decaymethod) and dry weight, total nitrogen and the organic nitrogencontent. The growth coefficient (0.69±0.01) was the samefor field bean, chick pea and lucerne and was unaffected bythe method of estimation. It was concluded that the dark decay method provided the bestestimate of the minimal maintenance requirements in the plantsstudied. Vicia faba L., Medicago sativa L., Cicer arientinum L., Phaseolus vulgaris L., Pisum sativum L., Nicotiana tobacum L., field bean, lucerne, chick pea, kidney bean, pea, tobacco, respiration, maintenance, growth, nitrogen content