Ultrastructure of the Lichen Xanthoria fallax

Ultrastructure of the Lichen Xanthoria fallaxAuthor(s): William C. Hayes and Daniel E. WujekSource: Transactions of the Kansas Academy of Science (1903-), Vol. 76, No. 3 (Autumn, 1973),pp. 234-243Published by: Kansas Academy of ScienceStable URL: http://www.jstor.org/stable/3627105 .

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Ultrastructure of the Lichen Xanthoria fallax

WILLIAM C. HAYES and DANIEL E. WUJEK Department of BioIogy, Central Michigan University, Mt. Pleasant, Michigan 48859

Abstract The phycobiont-mycobiont relationship in the lichen Xanthoria

fallax was studied by electron microscopy. The phycobiont, Trebouxia, has a la-ge central pyrenoid with many pyrenoglobuli. The mycobiont exhi,ited concentric bodies, an invaginated plasmalemma, a membrane system, and a laminated wall. Intra- cellular haustoria rJere demonstrated. Trans. Kans. Acad. Sci., 76 (3), 1973.

Introduction

Significant contribut ons to lichen ultrastructure have been made since 1960. Improvements iLl electron microscopic techniques have hastened work in this area. In 1960, Moore and McAlear studied the lichen associations in Clcgdonici an d Lecidea, demonstrating the presence of haustoria. This was followed by Bednar and Juniper's (1964) study of the microfibrillar structure in the fungal portions of the lichen Xa^thoria perzeoltzn.

Brown and Wilson (1968) studied the lichen Phyxcia aipolia. Ellip- soidal (concentric) bodies were observed in the mycobiont, in addition to an invaginated plasmalemma. The pyrenoid in the phycobiont, Tsebozlxia was also observed. No haustoria were demonstrated.

From 1968 to 1970, Peveling did a series of studies on lichen ultrastructure (1968, 1969, 1970a, 1970b). In one paper she discussed the classification of the T;^ebozlxia phycobiont based on electron microscopy (1969). In another she studied the mycobiont and its contact with the phycobiont (1970a ) .

In 1969, Jacobs and Ahmadjian (1969) studied the ultrastructure of ten lichens all with the phycobiont T^ebogxiag Concentric bodies were commonly observed in the mycobiont and pyrenoglobuli were found associated with the pyrenoid in all species except one. Haustoria were demonstrated . . n severa specles.

A lichen is commonly defined as a symbiotic relationship between a fungus (mycobiont) and alga (phycobiont). Hale (1967) reviews some of the concepts of lichen symbiosis which have been put forth over the years. Recently a new term, helotism or balanced parasitism, has been

Transactions of the Kansas Academy of Science, Vol. 76, No. 3, 1973. Published July 19 1974.

[234]



Ult} clst} rtsre o f Xanthoria fallax 2 3 5

coined to describe the association. The change is based on the presence or absence of haustoria (Brown and Wilson, 1968). That is, if haustoria are present the relationship is described as helotism.

An ultrastructural study of the lichen Xthoria fallax (Hepp) Arn. was undertaken to determine the interrelationship of the phycobiont and the mycobiont. The micrographs of lichenized phycobiont and mycobiont were compared with similar studies in the literature.

Materials and Methods Xaozthowia fallax was collected in January? 1972> from an elm tree

on the campus of Central Michigan University, Mt. Pleasant, Michigan. After collection the specimens were hydrated with distilled water and placed in a growth chamber with a 12/12 dark-light cycle at 21° C for 24 hours.

After the 12 hours light period the lichen thalli were removed from the groBTth chamber and hand shaken to remove loose dirt and debris. The thalli were washed in cold running water for 10 minutes. Small pieces of thallus were fixed at room temperature for two hours with 3SG

glutaraldehyde in O.1M phosphate buffer (pH 7.0). The tissue was rirlsed three times for five tninutes each in a phosphate buffer and postfixed for one hour with lCo OsO. in O.1M phosphate buffer (pH 7.0). The tissue was rinsed for five minutes in phosphate buffer and then dehydrated in a graded acetone series. The tissue was embedded using 3-step infiltration in an Epon-Araldite mixture (MollenhauserX 1964).

Sections were cut with a glass or diamond knife on a Porter-Blum MT-2 Ultramicrotome and mounted on copper grids. Some sections were stained with lead citrate (Reynolds, 1963). Sections were examined with a Philips EM 300 electron microscope.

Observations Phycobioslt-The phycobiont, StSeboxia, a unicellular green alga, is

a common phycobiont of lichens. The most conspicuous structure observed in Tl ebozxxia is a large axial chloroplast (Fig. 1 ) . Thylakoids are arranged in groups of three to eight stacks. The pyrenoid lies in the center of the cell and is penetrated by the chloroplast thylakoids (Fig. 2). Arranged along the penetrating thylakoids are many electron-dense granules pyrenoglobuli (Fig. 2) No starch grains were observed.

The cytoplasm (}ccurred as a thin rim between the chloroplast and the cell wall (Fig. 1, 2) The nucleus and mitochondria were observed near the periphery of ie cell. Nucleoli were observed in a few cells (Fig. 3).



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1 Ht:X0000<00 WN N

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pW 2 i t iv Figures 1-2. Section through the phycobiont of Xanthoria fallax. Fig. 1. Near-

median section through a cell. The chloroplast occupies most of the cell. X 7,000. Fig. 2.

Note the large central pyrenoid (Py), single thylakoids penetrating the pyrenoid and

pyrenoglobSi lining the thylakoids (arrow). Polyphosphate bodies (P) are evident. X 7,500.



lJlts ts sctvs e o f Xanthoria f allax 2 3 7

Endoplanmir ret CUlUEl and g31gi were not obServed. WIicrotubzlles were infrequently ob erved. Polyphosphate bodies, large electron-dense gran- ule, were located throughout the cell (Fig. 1).

llIcobwoslt In general the fungal hyphae exhibited less detail than ttle algae. Nuclei and nbLcleoli were observed in some cells (Fig. 4). Endoplasmic reticulum, golgi apparati and mitochondria were observed ill some cells (Fig. 4). Atitochondria appeared to be in greater abundance in cells in contact with the phycobiont.

Most of the fungal cells contained many membranous structures arranged in concentric rings or many within one membrane (Fig. 4). Some of the membranes appeared to be double while others appeared single. Also apparent were large electron-transparent areas which may be sorr.e type of vacuole (Fig. 4).

Concentric bodies, lumen-like structures which are peculiar to lichenized fungi were observed (Fig. 4, 5). They appeared in groups of 2-13 and were usually found near the nucleus and appeared to be in cloce proximity to the membrane system previously mentioned. They ranged in size from 0.08y-0.19ju on the outside to 0.03y-0.06ju on the inside. Their structure varied with the plane of sectioning. Some sections revealed a ray-like structure surrounding the lumen while others appeared as a dark halo (Fig. 4).

The cell wall ranged from 0.24y-0.04ju and it appeared to have two major layers with possible minor subdivisions (multilaminate) making up to five layers (Fig. 4). Invaginations of the plasmalemma were commonly observed (Fig. 6). Septal pores with associated Woronin bodies (Bfergin, 1973) were observed (Fig. 5).

Phycobiont-Mycobiont Relationship Two types of contact are demonstrated in Xarsthori. The first and

most common is simply by wall to wall contact. At these points the walls, particularly the fungal wall, appeared to be thinner (Fig. 6). The second type of contact was an intracellular haustorium. The haustoria penetrated the algal cell wall but not the membrane (Fig. 7). The cell walls in the area of the haustorium appeared different than in either the normal algal or fungal cell. The layers were not apparent and the algal wall much reduced. There also appeared to be some space between the two cells.

Bacteria were also observed in the fungal layer, on the lower surface of the thallus (Fig. 8). They appeared to be in a clear matrix.



Figure 3-4. Fig. 3. Section through the phycobiont. A conspicuous nucleus (N) with nucleolus is evident. Also observed are polyphosphate bodies. X 9,500. Fig. 4. Section through the fungus. Concentric bodies (Cb) lie in a common matrix. Also present are apparent vacuoles (V) nucleus, nucleolus and mitochondria. X 22,500.

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Ultrstrrt?re of Xanthoria fallax 239

Discussion

Ahmadjian (1967) classified species of Tsebos/xia into two groups based on the chromatophore. The Treboslxia sp. in the foliose lichen Xanthoria fallax, is probably the Group II species. Group II phycobionts (often isolated from foliose and crustose lichens) are spherical cells possessing chloroplasts not deeply lobed (Fisher, 1971a). Further classification of Trebogxia has been extended to the electron microscope level but this remains unclear. There are several reports in the literature on various aspects of TrebogXia both lichenized and in culture (Brown and Wilson, 1968; Fisher, 1971a; Fisher and Lang, 1971; Jacobs and Ahmadjian, 1969, 1971; Peveling, 1970a). Reports are usually based on the study of a single lichen which makes generalization difficult.

The pyrenoid, an interesting structure in TrebogXia, stands out due to the presence of numerous pyrenoglobuli in the pyrenoid matrix. The pyrenoglobuli appear to be arranged linearly along the thylakoid membranes which penetrate the pyrenoid. The pyrenoglobuli were observed throughout the pyrenoid and not just on the outside as some investigators have observed in the hydrated state (Brown and NVilson, 1968; Jacobs and Ahmadjian, 1971) .

The pyrenoglobuli of Trebogxid have been found to contain lipids. The lipid containing globules probably represent storage products whose formation and utilization undergo seasonal changes. It is also postulated that the globules are associated with resistance to strong light or protection from gamma radiation (Jacobs and Ahmadiian, 1969; Fisher, 1971a; Fisher and I ango, 1971) . A new hypothesis links pyrenoglobuli with membrane metabDlism. Fisher and Lang (1971) observed diSerences in the number and size of pyrenoglobuli and membranes as a function of light intensity.

The function of the pyrenoid is often discussed. In addition to the possible storage function, the pyrenvid may also function in synthesis or utilization of materials produced by the chloroplast (Griffiths, 1970).

Several investigators discuss the storage products of Tebo/xi. They found starch only in the hydrated state or in cells grown in an organic medium; starch granules are usually not observed in desiccated specimens (Brown and Wilson, 1968; Fisher and Lang, 1971; Jacobs and Ahmadjian, 1969, 1971). Specimens collected for this study were collected in January and hydrated for only 24 hours. The short period of hydration could account for the absence of starch.

In addition to the pyrenoglobuli, polyphosphate bodies were found randomly distributed throughout the cell. Fisher (l97lb) studied these bodies in Trebogxia erici and found that the image of these granules



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Figure 5-7. Fig. 5. Mycobiont. Oblique section through a portion of a septum. Several Woronin bodies (arrows) are apparent on either side of the septal pore. X 25,000. Fig. 6. Mycobiont. A thinning of the fungal wall in contact with the phycobiont. X 10,500. Fig. 7. Section through the phycobiont penetrated by a haustorium. X 9,OOo.



Ultrastructure of Xanthoria fallax 241

varies with fixation. Jacobs and Ahmadjian (1969) found polyphosphate bodies only in cultured cells of Trebogxia.

The fungus exhibited several interesting structures, the first being ie concentric membranes which appeared to be some type of storage vacuole. The inside of these stmctures was usually electron-light, but some had electron-dense areas (Fig. 4). Similar structures were observed by Jacobs and Ahmadjian (1969) and Peveling (1970a). They observed that the endoplasmic reticulum has encapsulated small vesides and that these structares may be involved in the production of lomasomes.

The second point of interest was the cell wall which appeared to be lamellated. Chervin and Baker (1968) observed a 2-layered wall in Usnea. Jacobs and Ahmadjian (1971) observed three layers in Cladonia and a multilaminated cell wall in Xanthoria parientina. Some of these layers may be the result of age, physical state (hydrated of desiccated), or fixation. Jacobs and Ahmadjian (1969) noted the thickness of the wall depended Oll the age of the hyphae and its position in the thallus.

The invaginated plasmalemma had been cbserved by cthers (Brown and Wilson, 1968; Peveling, 1970a; Jacobs and Ahmadjian, 1971). The invaginated plasmalemma points to a speciali7ed molecular basis for cxchange of materials between the lichen components. Peveling (1970a) found the invaginated plasmalemma only in hyphae in the algal tone and

Figure 8. Bacterial cells found within the lichen extracellular matrix. X 15,000.



247 7-t^&l}Ze$'lE'tiO}ll' / the K{191JA5 Azvlevg: sjf Scie}Zz-e

thus concluded that this was an adaptation to the symbiotic life; the expanded surface area provided for more nutrient absorption from the alga.

Concentric (ellipsoidal) bodies have been observed in mycobionts of at least 43 species of lichens (Griffiths and Greenwood, 1972). They have also been reported in two species of non-lichenized fungi. The origin7 development, and function of these bodies is not yet known. Brown and Wilson (1968) observed these bodies near vacuoles and the nucleus and because of Lnembrane profiles which were associated with these structures have im>licated these bodies with membrane synthesis or as an organelle conceed with transport of materials. Javvbs and Ahmadjian (1971 ) observed concentric bodies intimately connected to the internal membrane system of the cell in Cladoslia rwiwtatella Micro- graphs by Peveling (19-, 0a) also appear to show concentric bodies associated with membranes. Concerltric bodies were commonly observed in XXzthowia fallax and wese usually located near the nucleus and membranes.

The association of al algal and fungus in a Iichen is quite interesting, but as yet investigations are few. Until recently it has been main- tained that the relationship is symbiotic since the lichen survives where neither member could survive unassociated (Brown and Wilson, 1968). More recently the association has been described as a balanced parasitism or helotism. This argument is based on the presence or absence of haustoria (Brown and Wilson, 1968).

The relation of phycobiont and mycobiont cells display a wide range of response correlated with thallus development (Ahmadj ian, 1965 ) . Plessl (1963) classified lichens into three categories with respect to the physical contact of algal and fungal cells: (1) lichens with no haustoria (close contact and thin walls); (2) intracellular haustoria; and (3) intra- membranous haustoria (highly organized thallus ). Xaoztho?ia fczilw;x exhibited the first two types of contact. In the second type haustoria appeared to penetrate the algal protoplast and often extend to the middle of a cell. This type of haustorium though demonstrated was infrequent in Xvthofiia.

In comparison with previous studies of Trebogxia (Brown and Wilscon7 1968; Fisher, 1971a; Fisher and Lang, 1971; Peveling, 1969) n the phycobiont appeared little changed except for reduction in cell wall size where hasutoria penetrate. Fungal adaptation in the lichen is more apparent partictllarly in the concentric bodies and the invaginated pIasma- lemma. Some observers have also noted that there were more mitochondria in hyphae in contact with the algae. There was some evidence for this in Xasthofiid.



Ultrastrtlctgre ot Xantheria fallax 243

Acknowledgements We would like to thank David Howell for collecting, identification

and assistance in embedding the lichen. This study was funded in part from a grant from the C.M.U. Research and Creative Endeavc)rs Committee.

References AHMADJIAN, Y. 1965. Lichens. Ann. Rev. Microbiol., 19:1-20. AHMADJIAN, V. 1967. A guide to the algae occurring as lichen symbionts: isola-

tion, culture, cultural physiology, and identification. Phycologia, 6:127- 160.

BEDNAR, T. W. and B. E. JUNIPER. 1964. Microfibrillar structure in the fungal portions of the lichen XvxlDhoria parietiva (L.) Th. Fr. Exp. Cell Res., 36:68>683.

BRQWN, M. and R. WIESON 1968. Electron microscolpy of the lichen Physcia vipolia (Ehrn.) Nyl. J. Phycol., 4:230240.

CHERVIN, R. and G. BAKER. 1968. The ultrastructure of phycobiont and mycobiont in two species of Us?e. Can. J. Bot., 46:241-245.

FISHER, K. A. 1971a. Ultrastructure of the pyrenoid of Trebouxia in Ramaliva megziesii TUCk. J. PhYCO1., 7:25-37.

F1SHER, K. A. 197lb. Polyphosphate in a chlorococcalean alga. Phycologia, 10: 177182.

FISHER, K A. and N. J. LANG. 1 97 1. Comparative ultrastructure of cultured species of Trebo,uxia. J. Phycol., 7:155-165.

GRIFFITHS, D. J. 1970. The pyrenoid. Bot. Rev., 36: 2F58. GRIFFITHS, H. B. and A. D. GREENWOOD 1972. The concentric bodies of lichen-

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MOLLENHAUER, H. H. 1964 Plastic embedding mixtures for use in electron microscopy. Stain Tech., 39:111114.

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PEVELING, E. 1968. Elektronenoptische untersuchungen an flechten I. Struktur- veran derungen der algenzellen von Leconora muralis (Schreber) Rabenh. Z. Pflanzenphysiol., 59:172 183.

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PEVELING, E. 197Ob. Das vorkommen von starke in chlorphyceen-phycolbionten. Planta, 93:82-85.

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Ultrastructure of the Lichen Xanthoria fallax