Ultrastructural features of primula yellows mycoplasma-like organism (MLO) in cryosections of Catharanthus roseus leaves

JOURNAL

OF STRUCTURAL

BIOLOGY

56-64

107,

(1991)

Ultrastructural Features of Primula Yellows Mycoplasma-like Organism (MLO) in Cryosections of Ca tharan thus roseus Leaves GIOVANNID'AGOSTINO Istituto Received

di Fitovirologia

applicata

March

and in revised

8, 1991,

INTRODUCTION

Mycoplasma-like organisms (MLOs), in contrast to spiroplasmas and animal mycoplasmas, have not yet been isolated and cultured in vitro (Jacoli, 1981; Lee and Davis, 1986) and this has prevented their detailed characterization. Nevertheless, their morphology and structure have been the subject of many electron microscope investigations (see Hiruki, 1988; Maramorosch and Raychaudhuri, 1988; Whitcomb and Tully, 1989). The manipulation of biological samples prior to electron microscopy causes artifacts (see Crang and Klomparens, 1988). This is particularly true for plant samples and even more true for MLOs due to

Italy

May

20, 1991

MATERIALSAND Plant

Material,

MLO,

and

Tissue

METHODS Processing

Healthy C. roseus plants were grown under standard glasshouse conditions and grafted with C. roseus infected by PY MLO, a typical member of the aster yellows ML0 group. The infected plant was obtained from M. F. Clark (Clark et al., 1989). Six months after grafting, young leaves with evident symptoms 56

Inc. reserved.

form

Torino,

their location in the sieve tubes, for which adequate fixation techniques are not available. For this reason, more than one electron microscopy technique must be employed to determine the actual morphology of MLOs in situ (Braun, 1977; Florance and Cameron, 1978; Haggis and Sinha, 1978; Hearon et al., 1976; Petzold et al., 1977; Waters and Hunt, 1980). However, the main morphological characteristics of the known MLOs associated with plant diseases seem to be very similar. Immunological methods can be very helpful in establishing antigenic differences among individual mycoplasmas, but their application in this field meets with consistent difficulties. Only recently a few reports have appeared dealing with the application of these methods to the diagnosis of ML0 diseases in plants (Chen et al., 1989; Lherminier et al., 1990; Sinha, 1988). Cryoultramicrotomy applied after chemical fixation has proved to be very suitable for immunocytochemical applications (Boonstra et al., 1987; Tokuyasu, 19861, offering at the same time good fine structure preservation because it minimizes certain artifacts associated with heavy metal staining and avoids those due to dehydration and embedding (Tokuyasu, 1986; Zierold, 1987). However, there appear to be no reports on plant MLOs in cryosections. The present study was undertaken to check whether cryoultramicrotomy could furnish more information, compared to conventional techniques, on morphology and structure of primula yellows (PY> MLO-infected Catharanthus roseus leaves. Preliminary reports on these findings have already been presented (D’Agostino, 1989 and 1990).

Cryoultramicrotomy was applied to the study of primula yellows (PY) mycoplasma-like organism (MLO) in Catharanthus roseus leaves. The aim was to check whether this method could give better information on morphology and structure of MLOs, in comparison to conventional Epon-Araldite embedding. After aldehyde fixation, some of the samples were processed for conventional sectioning, and others were frozen in liquid propane and cryosectioned. Two predominant morphotypes were obsewed, spherical and filamentous, often associated or connected to each other. ML0 structural details were better preserved in cryosections. The electron transparency of cryosections even at a thickness of 200 nm made it easy to follow the morphology and spatial development of the filaments. The filaments were tubular, 3545 nm in diameter and about 1.2 pm long, or were partially or completely pinched into long chains of discrete oval or spherical bodies, 45-75 nm in diameter. The rounded MLOs, 200-250 nm in diameter, were sometimes connected in a chain by a continuous outer membrane. These observations suggest that the filamentous forms may be involved in growth and reproduction of the PY MLO, in a manner similar to that shown for analogous structures observed in other plant MLOs and animal mycoplasmas. 0 1991 Academic Press, Inc.

1047-8477191 $3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form

de1 CNR,

ULTRASTRUCTURAL

FEATURES

were cut into approximately &mm’ portions and fixed for 1 or 3 hr at 4°C in 2% glutaraldehyde + 1% acrolein in 0.15 M phosphate buffer, pH 7.2, containing 0.6 M sucrose. During fixation the samples, kept completely submerged, were trimmed using a razor blade to a final size of approximately 1 X 1 mm and then a gentle vacuum was applied to let the fixative penetrate the tissues. The samples fixed for 3 hr were processed for epoxy sections, the others for cryosections. Epoxy

Sections

The fixed samples were postfixed in 1% osmium tetroxide in the same buffer for 1 hr at 4°C and stained overnight at 4°C in 1% aqueous uranyl acetate. Samples were then dehydrated in an ethanol series, treated with propylene oxide and embedded in Epon-Araldite, according to conventional procedures (D’Agostino and Pennazio, 1985). Ultrathin sections were cut with a Diatome diamond knife in a Reichert OM-U2 ultramicrotome, placed on uncoated 200-mesh copper grids, and stained with lead citrate. Cryosections The procedure outlined by Tokuyasu (1986) was followed: the fixed samples were infused for 30 min in 2.1 M sucrose, then mounted on special aluminium specimen carriers (Reichert, Wien, Austria), and quickly frozen by injection into liquid propane with a Reichert KF 80 cryofixation unit. Using glass knives, cryosections were cut at - 80°C with an automatic advance of 90 or 200 nm and a cutting speed of 0.8-5.0 mm set-’ in a Reichert Ultracut-E ultramicrotome equipped with an FC 4 cryochamber. Frozen sections were collected by a loop with a droplet of 2.3 M sucrose and transferred to hexagonal loo-mesh Formvar-coated copper grids. These were floated for 10 min on buffered 0.02 M glycine + 2% gelatin on ice and rinsed in 0.15 M phosphate buffer, pH 7.2, followed by distilled water. Finally they were stained for 5 min with 2% uranyl acetate-oxalate, pH 7 (Tokuyasu, 1978), followed by a short wash in distilled water; then floated on a 1.8% solution of methylcellulose (25 cP; Fluka AG) containing 0.4% uranyl acetate on ice for 10 min; picked up on a loop; and, after removing excess solution, dried quickly in a chamber containing silica gel (Griffiths et al., 1984). Comparable leaf tissues from healthy plants were used as control in each experiment. All sections were examined at 60 kV in a Philips EM 201 electron microscope. RESULTS

Conventional

Epoxy Sections

In sections about 60 nm thick (silver in color) the MLOs showed the typical ultrastructural features reported for conventional transmission electron microscopy. For comparison with the structures seen in cryosections (see below), it is noted only that, in conventional sections, aggregated DNA-like fibrils occurred in the vacuolated central area (Fig. la). In thicker sections (about 150 nm thick; pale purple in color), the matrix of the large MLOs was so dense that the internal structure could not be discerned. However, it was possible to observe a much greater number of small (50-100 nm in diameter) spherical bodies, often in close contact with the large MLOs or associated with them in chains (Fig. lb). Cryosections

In cryosections, cells and subcellular organelles, such as nuclei, chloroplasts, mitochondria, and cell

OF ML0

IN CRYOSECTIONS

57

membranes, were found to be well preserved. MLOs were easily detected in sieve elements of infected leaves (Fig. 2a). The fine structure of the MLOs was also well preserved, and although varying in size and shape, they were less polymorphic than in conventional epoxy sections. Two predominant morphotypes were observed, rounded and filamentous, often associated or connected with each other: both were bounded by a well-defined unit membrane with an average thickness about 8 nm (Figs. 2a, 2b). Often the unit membrane of rounded MLOs appeared slightly shrunken (Fig. 2b). The rounded MLOs, ranging in diameter from 75 to 500 nm, contained more or less electron-dense cytoplasm. In some of them scattered ribosome-like granules but not aggregations of DNA-like fibrils could be distinguished; in others the matrix was so electron-dense that internal structure could not be discerned (Fig. 2b). The morphology and spatial development of the filamentous forms were easily seen in sections about 200 nm thick, especially in cells containing a limited number of the larger rounded organisms, where many extended filamentous forms were visible (Fig. 3). The filamentous structures had a diameter of 35 to 45 nm, and contained a moderately electron-dense matrix in which no ribosome-like granules were distinguished. The average length of filaments was about 1.2 km, but segments up to 2 km were observed (Fig. 3); the maximum length could not be established because no terminal structure was seen. The ultrastructural features of filaments could vary within a single filament. Initial and advanced stages of constriction at one or more points along the filament were seen to occur between areas of moderate to electron-dense condensation. Often the filaments were partially or completely pinched into chains of discrete oval or spherical bodies, and some chains contained up to 20 units (Fig. 3) and were variously branched (Figs. 3,4a). Others appeared clearly connected to the rounded MLOs and bounded by the same unit membrane (Figs. 4a, 4b). The bodies within a chain had approximately the same size with a diameter of 45-75 nm (Fig. 3); however, in some chains the bodies varied in size and electron density, and some of them appeared vacuolated (Fig, 4a). Sometimes also the rounded MLOs, 200-250 nm in diameter, were still arranged in chain-like structures (Fig. 3) or connected in a chain by continuous membrane structures (Fig. 4~). Thin fibrils, about lo-15 nm thick, were observed between rounded and filamentous MLOs and sometimes in close contact with them and the sieve element walls (Figs. 3, 5a, 5b). They are likely to represent P-protein, The frequency of rounded MLOs ranged from a few, mostly located near the sieve element wall and

FIG. 1. Epon-Araldite sections of primula yellows mycoplasma-like organisms (MLOs) in sieve cells. (a) Ultrathin section of a cell containing small spherical bodies (arrows), indistinct filamentous structures (arrow heads), and large, polymorphic MLOs containing ribosome-like granules in the peripheral cytoplasm and sometimes aggregations of DNA-like fibrils in the central area. (b) Thick section of a cell showing large rounded electron-dense MLOs, often in close contact with filamentous forms, and small spherical bodies associated in chains. x 48 000 (a,b). 58

FIG. 2. (a) Ultrathin cryosections of phloem cells. (Left) A companion cell showing mitochondria, a nucleus with and cytoplasm rich in ribosomes. (Right) A sieve element filled with MLOs. (b) At higher magnification, MLOs preserved. They are bounded by a well-defined unit membrane and contain a matrix of varying electron density. dense matrix, ribosome-like granules but not DNA-like fibrils can sometimes be distinguished. Filamentous forms CW, cell wall; M, mitochondrion; N, nucleus. x 26 000 (a); x 95 000 (b). 59

condensed chromatin, are seen to be wellIn MLOs with a less are also clearly seen.

FIG. 3. Thick cryosection of a sieve located near the cell wall. Filamentous filaments between areas of electron-dense times branched. The thin fibrils (arrows

cell containing a few rounded electron-dense MLOs, some of which are still arranged structures with an average length of about 1.2 pm are seen. Constrictions occur condensation. Often the filaments are partially or completely pinched into chains heads) among the MLOs are likely to represent P-protein. x 63 000. 60

in chains, along the and some-

FIG. 4. membrane, Filamentous in diameter,

Ultrastructural show some structures connected

features of filamentous structures branching. Along the chains some clearly connected to a rounded ML0 in a chain, together with filamentous

in thick cryosections. (a) The filaments, bounded by a well-defined bodies vary in size and electron density or appear vacuolated (arrow). and bounded by the same unit membrane. Cc) Rounded MLOs, 200-250 forms. x 105 000 (a); X 160 000 (b); X 90 000 Cc). 61

I mit (b) nm

FIG. 5. (a) Fibrils, lo-15 nm thick, probably P-protein, lying between rounded and the cell wall. (b) Detail of a sieve element filled with such fibrils. Note the concentration of rounded MLOs, 150-500 nm in diameter, occupying most of the indistinctly observed; thin cryosection. (d) Intermediate concentration of rounded nection of filamentous forms; thick cryosection. X 52 000 (a); x 160 000 (b); X 37

and filamentous MLOs and in close contact with them presence of a chain of small ovoidal bodies. (c) High cell lumen, in which filaments and chains are still MLOs, arranged in clusters, with extensive intercon000 (c); X 35 000 (d).

ULTRASTRUCTURAL

FEATURES

frequently in close contact with it (Figs. 2b, 3, 4a, 5a), to a high concentration, occupying most of the cell lumen (Figs. 2a, 5~). At intermediate concentration the rounded MLOs often appeared arranged in clusters with extensive interconnection of filamentous forms (Figs. 2b, 5d). In the sieve elements completely filled with MLOs the predominant morphotype was large and rounded, 150-500 nm in diameter, though filaments and chains could also be observed (Figs. 2a, 5~). Healthy

Control

MLOs were absent in epoxy sections and cryosections of comparable leaf tissue from healthy plants. DISCUSSION

Since it is not possible to extract MLOs from living plants without inducing modifications and structure artifacts, it is necessary to study their morphology in situ. Ultrathin serial sections have been used to determine the three-dimensional form of the MLOs associated with different plant diseases (Florance and Cameron, 1978; Waters and Hunt, 1980). In addition to ultrathin sectioning, generally the first step in ML0 identification, semithin sections are strongly recommended, since they often reveal filamentous morphology not distinguishable in ultrathin sections (Hearon et al., 1976; Thomas, 1979). However, ML0 profiles in conventional sections contain all the morphological artifacts caused by dehydration and resin embedding during the preparation of samples for electron microscopy (see Mollenhauer, 1988). Cryoultramicrotomy avoids these artifacts as well as those due to osmium tetroxide fixation (Tokuyasu, 1986; Zierold, 1987). In fact the morphological and structural preservation of PY ML0 is better in cryosections than in epoxy sections, even though wrinkling of the unit membrane was observed, probably caused by aldehyde fixation and by the osmolality chosen (see Bowers and Maser, 1988; De Leeuw et al., 1985). The DNA-like fibril aggregates present in the central area of large PY MLOs in epoxy sections, and absent in cryosections, are likely to have arisen during ethanol dehydration after aldehyde fixation (Kellenberger et al., 1981; Smith et al., 1976) or during osmium tetroxide fixation (Waters, 1982). The high polymorphism of PY MLOs observed only in epoxy sections, similar to that normally reported in the literature for other MLOs (Florance and Cameron, 1978; Sinha and Paliwal, 1969; Waters and Hunt, 1980), is probably due to artifacts associated with this technique. In cryosections two predominant morphotypes were observed, rounded and filamentous. As ribbons of ultrathin cryosections can seldom be obtained with our material, thicker cryosections were used to gain more spatial information. The preservation of PY ML0 filamen-

OF ML0

IN CRYOSECTIONS

63

tous forms was excellent and their spatial extent easily seen, thanks to the higher electron transparency of cryosections compared to analogous epoxy sections. Both rounded and filamentous MLOs have been observed using different techniques such as examination of semithin sections (Hearon et al., 1976), negatively stained ML0 suspensions (Sinha, 1974), freeze-fracture and transmission electron microscopy (Braun, 1977), or freeze-etching and scanning electron microscopy (Haggis and Sinha, 1978; Petzold et al., 1977). The observation of intact animal mycoplasmas taken from culture shows that, though occurring most frequently as simple spheres or filamentous bodies, they can branch freely or even give rise to mycelial complexes (Razin, 1978; Tully, 1978). Freundt (1960) proposed a reproductive cycle for Mycoplasma mycoides, in which the filaments, by constriction of the cell membrane, transform into chains of cocci, with subsequent fragmentation into young elementary bodies. For plant MLOs also, the studies of Sinha and Paliwal(l969) of clover phyllody and of Hirumi and Maramorosch (1973) of aster yellows (AY) indicate that filaments may be important in the growth and reproduction of such organisms. In the ultrastructural studies of ML0 associated with hydrangea virescence Hearon et al. (1976) noted that the filamentous forms observed resemble those of some cultured animal mycoplasmas, particularly those of M. mycoides as described by Freundt (1960). In the present work, PY-filamentous forms were also found to resemble those of some animal mycoplasmas, suggesting that the mode of PY ML0 replication may be similar to that of animal mycoplasmas (see Rodwell and Mitchell, 1979). In PY ML0 the different structural features observed from filament to filament or even within a single filament might reflect the transformation of the filament into a chain of cocci, as demonstrated by Bredt et al. (1973) in film sequences of cultured M. hominis. In the case of PY ML0 chain fragmentation into single coccoid cells seems to be much delayed. In fact rounded PY MLOs, 200-250 nm in diameter, were often seen connected in chains by a continuous unit membrane or clustered with extensive interconnection and crossing of filamentous forms, suggesting an early stage of a possible colony-like formation. In such heterogeneous populations it seems likely that all stages of the cell cycle are present, as suggested by Hirumi and Maramorosch (1973) for AY MLO. Even if the ultrastructural results obtained for PY ML0 in C. roseus leaves are open to improvement using other cryotechniques that avoid chemical fixation (Steinbrecht and Zierold, 1987), cryoultramicrotomy was shown to be a useful method of demonstrating the morphology of such an organism in situ, particularly in regard to the fragile filamentous forms.

64

GIOVANNI

The recent production of monoclonal antibodies against MLOs (see Chen et al., 1989; Clark et al., 1989) might allow profitable immunocytochemical investigations on cryosections (Lherminier et al., 19901, as these are very suitable for immunocytochemical applications (see Boonstra et al., 1987; Tokuyasu, 1986). I thank Dr. M. F. Clark for originally providing MLO-infected plants, Dr. A. Appian0 for helpful discussion, and Mr. R. Lenzi for propagation of the plants. REFERENCES BOONSTRA, J., VAN MAURIK, P., AND VERKLEIJ, A. J. (1987) in. STEINBRECHT, R. A., AND ZIEROLD, K. (Eds.), Cryotechniques in Biological Electron Microscopy, Immunogold Labelling of Cryosections and Cryofractures, pp. 217-230, Springer-Verlag, Berlin. BOWERS, B., AND MASER, M. (1988) in CRANG, R. F. E., AND KLOMPARENS, K. L. (Eds.), Artifacts in Biological Electron Microscopy: Artifacts in Fixation for Transmission Electron Microscopy, pp. 1342, Plenum, New York. BRAUN, E. J. (1977). J. Ultrastruct. Res. 60, 4651. BREDT, W., HEUNERT, H. H., HOFLING K. H., AND MILTHALER, B. (1973) J. Bacterial. 113, 1223-1227. CHEN, T. A., LEI, J. D., AND LIN, C. P. (1989) in WHITCOMB, R. F., AND TULLY, J. G. (Eds.), The Mycoplasmas, Vol. 5, Detection and Identification of Plant and Insect Mollicutes, pp. 393424, Academic Press, San Diego. CLARK, M. F., MORTON, A., AND Buss, S. L. (1989) Ann. Appl. Biol.

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STEINBRECHT, R. A., AND ZIEROLD, K. (1987) Cryotechniques in Biological Electron Microscopy, Springer-Verlag, Berlin. THOMAS, D. L. (1979) Phytopathology 69, 928-934. TOKUYASU, K. T. (1978) J. Ultrastruct. Res. 63, 287-307. TOKUYASU, K. T. (1986) J. Microsc. 143, 139-149. TULLY, J. G. (1978) in MCGARRITY et al. (Eds.), Biology of the Mycoplasmas: Mycoplasma Infection of Cell Cultures, pp. l-33, Plenum, New York. WATERS, H., AND HUNT, P. (1980) J. Gen. Microbial. 116, lll131. WATERS, H. (1982) in DANIEL& M. J., AND MARKHAM, P. G. (Eds.), Plant and Insect Mycoplasma Techniques: Light and Electron Microscopy, pp. 101-151, Croom Helm, London. WHITCOMB, R. F., AND TULLY, J. G. (1989) The Mycoplasmas, Vol. 5, Academic Press, San Diego. ZIEROLD, K. (1987) in STEINBRECHT, R. A., AND ZIEROLD, K. (Eds.), Cryotechniques in Biological Electron Microscopy, Cryoultramicrotomy, pp. 132-148, Springer-Verlag, Berlin.