The significance of chloroplast abnormalities associated with infection by turnip yellow mosaic virus

VIROLOGY

The

42,2X3-303

(1970)

Significance

of Chforoplast

Infection

by turnip

R. USHIYAMA Department

AND

Abnormalities Yellow

Associated

Mosaic

with

Virus

R. E. F. MATTHEWS

of Cell Biology, University of Auckland,

Auckland,

New Zealand

Accepted May 28, 19YO In cells infected with turnip yellow mosaic virus (TYMV), small vesicles about 50 nm in diameter were present under all circumstances tested, including very early times after inoculation. These vesicles are bounded by a double membrane, and some contain stranded material. Using a wilting procedure to induce crystallisation of virus in situ, intact virus particles were observed only in the cytoplasm. We suggest that the replicative form of the viral RNA is located in the peripheral vesicles of the chloroplasts and that RNA complements move from the vesicles to the cytoplasm where coat protein synthesis and particle assembly take place. The following pathological changes in chloroplasts appear to be secondary effects of infection by TYMV, since they do not occur in all cells where virus replication is occurring, and in particular they are not present at very earlv stages of infection: reduction in size and number of grana, chlorophyll content, 68 S ribosomes, and starch; increases in amounts of phytoferritin and numbers of osmiophilic globules; changes, other than swelling, in size, shape and arrangement of chloroplasts.

leaves (Babos, 1969; Babos and Shearer, 1969). Thus autoradiographic experiments with actinomycin D have not given unambiguous results. If a particular cytological or biochemical modification is directly involved in synthesis of a particular virus, it would be expected to be found under all conditions where synthesis of this virus occurs, In the work described here we have determined the conditions under which the various alterations to chloroplasts occur following TYMV infection. The only modifications found under all circumstances tested were the swelling of chloroplasts and the presence of small vesicles at their periphery. We suggest that the replicative form of the viral RNA may be located in these vesicles.

INTRODUCTION

Infection of Chinese cabbage by TYMV has been shown to cause a range of biochemical and cytological abnormalities in the chloroplasts (Reid and Matthews, 1966; Chalcroft and Matthews, 1966, 1967; Gerola et al. 1966; MiliEiE and Stefanac, 1967). Ralph and Clark (1966) obtained evidence suggesting that the replicative form of TYMV-RNA was associated with the chloroplast fraction, but this was questioned by Bove (1967), who considered that the replicative RNA may be in the nucleolus. Using autoradiography to follow the uptake of tritiated uridine into tissue treated with actinomycin D, Lafleche and BOX& (1968) and Bove et al. (1969) showed that accumulation of the isotope in virus-infected actinomycin D-treated tissue was associated with the nucleolus and with the cytoplasmic spaces between clumped chloroplasts. However it is by no means certain that actinomycin D blocks all host RNA synthesis in

MATERIALS

AND METHODS

~+us and Plants TYMV was cultivated in Chinese cabbage plants (Brassica pekinensis Rupr. var Wong 293

294

USHIYAMA

AND MATTHEWS

Bok), in pots in the glasshouse.Strains of TYMV were isolated from the stock culture. “White, ” “yellow green,” and “pale green” strains (Chalcroft and Matthews, 1967) were used for cytological examination. Inoculation of virus was made mechanically on two expanded leaves of Chinese cabbage and turnip plants (Brussica rupa L.) with three well-expanded leaves, using freshly extracted infective leaf sap as inoculum. Control leaves were rubbed with distilled water. Plants were then maintained in the glasshouse(approximately 21 f 3”C), and infected leaves were sampled at various times after inoculation, together with leaves of similar age and position on the control plants.

Sucrosedensity gradients. Amounts of virus and ribosomesin leaf extracts were estimated after sucrose density gradient fractionation aspreviously described (Reid and Matthews, 1966). Chlorophyll estimation. Chlorophyll was estimated spectrophotometrically following extraction in diethyl ether: methanol: water (10: 7: 3 v/v). Chlorophyll content was calculated using the equation given by French (1960). RESULTS

Chloroplast Abnormalities in the Lam&a following Injection with Three Strains of TYMV

To study the occurrence of abnormalities in chloroplasts, sets of plants were inoculated Light Microscopy and Electron Microscopy with the pale green, yellow green, and white For cytological examination by brightstrains of the virus. Samples from the inoefield light microscopy pieces of fresh leaf ulated leaves were taken at 6 and 12 days tissue, about 3 X 10 mm, were detached after inoculation and from a set of young sysfrom intact plants. Hand-cut sections of temically infected leaves at 12 and 18 days. these were mounted in Honda medium The 12-day systemic leaves were only 2 cm (Honda et al., 1966) as previously described long, so that there was insufficient material (Chalcroft and Matthews, 1967). For elec- for analysis of chlorophyll or ribosomes at tron microscopy small pieces of leaf tissue this time. (2 X 3 mm) were taken from various porTen photographs were examined for each tions of TYMV-infected plants or healthy sample representing five sections from each plants, and fixed for 60 min at room tem- of two blocks. Size and number of grana. No significant perature in 6% glutaraldehyde in halfstrength Millonig’s phosphate buffer (Pease, deviation from normal was seen in number 1964). These samples were washed in the of grana per chloroplast (Table 1) or in size same buffer at 4” overnight or for 20 hours. of grana as a consequenceof infection by any Postfixation was for 1 hour at room tempera- strain at 6 days. At 12 days, grana in chloroture in 1% osmium tetroxide in buffer. De- plasts in cells infected with the pale green hydration was carried out at 15-min intervals strain were normal. Grana in cells infected through a graded ethanol seriesbeginning at with the yellow-green and white strains were 70 % ethanol. reduced both in size and number compared After dehydration, the tissue was trans- with healthy tissue. The results for the systemically infected ferred to Epon via propylene oxide and finally embedded in Epon. Sections were cut leaves are shown in Fig. 1. At 12 days after with diamond or glass knives on an LKB inoculation infection had little effect on the Ultrotome 4800. Collection of sections giving grana except for a reduction in average size a paleOgoldor silver interference color (600- by the white strain. Although concentration 900 A thickness) was usually made on of TYMV had reached a plateau value in a unfilmed 400-mesh grids. Formvar-coated systemically infected leaf 2 cm long, virus “Veto L” 2 X 1 mm copper grids were used content per leaf was still increasing expofor serial sections. Thin sections stained with nentially (Faed and Matthews, unpuburanyl acetate and lead citrate were ex- lished) . At 18 days wide differences in grana amined with a Philips EM. 200 electron size and number were apparent among the microscope at 80 kV. three strains. The pale green strain had no

CHLOROPLAST

SOME

EFFECTS

OF THREE

ABNORMALITIES

STRAINS

TABLE TYMV ON

OF

I

AT VARIOUS 6 Days local

h 5 a m __~

viruscontent b&w wt) Gram (number in 10 chloroplast9) Chlorophyll content (re;/d 68 S ribosomes bdme, fresh wt) a Insufficient

0.0

0.50

0.63

191

222

199

80

75

67

0.07

0.08

TIMES 12 Days

2 c 4

5 2; Fz 0.72 190

0.01

0.0

1.2

295

INFECTION

1 IN CHINESE INOCULATION

CHLOROPLASTS

AFTER

12 Days

local

9 BB 26 oe J $ 2” $ -____ 1.1

1.1

CABBAGE

systemic

-

LEAVES

18 Days

systemic

-

fx 3 ii

_-i

_-

0.0

-a

0.0

0.93

1.1

0.53

155

144

98

77

94

77

173

216

42

0

90

59

55

47

4

-=

102

90

52

15

-=

-0

0.06

0.006

I material

IN TYMV

0.006

0.0

0.25

0.12

0.06

0.04

available

for measurement.

FIG. 1. Effect of infection with TYMV on size of grana in systemically infected leaves at 12 and 18 days after inoculation. H, healthy leaf; PG, pale green strain; YG, yellow green strain; W, white strain. Number of lamellaeper granawas estimatedin the grana seenin thin sectionsof 10 chloroplastsfor eachsample.

significant effect. The yellow green strain had caused a marked reduction in the number of grana per chloroplast and in the size of grana, while in cells infected with the white strain grana were absent. Chlorophyll content. Changesin chlorophyll content of the tissue followed the same general trend as reduction in grana size and number (Table 1).

Content of 68 S ribosomes.These were reduced substantially for all three strains at all stagestested, except for the pale green strain at 6 days in inoculated leaves where no reduction had occurred (Table 1). Starch grains. No major differences in number and size of starch grains were noted between healthy tissue and tissue infected with any of the three strains at the various

296

USHIYAMA

AND

times after infection, except that grains were smaller in tissue infected systemically with the white strain at 12 days and absent from such tissue at 18 days. Samples were harvested from the plants between 11 AN and 2 PM. Osmiophilic globules. These were about 95100 nm in diameter in healthy chloroplasts, and tended to be few at all sampling times. None were seen in the young 2-cm leaf. No marked changes in the size or number of globules was produced by virus infection except for the white strain in 12-day local lesions where a very large number of globules were present. Many of these were much larger than normal, with diameters greater than 200 nm, and were less densely stained than normal globules. These differences were not seen in tissues systemically infected with the white strain. Phytojerritin. Regular arrays of electron dense particles assumed to be phytoferritin from their lattice spacing and density (Craig and Williamson, 1969) were seen occasionally in thin sections of chloroplasts. Their frequency appeared to be little affected in inoculated leaves. They appeared more often in systemically infected leaves at both times, except for tissue infected by the pale green strain at 12 days, where none were seen. Size and shape and arrangement of chloroplasts. In inoculated leaves at both 6 and 12 days after inoculation, for all three strains, chloroplasts were enlarged and rounded, often having a cup-shaped appearance. They were clumped together in groups. By contrast at 12 days in young systemically infected leaves, chloroplasts were smaller than normal for all strains. They were also rounded rather than lens shaped, and were clumped together. The effects of the three strains were easily distinguished at 18 days. Infection by the pale green strain resulted in enlarged, rounded and clumped chloroplasts similar to those in inoculated leaves. The yellow green strain caused a striking fragmentation of chloroplasts into smaller bodies, containing grana, stroma lamellae, and starch grains. The white strain induced enlarged spherical chloroplasts with little normal internal structure remaining. Spaces between clumped chloroplasts, which appear roughly circular in section,

MATTHEWS

were observed in all infected tissues in the experiment noted above except for the white strain at 18 days. Vesicles. Previous workers have noted the appearance of vesicles of variable size in chloroplasts of tissue infected with TYMV, and have provided illustrations of them (e.g., Chalcroft and Matthews, 1966; Gerola et al., 1966). Our present observations on the size distribution, time of appearance, and location within the chloroplast suggest that these vesicles fall into two classes-small vesicles up to about 0.3 p, in diameter, and large vesicles about 0.4-1.2 p, in diameter-or larger (Fig. 2). The small vesicles were most numerous in tissue infected with the white strain and least numerous with the pale green strain. The data in Fig. 2 are for the number of vesicles seen in thin sections. From such numbers it is possible to calculate the approximate num-

FIG.

2. Number

and size

of vesicles

in chloro-

plasts of systemically infected Chinese cabbage leaves at 12 and 18 days after inoculation. Histograms show the frequency distribution of vesicle diameters for the pale green (PG), yellow green (YG), and white (W) strains. Thin sections of four chloroplasts were examined for each sample.

CHLOROPLAST

ABNORMALITIES

ber of vesicles per chloroplast, assuming that these are spheres. For example in tissue systemically infected with the yellow green strain at 18 days there would be roughly 250 small peripheral vesicles per chloroplast. The small vesicles are discussed in detail in later sections. The large vesicles were present in inoculated tissue infected by all three strains, at both 6 and 12 days after inoculation. They were also present in systemically infected tissue at 18 days but not at 12 days (Fig. 2). The large vesicles were usually surrounded by a membrane and were usually devoid of electron dense contents. They appeared anywhere in the body of the chloroplast. Vesicles with a similar size and structure were sometimes seen free in the cytoplasm of infected cells.

IN TYMV

INFECTION

297

several small isometric viruses. Such arrays are rarely seen in cells infected with TYMV (Chalcroft and Matthews, 1966; Gerola et al., 1966; Milne, 1967). Although Hills and Plaskitt (1968) have described improved staining procedures, it is difficult in the absence of crystallization to distinguish with certainty between virus particles and ribosomes. Milne (1967) observed that when leaves infected with sowbane mosaic virus were detached and left in air at about 24’ for up to 3 hours virus particles formed small crystals within infected cells. We have worked out a modified procedure for TYMV. Wilting whole leaves at room temperature (about 20”) for 15-18 hours or heating in an oven at 60 f 2’ for 5-7 min did not lead to crystallization of TYMV within cells. However a combination of these two treatments-heatChloroplast Abnormalities in Midrib Tissue ing at 60” for 5 min followed by 18 hours at Injected with TYMV room temperature before fixation-did lead Midrib tissue of systemically infected Chi- to crystallization of TYMV. nese cabbage leaves 6 cm long contains about The wilting procedure caused disorganizaone-third the virus concentration found in tion of cell structure to a variable extent. the lamina (E. M. Faed, unpublished re- Nuclei could not be identified in either sults). If the virus present in the midrib is healthy or virus-infected tissue. Chlorosynthesized there, then chloroplasts in the plasts became somewhat swollen in healthy midrib should possess any abnormalities es- tissue or in tissue from dark green areas of sential for virus replication. mosaic-infected leaves, but usually retained Midribs were sampled from leaves 1, 6, their membranes. In virus-infected cells, and 10 cm in length systemically infected membranes could not be seen in many chlowith the pale green strain of virus. Chlororoplasts. plasts were seen in only a small proportion Samples were taken from tissues infected of the cell sections examined, but all chloro- for various times with all three strains of plasts were clumped and rounded with en- virus. Crystalline arrays of virus were seen larged spaces between them and numerous regularly in the rounded spaces between small peripheral vesicles. chloroplasts (Fig. 3), in the cytoplasm, and Chloroplust Abnormalities in Turnips Injected sometimes apparently located in the vacuole. The interparticle distance in these arrays was with TYMV 28-30 nm. Batches of turnip plants were inoculated A careful search for the presence of virus with each of the three strains of TYMV and inside chloroplasts was made of sections from samples taken for electron microscopy at 6 many samples of wilted tissue. Intact virus days after inoculation from an inoculated particles in numbers sufficient to form microleaf and from a leaf showing early systemic symptoms. Rounding and clumping of chlo- crystals appear to be absent from the chlororoplasts, and small peripheral vesicles were plasts and the virus-induced vesicles they contain. Very occasionally we observed small present in all specimens examined. groups of virus particles in crystalline array Location of Virus Particles within Infected apparently lying within the stroma of the Cells chloroplast. However, these were seen only in Intracellular crystalline arrays of virus chloroplasts where the bounding membrane particles have been seen in cells infected with was partly or entirely absent. Thus we as-

298

USHIYAMA

AND MATTHEWS

FIG. 3. Crystalline aggregates of TYMP particles seen in a thin section of a cell systemically infected with the pale green strain 18 days after inoculation, and subjected to a wilting procedure before fixation. Crystalline aggregates are seen in the enlarged spaces between two chloroplasts. Scale line = 500 nm.

sumethat this occasional appearance of virus in chloroplasts was an artifact of the wilting procedure. Examination of wilted tissue suggested that most of the stained particles in the spaces between chloroplasts were TYNIV particles. We examined the distribution of these particles more closely in sets of serial sections of clumped chloroplasts from unwilted tissue. One such set is shown in Fig. 4. These serial sections showed that (i) the circular spaces seen in section are in fact roughly spherical; (ii) virus particles are usually more concentrated in these spacesthan in the narrow channels formed between the chloroplasts. (iii) Although small vesicles are present beneath all parts of the chloroplast surface, they are particularly abundant in association with the enlarged spaces, as was noted by Bove et al. (1969). Chloroplast hnormcdities at Early Times after Inoculation In the experiments described above the only abnormalities seenunder all conditions

tested were rounding and clumping of chloroplasts and the presence of small peripheral vesicles. The rounded spaces between clumped chloroplasts were seen, under all circumstances except for tissue infected with the white strain at 18 days, when virus synthesis might have ceased. Any changes that are essential for virus replication should be present as soon as or before virus production begins. The earliest time we have been able to detect-new intact virus in inoculated leaves using a sensitive radiochemical procedure is 48 hours after inoculation (Matthews, 1970). At this stage we can assumethat only a very small proportion of the cells will be infected and .producing virus. These cannot be located macroscopically since visible lesions do not appear until about 5 days. Thus to locate infected cells at early times it was necessary to scan large areas of tissue. For this reason, in preliminary experiments, we used light microscopy of hand-cut fresh sections and searched for rounding and clumping of chloroplasts. In one experiment these changeswere seen

FIQ. 4. Serial sections (A-D) showing enlarged, more or less spherical plaats in a leaf systemically infected with the pale green strain of TYMV inoculation. Scale line = 500 nm. 299

spaces between and sampled

two chloro18 days after

300

USHIYAMA

AND

at 3 days in some cells in leaves inoculated with each of the three virus strains. In a, more detailed test half-leaves were inoculated with water or the white strain of TYMV, and samples taken at 24,48,52, and 72 hours after inoculation. Inoculations were made on the upper leaf surface. Observations were confined to the two layers of palisade mesophyll cells. Chloroplasts in the spongy mesophyll are more difficult to observe and record in fresh sections. Between 950 and 1550 cells were observed for each sampling time, in a total of eight sections from four pieces of tissue. Rounding and clumping was first observed in some cells at 52 hours after infection. Affected cells were 1.6% of the total observed. By 72 hours this figure was 14%. Samples of leaf taken at 72 hours were examined by electron microscopy for the presence of chloroplast abnormalities. The chloroplasts of many of the cell sections examined appeared normal. In sections of some cells most chloroplasts were normal while one or two showed slight swelling and the presence of a few small peripheral vesicles. These vesicles were not confined to regions of contact between adjacent chloroplasts. To check whether intact virus was present in such cells, other sampleswere subjected to the wilting procedure described above which leads to intracellular crystallization of virus. Small crystalline arrays of virus particles were seenin cells where the only abnormalities observed in chloroplasts were slight swelling and the presence of peripheral vesicles.

MATTHEWS

membranes of some vesicles were seen as a continuation of the inner membrane of the chloroplast (Fig. 5A). It is impossible to determine what proportion of the vesicles were connected in this way to the inner chloroplast membrane. The sections used were 600-900 i thick and the commonly observed diamete;s of the vesicles were in the range 300-800 A. The diameter of the channels observed between the vesicles and the space between chloroplast membranes was about 150 A. Thus these channels may quite frequently be obscured by other material in the section. We obtained no evidence that the peripheral vesicles increase in diameter with time after infection. Those observed at 3 days after inoculation had approximately the same size distribution as those observed in systemically infected tissue at 18 days. The interior space of the small vesicles contained material of low electron density which lacked granularity or stained particles. Thus 68 S and 83 S ribosomes, virus particles, and empty viral protein shells appeared to be absent from these vesicles. Some vesiclescontained stranded material with the general shape and staining properties of double-stranded or aggregated nucleic acid (Schreil, 1964) (Fig. 5). These strands were seen in about 80% of the vesicles observed three days after inoculation and in a lower proportion of vesicles at later times. Changes

Other Than in Chloroplasts

Chalcroft and Matthews (1967) noted the presenceof “star-shaped” bodies in the cytoplasm of cells infected with a yellow green Fine Structure of Peripheral Vesicles strain of the virus. In the present work, we In transverse sections virtually all the have never observed such bodies. However, small vesicles were located close to the pe- in cells systemically infected with the yellow riphery of the chloroplast. They were present green strain at 12 days many cells contained both behind the faces forming the channels nearly spherical inclusion bodies composedof between clumped chloroplasts and behind electron dense material without visible subthose surfaces which face the open cyto- structure. These were about l-2 I.L,in diamplasm of the cell. They were particularly eter and could be seenby light microscopy. prominent around the enlarged cytoplasmic We believe that the star-shaped bodies spacesbetween chloroplasts (Fig. 4). These seenby Chalcroft and Matthews were probvesicles were almost always surrounded by ably these spherical bodies in a collapsed or two membranes. In many of them the inner deformed state. membrane seemedto be pulled away someThese spherical inclusions were seen only what from the outer membrane. The outer with the yellow green strain and only in

CHLOROPLAST

ABNORMALITIES

IN

TYMV

INFECTION

301

FIG. 5. Fine structure of peripheral vesicles induced in Chinese cabbage chloroplasts by TYMV infection. (A) Vesicle in which the outer membrane appears to be continuous with the inner membrane of the chloroplast. Stranded material is seen in this vesicle and in those shown in B. Thin sections of leaf systemically infected with pale green strain 18 days after inoculation. CH, chloroplast; CY, cytoplasm. Scale line = 100 nm.

in grana and the increase in osmiophilic globules in white tissue. In normal leaves osmiophilic globules are usually seen more frequently in older leaves or other plant parts. The lipid they contain probably comes from the breakdown of grana during senesDISCUSSION cence (e.g., Frey-Wyssling and Kreutzer, The following modifications of chloroplasts 1958; Thomson, 1966). Osmiophilic globules can be assumed to be secondary effects of were markedly increased in leaves that had TYMV infection since they do not always been inoculated with the white strain for 12 days. These cells had mature chloroplasts occur in cells where virus replication is taking place, and in particular they are not when infected, and the presence of excess osmiophilic globules probably resulted from present during the early stages of infection: reduction in size and number of grana; re- the breakdown of grana. In the young sysduction in content of chlorophyll, 68 S ribo- temically infected leaf the white strain presomes, and starch; increase in amounts of sumably blocks grana formation. The small peripheral vesicles were seenin phytoferritin and numbers of osmiophilic globules; changesin size, shape, and arrange- chloroplasts at early times after infection and ment of chloroplasts other than mere swell- before any other changes except slight swelling; spacesbetween chloroplasts; and large ing of the chloroplasts could be observed. They were also found under all other condivesicles in chloroplasts. Some of these nonessential changes are tions tested. Thus it is possible that they probably linked-for example, the decrease may play someessential role in virus productissue systemically infected 12 days. No other abnormalities were seen outside the chloroplasts, except for the presenceof virus particles and occasional large apparently “empty” vesicles in the cytoplasm.

302

USHIYAMA

AND MATTHEWS

tion. We suggestthat these vesicles might be the site-of viral RNA synthesis, and that the viral RNA strands migrate from the vesicles to the cytoplasm where virus protein is synthesized, and virus particleqare assembled. This ‘suggestion would fit the following points: (i) Ralph and Clark (1966) and Ralph and Wojcik (unpublished work) have shown that the double-stranded viral RNA isolated from infected tissue is associated. with the chloroplast fraction. (ii) We have observed strands in the vesicles which have the appearance and staining properties of a double-stranded or aggregated nucleic acid (Fig. 5): (iii) Lafl&che’ and BovB (1968) in their autoradiographic study of the incorporation of tritiated uridine into TYMVinfected cells treated with actinomycin D, point to the accumulation of silver grains over the cytoplasmic spaces between chloroplasts. Inspection of their plate 4 suggests that there is also a’ concentration of silver grains over the zone containing the peripheral vesicles. Much further work would be needed to demonstrate this since, with the resolution of the method as at present developed, it would be difficult’to distinguish between grains originating just inside or just outside the chloroplast. Salpeter et al. (1969) have shown for the photographic materials used by La&he and Bove (1968) that there is a “half distance” of 1650 nm. (iv) The number of peripheral vesicles in a cell appears to be reasonable for the suggestedrole as the site of viral RNA synthesis. For the yellow green strain at 18 days after infection, we calculated that there were about 250 peripheral vesicles per chloroplast. Assuming there are 25 chloroplasts per cell, this gives roughly 6000 vesicles per cell. We have calculated that there are about 2 X lo6 TYMV particles in a fully infected cell. This would mean that each vesicle would need to produce of the order of 300 RNA complements. In young systemically infected leaves most cells are infected at about the same time (within l-2 days). Over the period of rapid virus synthesis in such leav,es (lasting lo-15 days) there may be an average of about 5000 virus particles produced per cell per hour (Matthews, 1970). Thus each vesicle would produce about one RNA complement per hour on the average. Actual rates in individ-

ual vesicles could be much higher if production was asynchronous between vesicles. ACKNOWLEDGMENTS We wish to thank Dr. S. Bullivant for advice on electron microscopy. One of us (R. U.) was the holder of a New Zealand University Postgraduate Scholarship during the course of this work. REFERENCES P. (1969). Rapidly labeled RNA associated with ribosomes of tobacco leaves infected with tobacco mosaic virus. Virology 39,893-960. BABOS, P., and SHEARER, G. B. (1969). RNA synthesis in tobacco leaves infected with tobacco mosaic virus. VGoZogy39, 28~295. Bov$, J. M. (1967). These de Doctorat d’Etat es Sciences Naturelles, presentee B la Faculte des Sciences de Paris, No. d’enrigestrement au C.N.R.S. A01289. Bovl, J. M., LAFLI~CHE, D., M~CQUOT, B., and Bovl, C. (1969). Turnip yellow mosaic virusRNA synthesis in vivo and in vitro. Znt. Bot. Congr. llth, Abstr. p. 20. CHALCROFT, J. P., and MATTHEWS, R. E. F. (1966). Cytological changes induced by turnip yellow mosaic virus in Chinese cabbage leaves. Virology 28, 555-562. CHALCROFT, J. P., and MATTHEWS, R. E. F. (1967). Role of virus strains and leaf ontogeny in the production of mosaic patterns by turnip yellow mosaic virus. Firology 33, 659-673. CRAIG, A. S., and WILLIAMSON, K. I. (1969). Phytoferritin and virus infection. Fiirology 39, 616617. FRENCH, C. S. (196Oj. The chlorophylls in tivo and in vitro. In “Handbuch der Pflanzenphysiologie” (W. Ruhland, ed.), Vol. 5(l), pp. 252 297. FREY-WYSSLING, A., and KREUTZER, E. (1958). Die submikroskopische Entwicklung der Chromoplasten in den Bltiten von Ranunculus repens, L. Plunta 51,104-114. GEROLA, F. M., BASSI, M., and GIUSSANI, G. (1966). Some observations on the shape and localisation of different viruses in experimentally infected plants, and on the fine structure of host cells. III. Turnip yellow mosaic virus in Brassica chinensis, L. Caryologia 19, 457479. HILLS, G. J., and PLASKITT, A. (1968). A protein stain for the electron microscopy of small isometric plant virus particles. J. Ultrastruct. Res. BABOS,

25, 323-329. HONDA, S. I., HONGLADROM,

T., and LATIES, G. G. (1966). A new isolation medium for plant organelles. J. Exp. Bot. 17, 460-172. LAFL~CHE, D., and Bovr?, J. M. (1968). Sites

CHLOROPLAST

ABNORMALITIES

d’incorporation de l’uridine tritiee dans les cellules du parenchyme foliaire de Brassica chine&s, saines ou infect&e par le virus de la mosai’que jaune du navet. C. R. Acad. Sci. Ser. 1839-1841. MATTHEWS, R. E. F. (1970). “Plant Virology.” Academic Press, New York. 778~~. MILIEI~, II., and ~TEFANAC, Z. (1967). Plastidenveranderungen unter dem Einfluss der Wasserrtibengellmosaikvirus (turnip yellow mosaic virus). Phytopathol. Z. 59, X5-296. MILNE, R. G. (1967). Electron microscopy of leaves infected with sowbane mosaic virus and other small polyhedral viruses. ViroZogy 32, 589-600. PEASE,

D. (1964). “Histological Techniques for Electron Microscopy.” Academic Press, New York.

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R. K., and CLARK, M. F. (1966). Intracellular location of double-stranded plant viral ribonucleic acid. Biochim. Biophys. Acta 119, 29-36. 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. SALPETER, M. M., BACHMANN, L., and SALPETER, E. E. (1969). Resolution in electron microscope radioautography. J. Cell Biol. 41, l-20. SXXREIL, W. H., (1964). Studies on the fixation of artificial and bacterial DNA plasms for the electron microscopy of thin sections. J. Cell Biol. 22, l-20. THOMSON, W. W. (1966). Ultrastructural development of chloroplasts in Valencia oranges. Bot. Gaz. (C/&ago) 127, 133-139. RALPH,