Planta (1984)161:193-200
P l a n t a 9 Springer-Verlag 1984
Calcium-oxalate crystals and crystal cells in determinate root nodules of legumes J.M. Sutherland and J.I. Sprent Department of Biological Sciences, University of Dundee, Dundee DD1 4HN, U K
Abstract. Early reports of the presence of calcium-
oxalate crystals in the cortices of Phaseolus vulgaris root nodules have been confirmed. Crystals were found in all six genera examined (Cajanus, Desmodium, Glycine, Lespedeza, Phaseolus, Vigna) that have determinate nodules and export ureides. They were absent from six genera examined that have indeterminate nodules and export amides. The possible physiological significance of these structures is discussed. Key words: Calcium oxalate (root nodules) - Root
nodule.
Introduction
Calcium-oxalate crystals occur widely throughout the plant kingdom and have been found in most plant tissues (for review, see Franceschi and Horner 1980). However, early reports (Spratt 1919; Fraser 1942) of their occurrence in nodules of Phaseolus have been overlooked in recent studies. We report here on the identification of calciumoxalate crystals in the cortex of root nodules of Phaseolus vulgaris and other nodules with determinate growth and the absence of such crystals in indeterminate nodules. This finding is discussed in relation to other structural and functional differences between nodules of these types (Sprent 1980). Materials and methods Plant material. The plant species listed in Table I were grown in pots of vermiculite:perlite (1:1) and watered as necessary with deionized water or N-free nutrient solution. All were effectively nodulated with either indigenous U K rhizobia (Dorycnium, Ononis, Parochetus), or strains from CSIRO, Canberra
(Vigna spp.) or Rothamsted Experimental Station (all others). With the exception of Vigna spp., all plants were grown in a greenhouse in Dundee, with natural daylight supplemented with 40-W fluorescent tubes to give a 14-h daylength, and day temperature 20-30 ~ C, night temperature 15-25 ~ C. Vigna spp. were from plants grown in the Canberra phytotron, with a 14-h light period and a day/night temperature regime of 28/23 ~ C. Active nitrogen fixing nodules were harvested from vegetative plants of various ages.
Clearing. Nodules were cleared by heating in 5% N a O H at 200 kPa for 10 min in a pressure cooker, followed by soaking overnight in hydrogen peroxide. Sections for light microseopy. Nodules were fixed in 3% glutaraldehyde in 25 tool m-3 potassium-phosphate buffer, pH 6.8 for 3 h at room temperature or overnight at 4 ~ C. They were then dehydrated in an ethanol series, embedded in Spurr's (1969) or LR white resin (London Resin Co., Basingstoke, Hants., UK) and sectioned on a Reichert Ultratome III ultramicrotome (C. Reichert, Optische Werke, Vienna, Austria).
Toluidine blue staining. Spurr's resin sections were stained with 0.5% toluidine blue 0 in 12 tool m 3 sodium-carbonate solution (pH 11.1) and LR white resin sections were stained with 0.05% toluidine blue 0 in 20 tool m - 3 benzoate buffer, pH 4.4 (O'Brien and McCully 1981). Staining for calcium oxalate. The method of Yasue (1969) for the determination of calcium oxalate was carried out on hand sections of fresh nodules.
Transmission electron microscopy. Nodules were fixed as for light microscopy, rinsed in potassium-phosphate buffer and post-fixed in 1% osmium tetroxide for 1 h at room temperature, rinsed in buffer, dehydrated, embedded and sectioned on the ultramicrotome. Sections were stained with uranyl acetate and lead citrate and examined in a AEI 801 electron microscope (AEI/Kratos, Manchester, UK). Scanning electron microscopy. Glutaraldehyde-fixed nodules were dehydrated in an ethanol-water series, followed by an ethanol-freon series and then dried in a Polaron series II critical-point drying apparatus (Polaron Equipment, Watford, Herts. UK). They were then mounted on aluminium stubs, coated with gold-palladium in a Polaron E5100 coating unit and examined in a scanning electron microscope (JSM35; Jeol UK, London, UK).
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J.M. Sutherland and J.I. Sprent: Calcium oxalate crystals in leguminous root nodules
Edax analysis. Nodule pieces were plunged into liquid nitrogen, then placed on a metal block at liquid-nitrogen temperature for transfer to a freeze drier. Dried nodule pieces were transferred to carbon stubs, coated with carbon in a coating unit (E306A; Edwards High Vacuum, Crawley, Sussex, UK) and examined with an energy-dispersive X-ray microanalysis probe (Edax International, Prairie View; Ill., USA) fitted to a Jeol JSM35 scanning electron microscope.
Results
Crystals were found in the outer cortical cells of the nitrogen-fixing nodules of Phaseolus vulgaris. The various tissues of a typical P. vulgaris nodule are illustrated in the scanning electron micrograph (Fig. 1) of a longitudinal section of a nodule and transverse section of the subtending root. The central infected tissue is surrounded by a cortex in which lie the vascular bundles : the location of crystals is indicated by arrows. Their location is further illustrated in the light micrograph of a section of a plastic-embedded nodule (Fig. 2). Crystals were extracted during processing for microscopy, but the crystal-shaped holes which remained (arrows) can be seen between the large cortical cells and the lenticel. When nodules were cleared in sodium hydroxide followed by hydrogen peroxide, the crystals were extracted but the surrounding walls were retained. These cleared nodules, when halved and squashed, showed that the resistant crystal-surrounding walls were unevenly distributed in a reticulate pattern around the nodule (Fig. 3) and formed projections at either end where they joined to cortical cell walls (Fig. 4). There was no apparent relationship between the crystals and the nodule endodermis (identified by its staining with Sudan black), but crystals were often found immediately outside the endodermis of vascular bundles in nodules of Phaseolus vulgaris (Fig. 6) and Cajanus cajan (not shown). The crystals appeared to have been deposited within cortical cells and thereafter to be surrounded by new cell-wall material. A cortical cell layer in or near which they were located was retained in the cleared material and the cells of this layer were close packed and devoid of intercellular spaces (Fig. 4). The new wall material was unevenly deposited around the crystals and was thickened at either one side or corner (Fig. 7) or at opposite corners (Fig. 9). This was particularly evident when crystals were examined by scanning electron microscopy, when corners or lateral projections of the crystals often appeared to be exposed from the wall material (Fig. 8). Examination by transmission electron microscopy, however,
showed that, as well as the unevenly thick fibrillar material, the crystals were surrounded by a thin (approx. 100 nm) electron translucent layer (Figs. 10, 1 ~). The abrupt termination of this layer shown in the thin section in Fig. 11 indicates that its deposition may be localised. The fibrillar wall material when stained for light microscopy with toluidine blue, appeared either reddish-purple as did other cortical cell walls or a similar pale green colour to that of the lignifled walls of the sclereids (Fig. 7) and xylem. In hand sections of fresh material stained with Sudan black, a very thin layer immediately surrounding the crystals, probably equivalent to the electrontranslucent layer, was stained bluish-black indicating the presence of lipid or suberin. This layer stained a slightly darker green colour with toluidine blue than did the remainder of the wall. Since the suberised lamella of the endodermis also stains green with toluidine blue, there may be more than a superficial similarity between the layers. The presence in these crystal-surrounding walls of either lignin or suberin would explain their resistance to nodule clearing agents. They also prevented isolation of the crystals for identification by X-ray diffraction, so other means of identification were sought. The crystals were stained positively by the Yasue (1969) method for calcium oxalate, but this staining was irregular perhaps because of differences in the stage of development of the wall layers. After removal of soluble oxalates from slices of fresh nodules, heating with solid diphenylamine produced the blue colour of aniline blue when taken up in ethanol. This reaction is selective for oxalic acid and insoluble oxalates (Feigl and Frehden 1935; Hodgkinson 1977). X-ray microprobe analysis of nodule slices pre-
Fig. 1. Scanning electron micrograph showing a transverse section of root (R) and longitudinal section of a nodule of Phaseolus vulgaris. Crystals are generally found in the layers of the outer cortex indicated by arrows, x 40 Fig. 2. Light micrograph of a section of P. vulgaris nodule showing infected cells, a vascular bundle (V), a lenticel (L) and several crystals (arrows). LR white resin, toluidine blue 0 stain. x 150 Fig. 3. Light micrograph of a cleared, squashed P. vulgaris nodule showing the crystal-containing layer of the outer cortex and the cell walls which form around the crystals. The crystals were extracted by the clearing process, x 200 Fig. 4. Light-micrograph of tissue prepared as in Fig. 3 showing the crystal-surrounding cell walls with lateral projections at the ends. The layer of cortical cells shown does not contain intercellular spaces. Phase-contrast optics, x 700
J.M. Sutherland and J.I. Sprent: Calcium oxalate crystals in leguminous root nodules
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196
J.M. Sutherland and J.I. Sprent: Calcium oxalate crystals in leguminous root nodules
Figs. 5-7. Light micrographs of sections of nodules embedded in LR white resin and stained with toluidine blue 0. Fig. 5. Outer cortex of a Phaseolus vulgaris nodule showing a longitudinal section of a crystal surrounded by an irregular wall which has stained less darkly than the surrounding cell walls, x 550. Fig. 6. Part of a vascular bundle of P, vulgaris nodule showing a longitudinal section of a crystal (arrows) in a cell adjacent to the bundle endodermis (e). x 530. Fig. 7. Outer cortex of a Cajanus cajan nodule showing crystals (arrows) attached to the inner walls of parenchyma cells adjacent to layers of sclereids (5). One edge of the crystal is embedded in wall material stained similar to that of the lignified sclereid layer, x 760,
pared as for scanning electron microscopy showed that the crystals contained high levels of calcium. Though not directly quantitative, comparison of peak heights above background gave ratios for C a + + : K + of 0.2:1; 1.5:1 and 13:1 in infected cells, cortical cells and crystals respectively (Fig. 12). These data taken together were considered sufficient evidence for the identification of these crys-
tals as calcium oxalate. The species in which crystals were or were not found are listed in Table 1 (classification after Polhill and Raven 1981). Discussion
The crystals found here are similar in appearance to the calcium-oxalate crystals found in the leaves of Rhynchosia caribaea (Horner and Zindler-Frank
J.M. Sutherland and J.I, Sprent : Calcium oxalate crystals in leguminous root nodules
197
Fig. 8. Scanning electron micrograph of a freeze-dried section of a Phaseolus vulgaris nodule showing a crystal incompletely surrounded by wall material and suspended within a cortical cell. x 2,500 Figs. 9-11. Transmission electron micrographs of sections of Phaseolus vulgaris nodules showing holes from which crystals have been extracted during processing. Fig. 9. Thickened areas of fibrillar wall material appear at opposite corners of the hole (arrows). x 9,000. Fig. 10. The wall layer adjacent to the hole is similar in appearance to suberised wall layers found in other tissues. x 32,000. Fig. 11. The suberin-like layer appears incomplete (arrow). x 48,000
1982 a) and in Phaseolus vulgaris veins and Canavalia ensiformis epidermal cells (Homer and ZindlerFrank 1982b), but differ from those found in nonleguminous species. The electron-translucent layer surrounding the crystals described here is similar in appearance to the suberised lamellae of endodermal cells (Sutherland 1976; Scott and Peterson 1979) and also to the suberised layer surrounding styloid calciumoxalate crystals of Agave americana leaves (Wattendorff 1976; Espelie et al. 1982). It did not, how-
ever, show the lamellate substructure of the other suberised layers, but this may have been caused by the thickness of the sections required in order to preserve the integrity of the holes in the electron beam. Such elaborate packaging supports the view that calcium-oxalate crystals are a form of waste product. However, considerable evidence to the contrary exists (see Hodgkinson 1977; Franceschi and Horner 1980). Dwarte and Ashford (1982) showed that the calcium-oxalate crystals of celery endosperm, which are not surrounded by cell walls,
198
J.M. Sutherland and J.I. Sprent: Calcium oxalate crystals in leguminous root nodules
Infected cell
Cortex
Crystal
Na
PSCI
K Ca
Fig. 12. Typical energy spectra obtained by X-ray microanalysis of infected cells, cortical cells and crystals. The Ca § § :K § ratios are 0.2:1, 1.5 : 1 and 13 : 1, respectively
undergo degradation during germination. This prompts the suggestion that the presence or absence of surrounding walls may provide some indication of the function of the crystals in a particular situation since it seems clear that crystal synthesis serves more than one purpose. Frank (1975) suggested that there are two groups of oxalate-containing plants; those in which the oxalate content rises with increasing nutritional calcium, and those in which the oxatate is independent of calcium. The crystals observed in the present work are deposited in that area of the nodule where the calcium: potassium ratio is highest and they are frequently located in or near lignified and-or suberised cells which might provide a barrier to apoplastic transport, resulting in a local build-up of calcium (Harrison-Murray and Clarkson 1973). However, the calcium:potassium level of nodules which do not accumulate oxalate crystals is also high relative to that of the infected cells (Yousef 1982), so that further information is required to determine the dependence or otherwise of the oxalate crystals of determinate nodules on the calcium nutrition of the plant. The absence of intercellular spaces in the crystal-containing layer(s) of the cortex may provide the barrier to free gas diffusion, which Sinclair and Goudriaan (1981) have calculated must exist in the cortex if the observed rates of gas exchange are to be explained. The metabolic pathways of oxalate production in plants are discussed by Hodgkinson (1977), Raven etal. (1982) and Franceschi and H o m e r (1980). Of the four possible pathways discussed by these authors, we discount that involving glyoxylate production via the photosynthetic carbon oxidation cycle and, for the present, that involving ascorbate synthesis, since we have no information
Table 1. Features of species examined for the presence of calcium-oxalate crystals Tribe
Species
Crystals
Nodule growth
Export product
Phaseoleae
Cajanus cajan GIycine max Phaseolus vulgaris Vigna mungo Vigna radiata Desrnodiurn dillenii Lespedeza thunbergia Dorycniurn rectum ( = Lotus rectum) Pisurn sativurn Vicia faba Ononis repens Parochetus cornmunis Lupinus albus
Present Present Present Present Present Present Present Absent
Determinate Determinate Determinate Determinate Determinate Determinate Determinate Indeterminate
Ureides Ureides Ureides Ureides Ureides Ureides Ureides Amides
Absent Absent Absent Absent Absent
Indeterminate Indeterminate Indeterminate Indeterminate Indeterminate
Amides Amides Amides Amides Amides
Desmodieae Loteae Viceae Trifolieae Genisteae
J.M. Sutherland and J.I. Sprent: Calcium oxalate crystals in leguminous root nodules
on ascorbate synthesis in nodules. The other possible routes are (1) from glyoxylate produced during the glyoxylate cycle, and (2) from oxaloacetate. There is little information on the glyoxylate cycle in nodules. Johnson et al. (1966) could find little or no isocitrate lyase (EC 4.1.3.1) or malate synthetase (EC 4.1.3.2) in the nodule cytosol, nor was malate synthetase found in bacteroids of the fastgrowing species Rhizobium leguminosarum, R. meliloti or R. trifolii (all of which occur in indeterminate nodules). It was, however, present in the slowgrowing species R. phaseoli and R. japonicum, both of which occur in determinate nodules and R. lupini which infects lupins, producing a type of indeterminate nodule. Thus the presence of malate synthetase in bacteroids does not correlate exactly with the presence of calcium-oxalate crystals in nodules. In any case, it is difficult to imagine how the operation of the glyoxylate cycle in bacterioids might influence synthesis of calcium oxalate in the nodule cortex, although as Rawsthorne et al. (1980) have pointed out there is little information on the exchange of carbon compounds between the two symbionts. Synthesis of oxalate from oxaloacetate via oxaloacetase (EC 3.7.1.1) is a pathway for which there is no direct information in nodules. Both types of nodule can produce oxaloacetate by the action of phosphoenolpyruvate carboxylase (see for example Layzell et al. 1979), and much of this may be used for amino-acid synthesis. Since they do not export large quantities of amino acids (although they use them for ureide biosynthesis), determinate nodules may have excess oxaloacetate which could be turned into oxalate. A further possibility for oxalate production is linked to the facts that all the crystal-containing nodules are determinate and assimilate their fixed nitrogen into ureides for export. In such nodules, the first-formed products of nitrogen fixation are used for nodule expansion rather than export (see, for example, Wilson and Umbreit 1937). The presence of high levels of urease (EC 3.5.1.5) in the expansion phase of Cajanus cajan nodules (this information can be derived from the data of Luthra et al. 1983) is consistent with nodule cells using ureides for expansion growth. Breakdown of ureides in bacteria and in leaves of soybean uses allantoicase (EC 3.5.3.4) or ureido glycolase (EC 4.3.2.3) (for review, see Vogels and Van der Drift 1976; Thomas and Schrader 1981; Matsumoto et al. 1982) and one of the products is glyoxylate. There is, as yet, no evidence for these enzymes in nodules. However, if glyoxylate were produced in this way, xanthine oxidoreductase (EC 1.2.3.2),
199
which is present in nodules, can also oxidise glyoxylate to oxalate (Hodgkinson 1977). We should like to thank the Agricultural Research Council for financial support and Professor J.A. Raven and Dr. R.J. Thomas (Dundee) and Dr. T.M. Sinclair (Florida) for stimulating discussions.
References Dwarte, D., Ashford, A.E. (1982) The chemistry and microstructure of protein bodies in celery endosperm. Bot. Gaz. (Chicago) 143, 164-175 Espelie, K.E., Wattendorff, J., Kolattukudy, P.E. (1982) Composition and ultrastructure of the suberized cell wall of isolated crystal idioblasts from Agave americana L. leaves. Planta 155, 166-175 Frank, E. (1975) On the formation of the pattern of crystal idioblasts in Canavalia ensiformis D.C. VII. Calcium and oxalate content on the leaves in dependence of calcium nutrition. Z. Pflanzenphysiol. 77, 80-85 Feigl, F., Frehden, D. (1935) Nachweis organischer Verbindungen mit Hilfe von Tiipfelreaktion. X. Mikrochemie 18, 272-276 Fraser, H.L. (1942) The occurrence of endodermis in leguminous root nodules and its effect upon nodule formation. Proc. R. Soc. Edinburgh Sect. B 61,328-343 Franceschi, V.R., Horner, H.T. (1980) Calcium oxalate crystals in plants. Bot. Rev. 46, 361427 Harrison-Murray, R.S., Clarkson, D.T. (1973) Relationships between structural development and absorption of ions by the root system of Cucurbitapepo. Planta 114, 1 16 Hodgkinson, A. (1977) Oxalic acid in biology and medicine. Academic Press, London New York Homer, H.T., Zindler-Frank, E. (1982a) Calcium oxalate crystals and crystal cells in the leaves of Rhynchosia caribaea (Leguminosae: Papilionoidae). Protoplasma 111, 10-18 Homer, H.T., Zindler-Frank, E. (1982b) Histochemical, spectroscopic, and X-ray diffraction identifications of the two hydration forms of calcium oxalate crystals in three legumes and Begonia. Can. J. Bot. 60, 1021-1027 Johnson, G.V., Evans, H.J., Ching, T.M. (1966) Enzymes of the glyoxylate cycle in rhizobia and nodules of legumes. Plant Physiol. 41, 1330-1336 Layzell, D.B., Rainbird, R.M., Atkins, C.A., Pate, J.S. (1979) Economy of photosynthate use in nitrogen-fixing legume nodules. Observations on two contrasting symbioses. Plant Physiol. 64, 888-891 Luthra, Y.P., Sheoran, I.S., Rao, A.S., Singh, R. (1983) Ontogenetic changes in the level of ureides and enzymes in their metabolism in various plant parts of pigeonpea Cajanus cajan. J. Exp. Bot. 34, 1358-1370 Matsumoto, T., Blevins, D.G., Randall, D.D. (1982) Soybean leaf ureide metabolism: allantoiease, ureidogyleolase and urease (Abstr.). Plant Physiol. 69, Suppl., 117 O'Brien, T.P., McCully, M.E. (1981) The study of plant structure. Principles and selected methods. Termarcarphi Pty. Melbourne, Australia Polhill, R.M., Raven, P.H. (eds) (1981) Advances in legume systematics. Royal Botanic Gardens, Kew, UK Rawsthorne, S., Minchin, F.R., Summerfield, R.J., Cookson, C., Coombs, J. (1980) Carbon and nitrogen metabolism in legume root nodules. Phytochemistry 19, 341-355 Raven, J.A., Griffiths, H., Glidewell, S.M., Preston, T. (1982) The mechanism of oxalate biosynthesis in higher plants:
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investigations with the stable isotopes 1so and ~3C. Proc. R. Soc. London Ser. B 216, 87-101 Scott, M.G., Peterson, R.L. (1979) The root endodermis in Ranunculus acris 1. Structure and ontogeny. Can. J. Bot. 57, 1040-1062 Sinclair, T.M., Goudriaan, J. (1981) Physical and morphological constraints on transport in nodules. Plant. Physiol. 67, 143-145 Spratt, E.R. (1919) A comparative account of the root nodules of the Leguminosae. Ann. Bot. (London) 33, 181-191 Sprent, J.I. (1980) Root nodule anatomy, type of export product and evolutionary origin in some Leguminosae. Plant Cell Environ. 3, 35-43 Spurr, A.R. (1969) A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 31-43 Sutherland, J.M. (1976) Observations on the development of the endodermis in Zea mays roots. M.Sc. thesis, Carlton University, Ottawa
Thomas, R.J., Schrader, L.E. (1981) Ureide metabolism in higher plants. Phytochemistry 20, 361-371 Vogels, G.D., Van der Drift, C. (1976) Degradation of purines and pyrimidines by microorganisms. Bacteriol. Rev. 40, 403-468 Wattendorff, J. (1976) Ultrastructure of the suberized styloid crystal cells in Agave leaves. Planta 128, 163-165 Wilson, P.W., Umbreit, W.W. (1937) Fixation and transfer of nitrogen in the soybean. Zentralbl. Bakteriol. Parasitenkd. Infektionsk. Hyg. Abt. 2 96, 402-411 Yasue, T. (1969) Histochemical identification of calcium oxalate. Acta Histochem. Cytochem. 2, 83-95 Yousef, A.N. (1982) Effects of salt stress on symbiotic nitrogen fixation in Viciafaba (L.). Ph.D. thesis, University of Dundee, UK Received 29 November 1983; accepted 2 February 1984
P l a n t a 9 Springer-Verlag 1984
Calcium-oxalate crystals and crystal cells in determinate root nodules of legumes J.M. Sutherland and J.I. Sprent Department of Biological Sciences, University of Dundee, Dundee DD1 4HN, U K
Abstract. Early reports of the presence of calcium-
oxalate crystals in the cortices of Phaseolus vulgaris root nodules have been confirmed. Crystals were found in all six genera examined (Cajanus, Desmodium, Glycine, Lespedeza, Phaseolus, Vigna) that have determinate nodules and export ureides. They were absent from six genera examined that have indeterminate nodules and export amides. The possible physiological significance of these structures is discussed. Key words: Calcium oxalate (root nodules) - Root
nodule.
Introduction
Calcium-oxalate crystals occur widely throughout the plant kingdom and have been found in most plant tissues (for review, see Franceschi and Horner 1980). However, early reports (Spratt 1919; Fraser 1942) of their occurrence in nodules of Phaseolus have been overlooked in recent studies. We report here on the identification of calciumoxalate crystals in the cortex of root nodules of Phaseolus vulgaris and other nodules with determinate growth and the absence of such crystals in indeterminate nodules. This finding is discussed in relation to other structural and functional differences between nodules of these types (Sprent 1980). Materials and methods Plant material. The plant species listed in Table I were grown in pots of vermiculite:perlite (1:1) and watered as necessary with deionized water or N-free nutrient solution. All were effectively nodulated with either indigenous U K rhizobia (Dorycnium, Ononis, Parochetus), or strains from CSIRO, Canberra
(Vigna spp.) or Rothamsted Experimental Station (all others). With the exception of Vigna spp., all plants were grown in a greenhouse in Dundee, with natural daylight supplemented with 40-W fluorescent tubes to give a 14-h daylength, and day temperature 20-30 ~ C, night temperature 15-25 ~ C. Vigna spp. were from plants grown in the Canberra phytotron, with a 14-h light period and a day/night temperature regime of 28/23 ~ C. Active nitrogen fixing nodules were harvested from vegetative plants of various ages.
Clearing. Nodules were cleared by heating in 5% N a O H at 200 kPa for 10 min in a pressure cooker, followed by soaking overnight in hydrogen peroxide. Sections for light microseopy. Nodules were fixed in 3% glutaraldehyde in 25 tool m-3 potassium-phosphate buffer, pH 6.8 for 3 h at room temperature or overnight at 4 ~ C. They were then dehydrated in an ethanol series, embedded in Spurr's (1969) or LR white resin (London Resin Co., Basingstoke, Hants., UK) and sectioned on a Reichert Ultratome III ultramicrotome (C. Reichert, Optische Werke, Vienna, Austria).
Toluidine blue staining. Spurr's resin sections were stained with 0.5% toluidine blue 0 in 12 tool m 3 sodium-carbonate solution (pH 11.1) and LR white resin sections were stained with 0.05% toluidine blue 0 in 20 tool m - 3 benzoate buffer, pH 4.4 (O'Brien and McCully 1981). Staining for calcium oxalate. The method of Yasue (1969) for the determination of calcium oxalate was carried out on hand sections of fresh nodules.
Transmission electron microscopy. Nodules were fixed as for light microscopy, rinsed in potassium-phosphate buffer and post-fixed in 1% osmium tetroxide for 1 h at room temperature, rinsed in buffer, dehydrated, embedded and sectioned on the ultramicrotome. Sections were stained with uranyl acetate and lead citrate and examined in a AEI 801 electron microscope (AEI/Kratos, Manchester, UK). Scanning electron microscopy. Glutaraldehyde-fixed nodules were dehydrated in an ethanol-water series, followed by an ethanol-freon series and then dried in a Polaron series II critical-point drying apparatus (Polaron Equipment, Watford, Herts. UK). They were then mounted on aluminium stubs, coated with gold-palladium in a Polaron E5100 coating unit and examined in a scanning electron microscope (JSM35; Jeol UK, London, UK).
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J.M. Sutherland and J.I. Sprent: Calcium oxalate crystals in leguminous root nodules
Edax analysis. Nodule pieces were plunged into liquid nitrogen, then placed on a metal block at liquid-nitrogen temperature for transfer to a freeze drier. Dried nodule pieces were transferred to carbon stubs, coated with carbon in a coating unit (E306A; Edwards High Vacuum, Crawley, Sussex, UK) and examined with an energy-dispersive X-ray microanalysis probe (Edax International, Prairie View; Ill., USA) fitted to a Jeol JSM35 scanning electron microscope.
Results
Crystals were found in the outer cortical cells of the nitrogen-fixing nodules of Phaseolus vulgaris. The various tissues of a typical P. vulgaris nodule are illustrated in the scanning electron micrograph (Fig. 1) of a longitudinal section of a nodule and transverse section of the subtending root. The central infected tissue is surrounded by a cortex in which lie the vascular bundles : the location of crystals is indicated by arrows. Their location is further illustrated in the light micrograph of a section of a plastic-embedded nodule (Fig. 2). Crystals were extracted during processing for microscopy, but the crystal-shaped holes which remained (arrows) can be seen between the large cortical cells and the lenticel. When nodules were cleared in sodium hydroxide followed by hydrogen peroxide, the crystals were extracted but the surrounding walls were retained. These cleared nodules, when halved and squashed, showed that the resistant crystal-surrounding walls were unevenly distributed in a reticulate pattern around the nodule (Fig. 3) and formed projections at either end where they joined to cortical cell walls (Fig. 4). There was no apparent relationship between the crystals and the nodule endodermis (identified by its staining with Sudan black), but crystals were often found immediately outside the endodermis of vascular bundles in nodules of Phaseolus vulgaris (Fig. 6) and Cajanus cajan (not shown). The crystals appeared to have been deposited within cortical cells and thereafter to be surrounded by new cell-wall material. A cortical cell layer in or near which they were located was retained in the cleared material and the cells of this layer were close packed and devoid of intercellular spaces (Fig. 4). The new wall material was unevenly deposited around the crystals and was thickened at either one side or corner (Fig. 7) or at opposite corners (Fig. 9). This was particularly evident when crystals were examined by scanning electron microscopy, when corners or lateral projections of the crystals often appeared to be exposed from the wall material (Fig. 8). Examination by transmission electron microscopy, however,
showed that, as well as the unevenly thick fibrillar material, the crystals were surrounded by a thin (approx. 100 nm) electron translucent layer (Figs. 10, 1 ~). The abrupt termination of this layer shown in the thin section in Fig. 11 indicates that its deposition may be localised. The fibrillar wall material when stained for light microscopy with toluidine blue, appeared either reddish-purple as did other cortical cell walls or a similar pale green colour to that of the lignifled walls of the sclereids (Fig. 7) and xylem. In hand sections of fresh material stained with Sudan black, a very thin layer immediately surrounding the crystals, probably equivalent to the electrontranslucent layer, was stained bluish-black indicating the presence of lipid or suberin. This layer stained a slightly darker green colour with toluidine blue than did the remainder of the wall. Since the suberised lamella of the endodermis also stains green with toluidine blue, there may be more than a superficial similarity between the layers. The presence in these crystal-surrounding walls of either lignin or suberin would explain their resistance to nodule clearing agents. They also prevented isolation of the crystals for identification by X-ray diffraction, so other means of identification were sought. The crystals were stained positively by the Yasue (1969) method for calcium oxalate, but this staining was irregular perhaps because of differences in the stage of development of the wall layers. After removal of soluble oxalates from slices of fresh nodules, heating with solid diphenylamine produced the blue colour of aniline blue when taken up in ethanol. This reaction is selective for oxalic acid and insoluble oxalates (Feigl and Frehden 1935; Hodgkinson 1977). X-ray microprobe analysis of nodule slices pre-
Fig. 1. Scanning electron micrograph showing a transverse section of root (R) and longitudinal section of a nodule of Phaseolus vulgaris. Crystals are generally found in the layers of the outer cortex indicated by arrows, x 40 Fig. 2. Light micrograph of a section of P. vulgaris nodule showing infected cells, a vascular bundle (V), a lenticel (L) and several crystals (arrows). LR white resin, toluidine blue 0 stain. x 150 Fig. 3. Light micrograph of a cleared, squashed P. vulgaris nodule showing the crystal-containing layer of the outer cortex and the cell walls which form around the crystals. The crystals were extracted by the clearing process, x 200 Fig. 4. Light-micrograph of tissue prepared as in Fig. 3 showing the crystal-surrounding cell walls with lateral projections at the ends. The layer of cortical cells shown does not contain intercellular spaces. Phase-contrast optics, x 700
J.M. Sutherland and J.I. Sprent: Calcium oxalate crystals in leguminous root nodules
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J.M. Sutherland and J.I. Sprent: Calcium oxalate crystals in leguminous root nodules
Figs. 5-7. Light micrographs of sections of nodules embedded in LR white resin and stained with toluidine blue 0. Fig. 5. Outer cortex of a Phaseolus vulgaris nodule showing a longitudinal section of a crystal surrounded by an irregular wall which has stained less darkly than the surrounding cell walls, x 550. Fig. 6. Part of a vascular bundle of P, vulgaris nodule showing a longitudinal section of a crystal (arrows) in a cell adjacent to the bundle endodermis (e). x 530. Fig. 7. Outer cortex of a Cajanus cajan nodule showing crystals (arrows) attached to the inner walls of parenchyma cells adjacent to layers of sclereids (5). One edge of the crystal is embedded in wall material stained similar to that of the lignified sclereid layer, x 760,
pared as for scanning electron microscopy showed that the crystals contained high levels of calcium. Though not directly quantitative, comparison of peak heights above background gave ratios for C a + + : K + of 0.2:1; 1.5:1 and 13:1 in infected cells, cortical cells and crystals respectively (Fig. 12). These data taken together were considered sufficient evidence for the identification of these crys-
tals as calcium oxalate. The species in which crystals were or were not found are listed in Table 1 (classification after Polhill and Raven 1981). Discussion
The crystals found here are similar in appearance to the calcium-oxalate crystals found in the leaves of Rhynchosia caribaea (Horner and Zindler-Frank
J.M. Sutherland and J.I, Sprent : Calcium oxalate crystals in leguminous root nodules
197
Fig. 8. Scanning electron micrograph of a freeze-dried section of a Phaseolus vulgaris nodule showing a crystal incompletely surrounded by wall material and suspended within a cortical cell. x 2,500 Figs. 9-11. Transmission electron micrographs of sections of Phaseolus vulgaris nodules showing holes from which crystals have been extracted during processing. Fig. 9. Thickened areas of fibrillar wall material appear at opposite corners of the hole (arrows). x 9,000. Fig. 10. The wall layer adjacent to the hole is similar in appearance to suberised wall layers found in other tissues. x 32,000. Fig. 11. The suberin-like layer appears incomplete (arrow). x 48,000
1982 a) and in Phaseolus vulgaris veins and Canavalia ensiformis epidermal cells (Homer and ZindlerFrank 1982b), but differ from those found in nonleguminous species. The electron-translucent layer surrounding the crystals described here is similar in appearance to the suberised lamellae of endodermal cells (Sutherland 1976; Scott and Peterson 1979) and also to the suberised layer surrounding styloid calciumoxalate crystals of Agave americana leaves (Wattendorff 1976; Espelie et al. 1982). It did not, how-
ever, show the lamellate substructure of the other suberised layers, but this may have been caused by the thickness of the sections required in order to preserve the integrity of the holes in the electron beam. Such elaborate packaging supports the view that calcium-oxalate crystals are a form of waste product. However, considerable evidence to the contrary exists (see Hodgkinson 1977; Franceschi and Horner 1980). Dwarte and Ashford (1982) showed that the calcium-oxalate crystals of celery endosperm, which are not surrounded by cell walls,
198
J.M. Sutherland and J.I. Sprent: Calcium oxalate crystals in leguminous root nodules
Infected cell
Cortex
Crystal
Na
PSCI
K Ca
Fig. 12. Typical energy spectra obtained by X-ray microanalysis of infected cells, cortical cells and crystals. The Ca § § :K § ratios are 0.2:1, 1.5 : 1 and 13 : 1, respectively
undergo degradation during germination. This prompts the suggestion that the presence or absence of surrounding walls may provide some indication of the function of the crystals in a particular situation since it seems clear that crystal synthesis serves more than one purpose. Frank (1975) suggested that there are two groups of oxalate-containing plants; those in which the oxalate content rises with increasing nutritional calcium, and those in which the oxatate is independent of calcium. The crystals observed in the present work are deposited in that area of the nodule where the calcium: potassium ratio is highest and they are frequently located in or near lignified and-or suberised cells which might provide a barrier to apoplastic transport, resulting in a local build-up of calcium (Harrison-Murray and Clarkson 1973). However, the calcium:potassium level of nodules which do not accumulate oxalate crystals is also high relative to that of the infected cells (Yousef 1982), so that further information is required to determine the dependence or otherwise of the oxalate crystals of determinate nodules on the calcium nutrition of the plant. The absence of intercellular spaces in the crystal-containing layer(s) of the cortex may provide the barrier to free gas diffusion, which Sinclair and Goudriaan (1981) have calculated must exist in the cortex if the observed rates of gas exchange are to be explained. The metabolic pathways of oxalate production in plants are discussed by Hodgkinson (1977), Raven etal. (1982) and Franceschi and H o m e r (1980). Of the four possible pathways discussed by these authors, we discount that involving glyoxylate production via the photosynthetic carbon oxidation cycle and, for the present, that involving ascorbate synthesis, since we have no information
Table 1. Features of species examined for the presence of calcium-oxalate crystals Tribe
Species
Crystals
Nodule growth
Export product
Phaseoleae
Cajanus cajan GIycine max Phaseolus vulgaris Vigna mungo Vigna radiata Desrnodiurn dillenii Lespedeza thunbergia Dorycniurn rectum ( = Lotus rectum) Pisurn sativurn Vicia faba Ononis repens Parochetus cornmunis Lupinus albus
Present Present Present Present Present Present Present Absent
Determinate Determinate Determinate Determinate Determinate Determinate Determinate Indeterminate
Ureides Ureides Ureides Ureides Ureides Ureides Ureides Amides
Absent Absent Absent Absent Absent
Indeterminate Indeterminate Indeterminate Indeterminate Indeterminate
Amides Amides Amides Amides Amides
Desmodieae Loteae Viceae Trifolieae Genisteae
J.M. Sutherland and J.I. Sprent: Calcium oxalate crystals in leguminous root nodules
on ascorbate synthesis in nodules. The other possible routes are (1) from glyoxylate produced during the glyoxylate cycle, and (2) from oxaloacetate. There is little information on the glyoxylate cycle in nodules. Johnson et al. (1966) could find little or no isocitrate lyase (EC 4.1.3.1) or malate synthetase (EC 4.1.3.2) in the nodule cytosol, nor was malate synthetase found in bacteroids of the fastgrowing species Rhizobium leguminosarum, R. meliloti or R. trifolii (all of which occur in indeterminate nodules). It was, however, present in the slowgrowing species R. phaseoli and R. japonicum, both of which occur in determinate nodules and R. lupini which infects lupins, producing a type of indeterminate nodule. Thus the presence of malate synthetase in bacteroids does not correlate exactly with the presence of calcium-oxalate crystals in nodules. In any case, it is difficult to imagine how the operation of the glyoxylate cycle in bacterioids might influence synthesis of calcium oxalate in the nodule cortex, although as Rawsthorne et al. (1980) have pointed out there is little information on the exchange of carbon compounds between the two symbionts. Synthesis of oxalate from oxaloacetate via oxaloacetase (EC 3.7.1.1) is a pathway for which there is no direct information in nodules. Both types of nodule can produce oxaloacetate by the action of phosphoenolpyruvate carboxylase (see for example Layzell et al. 1979), and much of this may be used for amino-acid synthesis. Since they do not export large quantities of amino acids (although they use them for ureide biosynthesis), determinate nodules may have excess oxaloacetate which could be turned into oxalate. A further possibility for oxalate production is linked to the facts that all the crystal-containing nodules are determinate and assimilate their fixed nitrogen into ureides for export. In such nodules, the first-formed products of nitrogen fixation are used for nodule expansion rather than export (see, for example, Wilson and Umbreit 1937). The presence of high levels of urease (EC 3.5.1.5) in the expansion phase of Cajanus cajan nodules (this information can be derived from the data of Luthra et al. 1983) is consistent with nodule cells using ureides for expansion growth. Breakdown of ureides in bacteria and in leaves of soybean uses allantoicase (EC 3.5.3.4) or ureido glycolase (EC 4.3.2.3) (for review, see Vogels and Van der Drift 1976; Thomas and Schrader 1981; Matsumoto et al. 1982) and one of the products is glyoxylate. There is, as yet, no evidence for these enzymes in nodules. However, if glyoxylate were produced in this way, xanthine oxidoreductase (EC 1.2.3.2),
199
which is present in nodules, can also oxidise glyoxylate to oxalate (Hodgkinson 1977). We should like to thank the Agricultural Research Council for financial support and Professor J.A. Raven and Dr. R.J. Thomas (Dundee) and Dr. T.M. Sinclair (Florida) for stimulating discussions.
References Dwarte, D., Ashford, A.E. (1982) The chemistry and microstructure of protein bodies in celery endosperm. Bot. Gaz. (Chicago) 143, 164-175 Espelie, K.E., Wattendorff, J., Kolattukudy, P.E. (1982) Composition and ultrastructure of the suberized cell wall of isolated crystal idioblasts from Agave americana L. leaves. Planta 155, 166-175 Frank, E. (1975) On the formation of the pattern of crystal idioblasts in Canavalia ensiformis D.C. VII. Calcium and oxalate content on the leaves in dependence of calcium nutrition. Z. Pflanzenphysiol. 77, 80-85 Feigl, F., Frehden, D. (1935) Nachweis organischer Verbindungen mit Hilfe von Tiipfelreaktion. X. Mikrochemie 18, 272-276 Fraser, H.L. (1942) The occurrence of endodermis in leguminous root nodules and its effect upon nodule formation. Proc. R. Soc. Edinburgh Sect. B 61,328-343 Franceschi, V.R., Horner, H.T. (1980) Calcium oxalate crystals in plants. Bot. Rev. 46, 361427 Harrison-Murray, R.S., Clarkson, D.T. (1973) Relationships between structural development and absorption of ions by the root system of Cucurbitapepo. Planta 114, 1 16 Hodgkinson, A. (1977) Oxalic acid in biology and medicine. Academic Press, London New York Homer, H.T., Zindler-Frank, E. (1982a) Calcium oxalate crystals and crystal cells in the leaves of Rhynchosia caribaea (Leguminosae: Papilionoidae). Protoplasma 111, 10-18 Homer, H.T., Zindler-Frank, E. (1982b) Histochemical, spectroscopic, and X-ray diffraction identifications of the two hydration forms of calcium oxalate crystals in three legumes and Begonia. Can. J. Bot. 60, 1021-1027 Johnson, G.V., Evans, H.J., Ching, T.M. (1966) Enzymes of the glyoxylate cycle in rhizobia and nodules of legumes. Plant Physiol. 41, 1330-1336 Layzell, D.B., Rainbird, R.M., Atkins, C.A., Pate, J.S. (1979) Economy of photosynthate use in nitrogen-fixing legume nodules. Observations on two contrasting symbioses. Plant Physiol. 64, 888-891 Luthra, Y.P., Sheoran, I.S., Rao, A.S., Singh, R. (1983) Ontogenetic changes in the level of ureides and enzymes in their metabolism in various plant parts of pigeonpea Cajanus cajan. J. Exp. Bot. 34, 1358-1370 Matsumoto, T., Blevins, D.G., Randall, D.D. (1982) Soybean leaf ureide metabolism: allantoiease, ureidogyleolase and urease (Abstr.). Plant Physiol. 69, Suppl., 117 O'Brien, T.P., McCully, M.E. (1981) The study of plant structure. Principles and selected methods. Termarcarphi Pty. Melbourne, Australia Polhill, R.M., Raven, P.H. (eds) (1981) Advances in legume systematics. Royal Botanic Gardens, Kew, UK Rawsthorne, S., Minchin, F.R., Summerfield, R.J., Cookson, C., Coombs, J. (1980) Carbon and nitrogen metabolism in legume root nodules. Phytochemistry 19, 341-355 Raven, J.A., Griffiths, H., Glidewell, S.M., Preston, T. (1982) The mechanism of oxalate biosynthesis in higher plants:
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investigations with the stable isotopes 1so and ~3C. Proc. R. Soc. London Ser. B 216, 87-101 Scott, M.G., Peterson, R.L. (1979) The root endodermis in Ranunculus acris 1. Structure and ontogeny. Can. J. Bot. 57, 1040-1062 Sinclair, T.M., Goudriaan, J. (1981) Physical and morphological constraints on transport in nodules. Plant. Physiol. 67, 143-145 Spratt, E.R. (1919) A comparative account of the root nodules of the Leguminosae. Ann. Bot. (London) 33, 181-191 Sprent, J.I. (1980) Root nodule anatomy, type of export product and evolutionary origin in some Leguminosae. Plant Cell Environ. 3, 35-43 Spurr, A.R. (1969) A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 31-43 Sutherland, J.M. (1976) Observations on the development of the endodermis in Zea mays roots. M.Sc. thesis, Carlton University, Ottawa
Thomas, R.J., Schrader, L.E. (1981) Ureide metabolism in higher plants. Phytochemistry 20, 361-371 Vogels, G.D., Van der Drift, C. (1976) Degradation of purines and pyrimidines by microorganisms. Bacteriol. Rev. 40, 403-468 Wattendorff, J. (1976) Ultrastructure of the suberized styloid crystal cells in Agave leaves. Planta 128, 163-165 Wilson, P.W., Umbreit, W.W. (1937) Fixation and transfer of nitrogen in the soybean. Zentralbl. Bakteriol. Parasitenkd. Infektionsk. Hyg. Abt. 2 96, 402-411 Yasue, T. (1969) Histochemical identification of calcium oxalate. Acta Histochem. Cytochem. 2, 83-95 Yousef, A.N. (1982) Effects of salt stress on symbiotic nitrogen fixation in Viciafaba (L.). Ph.D. thesis, University of Dundee, UK Received 29 November 1983; accepted 2 February 1984
