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     

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 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     

The Effect of Irradiance on the Recovery of Soybean Nodules from Sodium Chloride-Induced Senescence

Soybean (Glycine max L. Merr) cv. Clarke plants inoculated withBradyrhizobium japonicum strain RCR3407 were grown either ina greenhouse with a low irradiance (200–400)µmolm^–2 s^–1) or in a controlled-environment growth cabinetwith a higher irradiance (600 µimol m^–2 s^–1).At 42 d plants were given a nitrogen-free nutrient solutioncontaining 50 mol m^–3 sodium chloride for 2 weeks andthen allowed to recover from salt-stress for a further 2 weeks. Salt treatment reduced plant growth by at least half in bothgrowth regimes, however, the controlled environment-grown (CEG)plants were five times larger than the greenhouse-grown (GG)plants in terms of dry weight and number/weight of nodules perplant, regardless of treatment. The structure of nodules, from both growth regimes, harvestedat the end of the 2 week salt-stress was similar to unstressedcontrol nodules. However, nodules harvested 1 week later fromboth CEG and GG plants had structural changes including degradationof bacteria in vacuoles around host cell nuclei, particularlyin the outer cell layers of the infected tissue. In addition,meristematic activity was seen in the cortex of some nodulesfrom GG plants. Young cells here contained infection threadsand newly-released bacteria. Nodules harvested 2 weeks after removal of the salt-stress fromCEG plants showed an apparent recovery from the stress. However,there was a very marked increase in the amount of starch inthe cortex which was not seen in equivalent GG nodules. In contrast,nodules from GG plants contained many vacuolate infected cellsand, consequently, a lowered bacteroid population. Further,meristematic activity was seen in a zone concentric to the infectedzone, newly-formed cells contained many large infection threadsand were interspersed with intercellular bacteria. The meristematicactivity increased the relative volume of cortical to infectedcells in these nodules. Growth conditions did not affect control nodule specific nitrogenaseactivity or oxygen diffusion resistance (R) and these parameterswere also not altered in CEG nodules exposed to salt plus the14 d recovery period. However, nitrogenase activity was greatlyreduced, and R increased by more than eight times in equivalentGG nodules exposed to salt plus recovery. It is hypothesized that the gross morphological changes werean attempt to counter salt toxicity and/or oxygen damage underconditions of reduced photosynthate supply to the nodules dueto the poor light levels in the greenhouse. However, soybeannodules supplied with adequate photosynthate were able to withstandand recover from long-term salt-stress with little alterationto their structural integrity. Key words: Soybean, sodium chloride, nitrogen fixation, light intensity, oxygen diffusion resistance     

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     

Some properties of the amine oxidase in Vicia faba seedlings

Growth of the Vicia faba seedling is accompanied by a rapid15-day increase in amine oxidase activity of the apical parts.Cotyledons and roots were found to be devoid of activity. Thepartially purified enzyme from leaves readily oxidized putrescine,cadaverine, agmatine and spermidine, while dopamine (3-hydroxytyramine)and L- and D-lysine were oxidized more slowly. The Km valueswere 1.9?10^–3 M for cadaverine, 3.7?10^–5 M for putrescine,7.8?10^–4 M for spermidine, and 5.9?10^–3 M for dopamine.Carbonyl reagents and copper-binding agents were effective inhibitorsof Vicia faba amine oxidase. The diethyldithiocarbamate-treatedenzyme could be reactivated specifically by cupric copper. (Received May 25, 1977; )     

Determination of apoplastic K+ in intact leaves by ratio imaging of PBFI fluorescence

The tetraammonium salt of the K⁺ binding fluorescent dye benzofuranisophthalate (PBFI) was used to investigate the influence ofpotassium nutrition (0.1–2.1 mol m^–3) on apoplasticK⁺ inVicia faba leaves by means of ratio imaging. As a referencethe infiltration-centrifugation method was used. Both methodsreflected the influence of K⁺ supply on apoplastic K⁺ concentration.The abaxial leaf side revealed significantly higher K⁺ concentrations(20-25 mol m^–3) than the adaxial side (5–8 mol m^–3).Application of CCCP led to an immediate increase in apoplasticK⁺ demonstrating the reliability of the PBFI method. Key words: Vicia faba, leaf, apoplast, K⁺, PBFI, ratio imaging, ratiometric fluorescence microscopy     

In Vitro Culture and Plant Regeneration in Vicia faba subsp. Equina (var. Spring Blaze)

Explants of stem, leaves, roots, and cotyledons from etiolatedaxenically grown Vicia faba seedlings were cultured on a rangeof media. Shoot organogenesis was only obtained with nodal stemand cotyledonary node explants when cultured on MS medium with3% sucrose, 2.0 mg 1^–1 BAP and 02 mg 1^–1 NAA. Callusproliferation accompanied shoot organogenesis from nodal stemexplants. Successive subculture of nodal stem callus resultedin proliferation of regenerative callus which contained severalshoot bud initials. The capacity for shoot regeneration fromthis callus was maintained for 9 months. Histological studiesreveal de novo formation of meristematic centres in callus andtheir further development into bud primordia. High frequencyrooting of these adventitious shoots was obtained on half-strengthMS medium with 1.5% sucrose, 0.1 mg 1^–1 NAA and 0.5 mg1^–1 kinetin. Key words: Vicia faba, adventitious shoots, axillary shoots, de novomeristem formation, organogenesis, tissue culture     

The Effect of Different Water Regimes on Growth and Nodule Development of Greenhouse-Grown Vicia faba

Plants of Vicia faba cv. Maris Bead were germinated under uniformconditions; some were then transferred to conditions of waterexcess or stress for the vegetative growth phase. Those on theexcess water regime grew best, produced most root nodules, andfixed most nitrogen. Microscopic examination showed that controland water-stressed plants did not develop all those noduleswhich had previously been initiated. Average nodule weight andspecific activity were unaffected, but nodules showed a progressiveincrease in size and a more open structure as water supply wasincreased. Transfer between treatments resulted in adaptationto the new environment within a few days. It is concluded that established plants of V. faba are far morelikely to be adversely affected by water stress than by excesswater.     

The Growth and Photosynthesis of Seedling Plants of White Clover at Low Temperature

The growth of white clover (Trifolium repens L.) in conditionstypical of April in Southern England (8 °C day/4 °Cnight, 12 h photoperiod of 90 J m^–2 s^–1 visibleradiation) was extremely slow, whether the plants were dependentfor nitrogen on fixation by their root nodules or were suppliedwith abundant nitrate; although growth was slower in the nodulatedplants. The reasons for slow growth were a large root: shootratio and a small leaf area, particularly in the nodulated plants,and a low photosynthetic rate in all plants. The probable effectsof these characteristics on the growth of white clover withgrasses in mixed pastures are discussed. Trifolium repens L, white clover, low temperature, leaf area, photosynthetic rate, nitrogen supply, growth     

Translocation of Nitrogenous Compounds in Symbiotic and Nitrate-Fed Amide-Exporting Legumes

Peoples, M. B., Sudin, M. N. and Herridge, D. F. 1987. Translocationof nitrogenous compounds insymbiotic and nitrate-fed amide-exportinglegumes.–J. exp. Bot. 38: 567–579. The transport of nitrogen from the roots and nodules of chickpea(Cicer anetinum L.), lentil (Lens culinaris Medic), faba bean(Vicia faba L.) and pea (Pisum sativum L.) was examined in glasshouse-grownplants supplied either with nitrate-free nutrients or with nutrientssupplemented with 1,2,4 or 8 mol m^-3153N-nitrate. A sixth treatmentcomprised uninoculated plants supplied with 8–0 mol m^-31513N-nitrate. For each species, more than 75% of the nitrogenwas exported from the nodules as the amides, asparagine andglutamine. In fully symbiotic plants, the amides also dominatednitrogen transport to the shoot When N2 fixation activity wasdecreased by the addition of nitrate to the rooting medium,the N-composition of xylem exudate and stem solutes changedconsiderably. The relative concentrations of asparagine tendedto increase in the xylem whilst those of glutamine were reduced;the levels of nitrate increased in both xylem exudate and thesoluble nitrogen pool of the stem with a rise in nitrate supply.The changes in relative nitrate contents reflected generallythe contributions of root and shoot to overall nitrate reductaseactivity at the different levels of nitrate used. The relationshipsbetween the relative contents of xylary or stem nitrate andamino nitrogen and the plants' reliance on N2 fixation (determinedby the ¹⁵N isotope dilution procedure) were examined. Data suggestthat compositional relationships based on nitrate may be reasonableindicators of symbiotic dependence for all species under studyexcept faba bean when greater than 25% of plant nitrogen wasderived from N2 fixation. Key words: Nitrogen, translocation, legumes     

Inhibitory effect of fusicoccin on cell division

Rapid interactions in cell division and cytodifferentiationare induced by hormone treatments in dark-cultured explantsof Jerusalem artichoke. Fusicoccin, at concentrations between10^–6 and 10^–5 M, markedly inhibited the division-promotingactivity induced by plant hormones. Further, fusicoccin-treatedmeristematic root tips of Vicia faba and Allium cepa showeda rapid decrease in the mitotic index. Fusicoccin seems to inhibitsome hormone-sensitive processes required during the inductionand regulation of cell division. (Received March 28, 1979; )     

Mechanism of Photosynthate Efflux from Vicia faba L. Seed Coats

In order to develop a tentative model of the mechanism of photosynthateefflux from the vascular region of Vicia faba L. seed coats,wash-out experiments were performed after removal of the embryo. The sulphydryl group modifiers, pCMBS and NEM, reduced ¹⁴C-photosynthateefflux by 40% and 50%, respectively. Their inhibitory effectcould be prevented or reduced (in the latter case) by includingDTT in the bathing solution. Maltose competed with sucrose forefflux; a concentration of 300 mol m^–3 inhibited ₁₄C-photosynthaterelease by 35%. The cations K⁺ , Na⁺ Mg²⁺ and TPP⁺ enhancedefflux significantly, whereas the countenon Cl^– had noeffect. The presence of the protonophore CCCP (0·1 molm^–3) led to a reduction of efflux by 50% net proton extrusiondropped by 34%. To a lesser extent, an efflux inhibition wasalso achieved by decreasing the cytoplasmic pH with the weakacid DM0. In contrast, alterations in the external pH causedonly a feeble response. The ATPase inhibitor, EB, decreasedphotosynthate efflux and H⁺ extrusion. DES reduced efflux slightly,presumably by affecting ATPase activity as well as energy metabolism. Based on these findings, it is proposed that a sucrose/protonantiport mechanism could be responsible for photosynthate effluxfrom Vicia faba seed coats. Key words: Photosynthate efflux, proton extrusion, proton/sucrose antiport, seed coat, Vicia faba L.     

A System for Measuring Effects of Sulphur Dioxide on Gas Exchange of Plants

Apparatus is described for exposing plants to low concentrationsof SO₂ (50–500µg m^–3 in air) and for measuringeffects on photosynthesis, dark respiration, and transpiration.Temperature, humidity, and irradiance in the chambers were controlledindependently, and fans ensured that leaf boundary layer resistanceswere low. Experiments with plants of Vicia faba in clean andpolluted air showed that: (i) a depression of net photosynthesisby 50 µg m^–3 SO₂ depended on boundary layer resistanceand on irradiance; (ii) stomatal resistance was increased ordecreased by 50 µg m^–3 SO₂ when relative humidityin the chambers was low (35% r.h., 22 °C) or high (50% r.h.,22 °C) respectively.     

Isolation of cDNA for a Plasma Membrane H+-ATPase from Guard Cells of Vicia faba L.

cDNA encoding the plasma membrane H⁺-ATPase of guard cells ofVicia faba L. was isolated. The clone encoded a 105-kDa polypeptide(956 amino acids) that was 79–85% identical in terms ofamino acid sequence to other plant H⁺-ATPases. High levels ofmRNA explain the high H⁺-ATPase activity of these plasma membranes. (Received December 24, 1994; Accepted April 12, 1995)     

Nitrogen Utilization in N-limited Barley During Vegetative and Generative Growth: III. POST-ANTHESIS KINETICS OF NET NITRATE UPTAKE AND THE ROLE OF THE RELATIVE ROOT SIZE IN DETERMINING THE CAPACITY FOR NITRATE ACQUISITION

Barley (Hordeum vulgare L., cvs Golf, Mette, and Laevigatum)was grown under nitrogen limitation in solution culture untilnear maturity. Three different nitrogen addition regimes wereused: in the ‘HN’ culture the relative rate of nitrate-Naddition (RA) was 0·08 d^–1 until day 48 and thendecreased stepwise to, finally, 0·005 d^–1 duringgrain-filling; the ‘LN’ culture received 45% ofthe nitrogen added in HN; the ‘CN’ culture was maintainedat RA 0·0375 d^–1 throughout. Kinetics of net nitrateuptake were measured during ontogeny at 30 to 150 mmol m^–3external nitrate. V_max (which is argued to reflect the maximuminflux rate in these plants) declined with age in both HN andLN cultures. A pronounced transient drop was observed just beforeanthesis, which correlated in time with a peak in root nitrateconcentration. Similar, but less pronounced, trends were observedin CN. The relative V_max (unit nitrogen taken up per unit nitrogenin plants and day) in all three cultures declined from 1·3–2·3d^–1 during vegetative growth to 0·1–0·7d^–1 during generative growth. These values are in HN andLN cultures 15- to more than 100-fold in excess of the demandset by growth rates throughout ontogeny. Predicted balancingnitrate concentrations (defined as the nitrate concentrationrequired to support the observed rate of growth) were below6·0 mmol m^–3 in HN and LN cultures before anthesisand then decreased during ontogeny. In CN cultures the balancingnitrate concentration increased during grain-filling. Apartfrom the transient decline during anthesis, most of the effectof ageing on relative V_max can be explained in terms of reducedcontribution of roots to total biomass (R:T). The loss in uptakeper unit root weight is largely compensated for by the declinewith time in average tissue nitrogen concentrations. The quantitativerelationships between relative V_max and R:T in ageing plantsare similar to those observed for vegetative plants culturedat different RAs. The data support the contention that the capacity for nitrateacquisition in N-limited plants is under general growth control,rather than controlled by specific regulation of the biochemicalpathway of nitrate assimilation. Key words: Barley, nitrogen concentration, root: total plant biomass ratio, V_max     

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     

Xylem fluxes of fixed N through nodules of the legume Acacia littorea and haustoria of an associated N-dependent root hemiparasite Olax phyllanthi

Nodulated 1-1.5-year-old plants of Acacia littorea grown inminus nitrogen culture were each partnered with a single seedlingof the root hemiparasite Olax phyllanthi. Partitioning of fixedN between plant organs of the host and parasite was studiedfor the period 4–8 months after introducing the parasite.N fluxes through nodules of Acacia and xylem-tapping haustoriaof Olax were compared using measured xylem flows of fixed Nand anatomical information for the two organs. N₂ fixation duringthe study interval (635 µg N g FW nodules^–1 d^–1)corresponded to a xylem loading flux of 0.20 µg N mm^–2d^–1 across the secretory membranes of the pencycle parenchymaof the nodule vascular strands. A much higher flux of N (4891µg mm^–2 d^–1) exited through xylem at the junctionof nodule and root. The corresponding flux of N from host xylemacross absorptive membranes of the endophyte parenchyma of Olaxhaustorium was 1.15 µg N mm^–1 d^–1, six timesthe loading flux in nodules. The exit flux from haustorium toparasite rootlet was 20.0 pg N mm^–1 d^–1, 200-foldless than that passing through xylem elements of the nodule.Fluxes of individual amino compounds in xylem of nodule andhaustorium were assessed on a molar and N basis. N flux valuesare related to data for transpiration and partitioning of Cand N of the association recorded in a companion paper. Key words: Olax phyllanthi, host-parasite relationships, N flux, Acacia, N₂ fixation     

Growth and Nitrogen Fixation of Phaseolus vulgaris L. at Two Irradiances II. Nitrogen Fixation

Phaseolus vulgaris cv. Glamis plants grown at 7 and 28 W m^–228 W m^–2 in controlled environment cabinets showed copiousnodulation and high levels of acetylene reducing activity. Earlydifferences in nodulation were apparent before differences inphotosynthesis and were attributed to an effect of far-red lighton nodule development. Total plant nitrogen content was greater at 28 W m^–2 thanat 7 W ^–2 but nitrogen content as a percentage of d. wtwas greater at the lower irradiance level. Total acetylene reducing activity (nmol. min^–1 root^–1)was greater at 28 W m^–2 than at 7 W ^–2, but therewas no irradiance effect on specific activity (nmol. min^–1g d. wt of pink nodules^–1 or nmol. min^–1 pink nodule^–1). Transfer of 40-day-old plants from 7 W m^–2 to 28 W m^–2resulted in increased nodulesize(due toincreased size of infectedcells), accompanied by increased total, but not specific, acetylenereducing activity. Transfer of plants from 28 W m^–2 to 7 W m^–2 resultedin a fall of total acetylene reducing activity within 24 h,and senescence of large nodules. Specific acetylene reducingactivity was unaffected The results are interpreted as an effect of light on the productionof nitrogen fixing tissue, rather than on nitrogenase activity.     

Nutrient Solution pH Control using Dipolar Buffers in Studies of Trifolium repens L. Nitrogen Nutrition

In studies of Trifolium repens nitrogen nutrition, the controlof nutrient solution pH using dipolar buffers, was evaluatedin tube culture under sterile conditions. Five buffers; MES,ADA, ACES, BES and MOPS with pK₂s (20 °C) of 6.15, 6.60,6.90, 7.15 and 7.20 respectively, at a concentration of 2.0mol m^–3, were provided to inoculated Trifolium repensgrowing in nutrient solution containing 7.13 mol m^–3 nitrogenas (NH₄)₂SO₄. Initial pH of each solution was adjusted to theappropriate buffer pK₂ Two buffers, ADA and ACES completelyinhibited plant growth. The remaining buffers had little effectin limiting pH change, although plant dry matter was higherand nodule numbers lower in the presence of these buffers. MESand MOPS were supplied to nutrient solutions with and without7.13 mol m^–3 (NH₄)₂SO₄, at concentrations ranging from0–12 mol m^–3. MES at 9 mol m^–3 and 12 molm^–3 reduced growth of plants reliant on the symbiosisfor providing nitrogen. The provision of MES to plants providedwith NH₄⁺ significantly increased plant yield and reduced nodulenumber at all concentrations. MOPS did not affect plant yieldor nodule number. The use of dipolar buffers in legume nitrogennutrition studies is considered in terms of buffering capacity,and the side effects on plant growth and symbiotic development. Key words: Ammonium, Dipolar buffer, Nitrogen nutrition, pH control, Symbiosis, Trifolium repens