J. gen. Virol. (1973), 2o, 413-426
413
Printed in Great Britain
Aggregates of Chloroplasts in Local Lesions induced in
Chenopodium quinoa Wild. by Turnip Mosaic Virus
(Accepted 24 May I973)
SUMMARY
Aggregates of up to ao chloroplasts occur in parenchyma cells from local
lesions induced in Chenopodium quinoa Wild. by turnip mosaic virus. Electron
microscopic examination revealed a single layer of virus particles parallel to the
surface of chloroplasts between most of the aggregated chloroplasts.
Cruciferous crops grown in S~.o Paulo State, Brazil, are commonly infected with turnip
mosaic virus (TuMV) (Costa, Kitajima & Nagai, I972), an elongated, aphid-borne virus
belonging to the potato virus Y (PVY) group (Tomlinson, 197o). Chenopodium quinoa Wild.
is a useful indicator plant, producing conspicuous chlorotic local lesions after mechanical
inoculation with sap from TuMV-diseased hosts. This communication reports a peculiar,
virus-mediated aggregation of the chloroplasts in the cells of these local lesions.
Chenopodium quinoaplants, about 2 to 3 weeks old, were inoculated mechanically with an
isolate of TuMV, obtained from and maintained in kale (Brassica oleraceaL. var. acephala).
Local chlorotic lesions were discernible in the inoculated leaves 5 to 7 days after inoculation.
Samples from these local lesions were fixed in 3 % glutaraldehyde and post-fixed in 1%
OsO4 (both in phosphate buffer), dehydrated in acetone and embedded in Epon. Sections
were stained with uranyl acetate and lead citrate, and examined in a Siemens Elmiskop i. For
comparative purposes the following leaf material was also processed and examined as described above: kale and radish (Raphanussativus L.) systemically infected with TuMV; local
lesions in Chenopodium amaranticolor Coste & Reyn. and tobacco (Nicotiana tabaeum L.)
inoculated with TuMV; local lesions in C. quinoa inoculated with tobacco mosaic,
Brazilian tobacco rattle, potato virus X, potato virus S, bidens mosaic (Kitajima, Carvalho
& Costa, I961), lettuce mosaic, and celery yellow mosaic (Kitajima & Costa, I968) viruses.
Light-microscopic examination of the tissues from the local lesions were made on hand-cut
unstained sections floated on buffered 3 % glutaraldehyde as well as on thick (I to 2 tim)
sections on Epon-embedded tissues, stained with a mixture of Azur II and methylene blue.
Most palisade and spongy mesophyll cells contained chlorotic chloroplasts forming large
clumps (Fig. I, inset), instead of being uniformly distributed along the cell periphery.
Chloroplast aggregation was rare in vascular parenchyma cells.
Chloroplast aggregates were best seen in thin sections examined in the electron microscope (Figs. I to 4)- Under low-power magnification they were seen to consist of up to
2o chloroplasts, tightly apposed to each other in a chain or irregularly grouped. Individual
chloroplasts usually contained several large osmiophilic granules, a poorly developed
lamellar system, few or no starch grains, and also had an irregular profile, depending on the
surrounding chloroplasts.
At higher magnifications, a layer of elongated particles was seen between most of the
adjacent chloroplasts in the aggregates. These particles were arranged parallel to each other,
with their long axes parallel to the chloroplast surface (Figs. 2 to 4). They also appeared
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Short communications
415
between the chloroplast and the tonoplast where these structures were closely apposed
(Fig. 3), as well as in narrow strands of cytoplasm projecting into the vacuole (Fig. a). In
several instances they were seen at the surfaces of the chloroplasts that were in direct contact
with the cytoplasm and not apposed to other chloroplasts (Figs. z, 3). Whenever elongated
particles were associated with the surface of the chloroplast they always appeared in a
single layer.
The diam. of the elongated particles was I o to 12 nm but their length was not measurable.
They probably represent TuMV because they are similar to the TuMV particles observed in
sections of purified preparations (Hill & Shepherd, I97z) and those pelleted by ultracentrifuging (Fig. 4, inset).
In addition to the chloroplast aggregates, most cells contained many lamellar inclusions,
with varied configurations depending on the plane of sectioning, which are characteristic of
cells infected with viruses of the PVY group (Edwardson, Purcifull & Christie, t968)
(Fig. x).
Similar, although much less conspicuous, chloroplast aggregates were observed in local
lesions from TuMV-infected Chenopodium amaranticolor, but not in tobacco (local lesions)
or radish and kale (systemically infected leaves). Chloroplast aggregates were not detected in
local lesions, from C. quinoa leaves induced by other elongated viruses (tobacco mosaic,
Brazilian tobacco rattle, potato viruses S and X) including some other members of the PVYgroup (bidens mosaic, lettuce mosaic, celery yellow mosaic). Previous studies on cytological
aspects of TuMV-infection in cruciferous hosts (Hayashi, Matsui & Yamaguchi, I965;
Kamei, Honda & Matsui, I969; Edwardson & Purcifull, I97o) do not mention aggregation
of chloroplasts.
Some degree of plastid aggregation associated with virus particles was reported in chinese
cabbage infected with turnip yellow mosaic virus (Chalcroft & Matthews, 1966) and in barley
infected with barley stripe mosaic virus (Carroll, 197o), but it was much less conspicuous than
the aggregation described here. Moreover, the rod-shaped particles of barley stripe mosaic
virus were attached to the chloroplast surface by their ends and not by their sides. A similar
difference was reported between a Brazilian strain of tobacco rattle virus (Harrison &
Roberts, I968; Kitajima & Costa, I969) and henbane mosaic virus (Kitajima & Lovisolo,
I972) in their association with mitochondria. The former attach to the mitochondrial surface by their ends, whereas the latter attach by their sides. Thus the pattern of plastid
aggregation in TuMV-infected Chenopodium quinoa is similar to the mitochondrial aggregate in henbane mosaic virus-infected Datura stramonium L. (Kitajima & Lovisolo, I972 )
and the mechanism of aggregation proposed for that system is probably applicable here. The
specific interaction between the outer surface of the chloroplast and the TuMV particles probably involves specific receptor sites. However, in preliminary trials, chloroplasts isolated
Fig. I. Electron micrograph of a thin section of a local lesion in Chenopodiumquinoa caused by
TuMV. Ceilsare somewhat shrunken and most chloroplasts (P) are aggregated. They usually contain
large osmiophilic granules, underdeveloped tamellar system, and irregular contour. Lamellar
inclusions (LI), typical for viruses of the PVY group can be seen abundantly in the cytoplasm. In the
inset a light micrograph of a similar but thicker section, showing chloroplast aggregates in parenchyma cells close to the vascular bundle. X, xylem; IS, intercellular space; vc, vacuole; W, cell wall.
Fig. 2. A chloroplast aggregate in which a layer of TuMV particles can be seen' sandwiched' between
adjacent plastids (P). Some virus particles can be seen in cytoplasmic strands projecting into the
vacuole (arrow).
Figs. 3, 4. A higher magnification of a chloroplast aggregate. Virus particles (arrows) can be seen in
transverse or longitudinal orientation. Inset of Fig. 4 shows a section of a purified TuMV preparation, pelleted by ultracentrifuging.
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Short communications
416
f r o m h e a l t h y C. quinoa failed to a g g r e g a t e w h e n a l l o w e d t o r e a c t w i t h p u r i f i e d T u M V & vitro.
P o s s i b l y t h e p h y s i o l o g i c a l state o f t h e c h l o r o p l a s t in t h e i n f e c t e d cell is i m p o r t a n t .
B e c a u s e h a n d - c u t sections m a y easily be p r e p a r e d a n d e x a m i n e d t h e d e t e c t i o n o f c h l o r o p l a s t a g g r e g a t e s in T u M V - i n f e c t e d Chenopodium quinoa m a y h a v e a useful specific diagn o s t i c value, p r o v i d e d t h a t this p h e n o m e n o n c o u l d be d e m o n s t r a t e d w i t h o t h e r strains o f
TuMV.
T h i s w o r k r e c e i v e d f i n a n c i a l s u p p o r t f r o m C o n s e l h o N a c i o n a l de P e s q u i s a s - C N P q
( T C I2175) a n d F u n d a ~ t o de A m p a r o a P e s q u i s a d o E s t a d o d e S~o P a u l o (C. A g r o n .
72]376). T h e a u t h o r s are r e s e a r c h fellows o f C N P q .
Virus Department
Instituto Agron6mico
I3. ~oo-Campinas, S P
Brazil
E . W . KITAJIMA*
A . S . COSTA
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* Present address: Dept. Biologia Celular, ICB, Universidade de Brasilia, 7o.ooo Brasilia, DF, Brazil.
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