VIROLOGY 28, 555-562 (1966)

Cytological Changes Induced by Turnip Yellow Mosaic Virus in

Chinese Cabbage Leaves

J. CHALCROFT AND R. E. F. MATTHEWS

Microbiology Department, University of Auckland, Auckland, New Zealand

Accepted October 26, 1965

Cells infected with TYMV have markedly abnormal chloroplasts. Nuclei and mitochondria appear normal. No particles the size of TYMV or 83 S ribosomes were observed inside chloroplasts. Irregular star-shaped membranous inclusion bodies were frequent,ly seen in infected cells. In yellow green areas from leaves showing mosaic, chloroplasts contained very few grana. Grana were more frequent and larger in size in pale green areas, and appeared normal in dark green areas. Organelles in cells from dark green areas showed no cytological abnormalities.

INTRODUCTION

Rezende-Pinto and Borges (1952)) using light microscopy, examined the chloroplasts in Chinese cabbage leaves systemically infected with turnip yellow mosaic virus (TYNV). They found no marked change in the granular structure of chloroplasts from green areas. In the “chlorotic” areas many chloroplasts had lost their granular struc- t’ure. Rubio (1956), using light microscopy, found that inclusion bodies in TYRIV-in- fected Chinese cabbage leaves were of the “X body” type. They appeared very com- pact’ and granular, but’ were sometimes vacuolate or reticulate. Their shape was generally ovoid, and t.hey were larger t’han the nuclei. He found fully developed X bodies only in the late stages of the disease. Rubio considered that the X bodies were formed from chloroplasts. He examined the inclusions by electron microscopy, but the techniques used were inadequate to reveal very much structural det’ail.

In the work reported here we have made an electron microscopic examination of cytological changes induced by TYMV in Chinese cabbage leaves. Chloroplast ab- normalities are the most striking changes seen in infected cells. Organelles in cells from dark green areas in leaves showing mosaic appear normal, supporting t,he view

555

(Reid and Matthews, 1966) that TYNV mult’iplication does not occur in these areas.

MATERIALS AND METHODS

TYMV was grown in Chinese cabbage plants as described by Reid and Matthews (1966). Tissue sections were prepared from leaves locally and systemically infected with TYNV and also from healthy control plants.

Small pieces of leaf tissue were cut from the leaves, care being taken to include only one type of area in the systemically in- fected leaves. The leaf pieces were prefixed at 4°C for 2 hours in 6.5 % glutaraldehyde in 0.1 M phosphate buffer pH 7.4 (Sabatini et al., 1963), stored overnight in buffer alone, then fixed with 2% osmium tetroxide in buffer. Dehydration was carried out at j$hour intervals t,hrough a graded ethanol series beginning at 70% ethanol. Specimens were embedded in Araldite (Glauert and Glauert, 1958). Sections were cut with an LKB Ultrotome 4800 by a diamond knife, and sections were usually stained with a methanolic solution of phosphotungstic acid (PTA) or of methanolic uranyl acetate (Stempak and Ward, 1964) or occasionally with lead citrate (Reynolds, 1963). Thick sections used for orientation purposes and light microscope study were stained with toluidine blue in sodium carbonate solution

556 CHALCROFT AND MATTHEWS

FIG. 1. Chloroplast from healthy tissue stained with phosphotungstic acid. Magnification: X 42,900.

(Trump et al., 1961). Thin sections (grey to pale gold interference colours) were usually mounted unsupported on grids and examined with a Philips EM 200 electron microscope at 80 kV.

RESULTS

Healthy Cell Tissue

The mesophyll cells of Chinese cabbage are very similar to those of other herbaceous leaves that have been examined (e.g., Shalla, 1964) (Fig. 1). With the procedures we have used we observed numerous bodies about the size expected for 83 S ribosomes in the cytoplasm. We were not able to resolve the 68 S ribosomes in chloroplasts.

Local Lesions

In tissue taken from the centre of the yellow local lesions which form following inoculation with TYMV, the chloroplasts appear globular and enlarged, and they contain large amounts of starch even when sampled early in the day. They are not clumped in the cell, and their general dis- tribution is like that in normal cells. The chloroplasts contain many small vesicles, mainly around the periphery (Fig. 2). The grana and stroma lamellae appear essentially normal. The nucleus, mitochondria, and cytoplasm appear normal.

Yellow Green Areas from Leaves Shofwing Mosaic

The yellow green areas usually predomi- nate in the mosaic pattern of systemically infected leaves. Chloroplasts are swollen and aggregated into large clumps into which individual chloroplasts fit closely without touching one another (Fig. 3A). Very little starch is observed compared with normal tissue harvested at midday.

A narrow gap separates the individual chloroplasts. This gap is commonly en- larged to form spaces which appear more or less circular in section. These spaces between chloroplasts are filled with particulate ma- terial of the same size and staining properties as the ground cytoplasm. We have found no indication of any membrane bounding these chloroplast aggregates in sections stained with PTA, uranyl acetate, or lead citrate. We therefore assume that the spaces be- tween chloroplasts in the aggregates are filled with ground cytoplasm.

Numerous vesicles of various size occur mainly near the margins of the chloroplast. These vesicles usually have no contents that take up stain. They are bounded by a mem- brane and sometimes show membranes within them (Fig. 3B). The vesicles show no opening to t,he cytoplasm. Occasionally vesicles appear to be partly in the cytoplasm

CYTOLOGICAL CHANGES INDUCED BY TYMV 557

FIG. 2. Chloroplasts in a cell from the center of a local lesion showing large amounts of starch (S), normal grana, and numerous small vesicles (V), mainly near their margins. M, mitochondria; P, phyto- ferritin. Stained with lead citrate, Magnification: X 9000.

and partly in the chloroplast. We assume that apparently empty membrane-bounded vesicles occasionally seen in the cytoplasm may have originated in the chloroplasts.

Stroma lamellae are widely scattered in the swollen chloroplasts but apparently retain their basic structure. Very few grana are seen, and those present are thinner than normal. Phytoferritin aggregates are seen in some chloroplasts.

We have not observed any particles of the size of TYMV or 83 S ribosomes within the abnormal chloroplasts in either PTA or uranyl acetate stained material. Since yellow green tissue contains 3-6 mg of TYMV per gram fresh weight we assume that com- pleted virus must accumulate in the cyto- plasm. However, we cannot at present distinguish between TYMV and 83 S ribosomes.

558 CHA~CR~~T AND MATTHEWS

The nucleus and mitochondria in cells from yellow green areas appear normal.

In sections of yellow green tissue highly irregular, roughly star-shaped membranous bodies are seen in many cells. Such a body can be seen in E’ig. 3A. Figure 4 shows another at higher magni~catio~. These bodies have not been observed in healt,hy tissue, They appear structurally similar following PTA, uranyl acetate, or lead staining. Particles the size of TYMV or 53 S ribosomes appear to be absent in these bodies. Up to three have been observed in a section of a single cell.

chloroplasts (Fig. 5). Very large vesicles are more common than in yellow-green areas. Vesicles are frequently seen partly in the cytoplasm and partly in the chloro- plast. No &arch grains are present. Fewer chloroplasts are seen per section than in yellow-green areas. Stroma lamellae are present but grana-like st~ctures are absent.

In some sections remains of disintegrated chloroplast,s can be observed (Fig. 5). Aggregates of ele&ron dense mat.erial as- sumed to be phytoferritin (Hyde et al., 1963) are seen more commonly in chloroplasts in sections of whit,e areas t.han in other diseased

White Areas of Leaves Showing Mosaic tissue.

White areas are much less common in the Pale Green Areas from Leaves Xhowing

mosaic pattern than yellow green. In cells Mosaic

from such areas the ~hlorop1~t.s are en- These areas are i~~termediate in colour larged and more or less spherical. They between dark green and yellow green. occasionally form clumps but mainly occur Abnormalities in the chloroplasts are very singly. There are numerous vesicles in the similar to t,hose in yellow green areas except,

FIG. 3A. Whole cell from a yellow green are8 in mosaic of a systemically infected leaf; N, nucleus; icI, mitochondrion; CA, ehloropl~t aggregate; SB, star-shaped body; V, large vesicles. Stained with phosphotungst,ic acid. Magnification: X 7900.

CYTOLOGICAL CHANGES INDUCED BY TYMV 559

FIG. 3B. Part of a chloroplast aggregate from a region similar to Fig. 3A showing gaps contain- ing cytoplasmic material between chloroplastjs (C). V, vesicle; L, stromatic lamellae. Stained with phosphotungstic acid. Magnification : X 28,ocQ.

that well developed grana are frequently seen. The number and thickness of these is intermediate between normal tissue (or dark green mosaic areas) and yellow green tissue. Phytoferritin aggregates are seen in some chloroplasts.

Green Areas from Leaves Showing Mosaic

Sections of cells from these areas show apparently normal structure in the chloro- plasts and other organelles (Fig. 6). Starch grains are present in t’issue sampled at midday.

DISCUSSION

The inclusion bodies of the “X-body” type observed by Rubio (1956) were almost certainly the clumps of abnormal chloro- plasts we have described above for yellow and pale green areas. We have been unable to demonstrate any membrane surrounding the clumps of chloroplasts. It is not clear what holds the clumps together, since the

individual chloroplasts are separated by narrow zones of ground cytoplasm.

The effects of TYMV infection on chloro- plast’s in cells from local lesions and the effects on those in yellow green areas of leaves showing mosaic differ in several ways: (1) chloroplasts in local lesions are not clumped; (2) they retain large amount’s of starch over the night period; (3) their grana appear normal. Since virus is produced in similar concentrations in local lesions and in yellow green areas, the clumping of chloro- plasts, lack of starch, and small grana in yellow green areas are not necessary COKE- comitants of TYMV production. In contrast the abnormal vesicles seen in chloroplasts of all types of infected cells may be more intimately connected with virus multi- plication. Similar vesicles have been ob- served in chloroplasts of cells infected with tobacco mosaic virus (HrE;el and BrE&k, 1964). It was suggested that these vesicles arose by invagination of the chloroplast membrane. We observed no conclusive evidence for such a process in TYMV-in- fected cells. The vesicles in TMV-infected

FIG. 4. Star-shaped body in cytoplasm of a cell from white area in the mosaic. Stained with uranyl acetate. Magnification: X 49,000,

560 CHALCROFT AND MATTHEWS

FIG. 5. Part of a cell from a white area in the mosaic showing two intact chloroplasts (C) with nu- merous vesicles (V). In the center are the remnants of a disintegrated chloroplast (DC) showing large and small vesicles and stromatic lamellae (L). M, mitochondrion. Stained with uranyl acetate. Magnifi- cation: X 25,000.

cells frequently showed osmiophilic granules or masses. We have seen these only rarely in vesicles in TYMV-infected cells.

The facts that the double-stranded TYMV RNA is associated with the chloroplast fraction (Ralph and Clark, personal com- munication) and that 68 S ribosome and fraction I protein concentrations drop in TYMV-infected tissue (Reid and Matthews, 1966) suggested that the chloroplasts may be intimately connected with TYMV

replication. Our cytological observations support this view. The chloroplasts are the only organelles showing abnormalities fol- lowing virus infection. Whatever part of the virus may be synthesized in the chloroplasts, intact TYMV does not accumulate within them.

The irregular star-shaped inclusion bodies seen in the cytoplasm appear to occur only in infected cells. Their origin, and signifi- cance if any, in virus replication is obscure.

FIG. EA. Low power eIectron micrograph of cells in a dark green arett of the mosaic; the chl~ropl~~t strurttwe appears normal. S, starch grain; M, mitochondrion. IJnstaincd. Magnification: X 7200.

FIG. GB. Chloroplrtsts in a dark green area of mosaic showing no abnormal structure. P, phytoferritin. Tinstained. Rlugnification: X 34,000.

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562 CHALCROFT AND MATTHEWS

However, if they remained intact in leaf homogenates, their size is such that they would be likely to contaminate chloroplast preparations isolated by centrifugation.

There is a correlation between the shade of green or yellow in tissue from a leaf showing mosaic and the size and frequency with which grana are seen in the chloroplasts.

Dark green areas in the mosaic appear quite normal cytologically. This supports other evidence that TYMV does not multi- ply in these areas (Reid and Matthews, 1966).

Phytoferritin is thought to be the reserve of iron from which growing plastids draw during development of the photosynthetic apparatus (Hyde et al., 1963). Although we have no quantitat’ive data, phytoferritin aggregates are more frequently seen in virus-infected cells than in normal or dark green areas of mosaic-affected leaves. It’ may be that virus infection interferes with the utilization of phytoferrit’in in the formation of grana.

REFERENCES

mosaic virus and cucumber virus 4. Virology 23, 252-258.

HYDE, B. B., HODGE, A. J., KAHN, A., and BIRN- STIEL, M. L. (1963). Studies on phytoferritin. I. Identification and localization. J. Ultra- struct. Res. 9, 248-258.

REID, M. S. and MATTHEWS, R. E. F. (1966). On the origin of the mosaic induced by turnip yellow mosaic virus. Virology 28, 563-570.

REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain in elec- tron microscopy. J. Cell Viol. 17, 208-212.

REZENDE-PINTO, M. C., and BORGES, M. de L. V. (1952). L’action pathologique du virus de la mosai’que jaune du Navet sur les chloroplasts de Brassica chinensis. Apron. Lusitana 14, 259- 267.

RUBIO, M. (1956). Origin and composition of cell inclusions associated with certain t,obacco and crucifer viruses. PhytopathoZogy 46, 553-556.

SABATINI, D. D., BENSCH, K., and BARRNETT, R. J. (1963). Cytochemistry and electron micros- copy. The preservation of cellular ultrastruc- ture and enzymatic activity by aldehyde fixa- tion. J. Cell Biol. 17, 19-58.

SHALLA, T. A. (1964). Assembly and aggregation of tobacco mosaic virus in tomato leaflets. J. Cell BioZ. 21, 253-264.

GLAUERT, A. M., and GLAUERT, R. H. (1958). STEMPAK, J. G., and WARD, R. T. (1964). An im-

Araldite as an embedding medium for electron proved staining method for electron microscopy.

microscopy. J. Biophys. Biochem. Cytol. 4, 191- J. Cell BioZ. 22, 697-701.

lQ4. TRUMP, B. F., SMUCKLER, E. A., and BENDITT,

HR~EL, I., and BREAK, J. (1964). Ultrastructural E. P. (1961). A method for staining epoxy sec-

changes in chloroplasts and cytoplasm caused t,ions for light microscopy. J. Ultrastruct. Res. 5,

by local infection of tobacco with tobacco 343348.