An ELISA Procedure for Quantification of Relative Amounts of Intercellular Glycoprotein in Legume Nodules

An enzyme-linked immunosorbant assay (ELISA) method based ona monoclonal antibody (MAC236) is described in which relativeamounts of an intercellular glycoprotein were quantified inextracts of whole legume nodules. This glycoprotein has recentlybeen shown to be an important component of the cortical oxygendiffusion barrier. The ELISA method is demonstrated on threeexamples of soybean (Glycine max L. Merr.) nodule systems whichhave been the subject of previously published investigations:(a), cv. Clarke inoculated with Bradyrhizobium japonicum RCR3442,nodulated root systems of which were subject to 10, 21 or 40%oxygen continuously for 28 d; (b), cv. Bragg and its supernodulatingmutant derivative (nts382) inoculated with Bradyrhizobium japonicumUSDA110; (c), cv. Clarke inoculated with Bradyrhizobium japonicumRCR3442 or RCR3407. ELISA results are related to oxygen diffusioncharacteristics defined in previous publications and show thatincreases in the amount of glycoprotein present correlated withincreases in supra-ambient (40%) levels of rhizosphere pO₂,in minimum gas diffusion resistance and in speed of diffusionbarrier response. Area data of component parts of nodule inner cortices suggestthat diffusion resistance control under sub-ambient (10%) oxygenlevels also involves cell expansion. The amount of MAC236 antigen in nodules is affected by bothhost plant genotype and rhizobial strain and the latter alsoappears to be involved in determining the morphological developmentof the nodule inner cortex.Copyright 1993, 1999 Academic Press Oxygen diffusion resistance, glycoprotein, nodules, Glycine max, ELISA     

Increase in the Formation and Nitrogen Fixation of Soybean Nodules by Vesicular-Arbuscular Mycorrhiza

Symbiotic interactions of the tripartite association of soybeanplant, vesiculararbuscular (VA) mycorrhizal fungus and Rhizobiumjaponicum were shown. Mycorrhizal plants absorbed more P, Ca and Mg and had higherP, Ca and Mg contents in their stems or leaves than non-mycorrhizalplants. Phosphorus concentration was also higher in the nodulesof mycorrhizal plants. VA mycorrhizae increased nodule number, nodule weight and acetylenereduction activity of nodules. Concomitantly seed productionand N content of leaves were enhanced. Both nodulating (A62-1) and non-nodulating (A62-2) cultivarsof soybean plants [Glycine max (L.) Merr.] were colonized byVA mycorrhizal fungi, identified as belonging to the genus Glomus. (Received August 12, 1985; Accepted January 14, 1986)     

A New Procedure for the Calculation of Oxygen Diffusion Resistance in Legume Nodules from Flow-through Gas Analysis Data

Plants of white lupin (Lupinus albus L cv Multolupa) and soyabean(Glycine max L cv Clarke) were grown in controlled-environmentcabinets, subjected to various stresses and their nodular nitrogenaseactivity and total root respiration measured When these measurementswere used to calculate nodular oxygen diffusion resistance,using a simplified equation for Fick's first law of diffusion,it was found that the apparent resistance of stressed nodulesincreased anomalously with decreases in external oxygen concentrationA new analysis procedure is proposed to alleviate this anomalyThis procedure also uses the simplified Fick's law equationbut includes a respiratory contribution to the total oxygenflux across the diffusion barrier which is not coupled to nitrogenaseactivity Also, resistance is modelled as an exponential functionof external oxygen concentration Use of this analysis procedureproduces realistic values for total resistance and providesa characterisation of this resistance into a minimum value andan adjustment factor for changes in external oxygen It is postulatedthat the additional respiration component represents the activityof nodule cortex cells involved in the diffusion barrier, particularlythat of vascular bundles Oxygen diffusion resistance, nodule, nitrogen fixation, respiration     

Microelectrode Measurements of Hydrogen Concentrations and Gradients in Legume Nodules

This paper describes the construction and operation of H₂ specificmicroelectrodes and their application to measurements of H₂concentrations, H₂ gradients and H₂ inhibition of N₂-fixationin legume root nodules. Electrode construction was similar to that of O₂ specific microelectrodespreviously reported. They comprise an outer casing drawn toa 2–20 µm tip plugged with silicone rubber and aconcentric inner electrode made from glass coated platinum wire.The exposed tip of the Pt wire was placed close to the siliconeplug and polarized positively at 0?4 V with respect to an internalAg reference electrode. With an internal electrolyte of KC1/HC1current flow through the electrode was proportional to H₂ concentrationand independent of CO₂ and O₂. With appropriate amplificationand screening the detection limit for this system was 0?0001atm H₂ (4?0 µmol m^–3). Within newly detached nodulesof Hup^–ve symbioses of soyabean, pea and clover H₂ concentrationvaried from 0?009 to 0?014atm compared with 0?021 atm in lupinnodules. In nodules formed by the Hup^–ve soyabean/RCR3442symbiosis internal pH₂ increased from 0?012 atm to 0?09 atmwhen external pO₂ was raised to 0?60 atm. Hydrogen could notbe detected within nodules of the Hup^+ve Clarke/RCR3407 symbiosiseven when N₂ in the gas phase was replaced with Ar and externalpO₂ was increased to 0?60 atm. An assessment of H₂ inhibition of nitrogen fixation in the soyabean(Clarke/RCR3442) symbioses involved measurements of H₂ productionat increasing internal H₂ levels, induced by stepped increasesin gas phase H₂ concentration. The initial relative efficiencyof 0?66 (calculated from the pH₂ of nodules exposed to air andAr/O₂ mixtures) started to decrease at an internal pH₂ of 0?02to 0?03 atm and fell by 80% to 0?18 at an internal pH₂ of 0?1atm. This threshold value for inhibition is above the measuredmean H₂ concentration for this symbiosis of 0?01 atm. Hydrogen gradients through the nodule showed a sharp increasein the region of the inner cortex, which was reciprocal to adecrease in O₂ concentration, and a shallow gradient throughthe infected zone. These results indicate that the inner airspaces in the nodule are interconnected and confirm that thebarrier to O₂ diffusion is located in the inner cortex. Key words: Root nodules, hydrogen, hydrogenase, oxygen     

Changes in Morphometry and Elemental Composition of Robinia pseudoacacia Pulvinar Motor Cells During Leaflet Movements

Changes in shape and size of Robinia pulvinar cortical cellsin relation to leaflet movements have been investigated usingan image processing system applied to drawings of transverseand longitudinal pulvinar sections. Both the size and shapeof cell sections underwent change during movement. The dorsal-leftside region of the cortex has been characterized as the extensorregion which increases turgor during opening. Morphometric changesoccur throughout the cortical motor cells except in the threeor four inner layers. K, Cl, S, and Ca distribution in cellwalls and protoplasts of inner and outer motor cells have beenmeasured with X-ray microanalysis. The distribution patternof K and Cl shows that these ions are mainly responsible forturgor changes. K and Cl were simultaneously depleted in apoplastand protoplast, which suggests that cell walls do not possessa high enough ionic reservoir during Robinia leaflet movements.Ca was always higher in flexor cell walls than in extensor regionsof closed pulvini. This fact could be related to a lower abilityto extend of flexor cells which underwent fewer morphomerticchangrs during movement.     

Magnesium uptake by soybeans

Magnesium contents of soybean (Glycine max) roots increase and the K and Ca contents decrease with increased MgCl₂ concentrations in ambient solutions. The Mg uptake is inhibited when both Ca and K are present in the solution, but not by K or Ca alone. Chloride uptake, which is very low from the MgCl₂ solution, is greatly enhanced by the presence of K. The selectivity against Mg imparted by K + Ca appears to be at an external barrier for cation uptake as shown by its dependence on the presence of Ca in the external solution. The Ca content of roots is influenced only slightly by changes in external Ca concentrations from 10⁻⁴ to 10⁻²m, but that of shoots is greatly enhanced as the Ca concentration is increased or the K concentration is decreased. These effects on Ca contents are explained as arising from transport to the shoot without involvement of vacuoles of root cells.     

Ion compartmentalization in frog oocytes as demonstrated by X-ray microanalysis

The distributions of K, Na, Mg and Ca within frog ovarian and oviductal oocytes were studied by electron probe wavelength dispersive X-ray microanalysis. An important heterogeneity could be found both in nuclear and jelly coated oocytes. The highest K, Mg and, to a lesser extent, Na concentrations were found in the pigmented area of the peripheral cytoplasm. There is a certain correlation between the distribution of K and Mg. The concentration of K (but not of Na) in the nucleus was higher than that in the non-pigmented cytoplasm. The distribution of Ca was rather uniform. The high amounts of K, Na and S determined in the oocyte jelly coat seem to have become accumulated by ion-exchange mechanism. Oocyte pigment granules are believed to be the site of ion compartmentalization and to play a role in regulation of intracellular ionic composition.     

Scion Control of Genotypic Differences in Mineral Salts Accumulation in Soyabean (Glycine max L. Merr.) Seed

Stem-root grafts of seedling plants were used to ascertain thatgenotypic differences in P, Mg, Mn, and B accumulation in soyabean(Glycine max L. Merr.) seeds are controlled by the scion ofthe plant. The effect of the graft per se on mineral accumulationwas negligible. These results are similar to those reportedfor Sr and Ca accumulation in soyabeans. Mechanisms which couldaccount for these observations are briefly discussed.     

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     

Alpha-Mannosidase II Isoenzyme in the Peribacteroid Space of Glycine max Root Nodules

Kinnback, A., Mellor, R. B. and Werner, D. 1987. Alpha-mannosidaseII isoenzyme in the peribacteroid space of Glycine max rootnodules.—J. exp. Bot. 38: 1373–1377. Three isoenzymes of alpha-mannosidase from soybean tissue wereseparated by isoelectric focussing. The vacuomal isoenzymesI and II accounted typically for over 90% of the activity inextracts from roots whether or not they were infected with Bradyrhizobiumjaponicum, tissue culture and effective nodules. Ineffectivenodules, on the other hand, contained up to 62% of the typicallyextracellular located isoenzyme III. The compartment betweenthe symbiotic partners (the peribacteroid space) contained onlyisoenzyme II. The bearing of isoenzyme distribution upon modelsof peribacteroid membrane biogenesis is discussed. Key words: Aipha-mannosidase, Bradyrhizobium japonicum, Glycine max, membrane flow/recycling, peribacteroid space, symbiosis     

Comparative Study of the Elemental Composition of Vegetative and Resting Microbial Cells

X-ray microanalysis showed that vegetative cells, viable resting forms, and nonviable forms (micromummies) of the bacteria Bacillus cereus and Micrococcus luteus and the yeast Saccharomyces cerevisiae differ in the content of elements S, P, Ca, and K and Ca/K and P/S ratios. Viable resting forms (cystlike refractive cells and bacillar endospores) had more calcium and less phosphorus and potassium than vegetative cells, the difference being higher for bacilli than for micrococci and yeasts. The distinctive feature of all viable resting microbial forms was their low P/S ratios and high Ca/K ratios. The differences revealed in the cellular content and ratios of elements probably reflect changes in ionic homeostasis accompanying the transition of vegetative microbial cells to the dormant state. Relevant potassium parameters indicate that the membranes of viable resting forms retain their barrier function. At the same time, the nonviable micromummies, even those morphologically intact, of B. cereus and S. cerevisiae exhibited an anomalously low content of potassium, while those of M. luteus had an anomalously high content of this element. This suggests that the cellular membranes of micromummies lose their barrier function, which results in a free diffusion of potassium ions across the membranes. The possibility of using the elemental composition parameters for the quick analysis of the physiological state of microorganisms in natural environments is discussed.     

Abscisic Acid and the Regulation of Water Loss in Plantlets of Brassica oleracea L. var. botrytisRegenerated Through Apical Meristem Culture

Water loss was studied in regenerated plantlets of Brassicaoleracea var. botrytis cv. Currawong derived through apicalmeristem culture. Hardening of plantlets was eliminated by asingle application of a polyvinyl resin (S600) sprayed immediatelyafter transplanting. Plantlets sprayed with S600 had highercuticular resistances than unsprayed plantlets; this treatmenthad no effect on stomatal resistance. Leaves formed during theculture period showed very little wax formation and using markedleaves it was found that only reduced levels of wax formed onthese leaves even after transplanting. New leaves formed aftertransplanting, showed typical wax formation compared to seedgrown plants. Abscisic acid (ABA) at 10^–4 M applied as a leaf sprayto transplants did not cause a substantial increase in stomatalresistance in leaves which had been initiated during the cultureperiod. Leaves of seed-grown plants as well as leaves of plantletsformed after transplanting did respond to a leaf spray of ABAat 10^–4 M by a large increase in stomatal resistance. Relative concentrations of K, Na, Ca, P, S and Mg in guard cellswere calculated for each leaf type by X-ray micro-probe analysis.K/Na values decreased in the order: seedling > leaves formedafter transplanting > leaves intiated during culture. A highpositive correlation was also found between K/Na and K/P forthe three leaf types. K:Mg and K:Ca ratios for leaves formedduring culture were low in comparison to the values obtainedfor leaves formed after transplanting and seedlings for whichthe values were similar. Brassica oleracea var. botrytis, cauliflower, regenerated plantlets, meristem culture, stomatal resistance, water loss, abscisic acid, X-ray micro-probe analysis     

Mineral partitioning in the phloem during autumn senescence of beech leaves

Summary During the period of leaf senescence in fall, the minerals Mg, Ca, K, P, Cl, S, and Si were compared for occurrence and density in tissue compartments of leaf blade, petiole, and subtending stem of beech (Fagus sylvatica L.). Measurements were made by energy-dispersive X-ray microanalysis. The plant material was collected on 2,9, 16 and 23 October, and showed green, greenyellow, yellow, and red-brown autumn leaf coloration. Mg, K, and P were retrieved from the leaf blade prior to shedding, and deposited mainly in cortex and pith tissues of the stem. S and Ca remained in the leaf, and Si and Cl appeared to accumulate in the leaf prior to shedding. During the four stages of leaf senescence, the phloem compartments of the petiole showed considerable changes in mineral content. In addition, leaf senescence in several cases was accompanied by ion shifting from symplastic to apoplastic compartments and vice versa.     

Seasonal Dynamics of the Accumulation, Distribution and Redistribution of Dry Matter and Mineral Nutrients in a Weedy Species of Gladiolus (Gladiolus caryophyllaceus)

The seasonal dynamics of the accumulation, distribution andredistribution of dry matter and 12 mineral nutrients by a weedyspecies of gladiolus (Gladiolus caryophyllaceus) were studiedat Perth, Western Australia, where it has colonized the nutrient-poorsandy soils. Parent corms sprouted in autumn, and the plantshad completed their growth cycle by early summer. The maturereplacement corm had 15-25% of the plant's P, Ca, Na, Zn andCu, 5-15% of its K, N, Cl, Mg, S and dry matter, and < 5%of its Fe and Mn. Seeds had 26% of the plant's dry matter, 60%of its N and P, 21-33% of its S, Mg, Cu and K, 5-20% of itsFe, Mn and Zn, and < 5% of its Ca and Na. The mature vegetativeshoot had 47% of the plant's dry matter and over 40% of eachnutrient, except for N, P and Cu. Phosphorus, K and N were redistributedfrom the parent corm with over 85% efficiency, S, Mg, Zn andCu with 60-70% efficiency, but there was < 10% redistributionof Ca, Na, Cl, Fe and Mn. The efficiency of redistribution fromthe leafy shoot was over 70% for N and P, 29-52% for K, Mg andCu, 16-20% for S, Zn and Cl, but negligible for Ca, Na, Fe andMn. Redistribution from the shoot could have provided the replacementcorm and seeds with 53-98% of their Cu, Mg, N, P and K, and29-38% of their S, Zn and dry matter. Seeds contained over 60%of each nutrient in a capsule, except for Ca, Na and Fe. Redistributionfrom the capsule walls could have provided 13-19% of the P,Cu and Zn, and 3-7% of the N, K, Mg and dry matter accumulatedby seeds. Each plant produced an average of 520 seeds. Removalof flowers and buds at first anthesis resulted in a larger replacementcorm containing a greater quantity of most nutrients, indicatingcompetition between the replacement corm and seeds for nutrients.Redistribution from parent to replacement cormlets in the absenceof shoot and root development was high, with over 50% of thedry matter and each nutrient, except for Ca, being transferred.Concentration of nutrients were low in all organs of G. caryophyllaceus,especially the replacement corm. It was concluded that the effectiveredistribution of key nutrients, such as N and P, to reproductivestructures and tolerance of low internal concentrations of nutrientscontribute to the capacity of G. caryophyllaceus to colonizeand persist on infertile soils.Copyright 1993, 1999 AcademicPress Gladiolus caryophyllaceus, corm, distribution, dry matter, gladiolus, mineral nutrients, nutrient accumulation, nutrient redistribution, seasonal growth, weed     

Distribution of Potassium, Calcium and Magnesium in the Gametophyte and Sporophyte Generations of Funaria hygrometrica Hedw

Analyses have been made of the K, Ca and Mg in separated sporophytes,spores and gametophytes of the moss Funaria hygrometrica duringsporophyte development. Degeneration of the gametophyte wasaccompanied by loss of K and a gain in Ca while the K contentof the developing sporophyte increased more rapidly than thatof Ca. The presence of an air–gap in the expanding capsulesignificantly influenced the observed cellular location of ions.Relative to the sporophyte the spores were shown to have a higherK and a lower Ca concentration. The behaviour of Mg was intermediatebetween K and Ca throughout. Funaria hygrometrica, potassium, calcium, magnesium, cation distribution, gametophyte, sporophyte     

Correlation between potassium and phosphorus content and their nonuniform distribution in Acanthamoeba castellanii

Summary Biologically important elements: K, Na, Mg, Ca, Cl, P, and S were analyzed in Acanthamoeba castellanii. A higher potassium content, as compared with other cations, was detected. Total content of the cation-forming elements: K, Na, Mg, and Ca was ca. 360 mmoles/kg dry weight of the cells. Phosphorus content was estimated as 492 mmoles/kg dry weight. Content of chlorine, a basic cellular anion, was 173 mmoles/kg dry weight. The low level of chlorine appears not the be sufficient to balance all the cations in Acanthamoeba.Distribution of potassium in Acanthamoeba cells was nonuniform and similar to that of phosphorus as shown by X-ray microanalysis technique. Quantitative correlation between phosphorus and potassium as well as the similar distribution of these elements suggests that in Acanthamoeba phosphorus is an essential anion which, being nonuniformly distributed in the cell, determines also a nonuniform distribution of potassium.     

The Locations and Amounts of Endogenous lons and Elements in the Cap and Elongating Zone of Horizontally Oriented Roots of Zea mays L: An Electron-probe EDS Study

We used quantitative electron-probe energy-dispersive x-raymicroanalysis to localize endogenous Na, Cl, K, P, S, Mg andCa in cryofixed and freeze-dried cryosections of the cap (i.e.the putative site of graviperception) and elongating zone (i.e.site of gravicurvature) of horizontally oriented roots of Zeamays. Ca, Na, Cl, K and Mg accumulate along the lower side ofcaps of horizontally oriented roots. The most dramatic asymmetriesof these ions occur in the apoplast, especially the mucilage.We could not detect any significant differences in the concentrationsof these ions in the central cytoplasm of columella cells alongthe upper and lower sides of caps of horizontally-oriented roots.However, the increased amounts of Na, Cl, K and Mg in the longitudinalwalls of columella cells along the lower side of the cap suggestthat these ions may move down through the columella tissue ofhorizontally-oriented roots. Ca also accumulates (largely inthe mucilage) along the lower side of the elongating zone ofhorizontally-oriented roots, while Na, P, Cl and K tend to accumulatealong the upper side of the elongating zone. Of these ions,only K increases in concentration in the cytoplasm and longitudinalwalls of cortical cells in the upper vs lower sides of the elongatingzone. These results indicate that (1) gravity-induced asymmetriesof ions differ significantly in the cap and elongating zoneof graviresponding roots, (2) Ca accumulates along the lowerside of the cap and elongating zone of graviresponding roots,(3) increased growth of the upper side of the elongating zoneof horizontally-oriented roots correlates positively with increasedamounts of K in the cytoplasm and longitudinal walls of corticalcells, and (4) the apoplast (especially the mucilage) may bean important component of the pathway via which ions move ingraviresponding rots of Zea mays. These results are discussedrelative to mechanisms for graviperception and gravicurvatureof roots. Corn, gravitropism (root), ions, x-ray microanalysis, Zea mays     

Quantitative X-ray microanalysis of the morula cell of the blood of the ascidian Halocynthia papillosa (Stolidobranchiata)

Summary The elemental composition of the morula cell of Halocynthia papillosa blood was studied by X-ray microanalysis with respect to the possible iron accumulation in this cell type. We found various amounts of Na, Mg, P, S, Cl, K, Ca, Fe and Br in the cytoplasm, nucleus and vacuoles. With the exception of a few cells, Ca, Fe and Br were not detected. Thus, the morula cells of the studied species are not iron-rich cells.     

Detection of Microorganisms in the Environment and the Preliminary Appraisal of Their Physiological State by X-ray Microanalysis

The paper deals with the X-ray microanalysis of the elemental composition of bacteriomorphic particles in 170000-year-old Antarctic permafrost sediments and in indoor dust. A comparative analysis of the phosphorus, sulfur, calcium, and potassium contents and the Ca/K and P/S ratios in these particles and in reference microbial cells occurring in different physiological states showed that the absence of P and/or S peaks in the X-ray spectrum of an object may indicate that it is abiotic. Resting microbial forms can be revealed on the basis of the following characteristic features: an increased content of Ca, a high Ca/K ratio, and a low P/S ratio. Model experiments with nonviable bacterial and yeast micromummies with alterations in the structural and barrier functions of the cytoplasmic membrane showed that micromummies can be recognized by a superhigh content of a marker element (e.g., P, K, or Si), accumulated due to facilitated diffusion along a deliberately created concentration gradient. Such an analysis of the permafrost sediment and dust made it possible to suggest the presence of mummified cells in these objects. The possibility of using X-ray microanalysis for the detection of microbial cells in natural habitats in order to enhance the efficiency of ecological monitoring of the environment is discussed.     

Legume Nodule Morphology with Regard to Oxygen Diffusion and Nitrogen Fixation

Soybean (Gtycine max (L.) Merr. cv. Fiskeby V), white clover(Trifolium repens L. cv Blanca) and lucerne (Medicago sativaL.cv. Europe) nodules grown in fluid culture of Perlite wereexamined by cryo-scanning electron microscopy. The surfacesof all three species consisted of loosely packed cortical cells,collapsed areas covering intact cells and in soybean, troughswhich lacked the layers of loosely packed cells and were coveredby an amorphous matrix. The superficial cortical cells werehydrophobic and their surfaces were covered by a solvent-extractablestippling. This stippling was absent from the surfaces of underlyingcells. Air-filled intercellular spaces in fractured noduleswere seen throughout the cross-section. Nitrogen fixation, intercellular pathways, hydrophobicity, oxygen diffusion, cryo-SEM, soybean, Glycine max (L.) Merr. cv. Fiskeby V, clover, Trifolium repens L. cv. Blanca, lucerne, Medicago saliva L. cv. Europe.