LOW-POWER ELECTRON MICROSCOPY OF THE ROOT CAP REGION OF EUCALYPT MYCORRHIZAS

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1 New Phytol. (1968) 67, LOW-POWER ELECTRON MICROSCOPY OF THE ROOT CAP REGION OF EUCALYPT MYCORRHIZAS BY G. A. CHILVERS Botany Department, School of General Studies, Australian National University, Canberra {Received 15 January 1968) SUMMARY Low-power electron micrographs of a eucalypt mycorrhiza are presented which show how root cap cells are progressively decomposed by invasion of fungal hyphae. The development of tannin-like deposits in cells of the root cap and epidermis is discussed briefly. INTRODUCTION The anatomy of eucalypt mycorrhizas has been studied previously with the light microscope (Chilvers and Pryor, 1964). An electron microscope study has been initiated to enlarge upon this earlier work and, in particular, to try to answer some questions raised by it. This preliminary report clarifies the fate of the root cap cells within the fungal sheath and describes briefly the development of tannin-like materials in the root cap and epidermal layers. For this study, active mycorrhizas were taken from a 2-year-old specimen of Eucalyptus btcostata Maiden, Blakely and Simmonds, during the spring growth period. Severed tips were fixed in 1.5% buflfered glutaraldehyde followed by a post-fixation in OSO4.. These were then dehydrated through an ethanol series containing uranyl acetate and transferred through propylene oxide to Araldite (Glauert and Glauert, 1958) for impregnation and embedding at 60^ C, Sections were cut with a Porter-Blum ultramicrotome and examined with a Japan Electron Optics JEM-T6 electron microscope. Although the tips were held in liquid Araldite for as long as possible, impregnation was incomplete with the result that blocks varied in hardness from place to place and often contained holes in the outer layers of the root tissue. Consequently it has not yet been possible to obtain large areas of section suitable for recording at high magnification. However, mosaics of low-magnification ( x 1200) electron micrographs have been obtained which reveal some useful information about the root cap region. Fig, i is a tracing of a median longitudinal section which shows the general relationship between root cap (shaded) and the adjacent epidermal and sheath tissues. Plate i reproduces a portion of the same mosaic. On the basis of light microscope studies, Clowes (1954) concluded from indirect anatomical evidence that the root cap cells of ectotrophic mycorrhizas are decomposed within the fungal sheath. The electron micrographs provide direct evidence confirming that this occurs. Fungal hyphae penetrate into the root cap cells and grow through them, subdividing the dark tannic substance within into successively smaller fragments until 663

664 G. A. CHILVERS only small slivers remain in the spaces between hyphae. The outermost root cap cells arc found dismembered in this way right up to the tip.

2 664 G. A. CHILVERS only small slivers remain in the spaces between hyphae. The outermost root cap cells arc found dismembered in this way right up to the tip. Backwards from the tip the invasion of root cap cells proceeds inwards, layer by layer, until the hyphae reach the more vital epidermal cells (at point marked X in both illustrations). Thus the root cap tissue is progressively transformed by invasion and dismemberment of the cells into the inner layers of the mantle. In this young mycorrhiza the invading hyphae are themselves very active and healthy looking. In older mycorrhizas they become impregnated with brown substances, presumably from the dead cell fragments, and appear moribund like those illustrated in the 'tannin layer' of Finus radiata D.Don, by Foster and Marks (1966). Differences in histogenesis apart, their 'tannin layer' of darkly pigmented plant cells together with the pockets of 'polyphenolic substances' found in the adjacent 'inner mantle' appear to be equivalent to the decomposing root cap tissue shown here. A further equivalence between the mycorrhizas of P. radiata and Eucalyptus bicostata occurs in the 'Hartig net' Fig. I. Tracing from a mosaic of low-power electron micrographs of the root cap region of a eucalypt mycorrhiza (root cap tissue shaded), fs. Fungal sheath; re, root cap; ep, epidermis; co, cortex;.x, first epidermal cell contacted by fungal hyphae. region. Foster and Marks show cortical cells enclosed by intercellular hyphae in which the thin cytoplasmic lining of the cell wall is itself overlain by a solid deposit of tanninlike material aggregated in some places into large 'coacervates'. In eucalypt mycorrhizas the Hartig net develops among the epidermal cells and similar coacervates are formed against the cytoplasmic lining of these. It can be seen in Plate i that these coacervates are actually formed before intercellular penetration by the fungus commences. The tannin-like materials of the epidermis and root cap are probably similar in composition. They originate in an identical manner soon after vacuolation commences. In the root cap cells nearly all the developing vacuoles become filled with a homogeneous brown material whose production keeps pace with the expansion of the vacuoles until the bulk of the cell is filled with it. It appears that the original vacuoles never become completely lost, since shrinkage of the dark material during fixation and dehydration reveals a pattern of cracks which are clearly related to the boundaries of vacuoles. In the epidermal cells only about one half of the developing vacuoles contain the pigment and these eventually all merge into one. Originally homogeneous in appearance, like

$THE NEW PHYTOLOGIST, 67, 3 PLATK I I art ot a mosaic ot low-power electron micrographs SIIDWHIK the outer la\ers of a eucilypt niycorrhiza to the rear of the root cap, F, Fungal h\ pliae inside more$

3 THE NEW PHYTOLOGIST, 67, 3 PLATK I I art ot a mosaic ot low-power electron micrographs SIIDWHIK the outer la\ers of a eucilypt niycorrhiza to the rear of the root cap, F, Fungal h\ pliae inside more or less entire root cap cells; R, residues of root root cap cap cells cells wnicn which na\x' h a \ e heen neark' repiacea replaced '"" (' ".>.l K.nl-,-.<.'-" ', i\, resiuues or neen neari\' ny lungai nypnae; >\, inner and outer walls ot a root cap cell heavily i n \ a d e d hy h\'phae; V, coacerxates <>( tannin-like material: X, the first epidermal cell in actual contact with tlie fungus this can be used to relate the plate to the text figure. CHIIAERS- ELECTRON MICROSCOPY OE MYCORRIII/.A [faaiin pa- t>(<4)

4 Electron 7nicroscopy of mycorrhiza 665 the root cap material, this tannin-like substance tends to form aggregates in the \ acuoles of radially elongating cells and finally condenses into the coacervates as shown in Plate I. The mass of small aggregates formed in the earlier vacuoles is quite possibly a preparation artifact which merely demonstrates a propensity for polymerization not yet realized in the living material. On the other hand, the coacervates are certainly real since they coincide with characteristic outgrowths of the cytoplasmic lining and in older mycorrhizas they frequently grow to a size where they can be seen readily under the light microscope. ACKNOWLEDGMENT I wish to thank Mrs A. Pettersson for her patient preparation of sections from very difficult material. RKFERENCES CHILV'ERS, G..\. & PRYOR, L. D. (1964). The structure of eucalypt mycorrhizas. Aust. J. Bot., 13, 245. CLOWES, F. A. L. (1954). The root cap of ectotrophic mycorrhizas. Netv Phytol., 53, 525. FOSTER, R. C. & MARKS, G. C. (1966). The tine structure of the mycorrhizas of Piiuis radiata D.Don. Aust. J. biol. Sci., 19, GLAUERT, A. ^^ & GLAUERT, G. H. (195S). Araldite as an embedding medium for electron microscopy. y. biophys. biochem. CytoL, 4, 191.

THE BEHAVIOUR OF CHLOROPLASTS DURING CELL DIVISION OF ISOETES LACUSTRIS L.

New Phytol (1974) 73, 139-142. THE BEHAVIOUR OF CHLOROPLASTS DURING CELL DIVISION OF ISOETES LACUSTRIS L. BY JEAN M. WHATLEY Botany School, University of Oxford (Received 2 July 1973) SUMMARY Cells in