Micron and Microscopica Acta, VoL 20, No. 1, pp. 33-36, 1989. 0739-6260/89 $3.00+0.00 Printed in Great Britain. Maxwell Pergamon Macmillan plc
SCANNING ELECTRON MICROSCOPY OF CALCIUM-RICH CRYSTALS IN FRANKIA ROOT NODULES OF ALNUS GLUTINOSA SAPLINGS
D. JONES,* W. J. MCHARDY,t M. J. WILSON* and G. I. COOPER S
*Division of Soil and Soil Microbiological Research and tAnalytical Division, The Macaulay Land Use Research Institute, Craigiebuckler, Aberdeen, AB9 2Q J, Scotland, U.K.
:~Crystallography Unit, Department of Geological Sciences, University College London, Gower Street, London WC l E 6BT, U.K.
(Received 23 January 1989)
Abstract--Birefringent clusters of crystals have been found in the cortex of root nodules in Alnus 91utinosa during a scanning electron microscopy study of cryofixed material. Such nodules are formed by nitrogen-fixing actinomycetes of the genus Frankia. The crystals were soluble in hydrochloric acid, insoluble in acetic acid and gave calcium only on energy dispersive X-ray micro-analysis. The evidence suggested that the crystals were composed of calcium oxalate. This appears to be the first report of oxalate in nitrogen-fixing nodules of non- leguminous species.
Index key words: Scanning electron microscopy, calcium oxalate, Frankia sp., Alnus glutinosa.
INTRODUCTION The Alnus-Frankia spp. actinorhizal symbio-
sis, leading to nitrogen-fixing processes beneficial to the tree host, is an important factor in forest management, according to Burgess and Peterson (1987), and it has been noted that the nitrogen fixed can surpass that fixed by Rhizobium and legume symbioses (Dawson, 1986). Information on this association between actinomycete and higher plant is desirable if we are to understand the physiological and biochemical intricacies between host and microbe symbionts.
During our studies on the ultrastructural features of cryofixed Frankia nodules on Alnus glutinosa, clusters of what appeared to be inor- ganic crystals were encountered and this note reports on their location and preliminary analy- sis. It would appear that no previous information on this aspect is available for Alnus, although crystals of calcium oxalate have been reported in the cortex of nitrogen-fixing root nodules of Phaseolus vulgaris and five other genera, Caja- nus, Desmodium, Glycine, Lespedeza and Vigna (Sutherland and Sprent, 1984), all leguminous plants. These authors were unable to provide
analysis data from X-ray diffraction techniques or infrared spectroscopy since the individual crystals were not sufficiently concentrated in the tissue. Their evidence for calcium oxalate was based on histochemical methods coupled with X-ray microprobe analysis.
MATERIALS AND METHODS Saplings of A. glutinosa L. Gaertn. with well
developed nodules, with a healthy appearance (Fig. 1), were obtained from The Forestry Com- mission Newton Nursery, Elgin, Moray, N.E. Scotland.
Nodules were prepared for scanning electron microscopy by two methods. The first involved no chemical fixatives, specimens being instantly frozen, by immersing in liquid nitrogen, prior to transfer to a Hexland cryo-stage attached to a Cambridge Instruments $4 scanning electron microscope (Jones and McHardy, 1985). The specimens were cryofractured and sputter-coated with gold before being examined in the frozen- hydrated state. The second method also involved cryofixation, with no chemical fixation, but the
34 D. Jones, W. J. McHardy, M. J. Wilson and G. I. Cooper
Fig. I. Root nodules (arrowedl on A. #lutinosa formed by the nitrogen-fixing actinomycete Frankia sp. Bar: 10 mm.
Fig. 2. Crystal cluster (arrowed) in cortex of cryofractured root nodule ofA. glutinosa. Frozen-hydrated, gold- coated. Bar: 40 um.
Fig. 3. Two crystal clusters in freeze-dried nodule. Secondary electron image of carbon-coated specimen, sliced before freezing. Bar: 10 rtm.
Fig. 4. Crystal cluster in freeze-dried nodule, previously freeze-fractured. Carbon-coated. Bar: 10 tim.
SEM of Crystals in Alnus glutinosa 35
specimens were allowed to freeze-dry completely on the Hexland cryo-stage. Some specimens were sliced into two halves prior to freezing, others were cryofractured on the cryo-stage. All were carbon-coated. Conventional secondary electron images and back-scattered images of the crystals were recorded. In addition, energy-dispersive spectra from point analyses of the crystals in carbon-coated specimens were recorded.
RESULTS AND DISCUSSION A cryofractured nodule illustrating the root
nodule cortex and conducting vessels is shown in Fig. 2. Surface ice was sublimed at -90C to reveal cellular structure. Extra-cellular aggre- gates of blocky or, perhaps, platy crystals from 10-15 ~tm dia and markedly birefringent, occurred frequently throughout the cortex. Intra-cellularly, the different morphological fea- tures of the actinomycete were apparent in the cortical cells and these included vesicles, sporan- gia and hyphae. Energy-dispersive spectra from electron probe analysis of the crystals in the variously prepared carbon-coated specimens revealed calcium with only a small amount of potassium (Fig. 5). A spectrum from a cortical cell with no crystals is given in Fig. 6 which reveals that the predominant detectable element is potassium. There may be some calcium present but the main K~ peak for calcium is overlapped by the K~ peak for potassium. This concentration of calcium was more clearly demonstrated by using back-scattered electron imagery where atomic number difference influences contrast. A conventional secondary electron image of a carbon-coated, freeze-dried specimen (sliced before freezing) showing crystal clusters, is illustrated in Fig. 3. The cellular structure of the nodule with a crystal cluster is more clearly defined in freeze-fractured specimens (Fig. 4). Histochemical tests were made because attempts to isolate crystal clusters from the nodules failed and no interpretable X-ray powder pattern or infrared spectra of the crystals in thin slices of the freeze-dried nodules was possible. The crystals proved to be soluble in 1 N HC1 but insoluble in 5% acetic acid indicating that the crystals were not calcium carbonate. Birkby and Preece (1988) have noted that Hodgkinson (1977) considers the best test for calcium oxalate is, firstly, to treat the specimen with 2 M acetic acid, followed by hydrochloric acid after drying the material over a
Fig. 5. Energy dispersive spectrum recorded from spot analysis of crystal.
KK -kCa K
X-ray Energy ~
Fig. 6. Energy dispersive spectrum recorded from spot analysis of cortical cell.
flame. The acid treatment results in the dissolv- ing of crystals with a resulting effervescence due to carbon dioxide being evolved. The process depends on the conversion of the oxalate to carbonate which is reactive with the acid. The crystals in the nodules certainly dissolved in the hydrochloric acid after treatment with the acetic acid, although effervescence was not positively observed. An attempt was made to analyse the crystal clusters by Fourier Transform infrared spectroscopy integrated with an optical micro- scope. Difficulties were encountered due to the cellular background components in hand-cut thin sections of the freeze-dried nodules, but an interpretable spectrum was obtained which iden- tified the crystals as calcium oxalate mono- hydrate (whewellite).
36 D. Jones, W. J. McHardy, M. J. Wilson and G. 1. Cooper
No crystals were observed in the non-nodular roots of the saplings. Neither were crystals found in non-nodular roots of much older trees which were characterized by having sheathing ectotro- phic mycorrhizal hyphae. On their decomposi- tion in soil, the nodules will provide a source of calcium to the growing plant on the microbiolo- gical decomposit ion of the oxalates because certain actinomycetes and bacteria can utilize calcium oxalate as their source of carbon. The function of the oxalate in the Alnus nodule is not clear. One of the few references attempting to explain the presence of calcium oxalate in plant tissue is that of Sanchez-Alonso and Lachica (1988) in which is described the synthesis of oxalic acid and its precipitation as the calcium salt in leaves in certain plants. It was suggested that this salt may prevent osmotic problems in nitrate assimilation processes. This may have some relevance to nitrogen fixation in Alnus nodules and the subsequent transportat ion of nitrogenous substances to the host plant.
Acknowledgements We thank Dr Richard Thomas for drawing our attention to pertinent publications, Dr Mike Proe for providing the nodulated Alnus,qlufinosa and Dr J. D. Russell and Mr A. R. Fraser for interpreting infrared spectra.
REFERENCES Birkby, K. M. and Preece, T. F., 1988. Calcium oxalatc
crystals on the sporangium of Pilobolus. Mycoloqist. 2: 68 69.
Burgess, D. and Peterson, R. L., 1987. Development of Alnu.~ .japonica root nodules after inoculation with Frankia strain HFP ArI 3. ('an. J. Bot., 65:1647 1657.
Dawson, J. O., 1986. Actinorhizal plants; their use in forestry and agriculture. Outlook on Agriculture, 15:202 208.
Hodgkinson, A., 1977. Oxalic" Acid in Bioloqy and Medicine, Academic Press, London.
Jones, D. and McHardy, W. J., 1985. Cryofixation tech- niques with special reference to soil fungi. Microhiol. Sci.. 2:225 230.
Sanchez-Alonso, F. and Lachica, M., 1988. Oxalate salts in the leaves of plum (Prunus salicina L.) and cherry (P. arium L.). Nee' Phytol., 108:505 508.
Sutherland, J. M. and Sprent, J. 1., 1984. Calcium-oxalate crystals and crystal cells in determinate root nodules of legumes. Planta, 161:193 200.