Download Characterization of Chloroplast Division Using the Arabidopsis

Plant Physiol. (1 996) 1 12: 149-1 59
Characterization of Chloroplast Division Using the
Arabidopsis Mutant arc5’
Elizabeth J. Robertson, Stephen M. Rutherford, and Rache1 M. Leech*
Department of Biology, The University of York, P.O. Box No 373, York, YO1 5YW, United Kingdom
arc5 is a chloroplast division mutant of Arabidopsis fhaliana. To
identify the role of ARC5 in the chloroplast replication process we
have followed the changes in arc5 chloroplasts during their perturbed division. A R G does not affect proplastid division but functions at a later stage in chloroplast development. Chloroplasts in
developing mesophyll cells of arc5 leaves do not increase in number
and all of the chloroplasts in mature leaf cells show a central
constriction. Young a r d chloroplasts are capable of initiating the
division process but fail to complete daughter-plastid separation.
Wild-type plastids increase in number to a mean of 121 after
completing the division process, but in the mutant arc5 the approximately 13 plastids per cell are still centrally constricted but much
enlarged. As the a r d chloroplasts expand and elongate without
dividing, the interna1 thylakoid membrane structure becomes flexed
into an undulating ribbon. We conclude that the ARCS gene is
necessary for the completion of the last stage of chloroplast division
when the narrow isthmus breaks, causing the separation of the
daughter plastids.
Chloroplast replication is a fundamental component of
normal chloroplast development. In higher plants the division of young chloroplasts in leaf mesophyll cells has
been identified in severa1 species (wheat, maize, bean, spinach, turnip, tobacco, and Arabidopsis thaliana) by increases
in chloroplast number and recognition of division profiles
(see Boffey, 1992, for a review). Chloroplasts divide by
binary fission. The initiation of the replication process is
first recognized as a centripetal invagination, followed by
extreme narrowing of the central constriction (isthmus),
then the separation of the two daughter chloroplasts (Leech
et al., 1981).A consistent feature of dividing chloroplasts is
an electron-opaque torus around the narrowing isthmus,
which is recognized in higher plants (Leech et al., 1981;
Hashimoto, 1986; Oross and Possingham, 1989; Modrusan
and Wrischer, 1990), algae (Mita et al., 1986; Mita and
Kuriowa, 1988; Hashimoto, 1992), and lower plants
(Tewinkel and Volkmann, 1987; Duckett and Ligrone,
1993). The function of the torus remains to be determined,
but in algae (Mita and Kuroiwa, 1988; Hashimoto, 1992)
and lower plants (Tewinkel and Volkmann, 1987) there is
evidence that suggests that it may contain actin.
Supported by an Agricultura1 and Food Research Council
(now BBSRC, UK) plant molecular biology grant (LR87/528) to
R.M.L. The authors wish to dedicate this paper to the memory of
Keith Partridge.
* Corresponding author; fax 44-1904-432860.
The chloroplast division process is under tight control
and in wheat has been shown to always follow the same
sequence of physical changes (Leech et al., 1981; Possingham and Lawrence, 1983). A11 of the chloroplasts in a
young wheat leaf cell undergo division synchronously.
ctDNA replication always takes place prior to chloroplast
division and ctDNA molecules segregate into the two
daughter chloroplasts at division (Scott and Possingham,
1980; Boffey and Leech, 1982; Possingham and Lawrence,
1983).
Chloroplast accumulation in developing leaves enormously affects photosynthetic efficiency, yet the control of
the chloroplast division process itself is one of the leastunderstood areas of chloroplast biology. Previously, the
lack of appropriate mutants has been a severe limitation in
studies of chloroplast replication, but our recent isolation
of a collection of mutants with extreme, specific lesions in
the chloroplast division process has remedied this deficiency (Pyke and Leech, 1992,1994; Pyke et al., 1994; Robertson et al., 1995). These arc (accumulation and replication
of chloroplasts) mutants are the first to be identified with
large, stable changes in chloroplast number, representing
recessive lesions of at least 10 independent nuclear alleles.
We have mutants in which chloroplast number per cell is
either greatly increased (+50%) or greatly reduced (-95%)
compared with wild type. These mutant plants grow normally and are fertile in controlled-growth conditions.
We now propose to use our unique Arabidopsis mutants to
determine the molecular mechanisms involved in the chloroplast division process. In one of these mutants, the chloroplasts of arc5 have a particularly interesting phenotype: they
are permanently constricted dumbbells and never complete
division into two daughter plastids. There are only 13 mature
chloroplasts per cell in arc5 (121 in wild type) and they are
6-fold larger than wild-type chloroplasts (Pyke and Leech,
1994). The chloroplast division process has been initiated but
not completed in the mutant arcfi, but the ARC5 gene clearly
has a critica1 role in normal chloroplast replication. To identify the precise lesion in arc5 chloroplast division and specify
the role of ARC5, we have followed in detail the changes in
a r d chloroplasts during their perturbed division. For comparison, the normal chloroplast division process in wild-type
Arabidopsis is also described.
MATERIALS A N D M E T H O D S
Plant Crowth
Wild-type Arabidopsis thaliana plants of the ecotype
Landsberg erecfa and plants of the arc5 mutant were grown
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Robertson et al.
150
in controlled conditions as described previously (Pyke and
Leech, 1991). The arc5 mutant was isolated from an ethyl
methanesulfonate-induced mutagenized Arabidopsis population in the background Ler (Lehle Seeds, Tucson, AZ)
(Pyke and Leech, 1994).
Scoring of Division Profiles
Slices of leaf tissue (1mm in thickness) were fixed for 1h
in 3.5% (v/v) glutaraldehyde and then the fixative was
replaced with 0.1 M Na,EDTA (pH 9). Incubation at 60°C in
fresh 0.1 M Na,EDTA (pH 9) for another 3 h ensured
adequate cell separation when tissue samples were teased
apart on glass slides (Pyke and Leech, 1991). Chloroplasts
in division within the individual fixed mesophyll cells
were counted by eye using Nomarski differential interferente contrast optics.
Confocal Scanning Laser Microscopy
First leaves from 19-d-old plants were harvested, cut into
2- to 3-mm-thick slices, fixed for 1h in 3.5% glutaraldehyde
(v/v), washed briefly in 0.1 M Na,EDTA (pH 9), and then
incubated in EDTA for 1h at 60°C. Intact isolated cells were
obtained by gentle maceration of the leaf tissue on a microscope slide. Nineteen-day-old plants were chosen by
light microscope examination, since mesophyll chloroplast
size was optimal for examination at this developmental
stage. Due to chlorophyll autofluorescence, chloroplasts
were visualized without the need for staining. Individual
cells were optically sectioned using a confocal laser scanning microscope (Axiovert 100, Zeiss) coupled to an inverted microscope (LSM 410, Zeiss) using a 488-nm blue
argon-ion laser. Cells were viewed at 4-pm intervals, sectioning a total depth of 28 pm in each cell scanned. Confocal images were captured and transferred to an image
processing program (Adobe Photoshop, Adobe Systems,
Mountain View, CA) on a Power Macintosh 8100 series
computer. Images were printed on a thermal transfer
printer (ColourMaster Plus model 6600PS, Calcomp LTD,
Vector House, Berkshire, UK).
lsolation of Plastids
One gram of fully expanded first leaf tissue of both wild
type and the a r d mutant were incubated in 10 mL of digest
medium (0.5 M sorbitol, 5 mM Mes, 1 mM CaCl,, pH 5.5)
containing pectolyase Y23 (O.l%, w / v ) and cellulase (2%,
w / v ) at 30°C for 3 h. After 3 h the enzyme medium was
carefully removed by suction through muslin attached
across the aperture of an inverted pipette. A11 subsequent
steps were carried out using ice-cold solutions. The segments were washed with 5 mL of washing medium (0.5 M
sorbitol, 5 mM Mes, 1 mM CaCl,, pH 6.0) three times,
releasing the digested leaf material by gentle shaking. After
each wash, the contents were filtered through a nylon tea
strainer. The filtrates were pooled and spun at lOOg for 5
min at 4°C. The pellet was resuspended in 5 mL of the
bottom layer of solution (0.5 M SUC,5 mM Mes, 1mM CaCl,,
pH 6.0), overlaid with 3 mL of a second layer (0.4 M SUC,0.1
M sorbitol, 5 mM Mes, 1 mM CaCl,, pH 6.0) and then 2 mL
Plant Physiol. Vol. 112, 1996
of washing medium, and was then spun at 1508 for 10 min
at 4°C. The purified protoplasts were collected between the
interface of the washing medium and the second layer
solution using a Pasteur pipette. The protoplasts were then
spun at 1508 for 5 min and placed in resuspension medium
(0.5 M sorbitol, 10 mM Na,EDTA, 25 mM Tricine [Hopkins
and Williams, Chadwell, UK], pH 8.4). The protoplasts
were gently broken on a glass microscope slide and the
isolated chloroplasts from wild-type and a r d cells were
viewed with Nomarski differential interference contrast
optics.
Ultrastructural Analysis
For ultrastructural analysis, whole seedlings from wild
type (Landsberg erecta) and a r d were harvested after 5, 7,
8, and 9 d of growth. First leaves from wild type (Landsberg erecta) and arc5 were harvested 10 and 27 d after
sowing. The shoot apex from the 5-d-old seedling, the first
leaf primordium of the young Arabidopsis seedlings, and
leaf tissue from the 10- and 27-d-old leaves were examined
after fixation and embedding in Spurr’s resin as previously
described for Arabidopsis (Pyke et al., 1994).
RESULTS
arc5 Chloroplasts
a r d mesophyll cells contain an average of 13 chloroplasts, compared with 121 in the leaf cells of wild-type
plants. a r d chloroplasts are also 6-fold larger than wildtype chloroplasts (Pyke and Leech, 1994). Scoring of
dumbbell-shaped chloroplasts within isolated mesophyll
cells in wild type and arc5 using differential interference
contrast optics (Nomarski) showed that in wild-type cells
(Fig. l a ) the majority of chloroplasts have completed division early in cellular development, but in arc5 mesophyll
cells the chloroplasts remain in suspended division until
cell maturity (Fig. lb). To establish the exact proportion of
the arc5 chloroplast population in which division is arrested and to enable chloroplasts along the cell edge to be
visualized accurately, the arc5 mesophyll cells were examined at 19 d using confocal scanning laser microscopy.
Optical sectioning of cells from arc5 and wild-type mature
mesophyll cells allowed the visualization of sequential
slices through isolated cells. In the arc5 cells the enlarged
plastids could easily be resolved and the entire chloroplast
population examined in detail. A11 of the arc5 plastids had
a definite central constriction (1-10 pm in diameter), in
contrast to the much more numerous, small, rounded chloroplasts in the wild-type cells. These images clearly illustrate the extreme size and constricted morphology of arc5
plastids (Fig. 2, top) compared with wild type (Fig. 2,
bottom), and confirm that a11 of the plastids in each cell are
indeed arrested in a late stage of chloroplast division.
To determine if the constriction of the enlarged arc5
plastids was a stable configuration, plastids in the process
of division were isolated from protoplasts of both arc5 (Fig.
3b) and wild-type (Fig. 3a) mesophyll cells. The isolated
plastids a11 stably retained their central constrictions. In the
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Arrested Plastid Division in the Arabidopsis Mutant arc5
ul
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a
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151
causes severe changes in both proplastid division and chloroplast division (Pyke et al., 1994; Robertson et al., 1995).
l h e Ultrastructure of arc5 Chloroplasts and Wild-Type
Chloroplasts during Development
0
k
O
1000 2000 3000 4000 5000 6000 7000 8000
O
1000 2000 3000 4000 5000 6000 7000 8000
Mesophyll Cell Plan Area (pmz)
Figure 1. The relationship between the proportion of the total number of chloroplasts in division per mesophyll cell and mesophyll cell
plan area (Fm2) from fully expanded leaves of w i l d type (Landsberg
erecta) (a, O)and arc5 (b, O). Each data point represents the mean of
a11 cell measurements per 500 pm2 range of cell plan area. SE bars are
shown for each point; in some cases the error bar is smaller than the
data point symbol. In wild-type cells (a), the proportion of the
chloroplasts in division increases dramatically in the early stages of
cell expansion to a peak of approximately 25% and then declines to
approximately 5 to 10%. In the arc5 mesophyll cells (b), the proportion of dumbbell-shaped chloroplasts increases in the early stages of
cell expansion until the majority of chloroplasts in each cell have a
central constriction.
mutant the greatly enlarged a r d chloroplasts never progressed beyond this stage of division, whereas a11 of the
wild-type chloroplasts completed this division process.
a r d Proplastids
Young meristematic tissue from 5-d-old seedlings of arc5
was examined to determine if the mutation affects proplastid development in young cells as well as chloroplast division in older cells. In Figure 4a (arc5) and in Figure 4c
(Landsberg erecta) meristematic cells are shown. The proplastids in cells from both plants are small, rounded organelles with very rudimentary, nonappressed thylakoid
membranes irregularly distributed throughout the stroma.
No significant difference in proplastid morphology can be
seen between the wild-type and the a r d chloroplasts. The
arc5 mutation, therefore, does not affect proplastid development. This is in contrast to the arc6 mutation, which
As proplastids mature into chloroplasts during normal
development they enlarge in size and the amount of thylakoid membrane increases and becomes more aligned.
Leaf primordial cells from arc5 are shown in Figure 4b and
cells from Landsberg erecta seedlings are shown in Figure
4d. The number of chloroplast profiles per cell is similar in
wild-type and arc5 cells. There is also little difference in
plastid morphology, internal thylakoid membrane structure, or thylakoid alignment when arc5 and wild-type plastids are compared. These observations suggest that the
wild-type and arc5 chloroplasts of leaf primordial cells
develop at a similar rate; the arc5 mutation is not yet
evident in leaf primordia, nor does the arc5 mutation lead
to any microscopically detectable changes in proplastid or
young chloroplast development.
Chloroplast division profiles have been repeatedly observed in developing cells of both arc5 and wild-type
plants. To our knowledge, this is the first time the chloroplast division mechanism has been described in wild-type
Arabidopsis. Chloroplast division in Arabidopsis occurs by
binary fission following a sequence of changes very similar
to those previously observed in wheat chloroplasts (Leech
et al., 1981). Using the observations on the sequence of
phases in chloroplast division in developing wheat cells as
a model, specific stages in the chloroplast division process
can also be identified in Arabidopsis chloroplasts. In Figure
5, three sequential stages in the division process are shown
for both a r d and wild-type chloroplasts. Dumbbell-shaped
plastids are very common in young leaves of both auc5 (Fig.
5a) and wild-type (Fig. 5e) leaf cells, and are indicative of
one of the first stages of plastid division in which a central
constriction forms a wide isthmus. An intermediate stage
in division is illustrated in Figure 5, b and f, where the
plastids of a r d and wild-type cells, respectively, are shown
to be elongated and twisted to give a much narrowed
isthmus and an L-shaped appearance in cross-section. Very
late stages in chloroplast division are illustrated in Figure
5, c and d, for arc5 and in Figure 5 g in wild-type cells. The
isthmus is extremely narrow in a11 the chloroplasts at this
time and separates the plastid into two distinct and equal
halves. Frequently, twisting of the internal thylakoid membrane fretwork is observed at this late stage of division.
Normally, the thylakoid membranes are oriented parallel
to the long axis of the plastid, but during the later stages of
chloroplast division the alignment of the thylakoid membrane typically changes and the longitudinal orientation of
the membrane is lost in one of the two halves of the plastid
(Fig. 5c). This occurs because the two halves of the dividing
plastid ultimately twist in opposite directions around the
central isthmus (Leech et al., 1981), resulting in the thylakoid membrane in the two halves of the plastid being
oriented at right angles to one another (Fig. 5g).
Another classic feature of chloroplasts in the process of
division is the torus, or opaque ring, around the narrow
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Robertson et al.
152
Figure 2. arc5 (top) and Landsberg erecta (bottom). 1, An
isolated mesophyll cell from a fully expanded leaf viewed
with differential interference contrast optics (Nomarski). 2
to 9, Sequential confocal scanning laser micrographs
through a comparable isolated mesophyll cell from a 19d-old first leaf. Optical sections were taken 4 fim apart
through a total depth of 28 /j,m of the cell. Note that all of
the plastids in the cell show a central constriction. Arrows
follow a plastid through images 2 to 5 on the upper surface
of the cell. Arrowheads follow a plastid through images 7
to 9 on the lower surface of the cell.
Plant Physiol. Vol. 112, 1996
arcS
Landsberg erecta
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Arrested Plastid Division in the Arabidopsis Mutant arc5
Landsberg erecta
153
old. In wild-type plants, the majority of the chloroplasts
have completed division and appear as small, ovoid
organelles around the periphery of the now-vacuolated
cells (Fig. 6, c and d) in 10-d-old plants. In contrast, the
chloroplasts in arc5 plants of similar age are much larger
and the central constriction is clearly observed (Fig. 6, a
and b). The diameter of the isthmus in the arc5 chloroplasts has increased from approximately 100 nm in 9-dold chloroplasts to approximately 200 nm in 10-d-old
chloroplasts. In both arc5 (Fig. 6b) and wild-type (Fig.
6d) plastids, the amount of thylakoid membrane has
greatly increased and the arrangement of the membranes
is more complex, with appressed and nonappressed
membranes occurring throughout the stroma. The thylakoid fretwork of membranes is oriented parallel to the
long axis of the chloroplasts in both arc5 and wild-type
plastids at this stage in development.
The Infrastructure of the Mature arc5 Chloroplast
Figure 3. Photomicrographs of isolated mesophyll cell chloroplasts
in the process of division. The chloroplasts were from broken protoplasts of fully expanded first leaves of wild-type (a) and arc5 mutant
(b) A. thaliana, ecotype Landsberg erecta, viewed with differential
contrast optics (Nomarski). Bar = 10 /j.m. Note the greatly enlarged
arc5 chloroplasts that never progress beyond this stage of development. All of the wild-type chloroplasts complete the division process
in the cell.
isthmus of the dividing plastid (Leech et al., 1981; Hashimoto, 1986; Modrusan and Wrischer, 1990). In the present
study, a torus was visible in both arc5 (Fig. 5d) and in
Landsberg erecta (Fig. 5g) plastids. The torus was approximately 10 nm in cross-sectional width and 100 nm in
cross-sectional length and located at the isthmus (also approximately 100 nm) of the dumbbell-shaped plastids. This
torus is thought to circumvent the isthmus of dividing
plastids in a transitory manner when the isthmus is close to
partitioning. Consequently, the torus is very rarely observed, since it is necessary to capture the plastids at exactly the right transitory moment in division and also to
section plastids exactly above the narrow, ring-like torus
for it to be resolved. Since arc5 dividing plastids showed
morphological changes during the early stages of the chloroplast division process that are very similar to those seen
in wild-type plastids, young arc5 plastids clearly must have
the capability to initiate and complete the early stages in
plastid division, as do wild-type plants. Since the majority
of the plastids in arc5 have extremely narrow isthmi, the
division of chloroplasts in the arc5 mutant is arrested at an
extremely late stage in the chloroplast division process.
After another 17 d of growth, the fully mature arc5
chloroplasts have undergone dramatic changes in their
morphology and internal membrane structure. In these
27-d-old plants ultrastructural examination of mature arc5
leaves reveals that all of the mutant plants have a contorted
morphology and membrane structure compared with wildtype plastids (Fig. 7). All of the arc5 plastids are also now
grossly enlarged (6-fold) compared with wild type. The
compacted, rotund, uniform appearance of the wild-type
plastids, which has changed very little since d 10, is very
different from that of the arc5 chloroplasts, which are extremely elongated in form and have an undulating surface.
The plastids are flattened between the vacuole and the cell
wall.
The arrangement of the thylakoid membranes in the arc5
plastids is also very different from that of the normal
wild-type membrane arrangement. Appressed and nonappressed membranes are both evident, but granal stacking is
more condensed in arc5 than in wild-type chloroplasts. The
orientation of the membrane within the arc5 plastids is also
extremely contorted. Instead of running parallel to the
chloroplast envelope membranes as in wild-type plastids
(Fig. 7d), the thylakoid membrane in arc5 is frequently bent
into loops and twisted into U-shaped segments at right
angles to each other within the plastid (Fig. 7b). The extreme irregularities in the thylakoid membrane alignment
of the arc5 chloroplasts resembles a similar but more complex membrane alignment, as seen in the two halves of a
normal dividing chloroplast.
The Chloroplasts in arc5 and Wild-Type Epidermal and
Vascular Cells
In contrast to the appearance in mesophyll cells, in the
epidermal and vascular cells of arc5 leaves the plastids
are similar in appearance to wild-type plastids (Fig. 8).
Identification of the First Phenotypic
In the epidermal cells, including the guard cells, arc5
Manifestation of the Mutation
plastids (Fig. 8, a and b) remain small and ovoid and no
The first visible signs of the arc5 mutant phenotype are
central constrictions are observed. In every way they
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evident in the mesophyll cells when
the
plants
are
10
d
appear
to All
wild-type
epidermal plastids (Fig. 8, d
Copyright © 1996 American Society of Plantsimilar
Biologists.
rights reserved.
Robertson et al.
154
Plant Physiol. Vol. 112, 1996
arcS
Figure 4. Electron micrographs of longitudinal sections through the Arabidopsis shoot apical meristem in arcS (a) and
Landsberg erecfa (wild type) (c) and through leaf primordia from 9-d-old Arabidopsis seedlings in arcS (b) and Landsberg
erecta (wild type) (d). All proplastids (a and c) and young chloroplasts (b and d) are identified by arrowheads. The arc5
mutant phenotype is not recognizable in the apical meristematic tissue nor in the leaf primordia after 9 d of growth.
Bar = 2 /xm.
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Arrested Plastid Division in the Arabidopsis Mutant arcS
155
arcS
Figure 5. Electron micrographs of chloroplast division profiles from leaf primordia of Arabidopsis seedlings after less than
10 d of growth in arcS (a-d) and in Landsberg erects (wild type) (e-f). All stages of plastid division are seen in both the arcS
mutant and wild-type cells. Arrowheads indicate position of isthmi. a and e, Early stage, dumbbell-shaped plastids with a
wide isthmus, b and f, Intermediate stage, elongated, L-shaped plastids with a narrowing of the isthmus, c, d, and g, Late
stage, two distinct halves with an extremely narrow isthmus. An opaque division ring can be seen at the isthmus of the
plastids in d and g. Bar = 500 nm.
and e). Chloroplasts in arc5 vascular cells (Fig. 8c) are
DISCUSSION
also comparable to wild-type vascular chloroplasts (Fig.
It is clear from the evidence presented in this paper
8f) and no central constrictions are evident. Therefore,
that all mature arc5 chloroplasts have a central constricthe manifestation of the arc5 mutation appears to be
mesophyll-cell-specific; only in these cells do the plastion. This phenotype is unparalleled in any other plant
tids maintain a central constriction through to maturity,
tissue and represents an extremely important lesion in
giving the appearance of chloroplasts arrested in the
the normal chloroplast division process. Using the disDownloaded from on June 14, 2017crimination
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final stages of division.
the confocal scanning laser microscope
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Robertson et al.
156
Plant Physiol. Vol. 112, 1996
arcS
Landsberg erecta ,
Figure 6. Electron micrographs of leaf mesophyll chloroplasts from 11-d-old first leaves of Arabidopsis. a and b, arcS; c and
d, Landsberg erecta (wild type). In the enlarged arcS chloroplast (b), arrowheads indicate the position of the central
constriction (isthmus). The smaller wild-type chloroplasts have completed division. Ap, Appressed thylakoid membrane;
CW, cell wall; N, nucleus; NAp, nonappressed thylakoid membrane; S, starch; V, vacuole. Bar = 500 nm.
sistently have external narrow isthmi about which the
(Fig. 2, top) to extend the light microscope observations
two halves of the plastid are twisted, suggesting that
of whole-leaf cells (Figs. 1 and 2, top and bottom) and
isolated chloroplasts, it can be clearly seen that chloroARCS influences the very final stages of chloroplast diplast division is initiated but not completed in arc5. We
vision. Absence of the influence of ARCS results in a few
very enlarged plastids that retain the central constriction
conclude that the ARCS gene functions during the final
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in mature
leaf cells.
stage of daughter-plastid separation.
arc5from
plastids
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Arrested Plastid Division in the Arabidopsis Mutant arc5
Chloroplast
Landsberg erecta
Chloroplasts
CW
Figure 7. Electron micrographs of leaf mesophyll chloroplasts from 27-d-old first leaves of Arabidopsis. a and b, arc5; c and
d, Landsberg erecta (wild type). Outlined areas in a and c are enlarged in b and d, respectively. The enlarged arc5 chloroplast
has an extremely contorted profile and internal structure (a) compared with wild-type chloroplasts (c). Note the unusual
organization of the thylakoid membrane in the arc5 chloroplast, with adjacent thylakoid membranes oriented at right angles
to one another (b). Ap, Appressed thylakoid membrane; NAp, nonappressed thylakoid membrane; CW, cell wall; P,
peroxisome; V, vacuole. Bar = 1 turn.
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157
Robertson et al.
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Plant Physiol. Vol. 112, 1996
arcS
v^^tm-
Figure 8. Electron micrographs of leaf cells of Arabidopsis in arcS (a-c) and in Landsberg erecta (wild type) (e and f).
Arrowheads point to all of the chloroplasts shown in a, c, d, and f. An epidermal chloroplast is shown in b and e.
Chloroplasts in the epidermal cells of arcS (a and b) and wild type (d and e) are small and poorly differentiated compared
with mesophyll chloroplasts. arcS chloroplasts located in the vascular tissue (c) are similar in size and ultrastructure to
wild-type plastids from the vascular tissue (f). Ep, Epidermal cell; Me, mesophyll cell; Va, vascular cell. Bar = 1 ^tm.
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Arrested Plastid Division in the Arabidopsis Mutant a r d
In contrast, in apical meristematic tissue and leaf primordia of the a r d mutant, proplastid development and replication is normal and resembles the sequence of events
observed in wild-type cells. The chloroplast number (13 in
postmitotic cells) in arc5 is similar to the estimated proplastid number (14) in wild-type cells of Arabidopsis (Pyke and
Leech, 1992, 1994). No significant difference in proplastid
morphology can be seen in meristematic a r d cells compared with wild type (Fig. 4, a and c). The young chloroplasts in leaf primordia are also similar in size and structure in a r d and wild-type cells. arc5 plastids arrive at the
final stages of plastid division on a time scale similar to that
of wild-type plastids, i.e. up to 9 d after germination, but
can go no further along the normal developmental pathway to form daughter plastids. This is in sharp contrast to
the ARC6 gene, which radically affects proplastid division
such that in arc6 plants, proplastid division is also interrupted (Robertson et al., 1995). The operation of AXC5 is
clearly restricted to young dividing chloroplasts in leaf
mesophyll cells; epidermal and vascular plastids in a r d
appear normal (Fig. 8). Therefore, ARC5 gene expression is
not only tissue-specific but cell-specific as well.
The extremely contorted form and internal membrane
arrangement of mature arc5 chloroplasts is very different
from the structure of other arc mutants having low numbers of enlarged plastids, for example, arc3 (Pyke and
Leech, 1994) and arc6 (Pyke et al., 1994). In these two
mutants, the plastids form a narrow sheet between the
plasma membrane a n d the vacuole, and the internal thylakoid membrane arrangement is similar to that of wild type,
with appressed and nonappressed membranes distributed
throughout the stroma and aligned parallel to the long axis
of the chloroplast. The reorientation of the thylakoid membrane observed in mature a r d chloroplasts is also seen in
the two opposite halves of normally dividing chloroplasts
and further identifies the phase affected by the ARC5 gene.
It is clear that after 9 d of growth, arc5 mesophyll chloroplasts differ considerably from wild-type chloroplasts in
their development: they are arrested in a late stage of
plastid division. a r d chloroplasts retain a central constriction and continue to expand. The internal membrane continues to accumulate and develop, but its alignment and
orientation is much perturbed. It twists and folds as in a
young dividing plastid, the result being an enlarged plastid
with a contorted outline due to the repeated folding and
twisting of the internal membrane. Preliminary immunogold labeling studies indicate a possible down-regulation
of actin within arc5 chloroplasts compared with wild-type
plastids. Down-regulation of this structural protein provides a possible explanation for the unusual organization
of the thylakoid membranes within mature a r d chloroplasts and J or the expansion of the torus. arc5 plastids are
suspended in the final stages of division and the contortion
of the mature plastid reflects the operation of incorrect
membrane alignment signals that are maintained into chloroplast maturity. The signals for the final stages of the
chloroplast division process remain t o be determined. It is
159
clear that the arc5 mutant offers a unique opportunity to
further unravel the mechanisms controlling the complex
and transient stage of isthmus partition in chloroplast
division.
ACKNOWLEDCMENTS
We wish to thank Dr. Kevin Pyke for valuable advice on this
work and Keith Partridge for growing the plants.
Received April 1, 1996; accepted June 5, 1996.
Copyright Clearance Center: 0032-0889/96/112/0149/ 11.
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