Dimensions and distribution of intercellular spaces in cryo-planed soybean nodules

The ability of legume nodules to regulate their permeability to gas diffusion has been attributed to physiological control over the size and distribution of gas-filed intercellular spaces within the nodule cortex. To examine the size and distribution of intercellular spaces and to determine whether they were filled with gas (high diffusion permeability) or liquid (low diffusion permeability), whole nodules were frozen in liquid nitrogen slush (-210°C), and then either cryo-fractured or cryo-planed before being examined by cold-stage scanning electron microscopy (SEM). The cryo-planed tissue was found to have many advantages over cryo-fractured nodules in providing images which were easier to interpret and quantify. Intercellular spaces throughout the nodule were examined in both tangential and medial planed faces. Since no differences were observed between views in either the size or shape of the open intercellular spaces, it was concluded that the intercellular spaces of nodules were not radially oriented as assumed in many mathematical models of gas diffusion. The inner cortex region in the nodules had the smallest intercellular spaces compared to other zones, and less than 10% of the intercellular spaces were occluded with any type of material in the central zone regions. Vacuum infiltration of nodules with salt solutions and subsequent cryo-planing for SEM examination showed that open and water-filled intercellular spaces could be differentiated. The potential is discussed for using this method to study the mechanism of diffusion barrier regulation in legume nodules.     

Oxygen Diffusion in Lupin Nodules: II. MECHANISMS OF DIFFUSION BARRIER OPERATION

The oxygen diffusion resistance of Lupinus albus (L.) cv. Multoluparoot nodules was increased by subjection to short-term stresses;lowering rhizosphere temperature from 25 to 16 °C (2 h),detopping plants (3 h), darkening plants (21 h) or exposingroots to 20 mol m^–3 KN03 for 2, 4 or 6 d. Microscopicobservations and measurements showed that this resulted in thearea of open intercellular spaces within the inner cortex beingreduced due to both cell expansion and increased productionof an occluding glycoprotein. Electrophoretic and Western Blotanalysis using the monoclonal antibodies MAC236 and MAC265 showedtwo distinct glycoprotein antigens with molecular weights of240 and 135 kDa, respectively. Both antigens are localized withinintercellular spaces of the inner cortex. The amount of glycoproteinwas determined using either ELISA, with MAC265, or quantificationof immunolabelling with MAC236. This immunolabelling also localizedthe glycoprotein within globules adhering to the inside of theinner cortical cell walls. Key words: Oxygen diffusion resistance, glycoprotein, nodules, nitrogen fixation, Lupinus albus     

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     

Detopping causes production of intercellular space occlusions in both the cortex and infected region of soybean nodules

A structural analysis was conducted to determine whether glycoprotein‐containing intercellular space occlusions are involved in medium‐term regulation of O₂ diffusion in soybean (Glycine max) nodules. Alterations in O₂ diffusion were induced by a 3 h detopping treatment, and glycoprotein was immunolocalized with the monoclonal antibodies MAC236 and MAC265. Western blots of unstressed nodules revealed that these antibodies recognize antigens with two different molecular weights in soybean nodules. Tissue printing of halved nodules showed that both antigens were present in fresh nodules from control and 3 h detopped plants. The main localization appeared to be the inner cortex, but some immunolabelling also occurred in the infected region. ELISAs demonstrated a significant increase in total nodule concentration of intercellular glycoprotein following detopping, and cryosections of fresh nodules from this treatment also showed localization of antigens within the intercellular spaces of the infected region. The production of intercellular space occlusions in both the mid‐cortex and infected regions after 3 h detopping was confirmed by light microscopy and silver‐enhanced immunolabelling; cortical changes were quantified by image analysis techniques. Electron microscopy revealed that the occlusions within the infected region were less dense and less heavily labelled than those in the cortex. These results are discussed in relation to O₂ diffusion regulation in soybean nodules     

Photosystem II and oxygen regulation in Sesbania rostrata stem nodules

The tropical wetland legume Sesbania rostrata Brem. produces nitrogen-fixing stem nodules which are green and contain chlorophyll, the chloroplasts being concentrated in a hand in the inner and mid-cortex close to the nitrogen-fixing cells. The photosystem II thylakoid membrane proteins D₁, D₂ and PsbO, which are essential for photo-synthetic O₂ evolution, were shown by immunoblotting to be present in extracts of leaves and stem nodules. Immunogold labelling confirmed their presence on stem nodule thylakoids and showed that labelling was most intense in well-developed chloroplasts in the mid-cortex and least intense in the smaller, less-abundant chloroplasts adjacent to the nitrogen-fixing cells. Concentrations of the oxygen-carrying protein leghaemoglobin (Lb) did not differ between stem and S. rostrata root nodules, and Lb was localized in bacteroid-containing cells, including those immediately adjacent to the cortex, in both nodule types. Moreover, nitrogenase component 2 was localized in bacteroids within the outermost layers of infected cells, suggesting that a low pO₂ was maintained, despite the nearby chloroplasts. Nodule extracts examined by ELISA and immunoblots, using the monoclonal antibody MAC265, showed greatly enhanced expression of a 139 kDa glycoprotein in stem compared to root nodules. Immunogold labelling showed that material containing the MAC265 antigen occluded intercellular spaces, and was present in cell walls, throughout the cortex of stem nodules (particularly in the chloroplasl-rich inner and mid-cortex), but was considerably less evident in root nodules.     

Electron microscopic investigation of water occlusions in intercellular spaces in the inner cortex of lucerne nodules.

It is unclear to what extent oxygen diffusion pathways through the cortex of the nitrogen-fixing zone of indeterminate nodules are liquid filled and whether a blockage of these pathways is involved in varying nodule oxygen permeability to control nitrogenase activity. We examined the proportion of water-filled intercellular spaces of lucerne (Medicago sativa L.) nodules with cryo-scanning electron microscopy. This technique allows for direct observation of water accumulation. Thirty percent of all intercellular spaces in the inner cortex of lucerne nodules were liquid filled. Decreasing the nodule oxygen permeability by detopping of the plant or by increasing the rhizospheric oxygen partial pressure to 80 kPa had no statistically significant effect on the water distribution in the intercellular spaces. Therefore, the hypothesis of a continuous aqueous diffusion barrier in the inner cortex could not be supported. The abundance of glycoproteins in intercellular spaces of the inner cortex was investigated with immunoelectron microscopy. No alteration due to detopping or after increase of the rhizospheric oxygen partial pressure was observed. Therefore, our results do not support the hypothesis of a short-term regulation of oxygen permeability by blockage of diffusion pathways through morphological changes in the cortex region of the nitrogen-fixing zone of lucerne nodules.     

Evidence that short-term regulation of nodule permeability does not occur in the inner cortex

Regulation of nodule permeability in response to short-term changes in environmental and physiological conditions is thought to occur by occlusion of intercellular spaces in the nodule inner cortex. To test this hypothesis, the permeability of legume nodules was altered by adapting them to either 20 or 80% O₂ over a 2.5-h period. The nodules were then rapidly frozen, cryo-planed and examined under cryo-scanning electron microscopy for differences in the number, area or shape factor of intercellular spaces. Comparisons were made between whole nodules and specific nodule zones (outer cortex, middle cortex, inner cortex and central zone) in each treatment. Gas analysis measurements indicated that nodules equilibrated at 20% O₂ had a 6.6-fold higher permeability than those equilibrated at 80% O₂ However, no significant differences were observed between pO₂ treatments in the number of open intercellular spaces, the cross-sectional area of those spaces, or the proportion of the tissue area present as open space in whole nodules or any nodule zone. Also, although nodules in both treatments possessed a boundary layer of tightly packed cells in the inner cortex, the total area of intercellular spaces between cells bordering this layer did not differ between treatments. Together these observations do not support the currently favored hypothesis that nodule permeability is regulated by opening or occlusion of intercellular spaces in the nodule inner cortex. Highly significant differences (P= 0.0006) were observed between O₂ treatments in the shape factor of the open intercellular spaces in all nodule zones. Nodules equilibrated at 80% O₂ had significantly more isodiametric spaces while those equilibrated at 20% O₂ had more long, narrow spaces. This observation suggests that the critical step in the regulation of nodule permeability to O₂ may be localized in the central, infected zone and involve changes in the ratio of the surface area of the intercellular space to the volume of the infected cell.     

Adenylate-coupled ion movement. A mechanism for the control of nodule permeability to O2 diffusion

In response to changes in phloem supply, adenylate demand, and oxygen status, legume nodules are known to exercise rapid (seconds to hours) physiological control over their permeability to oxygen diffusion. Diffusion models have attributed this permeability control to the reversible flow of water into or out of intercellular spaces. To test hypotheses on the mechanism of diffusion barrier control, nodulated soybean (Glycine max L. Merr.) plants were exposed to a range of treatments known to alter nodule O2 permeability (i.e. 10% O2, 30% O2, Ar:O2 exposure, and stem girdling) before the nodules were rapidly frozen, freeze dried, and dissected into cortex and central zone (CZ) fractions that were assayed for K, Mg, and Ca ion concentrations. Treatments known to decrease nodule permeability (30% O2, Ar:O2 exposure, and stem girdling) were consistently associated with an increase in the ratio of [K+] in cortex to [K+] in the CZ tissue, whereas the 10% O2 treatment, known to increase nodule permeability, was associated with a decrease in the [K+]cortex:[K+](CZ). When these findings were considered in the light of previous results, a proposed mechanism was developed for the adenylate-coupled movement of ions and water into and out of infected cells as a possible mechanism for diffusion barrier control in legume nodules.     

Temporal Relationships between Nitrogenase and Intercellular Glycoprotein in Developing White Lupin Nodules

The development of the N₂-fixing symbiosis between white lupin (Lupinus albusL.) cv. Multolupa andBradyrhizobiumstrain ISLU16 was followed using the acetylene reduction assay (ARA), immunoblots of protein extracts, and microscopy/immunogold labelling at 0, 8, 12, 17 and 20 d after infection. There was no ARA at 0, 8 and 12 d, although macroscopically visible nodule primordia had formed on roots by 8 d. The lack of nitrogenase at these times was confirmed by a negative signal to immunogold labelling with nitrogenase-specific antibodies. At 17 d three out of six plants had ARA, and nodules from these gave a positive signal with the nitrogenase antibody. By contrast, ARA⁻(fix⁻) nodules at 17 d were smaller (mean radius of 0.49 mm compared to 1.01 mm with fix⁺nodules) and gave a negative signal with the nitrogenase antibody. Western blots of nodule protein extracts using the monoclonal antibodies MAC236 and MAC265 (which recognize two epitopes on a glycoprotein which is considered to be involved in both rhizobial infection and the regulation of nodule oxygen diffusion) gave a strong signal with nodules (fix⁺) from 20 d plants and with 17 d fix⁺plants. The signal with MAC236/MAC265 was substantially weaker with nodules from 17 d fix⁻plants, and there was no signal apparent from nodules/nodulated roots from the 0, 8 and 12 d harvests. However, further investigation using immunogold labelling revealed that not only were MAC236 and MAC265 expressed within cortical intercellular spaces in 20 d and 17 d fix⁺/fix⁻nodules, but they were also strongly expressed in the developing cortex surrounding the newly-infected tissue in 8 d nodules, as well as in intercellular spaces within the cortex and infected tissue of 12 d nodules. These data demonstrate that the glycoprotein recognized by MAC236 and MAC265 is present before the onset of nitrogenase expression and function, but expression of the epitopes appears to be enhanced from the onset of N₂fixation. Nodules at all harvests were investigated for the presence of infection threads, as the MAC236/MAC265-recognized glycoprotein is also a component of the infection thread matrix in nodules from other legumes. Infection threads were not seen in nodules from any of the harvests except for the 20 d nodules, and then only after serial sectioning. The latter revealed occasional short wide infection threads entering and releasing rhizobia into small pockets of uninfected cells, within the infected tissue, but not within the meristems. The matrix of these infection threads labelled weakly, or not at all, with MAC236 and MAC265, and it was concluded that the majority of the MAC236/MAC265 detected in lupin nodule extracts originated from glycoprotein within cortical intercellular spaces.     

Time-course of changes involved in the operation of the oxygen diffusion barrier in white lupin nodules

Nodulated white lupins (Lupinus albus L. cv. Multolupa) weresubject to either darkening for 12 h, followed by 24 h recoveryin light, or to 50% O₂ for 30 min. For each treatment, noduleswere harvested at intervals for analysis by light and electronmicroscopy and determination of glycoprotein content using EnzymeLinked Immunosorbent Assays (ELISA). This allowed for an analysisof the sequence of events causing an increase in intercellularspace occlusion within the inner cortex. The temporal sequencein response to darkening appears to be: (1) an initial rapidincrease in the detectable levels of intracellular glycoprotein,due to either a state change or de novo synthesis, (2) a concomitantincrease in the volume of thickened cell walls, causing a reductionof intercellular space volume and (3) after 1–3 h a releaseof glycoprotein into the intercellular space network of theinner cortex, accompanied (and possibly spread) by the continuedconstriction of the spaces due to cell wall and cell contentexpansion. The results for exposure to 50% O₂ showed a similar,but much more rapid, sequence of events, operating within 15–30min. The main difference between the two sequences was the lackof expansion of thickened cell walls with increased pO₂. Also,it was possible to detect glycoprotein within cell walls followingexposure to 50% O₂ but not following darkening. These observationsare discussed in relation to proposed mechanisms for the operationof a variable oxygen diffusion barrier in legume nodules. Key words: Oxygen diffusion resistance, glycoprotein, nodules, Lupinus albus     

Gaseous diffusive properties of soybean nodules cultured with non-ambient pO2

Measurements of the short-term response of nodulated roots of soybean ( Glycine max L. Merr, cv. Harosoy: Bradyrhizobium japonicum USDA 16) to rapid changes in surrounding pO₂ indicate that their ability to reversibly adjust gaseous diffusive resistance is retained whether plants are cultured in rhizospheres of very low (2.8%) or very high (61.2%) pO₂. Thus the capacity for reversible short-term diffusion adjustment is additional to structural changes in the fixed diffusional barriers of nodules which allow their continued fixation of N₂ in unfavourably high or low external pO₂. Anatomical evidence, involving quantitative measurement of intercellular spaces in the cortical tissues using electron microscopy of thin sections, indicates that the major fixed diffusional barrier is a boundary layer of cells in the inner cortex which may be as small as one cell thick in nodules from 2.8% O₂ to 5 or 6 cells thick, and almost completely devoid of intercellular spaces, in those from 61.2% O₂. The data are interpreted to indicate that the variable diffusion harrier is distinct from the boundary layer and is most likely to be a property of cells and/or intercellular spaces inside the boundary layer of the nodule cortex.     

The Structure of Nitrogen Fixing Root Nodules on the Aquatic Mimosoid Legume Neptunia plena

Nodules of the aquatic mimosoid legume Neptunia plena (L.) Benth.were always found associated with roots but not stems. Theyappeared macroscopically 10 and 20 d after inoculation on plantsgrown hydroponically and in vermiculite, respectively. The developmentof nitrogen-fixing cells occurred in a series of stages notyet reported in legume nodule formation: initial infection wasapparently intercellular and rhizobia spread between cells andthrough intercellular spaces before penetrating individual hostcells by means of infection threads. Subsequently nodule developmentwas broadly similar to that described for indeterminate papilionoidnodules. The infection threads of Neptunia and pea nodules containeda matrix with a common epitope, which was, in Neptunia, extrudedfrom the infection thread at the point of bacterial release. The central tissue contained infected and interstitial cellsand was surrounded by a three-layered cortex and a phellem.Bounding the infected region was a layer two to three cellsthick with large, unoccluded intercellular spaces. Externalto this was a layer, one or more cells thick, in which the cellwalls were interlocked, reducing the number of radially orientedintercellular spaces. The outer layer, several cells thick,contained intercellular spaces many of which were occluded.These features did not vary with growth conditions in a waywhich might influence oxygen diffusion characteristics. However,the phellem of water-cultured nodules was much more aerenchymatousthan that of vermiculite-grown nodules. Aquatic legume, Neptunia plena, nitrogen fixation, oxygen, root nodules, Rhizobium     

Subcellular localization of glycoprotein epitopes during the development of lupin root nodules

Summary The monoclonal antibodies MAC236 and MAC265, raised against a soluble component of pea nodules, were used to elucidate the presence and subcellular localization of glycoprotein epitopes during the development of lupin (Lupinus albus L. cv. Multolupa) nodules, by means of immunocytochemistry and Western blot analysis. These antibodies recognize a single band of 95 kDa in pea, soybean and bean nodules, whilst two different bands of 240 and 135 kDa cross-react with MAC236 and MAC265 respectively in lupin nodules. This fact may indicate that the recognized epitopes can be present in different subcellular compartments and/or play different roles through the development of functional nodules. The results show that MAC265 is mainly associated with Bradyrhizobium infection and with the development of nodule primordium, in the first stages of nodulation. MAC265 is also detected when glycoprotein transport takes place across the cytoplasm and the cell wall, and also in the intercellular spaces of the middle cortex, attached to cell walls. The amount of MAC265 remains constant through nodule development. In contrast the amount of MAC236 increases with nodule age, parallel to the establishment of nitrogenase activity. This antibody is localized in cytoplasmic globules attached to the inner side of cell walls in the middle cortex, and mainly in the matrix filling the intercellular spaces of the middle and inner cortex. This main site of localization of MAC236 may indicate a role in the functioning of the oxygen diffusion barrier.     

The distribution of aquaporin subtypes (PIP1, PIP2 and gamma-TIP) is tissue dependent in soybean (Glycine max) root nodules

BACKGROUND AND AIMS The inner cortical cells (IC-cells) of legume root nodules have been previously shown to regulate the resistance to nodule O2 diffusion by a rapid contraction/expansion mechanism, which controls the volume of intercellular spaces and their occlusion by a liquid phase. The expression of aquaporins in IC-cells was also found to be involved in this nodule O2 diffusion mechanism. The aim of this study was to compare the expression of plasma membrane intrinsic proteins (PIP) aquaporin isoforms with tonoplast intrinsic protein (gamma-TIP) in both IC-cells and adjacent cell types. METHODS: Using immunogold labelling in ultra-thin sections of Glycine max nodules, the expression of two PIP isoforms was observed and compared with the gamma-TIP pattern. KEY RESULTS: The plasma membrane aquaporins PIP1 and PIP2 were expressed more in IC-cells and endodermis than in pericycle and infected cells. The tonoplast aquaporin gamma-TIP has shown a distribution pattern similar to that of the PIPs. CONCLUSIONS: PIPs and gamma-TIP aquaporins are highly expressed in both plasmalemma and tonoplast of nodule IC-cells. This distribution is consistent with the putative role of water fluxes associated with the regulation of nodule conductance to O2 diffusion and the subsequent ATP-dependent nitrogenase activity. In the endodermis, these aquaporins might also be involved in nutrient transport between the infected zone and vascular traces.     

The development and structure of root nodules on bambara groundnut [Voandzeia (Vigna)subterranea]

The infection of Vigna subterranea (formerly Voandzeia subterranea) by Bradyrhizobium strain MAO 113 (isolated from V. subterranea) was examined by light and transmission electron microscopy. Bacteria accumulated on the epidermis close to root hairs, and subsequently entered the latter via infection threads. Most of the steps involved in nodule formation were generally characteristic of determinate nodules, such as those which form on the closely related V. radiata. For example, nodule meristems were induced beneath the root epidermis adjacent to infected root hairs, but prior to infection of the meristem by rhizobia. Moreover, after the infection of some of the meristematic cells by the infection threads, and the release of the rhizobia into membrane-bound vesicles, the infection process ceased and dissemination of the rhizobia was by division of already-infected host cells. However, there were some aspects of this process in V. subterranea which have been more commonly described in indeterminate nodules. These include long infection threads entering a number of cells within the meristems simultaneously and a matrix within infection threads which was strongly labelled with immunogold monoclonal antibodies, MAC236 and MAC265, which recognize epitopes on an intercellular glycoprotein. The MAC236 and MAC265 antibodies also recognized material in the unwalled infection droplets surrounding bacteria which were newly-released from the infection threads. The amount of labelling shown was more characteristic of the long infection threads seen in indeterminate nodules such as pea (Pisum sativum) and Neptunia plena. The structure of mature V. subterranea nodules was similar to that described for other determinate nodules such as Glycine max, Vigna unguiculata and V.radiata, i.e. they were spherical and the infected zone consisted of both infected and uninfected cells. Surrounding the infected tissue was an inner cortex of uninfected cell layers containing the putative components of an oxygen diffusion barrier (including glycoprotein-occluded intercellular spaces), and an outer cortex with cells containing calcium oxalate crystals.     

Diffusion Limitation of Oxygen Uptake and Nitrogenase Activity in the Root Nodules of Parasponia rigida Merr. and Perry

Parasponia is the first non-legume genus proven to form nitrogen-fixing root nodules induced by rhizobia. Infiltration with India ink demonstrated that intercellular air spaces are lacking in the inner layers of the nodule cortex. Oxygen must diffuse through these layers to reach the cells containing the rhizobia, and it was calculated that most of the gradient in O₂ partial pressure between the atmosphere and rhizobia occurs at the inner cortex. This was confirmed by O₂ microelectrode measurements which showed that the O₂ partial pressure was much lower in the zone of infected cells than in the cortex. Measurements of nitrogenase activity and O₂ uptake as a function of temperature and partial pressure of O₂ were consistent with diffusion limitation of O₂ uptake by the inner cortex. In spite of the presumed absence of leghemoglobin in nodules of Parasponia rigida Merr. and Perry, energy usage for nitrogen fixation was similar to that in legume nodules. The results demonstrate that O₂ regulation in legume and Parasponia nodules is very similar and differs from O₂ regulation in actionorhizal nodules.     

The development and structure of root nodules on bambara groundnut [Voandzeia (Vigna)subterranea]

The infection of Vigna subterranea (formerly Voandzeia subterranea) by Bradyrhizobium strain MAO 113 (isolated from V. subterranea) was examined by light and transmission electron microscopy. Bacteria accumulated on the epidermis close to root hairs, and subsequently entered the latter via infection threads. Most of the steps involved in nodule formation were generally characteristic of determinate nodules, such as those which form on the closely related V. radiata. For example, nodule meristems were induced beneath the root epidermis adjacent to infected root hairs, but prior to infection of the meristem by rhizobia. Moreover, after the infection of some of the meristematic cells by the infection threads, and the release of the rhizobia into membrane-bound vesicles, the infection process ceased and dissemination of the rhizobia was by division of already-infected host cells. However, there were some aspects of this process in V. subterranea which have been more commonly described in indeterminate nodules. These include long infection threads entering a number of cells within the meristems simultaneously and a matrix within infection threads which was strongly labelled with immunogold monoclonal antibodies, MAC236 and MAC265, which recognize epitopes on an intercellular glycoprotein. The MAC236 and MAC265 antibodies also recognized material in the unwalled infection droplets surrounding bacteria which were newly-released from the infection threads. The amount of labelling shown was more characteristic of the long infection threads seen in indeterminate nodules such as pea (Pisum sativum) and Neptunia plena. The structure of mature V. subterranea nodules was similar to that described for other determinate nodules such as Glycine max, Vigna unguiculata and V.radiata, i.e. they were spherical and the infected zone consisted of both infected and uninfected cells. Surrounding the infected tissue was an inner cortex of uninfected cell layers containing the putative components of an oxygen diffusion barrier (including glycoprotein-occluded intercellular spaces), and an outer cortex with cells containing calcium oxalate crystals.     

Gas diffusion pathway in nodules ofCasuarina cunninghamiana

The gas diffusion pathway in nodules was traced by vacuum infiltration with India ink or aniline blue and by electron microscopy. India ink infiltration was observed in the outermost and the innermost cortex in sliced nodules, but not in intact nodules. With aniline blue infiltration, it was observed that intercellular air spaces in the outermost and the innermost cortex were connected to those in nodule roots. No air spaces were in contact with walls of infected cells, although intercellular air spaces existed in some groups of uninfected cells within the infected zone. Infiltration with either India ink or aniline blue could not be observed in the infected zone in essentially all cases. Thus it is suggested that the discontinuity of the intercellular air spaces represents a major resistance to O₂ diffusion in nodules ofCasuarina cunninghamiana.     

豆科根瘤内气体交换和气体扩散模型研究

近年来的研究表明根瘤皮层内存在着可调节的气体扩散屏障,它是由根瘤皮层内的一层细胞及填充在胞间隙的水层构成的,而根瘤是通过改变填充该层胞间隙的水层厚度来调节对气体扩散的阻力。本文概述了关于模拟豆科根瘤内气体交换和气体扩散的数学模型研究,阐明调节根瘤内含类菌体细胞维持低氧分压的有关问题。模型研究使我们获得了对共生固氮根瘤内极为复杂的微生态环境的初步认识,有待于通过改进试验和借助其他理论进一步探索根瘤气体交换和气体扩散的本质。     

OXYGEN CONCENTRATION WITHIN THE NITROGEN-FIXING ROOT NODULES OF MYRICA GALE L.

Microelectrodes were used to study the oxygen concentration within Myrica gale L. nodules. Low oxygen concentrations were found only in the region of the mature, nitrogen-fixing endophyte, and appeared to correspond to clusters of infected host cells. The oxygen concentration in the remainder of the nodule was much higher. Interconnected intercellular air spaces were demonstrated by infiltration with India ink. Infiltration of the spaces with water greatly reduced oxygen concentration throughout the nodule, indicating that they function in supplying oxygen to the infected cells and remainder of the nodule. These results differ from those found previously for soybean nodules and provide evidence that legume and actinorhizal nodules have different mechanisms for protecting nitrogenase from oxygen.