THE FREQUENCY OF HETEROCYSTS IN THE NOSTOC PHYCOBIONT OF THE LICHEN PELTIGERA CANINA WILLD.

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1 New Phytol. (1972) 71, THE FREQUENCY OF HETEROCYSTS IN THE NOSTOC PHYCOBIONT OF THE LICHEN PELTIGERA CANINA WILLD. BY H. BRONWEN GRIFFITHS, A. D. GREENWOOD AND J. W. MILLBANK Department of Botany, Imperial College of Science and Technology, London, S.W."] {Received i July 1971) SUMMARY A reliable estimate of 3.27% for the frequency of heterocysts in the cell population of the Nostoc phycobiont present in the thallus of the lichen Peltigera canina has been obtained by electron microscopy at low magnification. INTRODUCTION As part of an investigation of nitrogen fixation and metabolism in lichens (Millbank, 1972) it became necessary to estimate the number of heterocysts generally present in samples of lichen thallus, and to relate this to the total population of vegetative cells of the Nostoc phycobiont. Observations made by phase contrast and bright field microscopy on freeze-microtome sections of the thallus and thallus homogenates showed that in a large number of cases, distinguishing heterocysts from vegetative cells was unexpectedly difficult and uncertain. Routine haemacytometric estimates of the proportion of heterocysts present were, therefore, extremely tedious to carry out and unacceptably inaccurate. Drew and Smith (197) did not observe any heterocysts in Peltigera polydactyla most certainly for this reason, and Peat (198) reported that they were few in number and not frequently observed. The possible usefulness of triphenyl-tetrazolium chloride (TTC) to identify heterocysts (Fay et al., 198) depends upon the reductive vigour of the nitrogenase system, and if this system is inactive or of reduced activity, confusing results due to the other oxidation-reduction systems present in heterocysts and vegetative cells are almost certain to be obtained. In the present context it could not be assumed that production of formazan would be due exclusively to nitrogenase activity. Moreover, the homogenization necessary before counting in a haemacytometer almost completely stops nitrogenase activity, and although this may possibly be restored by the addition of sodium dithionite and ATP (Stewart, Haystead and Pearson, 199), it is unlikely that consistent and complete restoration of activity in all the heterocysts can be achieved. For these reasons use of the light microscope was discarded in favour of observations made with the electron microscope on thin sections of fixed, resin-embedded samples of the intact thallus. By this means heterocysts would be identified readily and unequivocally at comparatively low magnifications, and the numbers of heterocysts and vegetative cells in a series of sections could be accurately counted directly on the viewing screen, thus permitting a check at higher magnification in difficult cases. II

2 12 H. B. GRIFFITHS, et al. METHODS AND RESULTS Small pieces of moist thallus of Peltigera canina Willd. were fixed in 2% or 2.% glutaraldehyde followed by 2% osmium tetroxide in o.i M sodium cacodylate huffer, ph 7.0, or in 1% osmium in veronal-acetate buffer, ph.1 (Kellenberger, Ryter and Sechaud, 198), dehydrated in ethanols, and embedded in Epon. Thin sections stained with lead citrate or uranyl acetate followed by lead citrate were examined in the electron microscope at low magnifications ( x 00 to x 200), and the numbers of heterocysts and vegetative cells were counted. The electron micrograph, Plate i. No. i, is representative of the sections which were examined. Table i. Cell counts on sections of Peltigera canina Sample no. and date 2307C E D B 271C S2B 272C 27S2D 3038A rotal Cell counts Heterocysts 0 I S 7 I on sections Vegetative cells s 123 1s Heterocyst frequency 3-70% 3-2% 3-23% 2.27% -92% 2-78% -2% -7% - /o 3-08% -19% 3-33% 3-91% 2-2% 2.08% 3-7% 1-1% 1-9% 2-7% 2-% 3-77% Overall frequency Heterocyst frequency (average from total counts) 3-1% 2-27% -92% -0% 3-2% 2-99% 3-7% 2.% 3-27% Heterocysts can be distinguished from vegetative cells in thin sections by a relatively large number of diagnostic features in both the cytoplasm and the cell surface layers. These features notably include: the absence of structured granules (Plate i, Nos. i and 2; sg); the smaller number and absence at maturity of the polyhedral bodies (Plate i. No. 2; pb); the change in appearance of the photosynthetic lamellae, which are frequently inflated and may be rearranged to form a contorted reticulate configuration (Plate I, No. I; R) prominent at the polar ends of the heterocyst (Lang, 19); and the presence of the heterocyst envelope (Plate i, No. 2; E), which has a distinctive texture and is also considerably thickened (Plate i, No. 2; arrows) at the plugged heterocyst pore. More than one of these criteria can be found even in sections which do not pass through the characteristic heterocyst pore. The only cells excluded from the counts were those

3 THE NEW PHYTOLOGIST, 71, i PL.ATE I ^-B- GRIFFITHS, A. D. GREENWOOD AND J. W. MlLhB.M<iK HETEROCySTFREQUENCY J^ NOSTOC FROM PELTIGERA (facing page 12)

4 Heterocyst frequency in Nostoc from peltigera 13 visible as extreme tangential profiles, since sections in such planes do not permit conclusive identification of the cell type. Table i gives the counts made on sections cut from nine samples of embedded P. canina,fixedshortly after collection in the summer of three different years. The heterocysts amount, at most, to only about % of the total xvo^^oc cell population within the thallus, most values obtained being lower, in the region of 3%. The average percentage of heterocysts in the total number of cells counted is 3.27%. CONCLUSIONS Accurate values for the proportion of heterocysts in the thallus as a whole have up to now not been easily obtainable. A reliable estimate is provided by the method used here, and the average result of 3.27% is in line with the few references in the literature to suggest that the frequency of heterocysts in lichen thalli may generally be low. The method can readily be adapted by use of appropriate sampling procedures to obtain more complete information on the distribution of different cell types in lichens. ACKNOWLEDGMENTS We should like to thank the Science Research Council for financial support in this investigation. REFERENCES DREW, E. A. & SMITH, D. C. (197). Studies in the physiology- of lichens. VIIL Movement of glucose from alga to fungus during photosynthesis in the thalius of Peltigera polydaciyla. Neiv Phytol.,, 389. FAY, P., STEWART, W. D. P., WALSBV, A. E. & FOGG, G. E. (198). Is the heterocyst the site of nitrogen fixation in blue-green algae? Nature, Lond., 220, 810. KELLENBERGER, E., RYTEH, A. & S^CHAUD, J. (198). Electron microscope study of DN.-^-containing plasms. IL Vegetative and mature phage DN. as compared with normal bacterial nucleoids in different physiological states. J. biophys. biochem. CytoL,, 71. LANG, N. J. (19). Electron microscopic study of heterocyst development in Anabaena azollae Strasburger. J. PhycoL, I, 127. MILLBANK, J. W. (1972). Nitrogen metabolism in lichens. IV. The nitrogenase activity of the Nostoc phycobiont in Peltigera canina. New Phytol., 71, i. i'eat. A. (198). Fine structure of the vegetative thallus of the lichen, Peltigera polydactyla. Arch. Mikrohiol., 1, 212. STEWART, W. D. P., HAYSTEAD, A. & PEARSON, H. W. (199). Nitrogenase activity in heterocj'sts of bluegreen algae. Nature, Lond., 22, 22. EXPLANATION OF PLATE No. 1. Part of a vertical section through the thallus of Peltigera canina, showing cells of the Nostoc phycobiont and associated fungal hyphae. The Nostoc vegetative cells contain prominent electron-dense structured granules (;sg), which are absent from the three heterocysts (H). The lowermost heterocyst shows a reticulate configuration (R) of the internal lamellae. The three heterocysts are each enclosed in a heterocyst envelope. Osmium fixation (Kellenberger), uranyl/lead stain, x 000. No. 2. Part of a section of Peltigera canina similar to No. i, showing at higher m-agnification the pore of a Nostoc heterocyst and part of an adjacent vegetative cell. Structured granules (sg) and polyhedral bodies (pb) are seen in the vegetative cell. The heterocyst has an electrondense pore plug (P), and an outer envelope (E) which is characteristically swollen (arrows) at the pore. Both cells contain lamellae, ribosomes, and electron-dense )!-granules (P). Osmium fixation (Kellenberger), uranyl/lead stain, x 20,000.

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