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Electron microscopy of virus-infected sunflower leaves
© 1967by AcademicPressInc. J. ULTRASTRUCTURE RESEARCH 19, 173--195
(1967)
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E l ectron Microscopy of Vi rus-I nfected Su nflower L e a v e s 1,: HOWARD J. ARNOTT AND KENNETH M. SMITH The Cell Research Institute and The Department of Botany, The University o f Texas, Austin, Texas 78712 Received January 5, 1967
A short description is given of an apparently new virus disease of the mosaic type attacking the wild sunflower (Helianthus annuus L.). Its rapid spread in the field denotes an insect-vector, probably an aphid, but its identity is not yet established. Four major types of pathological changes were observed in the mesophyll cells: (a) cytoplasmic inclusions; (b) changes in the structure and type of plastid; (c) abnormalities in the nucleus; (d) presence of virus-like particles within the leaf cells. Crystalline inclusions were also present in both normal and infected cells. The cytoplasmic inclusions are of three types: whorls resembling "pinwheels"; long rods of composite structure attached to the pinwheels and resembling a "cat-o-nine-tails"; and circular inclusions of lamellate construction. Evidence suggesting that these inclusions do not consist of virus is given; but the possibility that they may consist of viral protein is considered. The pinwheels and rods are shown to have an intimate connection with the endoplasmic reticulum. The plastids undergo longitudinal division with the production of a fifth intervening layer, a phenomenon not hitherto recorded. The nucleus becomes swollen and completely filled with spherical or near-spherical particles with which are associated numerous fine threads. The virus particles are long flexible rods measuring at least 480 × 13 m/z; they are few in number and occur in association with the outer surface of the plastids. A discussion of these findings is appended. Wild sunflower plants (Helianthus annuus L.) growing in the vicinity of Austin, Texas, were f o u n d to be infected with a virus of the mosaic type. The first sign of infection was a mild mosaic mottling in the leaves but as the disease progressed a severe necrosis of leaves and the stem developed which caused the death of the upper part of the plant. Where the necrosis of the stem ended, a few very small distorted leaves were formed and further distorted leaves were produced f r o m the axillary buds. During the m o n t h of June, the virus spread rapidly a m o n g the wild sunflowers in the vicinity. The identities of the virus and of its vector have n o t yet been determined, however, we have ascertained that the virus is rod-shaped and suggest that 1 This investigation was supported in part by grant NIH GM-07289. The authors thank W. Gordon Whaley for his advice and support and Mrs. Linda Bourianoff for her technical assistance. 2 A preliminary report of this paper was presented at the Sixth Annual Meeting of the American Society for Cell Biology in Houston, Texas, 1966.
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the rapid spread is probably due to an insect vector, probably an aphid. Experiments on the mechanical transmission of the virus to young sunflower plants have so far been negative. The present report will present observations on the effects of this disease on the ultrastructure of sunflower mesophyll cells. MATERIALS AND METHODS Samples of leaves and petioles in both early and late stages of the disease, together with comparable samples from healthy plants, were fixed in a 1 : 1 mixture of 6 % glutaraldehyde and 6% acrolein with 0.2 M cacodylate buffer for 1 hour at pH 7.2 (13). They were then rinsed with the same buffer three times during 1 hour and postfixed in 4 % osmium tetroxide mixed 1 : 1 with buffer for 1 hour. The samples were then washed twice with buffer, twice with distilled water and then placed in 0.5 % uranyl acetate overnight in the refrigerator. This was followed by three washings with distilled water and dehydration in ethyl alcohol and acetone. The samples were then gradually infiltrated with Epon, evacuated, and put in a 60 ° oven overnight. Sections were cut with a diamond knife on a MT-2 "Porter-Blum" ultramicrotome and poststained in uranyl acetate and lead citrate. Micrographs were taken on RCA EMU 3F and Siemens Elmiskop I electron microscopes. OBSERVATIONS Four major types of pathological changes were observed in the mesophyll cells of the infected sunflower leaves: (a) cytoplasmic inclusions; (b) changes in the structure and type of plastid; (c) abnormalities in the nucleus, (d) presence of virus-like particles within the leaf cells. None of these changes were found uniformly throughout an infected leaf. Examination of tissues of different colors from the same leaf (Table I) revealed that only yellow tissues contain cytoplasmic inclusions and virus-like particles. Nuclear abnormalities are likewise confined to the yellow tissues, however, plastid differences occur in all tissues of the infected leaf. Our discussion will be limited to observations of palisade parenchyma cells in mature leaves. These cells are cylindrical in shape, being several times longer than their diameter which is about 7-10/z (Fig. 1). The cells are orientated with their long axis perpendicular to the leaf surface; in their mature state these cells are highly vacuolate, the central vacuole being surrounded by a peripheral cytoplasmic layer in which the nucleus and other cytoplasmic components are found. Although the cell shown in Fig. 1 shows m a n y symptoms of the disease, in organization it is similar to a normal palisade parenchyma cell.
IntraceIlular inclusions The most conspicuous intracellular inclusion takes the form of a cylinder with internal septa which radiate out from a small central cylinder; when cut transversely
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TABLE I P A T H O L O G I C A L C H A N G E S IN THE S U N F L O W E R LEAF TISSUES
Tissue Color
Infected green Infected yellow-green Infected yellow Uninfected (control)
Virus Present
Cytoplasmic Inclusions Present
Plastid Modification
Nuclear Abnormalities
+ -
- (+) + -
+ + + -
+ or -
it appears as a "pinwheel" or like an ophiuroid starfish (Figs. 1-3). Matsui and Yamaguchi (14) were probably the first to describe inclusions like these in their study of host cells of tobacco infected with tobacco etch virus; they called them "leoped profiles" and suggested that they corresponded to a coil of filamentous virus particles. While the present study was in progress, Edwardson (7, 8) published the results of an investigation of cytoplasmic inclusions associated with rod-shaped viruses. He described bodies of several types, laminated, circular and bundle inclusions. The one which was common to all the rod-shaped viruses studied, however, was the pinwheel. Edwardson (7) suggests that this type of inclusion body is diagnostic of infection with rod-shaped viruses of the potato virus Y group.
Structure of the inclusions The pinwheel inclusions are cylindrical bodies made up of approximately 9 to 12 septa radiating from a cylindrical inclusion center (Figs. 2 and 3). As the septa radiate they bend, forming a figure which looks like a pinwheel. The terminal portion of the septa may fuse with those of adjacent septa, thus forming a locule, or they may fuse with septa from adjacent inclusions to form an interconnected system of inclusions such as Edwardson (8) has shown in other cases (Fig. 2). The septa may be single or compound with as m a n y as three units fused together to produce a single compound septum. Apparently the locules are free at one or perhaps both ends (Fig. 6). The central cylinder from which the septa radiate may be "hollow" or it may contain a small dense core. In many cases it is possible to show that the structure of the septum is different from that of the membrane making up the endoplasmic reticulum (ER) or other cytoplasmic membranes (Fig. 3). A clear "unit m e m b r a n e " structure could be made out for the ER while a similar structure could not be observed in the septa. Our micrographs indicate that the septa do not have the structure of a unit membrane. If there are subunits which make up the septum they must be quite small; it seems
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unlikely that the septa are composed of an aggregation of complete virus particles. Examination of longitudinal or slightly oblique sections of the pinwheel inclusions show the septa in face view; in all of these which we have examined we have not been able to see any longitudinal striations. The thickness of these layers which make up or compose the septa are between 80-100 A units or only about 0.7 the thickness of the virus-like rods seen in infected cells which average about 130 A units (Fig. 16-18). I n the sunflower leaf cells there were two other types of inclusions which are associated with the pinwheels; their exact relationship to the pinwheels is not yet clear. One is a long rod which appears to be a composite structure made up of as m a n y as six layers, each layer being approximately 80-100 A units in thickness; these m a y be similar to the "lamellate inclusions" or the "dense b a n d s " reported by others (7, 14). In the sunflower the long rods sometimes appear to be connected at one end to a pinwheel, the whole structure closely resembling a "cat-o-nine-tails" (Fig. 5). A third type of inclusion b o d y is circular and seems to have a lamellate composition similar to that of the long rods. There m a y be as m a n y as f r o m three to five layers composing these inclusions and again the laminations appear t o be 80-100 A units in thickness. The layers m a y interconnect with m o r e than one of the circular inclusions, forming a more massive inclusion composed of anastomosing layers f r o m several circular bodies (Fig. 4). The pinwheel inclusions are associated in an intricate fashion with profiles of endoplasmic reticulum (Figs. 2, 3, and 6). In longitudinal sections of these pinwheel inclusions, E R profiles can be seen " a t t a c h e d " to the base of successive inclusion bodies (Fig. 6). The E R not only is associated with the base of the cylindrical pinwheel inclusions but penetrates into the locules or between the septa of the inclusions.
Key to abbreviations
C
plastid O osmiophilic globule CR crystal (contained within a single membrane) P plasmalemma E R endoplasmic reticulum S starch G granum SE septum GR nuclear granule T tonoplast I inclusion T H thylakoid IA intercellular air space V vacuole L locule VR virus-like rod M mitochondrion W wall N nucleus Fro. 1. Section of a leaf of Helianthus annuus infected with the sunflower mosaic virus (SMV). The section was made parallel to the surface of the leaf and passes through a palisade parenchyma cell perpendicular to its long axis; the cell is associated with other palisade parenchyma ceils and an intercellular airspace system. The section is from yellow tissue of a mature leaf, the level of section passes through the nucleus and a small portion of the large vacuole. Six plastids, two crystal-containing bodies bounded by a single membrane, several mitochondria, and rough ER are seen in addition to the cytoplasmic inclusions which are characteristic of the disease. Two pairs of plastids which may have undergone longitudinal division are shown. × 20,000.
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I n Fig. 2 each of the inclusions is penetrated by strands of E R ; in some cases the E R profiles are small a n d circular, i n others they are somewhat flattened a n d some septa m a y c o n t a i n more t h a n one profile of ER. The rod-shaped inclusions are also associated with ER; very c o m m o n l y the ends of the rods are " i n t e r c o n n e c t e d " with E R m e m b r a n e s that seem to f o r m direct contacts, in other cases the rod appears to be growing out of the ER, a n d at other p o i n t s the E R m a y be merely adjacent to the rods (Fig. 5). The E R which is associated with the inclusions can be s m o o t h or r o u g h (Figs. 2-5). Bodies similar to ribosomes are also present within the locules of the pinwheel inclusions. The matrix contained within the locules, a l t h o u g h it m a y be composed of a similar g r a n u l a r material, is generally less dense t h a n that of the cytoplasm. N o regular association of other organelles with these inclusions has been observed.
Changes in the chloroplasts I n cells of infected leaves conspicuous changes occur in the structure of the plastids. I n green a n d yellow-green areas of these leaves the changes are m a i n l y in the reduction of the n u m b e r of lamellae f o r m i n g a g r a n u m a n d some increase in the size of the osmiophilic globules. I n the yellow areas of the leaf, however, the change in the plastids is m u c h greater. I n general this change is similar to that which some fruits u n d e r g o during the f o r m a t i o n of chromoplasts, a n d in fact the plastids in the yellow area of the infected leaf would have to be classified as chromoplasts.
Normal plastids. Before a t t e m p t i n g to describe the changes which occur with the disease, we will describe the c o n d i t i o n of the plastids in n o r m a l leaves. A m a t u r e chloroplast in sunflower is a disk-shaped body separated from the cytoplasm by two FIG. 2. Peripheral cytoplasm of a cell from a leaf infected with the sunflower virus (SMV) showing four pinwheel inclusions. The central cylinder from which the septa extend is most clearly seen in the lower inclusion. Septa, composed of multiple layers, are seen in each inclusion; three partly separate septa fuse into one in the lower inclusion. Tubules of endoplasmic reticulum can be seen associated with each inclusion; over thirty profiles of ER are shown in or near the four inclusions. × 125,060. FIQ. 3. Pinwheel inclusion made up of 9 septa radiating from the central cylinder. Successive septa fuse with each other forming a series of closed locules; four locules contain profiles of ER. Note the structure of the septa as opposed to that of the ER and plasmalemma; the latter two clearly show a "unit membrane" structure whereas the septa seem to be constructed in seine other manner, x 154,000. FK6.4. Circular inclusions in the peripheral cytoplasm of a cell of the yellow portion of a leaf infected with SMV. Circular inclusions are interconnected by "septa-like" layers. The inclusions are composed of from one to five layers and like the pinwheel inclusions are associated with ER. × 82,500. FIG. 5. Rod-shaped inclusions in the cytoplasm of a cell infected with SMV. Note close association of ER with the inclusions. Arrow indicates an inclusion which seems,to be "growing out of" rough ER. × 135,000. F~6. 6. Pinwheel inclusions in the cytoplasm of a cell infected with SMV. Three inclusions are sectioned longitudinally, the other transversely. An end of each of the three inclusions shown in longitudinal section is in contact with ER. Virus-like rods are seen associated with the plastid in the upper right. Note that the face views of the septa show no structure similar to that of the virus-like rodsb x 70,000.
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unit membranes; in sectional view it has an elongated spindle shape (Figs. 7 and 8). Within these two membranes which make up the plastid envelope a series of thylakoids run for various distances; these flattened sacs combine at certain points to f o r m stacks or grana. In addition to the thylakoids there are m a n y plastid ribosomes, a few small osmiophilic globules, and one or two starch grains embedded in the plastid matrix. As m a n y as 35 or more thylakoids m a y be associated in a single gran u m ; however, grana with fewer units are c o m m o n . Almost every section of a mature chloroplast will have grana with 20 or more lamellae (thylakoids) as components (Fig. 7, 8). In our preparations a thin, very dense layer of material is f o u n d in the lumen of each thylakoid,whether it is a part of a g r a n n m or not. Plastids in green infected tissue. The changes which occur in plastids in the green areas of infected leaves are a matter of degree rather than of kind, as we shall see is the case in the yellow infected tissue. Three changes can be noted: (a) a reduction in the n u m b e r of thylakoids forming a g r a n u m (this is the most obvious difference in green areas of an infected leaf). These plastids seldom have as m a n y as 10 thylakoids in a granum, and the general impression one obtains is that the total n u m b e r of thylakoids in the plastid is reduced; (b) the n u m b e r and the size of the osmiophilic globules increase, the increase is relatively slight, however, when c o m p a r e d with that in the yellow tissues; (c) the shape of the plastids seems to change toward a m o r e spherical form. The ratio between the length and the height of the plastid, as seen in sectional view, is reduced. Plastids in yellow infected tissue. Most, but perhaps not all, of the plastids in this area of the leaf take on all the characteristics of chromoplastids, with the possible exception that they often contain several large starch grains. They still retain the two unit membranes which f o r m their envelope; ribosomes are still plentiful in the matrix but m a n y differences can be shown between them and the n o r m a l chloroplasts in uninfected tissue. There are two prominent features which are immediately apparent, both are continuations in the changes already denoted for the plastids in green infected tissue. The first concerns the further reduction in the total n u m b e r of thylakoids which participate in the formation of a g r a n u m (Figs. 9-13). The second concerns the spectacular increase in the n u m b e r of osmiophilic globules (Figs. 9 and 11). In addition to these changes the plastids in this tissue appear to be smaller than those f r o m n o r m a l cells (compare Figs. 7 and 8 with Figs. 9 and 10). Fins. 7 and 8. Normal plastids from the mature leaf of an uninfected plant of the sunflower. Note small number of osmiophilic globules, large number of thylakoids associated in each granum and the thinness of the thylakoids in both the grana and stroma in comparison with those from infected plants (Figs. 9-13). Fig. 7, x 30,700. Fig. 8, x 20,800. FIGS. 9 and 10. Plastids from yellow areas of leaves infected with SMV (compare these with the normal plastids seen in Figs. 7 and 8). Note the large number of osmiophilic globules, the thickness of the thylakoids, and the lower number of thylakoids per granum. As shown in Fig. 10 the plastids from this area of infected leaves often contain large starch grains. Fig. 9, x 48,000. Fig. 10, x 50,000.
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The change in n u m b e r of thylakoids as well as the reduction in the n u m b e r of thylakoids participating in grana formation is accompanied by a dramatic increase in the thickness of the layer of dense material contained in the lumen of the thylakoids. This material is as electron dense as that of the osmiophilic globules and like these bodies the substance deposited in the thylakoids does not show any apparent substructure. The thylakoid lumen in some cases becomes inflated by an accumulation of this electron dense material; when this inflation occurs there seems to be a concomitant decrease in the n u m b e r of osmiophilic globules (Fig. 13). The n u m b e r of osmiophilic globules seen in the plastids of yellow tissues is greatly in excess of those seen in n o r m a l chloroplasts. F r o m 15 to 20 of these dense bodies can be seen in a single section of a plastid. N o t only is there an increase in n u m b e r but the diameter, and hence the volume, increases in these plastids (Fig. 9). The characteristics of the plastids f o u n d in these different tissues are summarized in Table II. A n o t h e r interesting feature of the plastids in the yellow infected tissues is the appearance of plastids which appear to have undergone longitudinal division. In these cases the " d a u g h t e r " plastids are approximately the same size and are f o u n d juxtaposed longitudinally, a situation which was never seen in healthy tissues. Close observation of these plastids shows a dense line a b o u t 100-110 A units in thickness between the chloroplast envelopes of the " d a u g h t e r " plastids. F o r example, in Fig. 12 five lines are seen separating the two plastids, the upper two represent the two membranes of the upper plastid and the lower two represent the two membranes of the lower plastid; the central line represents the dense line mentioned above. This unique "fifth" layer between the plastids has not been reported before and is never present in plastids which are juxtaposed in other positions (end-to-end) or in the uninfected tissues. In some of the plastid pairs it was impossible to be sure that the two plastids were completely separate; what m a y be a stage in the division of plastids by a longitudinal division process m a y be seen in Fig. 11. In this case careful study seems to indicate that a portion of the two plastid interiors is still in connection (arrows).
Changes in the nucleus Conspicuous changes take place in the nuclei of some cells in the virus-infected sunflower leaves (Figs. 14 and 15). These changes only occur in the yellow tissues and FIG. 11. Longitudinally "divided" plastid in a cell from an infected leaf. The upper plastid is separated from the lower one by a curving line which crosses through the center of the pair complex and curves back at the upper right of the upper plastid, thus the lower plastid appears to enfold the upper one. Through most of this line of demarcation the two plastids are separate; however, between the two arrows it is not clear whether they are separate or not. Study of the micrograph seems to indicate that they are not separated and that this represents a stage in the division of the plastid. × 43,000. FIG. 12. "Divided" plastids in a cell from a leaf infected with SMV; the two membranes of both plastids are visible along with a "fifth" layer which separates the two plastid envelopes. Note the inflated thylakoids in these plastids. × 75,000.
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then have only been irregularly encountered. At an early stage in the infection of a cell, the nucleus appears normal even though the chloroplasts have undergone significant modifications and the pinwheel inclusions are apparent in the cytoplasm (Fig. 1). Within what seems to be a normal nuclear envelope the contents are similar to those seen in uninfected plants (Fig. 1). Figs. 14 and 15 show changes which occur in the nuclei of some infected leaf cells, especially near the vascular system. The nucleus becomes completely filled with spherical or near-spherical particles measuring about 40-60 m#. At this time, the nucleus appears to be swollen and an increase can be observed in the size of the nuclear pores; additionally, the dense material forming the annulus of the pore in normal tissues is missing in these cells. At a still later stage in nuclear change the nuclear envelope may be broken at m a n y points. At this time there are numerous fine threads associated with the particles (Figs. 14 and 15). The threads measuring about 20-25 A units in diameter form an anastomosing reticulate network between the particles. The threads are associated with small dense points measuring about 60 A units; together they sometimes appear like beads on a string. These dense points about 60 A units in diameter also seem to be components of the larger granules seen in the nucleus (Fig. 15).
Virus-like particles within the cells After considerable searching, our efforts were finally rewarded in the finding of long flexuous rods in the cells of infected plants. Although m a n y phosphotungstic acid (PTA) preparations were made, no virus-like particles were ever found. It was only by careful scrutiny of our micrographs of sectioned materials that we were able to find these bodies. Location of the rods. The rod-shaped inclusions have only been found in association with plastids (with one exception, see Fig. 6); in fact it was through the examination of oblique sections of plastids that we were first able to see these rods. The rods are always found between the outer plastid membrane and either the plasmalemma or the tonoplast. In these large vacuolate cells it is common for these membranes to be separated by only a few hundred Angstrom units and it is in this space, between two membranes, where the rods are found (Figs. 6, 16, and 18). In some cases rods are present between the outer plastid membrane and both the tonoplast and the plasmalemma (Figs. 16 and 18). Structure of the rods. The rods are long flexous structures which can be straight or bent in a shallow arc. The length is difficult to establish from sectioned material, but FIG. 13. Plastid in a cell from a leaf infected with SMV showing severely inflated thylakoids. The electron dense material seen in the lumen of the thylakoids is very similar in electron density to that of the osmiophilic globules which are also seen in this plastid. Particles approximately the size and having the general nature of the virus-like rods are seen between the two mitochondria and the plasmalemma (see circles) in the lower part of the micrograph, x 86,000.
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TABLE II SUMMARY OF MAJOR CHARACTERISTICS OF NORMAL AND INFECTED SUNFLOWER LEAF TISSUE Characteristic
Normal
Virus-like rods, 13 m/z diameter Inclusions Nuclear abnormalities Crystals Number of thylakoids in grana Number of osmiophilic globules in plastids Size of osmiophilic globules
Absent Absent Absent Present 15 or more Few Small
Green Infected
Absent Absent Absent Present Reduced Few Slightly larger than normal
Yellow Infected
Present in some cells Present in many cells Present in a few cells Present Severely reduced Many Twice that of normal
we find t h a t the m i n i m u m is 480 m#. T h e d i a m e t e r is m o r e easily m e a s u r e d a n d averages a b o u t 13 m#. O u r best m i c r o g r a p h s indicate t h a t the r o d s are h e x a g o n a l in cross section a n d t h a t a central dense core m a y be present (Figs. 16 a n d 18). A s u m m a r y of the m a j o r characteristics f o u n d in n o r m a l a n d infected tissues of the sunflower is shown in T a b l e II. Crystalline inclusions m a d e u p of particles regularly a r r a n g e d in rows a n d cont a i n e d within a m e m b r a n e are f o u n d in tissues of b o t h infected a n d n o r m a l leaves in a p p r o x i m a t e l y equal n u m b e r s (Figs. 1, 10, a n d 11). These crystals have a r e p e a t spacing of a b o u t 2 0 0 / ~ units a n d a p p e a r as squares, d i a m o n d s , rectangles, a n d triangles when seen in thin sections: A l l these sections m i g h t be i n t e r p r e t e d as r e p r e s e n t a tive of a cubic crystal. DISCUSSION One of the results of this research is to question the n a t u r e of the c y t o p l a s m i c inclusions. M a t s u i a n d Y a m a g u c h i (14) a n d H a y a s h i et al. (12) believe t h a t similar inclusions in the cells of the p l a n t s they have studied represent the virus particles in v a r i o u s types of a r r a n g e m e n t . O u r observations d o n o t l e a d us to a similar view f o r the case we have examined; on the c o n t r a r y , it seems clear t h a t the pinwheel, circular, o r r o d - s h a p e d inclusions are n o t m a d e up of particles similar in size to those of the
FIG. 14. A nucleus which is apparently undergoing breakdown in a cell from a leaf infected with SMV. Many granules of two sizes and fibrils are seen throughout the nucleus; the nuclear membrane has already undergone partial breakdown. Other organelles, e.g., mitochondria, appear more or less normal. Transverse sections of the virus-like rods are seen in the upper left where they are associated with a chloroplast, x 80,000. Fie. 15. Enlargement of the particles and fibrils in the nucleus seen in Fig. 14. The fibrils branch and anastomose forming a network, at some points [hey appear to enter and perhaps form a part of the larger particles. Small particles about 60 dk units in diameter are associated with the fibrils. × 165,000.
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presumed virus which infects the tissues. This is particularly clear from examination of longitudinal sections through the pinwheel inclusions (Fig. 6). In this plane of section many of the septa can be seen in face view; none have shown longitudinal striations which would indicate that they are made up of a series of rod-shaped virus particles in a two-dimensional array, even though the virus-like rods are clearly seen in this same figure. In a study of Vicia faba infected with bean yellow mosaic virus, Weintraub and Ragetli (24) found inclusions made up of dense bands, crystals, and small aggregates of virus. They found "the dimensions of the dense bands and crystalline inclusions were difficult to reconcile with BYMV particles." Shalla (17) illustrates bodies which may be similar to the inclusions under discussion, together with the virus particles in tomato tissues infected with tobacco mosaic virus. While our observations seem to indicate that the inclusions are not made up of complete virus particles it is possible to conceive of these inclusions as viral component(s); perhaps they may represent the protein portion of the virus in a paracrystalline array. The inclusions could be a storage mechanism through which the viral protein could be accumulated, or the inclusions might represent an overproduction of a viral component, the units of which undergo assembly into the various morphological structures which are seen in the infected cells. If these inclusions do represent a viral component this would help to explain the similarity of the morphology of inclusions seen in a wide range of host plants but always associated only with a certain group of viruses. For the time all that can be said is that the sunflower inclusions do not appear to be arrays of complete virus particles. Based on the work of Matsui and Yamauchi (14), Edwardson (7, 8), Weintraub and Ragetli (24) it is apparent that the inclusions seen in the present case (Figs. 2-6) are a diagnostic characteristic for the disease caused by the rod-shaped R N A viruses of the potato virus Y group. Edwardson (7) could not find inclusions in the infected tissues when viruses from the potato virus X group, TMV group, or tobacco rattle virus group were inoculated. Lee (26) illustrates inclusions similar to those shown here in a disease caused by the wheat streak mosaic virus (WSMV). The systematic classification of this virus (WSMV) has placed it in the potato virus S group; however, Brandes and Bercks (27) indicate that it is intermediate in length between the potato FIG. 16. Virus-like rods cut transversely. Note their hexagonal shape and the dense "core". x 440,000. FIG. 17. Tangential section of a plastid in a cell infected with SMV showing the virus-like rods which are usually found in association with the plastids. The plastid goes out of section approximately at the middle of the micrograph. Note the curved nature of the virus-like rods. FIG. 18. Section of a plastid in a cell infected with SMV showing associated virus-like rods cut in transverse section. The rods are shown between the plastid membrane and the tonoplast on the left and between the plastid membrane and the plasmalemma on the right. Many of these rods seem to show a hexagonal structure and seem to contain a central dense "core." × 197,000.
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virus S group and the potato virus Y group and could easily belong to the latter. In the sunflower the inclusions were discovered before the virus-like particles; with the present knowledge [Edwardson's paper (7) came out as we were pursuing this research], we might have predicted the nature of the virus with some degree of accuracy. The three-dimensional nature of inclusions like those in sunflower has been considered by Edwardson (8) through the reconstruction of these bodies from serial sections; he is of the opinion that the pinwheels and the bundles are different aspects of a single type of inclusion. This inclusion is assumed "to be cylindrical in shape and composed of curved plates with their inner edges converging around the central axis of the cylinder." He found, in examination of serial sections, that many pinwheels and bundles that are clearly separated in individual sections appear to be interconnected. Our micrographs certainly show that many "bundles" and pinwheels are interconnected, and further that several inclusion bodies may also be interconnected, even in a single section. Perhaps all the inclusions are interconnected. Previous authors have not noted that the inclusions often have obvious interconnections and/or interrelationships with the endoplasmic reticulum (ER). In our study we find this relationship is almost universal. Some of Edwardson's (7) micrographs show what appears to be a similar condition in the examples he has illustrated. The ER in association may be either rough or smooth. If the inclusions are protein (whether they are viral or "cellular" protein is incidental) the close association with rough ER seems to warrant careful study. One could suggest that the origin or the supply lines bringing materials to these inclusions is the ER; however, the functional aspects of these inclusions will have to await further research. The dramatic changes in the ultrastructure of the plastids which occur in infected cells represents one of the most interesting aspects in the cytological aspects of this disease, Shalla (17) observed the distortion and vacuolation of chloroplasts in tomato infected with TMV; the virus appeared within some of the vacuoles in the chloroplasts, and in other plastids extrusions were formed. Similar extrusions were reported in the chloroplasts of Vicia, but otherwise only little change was found in infected plants (24). Chalcroft and Matthews (3) found that a variety of changes occur in the leaves of the Chinese cabbage infected with turnip yellow mosaic virus (TYMV); the yellow and yellow-green areas of the infected leaf showed many changes, but the green areas appeared to be more or less normal. This is a situation identical to that which we have found in the present case. In the yellow-green areas they found that the plastids were swollen and aggregated into large clumps; there were few grana and those present were thinner than usual, a condition quite unlike that which we found in sunflower. In the yellow areas of the Chinese cabbage leaves they found the plastids contained large numbers of starch grains--here the situation was similar in the sun-
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flower. Because only the plastids showed abnormalities following virus infection and because of certain associated biochemical data, it was suggested (3) that some part of the virus may be synthesized in the chloroplasts; however, intact T Y M V particles were not found to accumulate within the plastids. In Beta vulgaris the lipid inclusions (osmiophilic globules) in the chloroplasts were sometimes unusually large when the cells were infected with beet yellows virus (6) and also inclusions consisting of elongated "virus-like" particles were present in the chloroplasts. These inclusions were found either free in the stroma, where they formed complicated patterns of parallelly oriented particles arranged in curved rows, or closely associated with lipid globules; otherwise the plastids were more or less normal. We have not found vacuolation, extrusion formation, or the presence of virus-like particles in the plastids of sunflower. The changes which appear in the plastids of the sunflower after infection are very similar to those which are reported in a variety of ontogenetic systems where plastids pass from the chloroplast to the chromoplast state of development. One of us (H. A.) has seen analogous changes in the normal development of the plastids of Malpighia glabra fruits as they ripen (unpublished). Similar changes as a result of normal development are reported in the petals of Ranunculus (9), in Aloe flowers (20), and recently in the development of the chromoplasts of the Valencia orange (21); with respect to the development of the osmiophilic globules and the reduction in the number of thylakoids (both in the grana and stroma) the latter case is almost identical to that reported herein for the sunflower. Another striking similarity to the changes in the sunflower has been shown in the chloroplasts of Abutilon infected with a virus; however, Sun (22) considers these changes to be degenerative in nature. In the cases reported in the literature (9, 20, 21, 22) the changes which occur during chromoplastogenesis do not involve increases of dense materials within the lumen of the thylakoids, such as is the case in the sunflower, rather the materials which cause density to appear in the plastids appear to be deposited on the surface of the thylakoids or between the surfaces of adjacent thylakoids. The changes seen in the sunflower with respect to the accumulation of osmiophilic globules, however, are very like that seen in the normal development of many chromoplasts. There are not many papers dealing with the division of plastids in higher plants (10, 15) but these usually show plastids dividing transversely to the long axis of the plastid. The possibility of longitudinal division must now be considered, even if only in connection with abnormal tissues. Our micrographs (Figs. 1, 11, and 12) illustrate "daughter" plastids associated together in such a manner as to be very suggestive of such a longitudinal division process. The origin of the "fifth" layer, that is, the layer between the plastid envelopes, may be a result of such a division process; such a layer has not previously been reported in the literature and appears only in connec13
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tion with paired "daughter" plastids. Stages in this division process have been seen, but at the present stage of our knowledge we cannot be absolutely sure that division is occurring. The modifications of the nucleus which we have seen in isolated cells in the yellow part of infected sunflower leaves have not been of the kind observed by others, e.g., deep grooves or cytoplasmic channels in the nucleus (19), the presence of virus particles in the nucleus (6). Although the nuclear pores become enlarged we have no evidence that materials pass from the nucleus to the cytoplasm as others have reported (25). Whether or not any viral component is manufactured in the nucleus of infected sunflower cells is not known, however, from our observations it seems more likely that the nuclei are merely undergoing some kind of breakdown or denaturation. The exposure of fibrils approximately 20 N units in diameter, hence approximately the size of the D N A molecule, as well as the larger 60 and 400-600 A units particles may represent nothing more than the "uncoiling" of the chromatin due to the disease. Dense granules about 200 A units in diameter were detected within chromatin clumps in the nuclei of cells affected with tobacco etch virus (14), but the relationship to the structures we have seen is not clear. The relationship of the rod-shaped, virus-like particles to the membranes of the plastids (and occasionally those of the mitochondria) and plasmalemma or tonoplast is difficult to understand. In every case where we have seen the virus-like particles they are found situated in these restricted spaces. The question whether the membranes have something to do with the production of the virus is of great interest. Recently Smith et al. (18) have shown that virus particles are attached to photosynthetic lamellae at certain stages in the development of the blue green algal virus within the cells of Plectonema. In the present case it may be of interest to note that the viruslike particles are always present in monolayer between the adjacent membranes (Figs. 16 and 18). The only exception to this was seen when six particles were found associated together in a small array. The measurements we have made indicate that the minimum length of the viruslike rods is 480 mk~; this is not to say that they are not much longer, which we strongly suspect, but sectioned materials are not the best for measuring size. We have not been able to see the rods in PTA preparations despite several efforts, perhaps this is due to their low concentration within the sunflower cells. Since the rods are probably somewhere near the size of those seen in the potato virus Y group (their diameter is close), we should not exclude the possibility that the present case in sunflower also represents an example of the cytological effects caused by a virus of that group. We find identical crystalline inclusions in infected and normal cells of the sunflower. These crystalline inclusions are contained within a membrane and are made of regularly arranged particles (Figs. 1, 10 and 11). Morphologically similar crystal-
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line inclusions have been seen in a variety of tissues (1, 2, 5, 11, 23). The crystals in sunflower are similar to the "proteosomes" described in the storage parenchyma cells of Helianthus tuberosus after experimentally induced greening (11). Similar crystals have been found in both the normal and virus infected cells of Citrus by Price (16). Chambers et al. (4) illustrate crystals very different in lattice parameters from the virus rods illustrated in the same micrograph of lettuce cells infected with necrotic yellows virus. It seems clear that m a n y crystalline inclusions in plant cells do not have anything to do with virus aggregations (1, 2, 5, 11, 23) or with virus infections. The possibility of enzyme storage or light reception has been discussed by Cronshaw (5) and Thornton and Thimann (23).
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.
ARNOTT,H. J., Am. J. Botany 53, 603 (1966). ARNOTT,H. J. and DAUWALDER,M., Am. J. Botany 53, 611 (1966). CHALCROET,J. and MATTHEWS,R. E. F., Virology 28, 555 (1966). CHAMBERS,T. C., CROWLEY,N. C. and FRANCKI, R. I. B., Virology 27, 320 (1965). CRONSHAW,J., Protoplasrna 59, 318 (1964). CRONSHAW,J., HOEEERT,L. and ESAU, K., J. Cell Biol. 31, 429 (1966). EDWARDSON,J. R., Am. J. Botany 53, 359 (1966). -Science 153, 883 (1966). FREY-WYSSLING,A. and KREUTZER,E., Planta 51, 104 (1958). GANTT,E. and ARNOTT, H. J., J. Cell Biol. 19, 446 (1963). GEROLA,F. M. and BASSI, M., Caryologia 17, 399 (1964). HAYASHI,T., MATSUI, C. and YAMAGUCHI,A., Phytopathology 55, 458 (1965). HEss, W. M., Stain Technol. 41, 27 (1966). MATSUI, C. and YAMAGUCHI,A., Virology 22, 40 (1964). MOHLETHALER,K., Z. Wiss. Mikroskopie 64, 444 (1960). PRICE, W. C., Virology 29, 285 (1966). SHALL&T. A., J. CellBioI. 21, 253 (1964). SMITH, K. M., BROWN, R. M., GOLDSTEIN,D. A. and WALNE, P. L., Virology 28, 580 (1966). SOL~ERG,R. A. and BALD, J. G., Am. J. Botany 49, 149 (1962). STEEFEN,K. and WALTER,F., Plants 50, 640 (1958). THOMPSON,W. W., Botan. Gaz. 127, 133 (1966). SUN, C. N., Protoplasma 60, 426 (1965). TrtORNTON,R. M. and THIMANN,K. V., J. Cell Biol. 20, 345 (1964). WEINTRAUB,M. and RAGETLI,H. W. J., Virology 28, 290 (1966). WETTSTEIN,D. V. and ZECH, M., Z. Naturforsch. 17 B, 376 (1962). LEE, P. E., J. Ultrastruct. Res. 13, 359 (1965). BRANDES,J. and BERCKS, R., Advan. Virus Res. 11, 1 (1965).
(1967)
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E l ectron Microscopy of Vi rus-I nfected Su nflower L e a v e s 1,: HOWARD J. ARNOTT AND KENNETH M. SMITH The Cell Research Institute and The Department of Botany, The University o f Texas, Austin, Texas 78712 Received January 5, 1967
A short description is given of an apparently new virus disease of the mosaic type attacking the wild sunflower (Helianthus annuus L.). Its rapid spread in the field denotes an insect-vector, probably an aphid, but its identity is not yet established. Four major types of pathological changes were observed in the mesophyll cells: (a) cytoplasmic inclusions; (b) changes in the structure and type of plastid; (c) abnormalities in the nucleus; (d) presence of virus-like particles within the leaf cells. Crystalline inclusions were also present in both normal and infected cells. The cytoplasmic inclusions are of three types: whorls resembling "pinwheels"; long rods of composite structure attached to the pinwheels and resembling a "cat-o-nine-tails"; and circular inclusions of lamellate construction. Evidence suggesting that these inclusions do not consist of virus is given; but the possibility that they may consist of viral protein is considered. The pinwheels and rods are shown to have an intimate connection with the endoplasmic reticulum. The plastids undergo longitudinal division with the production of a fifth intervening layer, a phenomenon not hitherto recorded. The nucleus becomes swollen and completely filled with spherical or near-spherical particles with which are associated numerous fine threads. The virus particles are long flexible rods measuring at least 480 × 13 m/z; they are few in number and occur in association with the outer surface of the plastids. A discussion of these findings is appended. Wild sunflower plants (Helianthus annuus L.) growing in the vicinity of Austin, Texas, were f o u n d to be infected with a virus of the mosaic type. The first sign of infection was a mild mosaic mottling in the leaves but as the disease progressed a severe necrosis of leaves and the stem developed which caused the death of the upper part of the plant. Where the necrosis of the stem ended, a few very small distorted leaves were formed and further distorted leaves were produced f r o m the axillary buds. During the m o n t h of June, the virus spread rapidly a m o n g the wild sunflowers in the vicinity. The identities of the virus and of its vector have n o t yet been determined, however, we have ascertained that the virus is rod-shaped and suggest that 1 This investigation was supported in part by grant NIH GM-07289. The authors thank W. Gordon Whaley for his advice and support and Mrs. Linda Bourianoff for her technical assistance. 2 A preliminary report of this paper was presented at the Sixth Annual Meeting of the American Society for Cell Biology in Houston, Texas, 1966.
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the rapid spread is probably due to an insect vector, probably an aphid. Experiments on the mechanical transmission of the virus to young sunflower plants have so far been negative. The present report will present observations on the effects of this disease on the ultrastructure of sunflower mesophyll cells. MATERIALS AND METHODS Samples of leaves and petioles in both early and late stages of the disease, together with comparable samples from healthy plants, were fixed in a 1 : 1 mixture of 6 % glutaraldehyde and 6% acrolein with 0.2 M cacodylate buffer for 1 hour at pH 7.2 (13). They were then rinsed with the same buffer three times during 1 hour and postfixed in 4 % osmium tetroxide mixed 1 : 1 with buffer for 1 hour. The samples were then washed twice with buffer, twice with distilled water and then placed in 0.5 % uranyl acetate overnight in the refrigerator. This was followed by three washings with distilled water and dehydration in ethyl alcohol and acetone. The samples were then gradually infiltrated with Epon, evacuated, and put in a 60 ° oven overnight. Sections were cut with a diamond knife on a MT-2 "Porter-Blum" ultramicrotome and poststained in uranyl acetate and lead citrate. Micrographs were taken on RCA EMU 3F and Siemens Elmiskop I electron microscopes. OBSERVATIONS Four major types of pathological changes were observed in the mesophyll cells of the infected sunflower leaves: (a) cytoplasmic inclusions; (b) changes in the structure and type of plastid; (c) abnormalities in the nucleus, (d) presence of virus-like particles within the leaf cells. None of these changes were found uniformly throughout an infected leaf. Examination of tissues of different colors from the same leaf (Table I) revealed that only yellow tissues contain cytoplasmic inclusions and virus-like particles. Nuclear abnormalities are likewise confined to the yellow tissues, however, plastid differences occur in all tissues of the infected leaf. Our discussion will be limited to observations of palisade parenchyma cells in mature leaves. These cells are cylindrical in shape, being several times longer than their diameter which is about 7-10/z (Fig. 1). The cells are orientated with their long axis perpendicular to the leaf surface; in their mature state these cells are highly vacuolate, the central vacuole being surrounded by a peripheral cytoplasmic layer in which the nucleus and other cytoplasmic components are found. Although the cell shown in Fig. 1 shows m a n y symptoms of the disease, in organization it is similar to a normal palisade parenchyma cell.
IntraceIlular inclusions The most conspicuous intracellular inclusion takes the form of a cylinder with internal septa which radiate out from a small central cylinder; when cut transversely
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TABLE I P A T H O L O G I C A L C H A N G E S IN THE S U N F L O W E R LEAF TISSUES
Tissue Color
Infected green Infected yellow-green Infected yellow Uninfected (control)
Virus Present
Cytoplasmic Inclusions Present
Plastid Modification
Nuclear Abnormalities
+ -
- (+) + -
+ + + -
+ or -
it appears as a "pinwheel" or like an ophiuroid starfish (Figs. 1-3). Matsui and Yamaguchi (14) were probably the first to describe inclusions like these in their study of host cells of tobacco infected with tobacco etch virus; they called them "leoped profiles" and suggested that they corresponded to a coil of filamentous virus particles. While the present study was in progress, Edwardson (7, 8) published the results of an investigation of cytoplasmic inclusions associated with rod-shaped viruses. He described bodies of several types, laminated, circular and bundle inclusions. The one which was common to all the rod-shaped viruses studied, however, was the pinwheel. Edwardson (7) suggests that this type of inclusion body is diagnostic of infection with rod-shaped viruses of the potato virus Y group.
Structure of the inclusions The pinwheel inclusions are cylindrical bodies made up of approximately 9 to 12 septa radiating from a cylindrical inclusion center (Figs. 2 and 3). As the septa radiate they bend, forming a figure which looks like a pinwheel. The terminal portion of the septa may fuse with those of adjacent septa, thus forming a locule, or they may fuse with septa from adjacent inclusions to form an interconnected system of inclusions such as Edwardson (8) has shown in other cases (Fig. 2). The septa may be single or compound with as m a n y as three units fused together to produce a single compound septum. Apparently the locules are free at one or perhaps both ends (Fig. 6). The central cylinder from which the septa radiate may be "hollow" or it may contain a small dense core. In many cases it is possible to show that the structure of the septum is different from that of the membrane making up the endoplasmic reticulum (ER) or other cytoplasmic membranes (Fig. 3). A clear "unit m e m b r a n e " structure could be made out for the ER while a similar structure could not be observed in the septa. Our micrographs indicate that the septa do not have the structure of a unit membrane. If there are subunits which make up the septum they must be quite small; it seems
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unlikely that the septa are composed of an aggregation of complete virus particles. Examination of longitudinal or slightly oblique sections of the pinwheel inclusions show the septa in face view; in all of these which we have examined we have not been able to see any longitudinal striations. The thickness of these layers which make up or compose the septa are between 80-100 A units or only about 0.7 the thickness of the virus-like rods seen in infected cells which average about 130 A units (Fig. 16-18). I n the sunflower leaf cells there were two other types of inclusions which are associated with the pinwheels; their exact relationship to the pinwheels is not yet clear. One is a long rod which appears to be a composite structure made up of as m a n y as six layers, each layer being approximately 80-100 A units in thickness; these m a y be similar to the "lamellate inclusions" or the "dense b a n d s " reported by others (7, 14). In the sunflower the long rods sometimes appear to be connected at one end to a pinwheel, the whole structure closely resembling a "cat-o-nine-tails" (Fig. 5). A third type of inclusion b o d y is circular and seems to have a lamellate composition similar to that of the long rods. There m a y be as m a n y as f r o m three to five layers composing these inclusions and again the laminations appear t o be 80-100 A units in thickness. The layers m a y interconnect with m o r e than one of the circular inclusions, forming a more massive inclusion composed of anastomosing layers f r o m several circular bodies (Fig. 4). The pinwheel inclusions are associated in an intricate fashion with profiles of endoplasmic reticulum (Figs. 2, 3, and 6). In longitudinal sections of these pinwheel inclusions, E R profiles can be seen " a t t a c h e d " to the base of successive inclusion bodies (Fig. 6). The E R not only is associated with the base of the cylindrical pinwheel inclusions but penetrates into the locules or between the septa of the inclusions.
Key to abbreviations
C
plastid O osmiophilic globule CR crystal (contained within a single membrane) P plasmalemma E R endoplasmic reticulum S starch G granum SE septum GR nuclear granule T tonoplast I inclusion T H thylakoid IA intercellular air space V vacuole L locule VR virus-like rod M mitochondrion W wall N nucleus Fro. 1. Section of a leaf of Helianthus annuus infected with the sunflower mosaic virus (SMV). The section was made parallel to the surface of the leaf and passes through a palisade parenchyma cell perpendicular to its long axis; the cell is associated with other palisade parenchyma ceils and an intercellular airspace system. The section is from yellow tissue of a mature leaf, the level of section passes through the nucleus and a small portion of the large vacuole. Six plastids, two crystal-containing bodies bounded by a single membrane, several mitochondria, and rough ER are seen in addition to the cytoplasmic inclusions which are characteristic of the disease. Two pairs of plastids which may have undergone longitudinal division are shown. × 20,000.
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I n Fig. 2 each of the inclusions is penetrated by strands of E R ; in some cases the E R profiles are small a n d circular, i n others they are somewhat flattened a n d some septa m a y c o n t a i n more t h a n one profile of ER. The rod-shaped inclusions are also associated with ER; very c o m m o n l y the ends of the rods are " i n t e r c o n n e c t e d " with E R m e m b r a n e s that seem to f o r m direct contacts, in other cases the rod appears to be growing out of the ER, a n d at other p o i n t s the E R m a y be merely adjacent to the rods (Fig. 5). The E R which is associated with the inclusions can be s m o o t h or r o u g h (Figs. 2-5). Bodies similar to ribosomes are also present within the locules of the pinwheel inclusions. The matrix contained within the locules, a l t h o u g h it m a y be composed of a similar g r a n u l a r material, is generally less dense t h a n that of the cytoplasm. N o regular association of other organelles with these inclusions has been observed.
Changes in the chloroplasts I n cells of infected leaves conspicuous changes occur in the structure of the plastids. I n green a n d yellow-green areas of these leaves the changes are m a i n l y in the reduction of the n u m b e r of lamellae f o r m i n g a g r a n u m a n d some increase in the size of the osmiophilic globules. I n the yellow areas of the leaf, however, the change in the plastids is m u c h greater. I n general this change is similar to that which some fruits u n d e r g o during the f o r m a t i o n of chromoplasts, a n d in fact the plastids in the yellow area of the infected leaf would have to be classified as chromoplasts.
Normal plastids. Before a t t e m p t i n g to describe the changes which occur with the disease, we will describe the c o n d i t i o n of the plastids in n o r m a l leaves. A m a t u r e chloroplast in sunflower is a disk-shaped body separated from the cytoplasm by two FIG. 2. Peripheral cytoplasm of a cell from a leaf infected with the sunflower virus (SMV) showing four pinwheel inclusions. The central cylinder from which the septa extend is most clearly seen in the lower inclusion. Septa, composed of multiple layers, are seen in each inclusion; three partly separate septa fuse into one in the lower inclusion. Tubules of endoplasmic reticulum can be seen associated with each inclusion; over thirty profiles of ER are shown in or near the four inclusions. × 125,060. FIQ. 3. Pinwheel inclusion made up of 9 septa radiating from the central cylinder. Successive septa fuse with each other forming a series of closed locules; four locules contain profiles of ER. Note the structure of the septa as opposed to that of the ER and plasmalemma; the latter two clearly show a "unit membrane" structure whereas the septa seem to be constructed in seine other manner, x 154,000. FK6.4. Circular inclusions in the peripheral cytoplasm of a cell of the yellow portion of a leaf infected with SMV. Circular inclusions are interconnected by "septa-like" layers. The inclusions are composed of from one to five layers and like the pinwheel inclusions are associated with ER. × 82,500. FIG. 5. Rod-shaped inclusions in the cytoplasm of a cell infected with SMV. Note close association of ER with the inclusions. Arrow indicates an inclusion which seems,to be "growing out of" rough ER. × 135,000. F~6. 6. Pinwheel inclusions in the cytoplasm of a cell infected with SMV. Three inclusions are sectioned longitudinally, the other transversely. An end of each of the three inclusions shown in longitudinal section is in contact with ER. Virus-like rods are seen associated with the plastid in the upper right. Note that the face views of the septa show no structure similar to that of the virus-like rodsb x 70,000.
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unit membranes; in sectional view it has an elongated spindle shape (Figs. 7 and 8). Within these two membranes which make up the plastid envelope a series of thylakoids run for various distances; these flattened sacs combine at certain points to f o r m stacks or grana. In addition to the thylakoids there are m a n y plastid ribosomes, a few small osmiophilic globules, and one or two starch grains embedded in the plastid matrix. As m a n y as 35 or more thylakoids m a y be associated in a single gran u m ; however, grana with fewer units are c o m m o n . Almost every section of a mature chloroplast will have grana with 20 or more lamellae (thylakoids) as components (Fig. 7, 8). In our preparations a thin, very dense layer of material is f o u n d in the lumen of each thylakoid,whether it is a part of a g r a n n m or not. Plastids in green infected tissue. The changes which occur in plastids in the green areas of infected leaves are a matter of degree rather than of kind, as we shall see is the case in the yellow infected tissue. Three changes can be noted: (a) a reduction in the n u m b e r of thylakoids forming a g r a n u m (this is the most obvious difference in green areas of an infected leaf). These plastids seldom have as m a n y as 10 thylakoids in a granum, and the general impression one obtains is that the total n u m b e r of thylakoids in the plastid is reduced; (b) the n u m b e r and the size of the osmiophilic globules increase, the increase is relatively slight, however, when c o m p a r e d with that in the yellow tissues; (c) the shape of the plastids seems to change toward a m o r e spherical form. The ratio between the length and the height of the plastid, as seen in sectional view, is reduced. Plastids in yellow infected tissue. Most, but perhaps not all, of the plastids in this area of the leaf take on all the characteristics of chromoplastids, with the possible exception that they often contain several large starch grains. They still retain the two unit membranes which f o r m their envelope; ribosomes are still plentiful in the matrix but m a n y differences can be shown between them and the n o r m a l chloroplasts in uninfected tissue. There are two prominent features which are immediately apparent, both are continuations in the changes already denoted for the plastids in green infected tissue. The first concerns the further reduction in the total n u m b e r of thylakoids which participate in the formation of a g r a n u m (Figs. 9-13). The second concerns the spectacular increase in the n u m b e r of osmiophilic globules (Figs. 9 and 11). In addition to these changes the plastids in this tissue appear to be smaller than those f r o m n o r m a l cells (compare Figs. 7 and 8 with Figs. 9 and 10). Fins. 7 and 8. Normal plastids from the mature leaf of an uninfected plant of the sunflower. Note small number of osmiophilic globules, large number of thylakoids associated in each granum and the thinness of the thylakoids in both the grana and stroma in comparison with those from infected plants (Figs. 9-13). Fig. 7, x 30,700. Fig. 8, x 20,800. FIGS. 9 and 10. Plastids from yellow areas of leaves infected with SMV (compare these with the normal plastids seen in Figs. 7 and 8). Note the large number of osmiophilic globules, the thickness of the thylakoids, and the lower number of thylakoids per granum. As shown in Fig. 10 the plastids from this area of infected leaves often contain large starch grains. Fig. 9, x 48,000. Fig. 10, x 50,000.
i~~ ~ ~
~ii
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The change in n u m b e r of thylakoids as well as the reduction in the n u m b e r of thylakoids participating in grana formation is accompanied by a dramatic increase in the thickness of the layer of dense material contained in the lumen of the thylakoids. This material is as electron dense as that of the osmiophilic globules and like these bodies the substance deposited in the thylakoids does not show any apparent substructure. The thylakoid lumen in some cases becomes inflated by an accumulation of this electron dense material; when this inflation occurs there seems to be a concomitant decrease in the n u m b e r of osmiophilic globules (Fig. 13). The n u m b e r of osmiophilic globules seen in the plastids of yellow tissues is greatly in excess of those seen in n o r m a l chloroplasts. F r o m 15 to 20 of these dense bodies can be seen in a single section of a plastid. N o t only is there an increase in n u m b e r but the diameter, and hence the volume, increases in these plastids (Fig. 9). The characteristics of the plastids f o u n d in these different tissues are summarized in Table II. A n o t h e r interesting feature of the plastids in the yellow infected tissues is the appearance of plastids which appear to have undergone longitudinal division. In these cases the " d a u g h t e r " plastids are approximately the same size and are f o u n d juxtaposed longitudinally, a situation which was never seen in healthy tissues. Close observation of these plastids shows a dense line a b o u t 100-110 A units in thickness between the chloroplast envelopes of the " d a u g h t e r " plastids. F o r example, in Fig. 12 five lines are seen separating the two plastids, the upper two represent the two membranes of the upper plastid and the lower two represent the two membranes of the lower plastid; the central line represents the dense line mentioned above. This unique "fifth" layer between the plastids has not been reported before and is never present in plastids which are juxtaposed in other positions (end-to-end) or in the uninfected tissues. In some of the plastid pairs it was impossible to be sure that the two plastids were completely separate; what m a y be a stage in the division of plastids by a longitudinal division process m a y be seen in Fig. 11. In this case careful study seems to indicate that a portion of the two plastid interiors is still in connection (arrows).
Changes in the nucleus Conspicuous changes take place in the nuclei of some cells in the virus-infected sunflower leaves (Figs. 14 and 15). These changes only occur in the yellow tissues and FIG. 11. Longitudinally "divided" plastid in a cell from an infected leaf. The upper plastid is separated from the lower one by a curving line which crosses through the center of the pair complex and curves back at the upper right of the upper plastid, thus the lower plastid appears to enfold the upper one. Through most of this line of demarcation the two plastids are separate; however, between the two arrows it is not clear whether they are separate or not. Study of the micrograph seems to indicate that they are not separated and that this represents a stage in the division of the plastid. × 43,000. FIG. 12. "Divided" plastids in a cell from a leaf infected with SMV; the two membranes of both plastids are visible along with a "fifth" layer which separates the two plastid envelopes. Note the inflated thylakoids in these plastids. × 75,000.
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then have only been irregularly encountered. At an early stage in the infection of a cell, the nucleus appears normal even though the chloroplasts have undergone significant modifications and the pinwheel inclusions are apparent in the cytoplasm (Fig. 1). Within what seems to be a normal nuclear envelope the contents are similar to those seen in uninfected plants (Fig. 1). Figs. 14 and 15 show changes which occur in the nuclei of some infected leaf cells, especially near the vascular system. The nucleus becomes completely filled with spherical or near-spherical particles measuring about 40-60 m#. At this time, the nucleus appears to be swollen and an increase can be observed in the size of the nuclear pores; additionally, the dense material forming the annulus of the pore in normal tissues is missing in these cells. At a still later stage in nuclear change the nuclear envelope may be broken at m a n y points. At this time there are numerous fine threads associated with the particles (Figs. 14 and 15). The threads measuring about 20-25 A units in diameter form an anastomosing reticulate network between the particles. The threads are associated with small dense points measuring about 60 A units; together they sometimes appear like beads on a string. These dense points about 60 A units in diameter also seem to be components of the larger granules seen in the nucleus (Fig. 15).
Virus-like particles within the cells After considerable searching, our efforts were finally rewarded in the finding of long flexuous rods in the cells of infected plants. Although m a n y phosphotungstic acid (PTA) preparations were made, no virus-like particles were ever found. It was only by careful scrutiny of our micrographs of sectioned materials that we were able to find these bodies. Location of the rods. The rod-shaped inclusions have only been found in association with plastids (with one exception, see Fig. 6); in fact it was through the examination of oblique sections of plastids that we were first able to see these rods. The rods are always found between the outer plastid membrane and either the plasmalemma or the tonoplast. In these large vacuolate cells it is common for these membranes to be separated by only a few hundred Angstrom units and it is in this space, between two membranes, where the rods are found (Figs. 6, 16, and 18). In some cases rods are present between the outer plastid membrane and both the tonoplast and the plasmalemma (Figs. 16 and 18). Structure of the rods. The rods are long flexous structures which can be straight or bent in a shallow arc. The length is difficult to establish from sectioned material, but FIG. 13. Plastid in a cell from a leaf infected with SMV showing severely inflated thylakoids. The electron dense material seen in the lumen of the thylakoids is very similar in electron density to that of the osmiophilic globules which are also seen in this plastid. Particles approximately the size and having the general nature of the virus-like rods are seen between the two mitochondria and the plasmalemma (see circles) in the lower part of the micrograph, x 86,000.
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TABLE II SUMMARY OF MAJOR CHARACTERISTICS OF NORMAL AND INFECTED SUNFLOWER LEAF TISSUE Characteristic
Normal
Virus-like rods, 13 m/z diameter Inclusions Nuclear abnormalities Crystals Number of thylakoids in grana Number of osmiophilic globules in plastids Size of osmiophilic globules
Absent Absent Absent Present 15 or more Few Small
Green Infected
Absent Absent Absent Present Reduced Few Slightly larger than normal
Yellow Infected
Present in some cells Present in many cells Present in a few cells Present Severely reduced Many Twice that of normal
we find t h a t the m i n i m u m is 480 m#. T h e d i a m e t e r is m o r e easily m e a s u r e d a n d averages a b o u t 13 m#. O u r best m i c r o g r a p h s indicate t h a t the r o d s are h e x a g o n a l in cross section a n d t h a t a central dense core m a y be present (Figs. 16 a n d 18). A s u m m a r y of the m a j o r characteristics f o u n d in n o r m a l a n d infected tissues of the sunflower is shown in T a b l e II. Crystalline inclusions m a d e u p of particles regularly a r r a n g e d in rows a n d cont a i n e d within a m e m b r a n e are f o u n d in tissues of b o t h infected a n d n o r m a l leaves in a p p r o x i m a t e l y equal n u m b e r s (Figs. 1, 10, a n d 11). These crystals have a r e p e a t spacing of a b o u t 2 0 0 / ~ units a n d a p p e a r as squares, d i a m o n d s , rectangles, a n d triangles when seen in thin sections: A l l these sections m i g h t be i n t e r p r e t e d as r e p r e s e n t a tive of a cubic crystal. DISCUSSION One of the results of this research is to question the n a t u r e of the c y t o p l a s m i c inclusions. M a t s u i a n d Y a m a g u c h i (14) a n d H a y a s h i et al. (12) believe t h a t similar inclusions in the cells of the p l a n t s they have studied represent the virus particles in v a r i o u s types of a r r a n g e m e n t . O u r observations d o n o t l e a d us to a similar view f o r the case we have examined; on the c o n t r a r y , it seems clear t h a t the pinwheel, circular, o r r o d - s h a p e d inclusions are n o t m a d e up of particles similar in size to those of the
FIG. 14. A nucleus which is apparently undergoing breakdown in a cell from a leaf infected with SMV. Many granules of two sizes and fibrils are seen throughout the nucleus; the nuclear membrane has already undergone partial breakdown. Other organelles, e.g., mitochondria, appear more or less normal. Transverse sections of the virus-like rods are seen in the upper left where they are associated with a chloroplast, x 80,000. Fie. 15. Enlargement of the particles and fibrils in the nucleus seen in Fig. 14. The fibrils branch and anastomose forming a network, at some points [hey appear to enter and perhaps form a part of the larger particles. Small particles about 60 dk units in diameter are associated with the fibrils. × 165,000.
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presumed virus which infects the tissues. This is particularly clear from examination of longitudinal sections through the pinwheel inclusions (Fig. 6). In this plane of section many of the septa can be seen in face view; none have shown longitudinal striations which would indicate that they are made up of a series of rod-shaped virus particles in a two-dimensional array, even though the virus-like rods are clearly seen in this same figure. In a study of Vicia faba infected with bean yellow mosaic virus, Weintraub and Ragetli (24) found inclusions made up of dense bands, crystals, and small aggregates of virus. They found "the dimensions of the dense bands and crystalline inclusions were difficult to reconcile with BYMV particles." Shalla (17) illustrates bodies which may be similar to the inclusions under discussion, together with the virus particles in tomato tissues infected with tobacco mosaic virus. While our observations seem to indicate that the inclusions are not made up of complete virus particles it is possible to conceive of these inclusions as viral component(s); perhaps they may represent the protein portion of the virus in a paracrystalline array. The inclusions could be a storage mechanism through which the viral protein could be accumulated, or the inclusions might represent an overproduction of a viral component, the units of which undergo assembly into the various morphological structures which are seen in the infected cells. If these inclusions do represent a viral component this would help to explain the similarity of the morphology of inclusions seen in a wide range of host plants but always associated only with a certain group of viruses. For the time all that can be said is that the sunflower inclusions do not appear to be arrays of complete virus particles. Based on the work of Matsui and Yamauchi (14), Edwardson (7, 8), Weintraub and Ragetli (24) it is apparent that the inclusions seen in the present case (Figs. 2-6) are a diagnostic characteristic for the disease caused by the rod-shaped R N A viruses of the potato virus Y group. Edwardson (7) could not find inclusions in the infected tissues when viruses from the potato virus X group, TMV group, or tobacco rattle virus group were inoculated. Lee (26) illustrates inclusions similar to those shown here in a disease caused by the wheat streak mosaic virus (WSMV). The systematic classification of this virus (WSMV) has placed it in the potato virus S group; however, Brandes and Bercks (27) indicate that it is intermediate in length between the potato FIG. 16. Virus-like rods cut transversely. Note their hexagonal shape and the dense "core". x 440,000. FIG. 17. Tangential section of a plastid in a cell infected with SMV showing the virus-like rods which are usually found in association with the plastids. The plastid goes out of section approximately at the middle of the micrograph. Note the curved nature of the virus-like rods. FIG. 18. Section of a plastid in a cell infected with SMV showing associated virus-like rods cut in transverse section. The rods are shown between the plastid membrane and the tonoplast on the left and between the plastid membrane and the plasmalemma on the right. Many of these rods seem to show a hexagonal structure and seem to contain a central dense "core." × 197,000.
!~'~i!~i"i~ i~i~~ ~i~i.........
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A R N O T T A N D K . M. SMITH
virus S group and the potato virus Y group and could easily belong to the latter. In the sunflower the inclusions were discovered before the virus-like particles; with the present knowledge [Edwardson's paper (7) came out as we were pursuing this research], we might have predicted the nature of the virus with some degree of accuracy. The three-dimensional nature of inclusions like those in sunflower has been considered by Edwardson (8) through the reconstruction of these bodies from serial sections; he is of the opinion that the pinwheels and the bundles are different aspects of a single type of inclusion. This inclusion is assumed "to be cylindrical in shape and composed of curved plates with their inner edges converging around the central axis of the cylinder." He found, in examination of serial sections, that many pinwheels and bundles that are clearly separated in individual sections appear to be interconnected. Our micrographs certainly show that many "bundles" and pinwheels are interconnected, and further that several inclusion bodies may also be interconnected, even in a single section. Perhaps all the inclusions are interconnected. Previous authors have not noted that the inclusions often have obvious interconnections and/or interrelationships with the endoplasmic reticulum (ER). In our study we find this relationship is almost universal. Some of Edwardson's (7) micrographs show what appears to be a similar condition in the examples he has illustrated. The ER in association may be either rough or smooth. If the inclusions are protein (whether they are viral or "cellular" protein is incidental) the close association with rough ER seems to warrant careful study. One could suggest that the origin or the supply lines bringing materials to these inclusions is the ER; however, the functional aspects of these inclusions will have to await further research. The dramatic changes in the ultrastructure of the plastids which occur in infected cells represents one of the most interesting aspects in the cytological aspects of this disease, Shalla (17) observed the distortion and vacuolation of chloroplasts in tomato infected with TMV; the virus appeared within some of the vacuoles in the chloroplasts, and in other plastids extrusions were formed. Similar extrusions were reported in the chloroplasts of Vicia, but otherwise only little change was found in infected plants (24). Chalcroft and Matthews (3) found that a variety of changes occur in the leaves of the Chinese cabbage infected with turnip yellow mosaic virus (TYMV); the yellow and yellow-green areas of the infected leaf showed many changes, but the green areas appeared to be more or less normal. This is a situation identical to that which we have found in the present case. In the yellow-green areas they found that the plastids were swollen and aggregated into large clumps; there were few grana and those present were thinner than usual, a condition quite unlike that which we found in sunflower. In the yellow areas of the Chinese cabbage leaves they found the plastids contained large numbers of starch grains--here the situation was similar in the sun-
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193
flower. Because only the plastids showed abnormalities following virus infection and because of certain associated biochemical data, it was suggested (3) that some part of the virus may be synthesized in the chloroplasts; however, intact T Y M V particles were not found to accumulate within the plastids. In Beta vulgaris the lipid inclusions (osmiophilic globules) in the chloroplasts were sometimes unusually large when the cells were infected with beet yellows virus (6) and also inclusions consisting of elongated "virus-like" particles were present in the chloroplasts. These inclusions were found either free in the stroma, where they formed complicated patterns of parallelly oriented particles arranged in curved rows, or closely associated with lipid globules; otherwise the plastids were more or less normal. We have not found vacuolation, extrusion formation, or the presence of virus-like particles in the plastids of sunflower. The changes which appear in the plastids of the sunflower after infection are very similar to those which are reported in a variety of ontogenetic systems where plastids pass from the chloroplast to the chromoplast state of development. One of us (H. A.) has seen analogous changes in the normal development of the plastids of Malpighia glabra fruits as they ripen (unpublished). Similar changes as a result of normal development are reported in the petals of Ranunculus (9), in Aloe flowers (20), and recently in the development of the chromoplasts of the Valencia orange (21); with respect to the development of the osmiophilic globules and the reduction in the number of thylakoids (both in the grana and stroma) the latter case is almost identical to that reported herein for the sunflower. Another striking similarity to the changes in the sunflower has been shown in the chloroplasts of Abutilon infected with a virus; however, Sun (22) considers these changes to be degenerative in nature. In the cases reported in the literature (9, 20, 21, 22) the changes which occur during chromoplastogenesis do not involve increases of dense materials within the lumen of the thylakoids, such as is the case in the sunflower, rather the materials which cause density to appear in the plastids appear to be deposited on the surface of the thylakoids or between the surfaces of adjacent thylakoids. The changes seen in the sunflower with respect to the accumulation of osmiophilic globules, however, are very like that seen in the normal development of many chromoplasts. There are not many papers dealing with the division of plastids in higher plants (10, 15) but these usually show plastids dividing transversely to the long axis of the plastid. The possibility of longitudinal division must now be considered, even if only in connection with abnormal tissues. Our micrographs (Figs. 1, 11, and 12) illustrate "daughter" plastids associated together in such a manner as to be very suggestive of such a longitudinal division process. The origin of the "fifth" layer, that is, the layer between the plastid envelopes, may be a result of such a division process; such a layer has not previously been reported in the literature and appears only in connec13
671825 d. Ultrastructure Research
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H . J. A R N O T T A N D K . M. S M I T H
tion with paired "daughter" plastids. Stages in this division process have been seen, but at the present stage of our knowledge we cannot be absolutely sure that division is occurring. The modifications of the nucleus which we have seen in isolated cells in the yellow part of infected sunflower leaves have not been of the kind observed by others, e.g., deep grooves or cytoplasmic channels in the nucleus (19), the presence of virus particles in the nucleus (6). Although the nuclear pores become enlarged we have no evidence that materials pass from the nucleus to the cytoplasm as others have reported (25). Whether or not any viral component is manufactured in the nucleus of infected sunflower cells is not known, however, from our observations it seems more likely that the nuclei are merely undergoing some kind of breakdown or denaturation. The exposure of fibrils approximately 20 N units in diameter, hence approximately the size of the D N A molecule, as well as the larger 60 and 400-600 A units particles may represent nothing more than the "uncoiling" of the chromatin due to the disease. Dense granules about 200 A units in diameter were detected within chromatin clumps in the nuclei of cells affected with tobacco etch virus (14), but the relationship to the structures we have seen is not clear. The relationship of the rod-shaped, virus-like particles to the membranes of the plastids (and occasionally those of the mitochondria) and plasmalemma or tonoplast is difficult to understand. In every case where we have seen the virus-like particles they are found situated in these restricted spaces. The question whether the membranes have something to do with the production of the virus is of great interest. Recently Smith et al. (18) have shown that virus particles are attached to photosynthetic lamellae at certain stages in the development of the blue green algal virus within the cells of Plectonema. In the present case it may be of interest to note that the viruslike particles are always present in monolayer between the adjacent membranes (Figs. 16 and 18). The only exception to this was seen when six particles were found associated together in a small array. The measurements we have made indicate that the minimum length of the viruslike rods is 480 mk~; this is not to say that they are not much longer, which we strongly suspect, but sectioned materials are not the best for measuring size. We have not been able to see the rods in PTA preparations despite several efforts, perhaps this is due to their low concentration within the sunflower cells. Since the rods are probably somewhere near the size of those seen in the potato virus Y group (their diameter is close), we should not exclude the possibility that the present case in sunflower also represents an example of the cytological effects caused by a virus of that group. We find identical crystalline inclusions in infected and normal cells of the sunflower. These crystalline inclusions are contained within a membrane and are made of regularly arranged particles (Figs. 1, 10 and 11). Morphologically similar crystal-
ELECTRON MICROSCOPYOF VIRUS-INFECTEDSUNFLOWER LEAVES
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line inclusions have been seen in a variety of tissues (1, 2, 5, 11, 23). The crystals in sunflower are similar to the "proteosomes" described in the storage parenchyma cells of Helianthus tuberosus after experimentally induced greening (11). Similar crystals have been found in both the normal and virus infected cells of Citrus by Price (16). Chambers et al. (4) illustrate crystals very different in lattice parameters from the virus rods illustrated in the same micrograph of lettuce cells infected with necrotic yellows virus. It seems clear that m a n y crystalline inclusions in plant cells do not have anything to do with virus aggregations (1, 2, 5, 11, 23) or with virus infections. The possibility of enzyme storage or light reception has been discussed by Cronshaw (5) and Thornton and Thimann (23).
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