CHLOROPLAST
IN A BROWN
AND
MITOCHONDRIAL
ALGA EGREGIA
T. BISALPUTRA
DNA
MENZIESII
and A. A. B I S A L P U T R A
From the Department of Botany, University of British Colmnbia, Vancouver, Canada
ABSTRACT
Chloroplasts and mitochondria of the brown alga Egregia menziesii were studied with the
electron microscope. In both organelles, 15-25-A fibrils with D N A characteristics are found
within areas of electron transparency. In each chloroplast there are two DNA-containing
areas, one at each tip of the chloroplast. This localization, the shape and size of each D N A containing area, and its close association with lamellae in a nondividing chloroplast are
noted. O n e or occasionally two DNA-containing areas are found within the mitochondrion
and they are compared with a similar structure in the chloroplast.
INTRODUCTION
In a previous paper (2), evidence was presented
for the occurrence of D N A fibrils in chloroplasts
of the red alga Laurencia spectabilis. The fibrils were
found within electron-transparent areas which
occur in random distribution within the chloroplast matrix. It was shown that these fibrils were
digested by DNase and have dimensions similar to
the 25-A D N A fibrils found in the chloroplast of
Chlamydomonas moewusii (17). There is no detailed
electron microscopic study of chloroplast D N A in
brown algae although areas of electron transparency were observed in this organelle by many
workers (cf. 9). Bouck (4) was the first to report
the occurrence of fibrils with D N A characteristics
in chloroplasts of Giffordia, Chorda, and Fucus and,
in his diagram of a hypothetical brown algal cell,
D N A fibers were shown to occur at each end of the
elongated chloroplasts. In higher plants, Kislev
et al. have shown, by Feulgen staining, radioautography, and DNase treatment, the presence of D N A
areas in chloroplasts of Swiss chard (I0). When
observed under the electron microscope, these
areas were found to contain fibrillar components
which were extractable with DNase (10). They
appear to have a random distribution within the
chloroplast matrix.
More detailed information is available for the
mitochondrial D N A as seen with the electron
microscope (10, 12-14). It has been shown in
these earlier works that D N A fibrils also occur in
electron-transparent areas within the matrix of
mitochondria and that when the fibrils appear in a
dispersed state they are of the order of 20-50-A in
diameter. In an extensive survey, Nass, Nass, and
Afzelius (14) found mitochondrial D N A in 20
species of plants and animals belonging to 16
classes and nine phyla; the plant species were
Allium cepa and Ceratium tripos. Those authors coneluded that D N A is an integral part of most or all
mitochondria.
The present work describes the morphology and
distribution of D N A fibrils in these two important
organelles of a brown alga, Egregia menziesii
(Turn.) Aresh. For this study, relatively well
differentiated regions of the blade and stipe were
used.
MATERIAL
AND METHODS
Material of Egregia menziesii was collected from Widbey Island, Washington. Blades and stipes of the alga
were fixed in a 6% solution of glutaraldehyde in equal
amounts of Dalton's K2Cr20~ (Potassium dichromate)
511
FIOURES 1 and ~ Chloroplasts in meristodermal cells showing the electron-transparent areas at the tips
(arrows); F V , fucosan vesicles; L, Chloroplast lamellae; m, mitochondrion; n, nucleus; s, starch; w, cell
wall. X 8,000.
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THE JOURNAL OF CELL BIOLOGY • VOL~E 33, 1967
FIOURE 8 Electron-transparent DNA-eontaining areas in a chloroplast and a mitochondrion of an outer
cortical cell. In the chloroplast, such an area is contained within a pocket of lamellae (L) at each end of
the organelle. Note also the parallel bands of photosynthetic lamellae (L) within the chloroplast. D N A
area within mitochondrion (m) is indicated by an arrow. E, chloroplast envelope; o, osmiophilic granules; pro, plasma membrane; w, cell wall X 26,000.
513
buffer (6) a n d sea water at p H 7.2 according to the
m e t h o d described previously (2). T h e material was
fixed for 1 h r at 4 ° C a n d subdivided into three portions. O n e p o r t i o n was w a s h e d t h o r o u g h l y in a buffer
solution m a d e u p of e q u a l v o l u m e s of K2Cr207 buffer
a n d sea w a t e r a n d postfixed in D a l t o n ' s fixative (6) for
1 hr. T h e second p o r t i o n of m a t e r i a l was destined for
D N a s e digestion. It was w a s h e d in distilled water a n d
treated w i t h D N a s e (1.0 m g of crystalline e n z y m e
per ml) for 6 h r at 40°C. T h i s w a s followedhy postfixation in D a l t o n ' s fixative as described a b o v e . T h e third
portion served as a control for D N a s e t r e a t m e n t . It
was w a s h e d a n d left in distilled w a t e r while being inc u b a t e d at 40°C for 6 a n d 12 hr. All portions were deh y d r a t e d in a n ethanol-propylene oxide series a n d
e m b e d d e d in M a r a g l a s (3). Polymerization was car-
Fm~-RE 4 Tips of chloroplast at higher magnification showing the occurrence of fibrils (arrows) within
the electron-transparent areas inside the lamellar pockets (L). X 60,000.
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T H E JOURNAL OF CELL BIOLOGY • VOLUME 38, 1967
FiounEs 5-7 Sections of chloroplasts cut tangential to the tips at the following levels: above the electron-transparent area (Fig. 5); through the electron-transparent area (Fig. 6); and below the electrontransparent area (Fig. 7), respectively. In Fig. 5, note the tangential view of the lamellae (L) of the pocket
which caps the electron-transparent area. Fig. 6 shows the fibrillar components (arrow) of the electrontransparent area. X 80,000, X 30,000, and X ~0,000, respectively.
T. BISALPUTm~ X~D A. A. Bis~LPu'ra.~
Chloroplastand Mitochondrial DNA
515
ried out in a vacuum oven at 65°C for a period of
18-24 hr. Thin sections were cut on a Porter-Blum
MT1 or an L.K.B. Ultrotome microtome with a du
Pont diamond knife. Sections were stained in uranyl
acetate for 20 rain followed by 10 min in lead citrate
(15). A Hitachi H U 11A microscope was employed
for the observation of sections.
OBSERVATIONS
The cytoplasm of a meristodermal cell of the
is dense and rich in organelles (Figs.
1 and 2). Chloroplasts, mitochondria, and large
intensely stained fucosan vesicles are prominent
features of the cytoplasm. Starch grains occurring
within the cytoplasm appear as membranebounded structures of medium electron opacity
with a large central clear area. The cell wall
stains dark and shows microfibrils oriented in an
orderly manner. The fine structure of the cell wall
of this alga will be described in a later paper.
Egregia blade
Chloroplast D N A
The majority of the chloroplasts in a blade or
stipe are found within the meristoderm and outer
cortical regions. They are oval-shaped bodies
located mainly along the periphery of the cyto-
plasm and contain well developed internal lamellae (L, Figs. 1 and 3). These photosynthetic
lamellae appear in parallel bands of three closely
associated discs (Fig. 3) similar to those of other
brown algae described previously by various
authorities (1, 4, 7-9, 11). These bands of discs
extend from one end of the chloroplast to the
other, but discs may divert from one band to the
neighboring band or bands (Fig. 3). Each disc is
approximately 180A wide, and a band of lamellae
(L) of three discs is about 65 m/z wide. The space
separating each disc in a band is approximately
40-50 A wide. The granular chloroplast matrix is
slightly denser than the cytoplasm, and osmiophilic
granules (0, Fig. 3) can be observed between
lamellar bands. O f significance is the occurrence
of an electron-transparent area within the matrix
at each end of the chloroplast (arrows, Figs. 1-9).
Each such area does not have any bounding m e m brane, but always occurs within a small pocket of
chloroplast matrix surrounded on three sides by
chloroplast lamellae. This is clearly illustrated by
Fig. 3. It can be observed that the band of lamellae
(L, Fig. 3) which forms the boundary of the inverted pocket at each tip is formed from discs of
several lamellar bands on either side of the pocket.
FIGURE 8 DNA-containing areas at tips of two chloroplasts of a meristodermal cell. Fibrils with DNA
characteristics are indicated by arrows. L, pocket of chloroplast lamellae. X 70,000.
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THE JOURNAL OF CELIa BIOLOGY • VOLUME33, 1967
FIGURES9 and 10 DNA-containing areas in a chloroplast and a mitochondrion, respectively, after 6 hr
of DNase treatment. X 60,000, and X 40,000, respectively.
The occurrence of the electron-transparent area
within such a pocket is a consistent feature of the
Egregia chloroplast. Well differentiated and nondividing chloroplasts are typified by deep cupshaped pockets containing circular or slightly
ovate electron-transparent areas (Figs. 1, 3, 8).
Chloroplasts with obtuse tips have, as a rule,
shallow pockets containing either one elongated or
two small electron-transparent areas (Figs. 2 and
4). The latter type of chloroplasts indicate a stage
in the process of chloroplast fission which will be
described in some detail in a separate report.
The circular electron-transparent areas of nondividing chloroplasts vary in size, b u t on the
average they are approximately 180-300 m/z in
diameter. The oval areas measure 180-300 m~ in
length and 120-150 m/~ in width (Figs. 3, 8). Figs.
5-7 are sections cut tangential to the tips of the
chloroplasts at the level above the electrontransparent area, through the area, and below the
area, respectively. From this series of micrographs
it is clear that the electron-transparent area at
each tip of the chloroplast is a localized and isolated area.
At higher magnification, it can be resolved that
each electron-transparent area at the tip contains
networks of fibrillar structure (arrows, Figs. 4, 6,
8). The smallest fibrils are of the order of 15-25 A
thick, and these were seen radiating from larger
fibrils of up to 120 A. The large fibrils may result
from the intertwining and clumping of the smaller
ones, b u t the large rod-shaped structure observed
previously in the chloroplast of the red alga
Laurencia (2) is not present, indicating that the
fibrils in Egregia are not so heavily clumped. No
other structure is discernible in the area around the
fibrils. Fibrils can be removed by DNase (Fig. 9)
in the same m a n n e r as that shown previously in
chloroplasts of other plants (2, 10, 17). However,
the fibrils are not removed by the control extraction treatment after either 6 or 12 hr (Figs. 11, 12).
At 12 hr, the cytoplasm shows considerable effect
of extraction but the fibrils remain distinct (Fig.
12).
Preliminary work has shown that the distribution and morphology of DNA-containing areas in
the chloroplasts of other brown algae, Fucus, Alaria,
and Sphaecelaria, are similar to that of Egregia.
M itoehondrial D N A
The general structure of mitochondria of Egregia
is shown in Figs. 2, 3, and 13). M a n y mitochondria
of the meristoderm cells are cup-shaped structures
with tubular cristae. A section parallel to the rim
of the cup would reveal the mitochondria as
doughnut-shaped structures, each with a central
T. BISALPUTRAAND A. A. BISALPUTRA Chloroplastand Mitochondrial DNA
517
FIGURES 11 and 1~ Micrographs showing the condition of the fibrillar structure in the electron-transparent areas of a chloroplast and a mitochondria after 6 and 1£ hr of extraction control, respectively. In
both cases, the fibrils (arrows) remain distinct while progressive deterioration of the cytoplasm becomes
noticeable. Both, X 40,000.
core of cytoplasm (Fig. 3). Electron-transparent
areas containing fibrils in mitochondria (arrows,
Figs. 3, 13) are similar in appearance and dimension to those described for the chloroplast. These
areas also have no bounding membrane, and the
fibrils within them again occur as networks of
central coarse (80-120 A) and radiating fine
fibrils of 15-25 A (arrows, Fig. 13). It is not possible to demonstrate whether these small fibrils
penetrate the surrounding matrix and become
closely associated with cristae as described by Nass
et al. (14). The fibrils are sensitive to DNase
treatment (Fig. 10), but resist the extraction of the
control treatment in the same manner as the
chloroplast fibrils (Figs. 11, 12).
DISCUSSION
Evidence from electron microscopic observations
(2, 4, 10, 17) seems to indicate that fibrils with
D N A characteristics can be detected within electron-transparent areas in the chloroplasts of
518
several plant groups. Ris and Plaut (17) have
shown by Feulgen reaction, fluorescence, and
electron microscopy that DNA-containing areas
are found scattered in the vicinity of the pyrenoid
in Chlamydomonas. Sager and Ishida (18) extracted
D N A from Chlamydornonas chloroplasts and coneluded that it may represent the D N A reported by
Ris and Plaut (17). In the red alga Laurencia and
higher plants, similar DNA-containing areas are
found scattered wihin the chloroplast (2, 10).
As the result of DNase treatment and electron
microscopy demonstrated in this report, it appears
that chloroplast D N A of Egregia occurs as 15-25-A
fibrils in the electron-transparent areas similar to
those of Chamydomonas and Laurencia, and the finding of Bouck (4) is confirmed. The chloroplast
D N A fibrils of these plants seem to have a similar
morphology, but, perhaps, the most significant
departure of Egregia and other brown algae from
the other groups of plants lies in the definite distribution of the fibrils within the organelle. There
THE JOURNAL OF CELL BIOLOGY • VOLUME33, 1967
FIGURE 13 Mitochondria within a transcelhflar strand of an outer cortical cell. Arrows indicate D N A
fibrils within the electron-transparent areas. V, vacuole. X 75,000.
T. BISALPUTRA AND A. A. BISALPUTRA Chloroplast and Mitochond~'ial D N A
519
are two DNA-containing areas in each chloroplast
of Egregia, one at each tip of the chloroplast. Previous workers had reported the occurrence of electron-transparent areas aptly termed "vacuoles"
(cf. reference 9) at the tips of chloroplasts of
brown algae, but the presence of the fibrils within
the "vacuoles" was not reported until much later
(4). In Egregia these so called "vacuoles" are, in
fact, the DNA-containing areas described above.
O f particular importance is the definite localization of these D N A areas and their relationship
with the lamellar pocket at the tips of the chloroplasts. This observation, together with the fact
that chloroplasts are self-duplicating organelles,
will allow one to study the replication, if any, and
the subsequent distribution of the D N A areas
during the chloroplast division. This important
process will be described and discussed in a separate report.
Although no quantitative analysis of chloroplast D N A in Egregia has been undertaken, it is
clear by comparing the number and dimension of
D N A areas and the over-all size of chloroplasts
that the amount of D N A per chloroplast or per
volume of chloroplasts in Egregia is less than that
of the red alga Laurencia and perhaps Chlamydomonas.
The DNA-containing areas of mitochondria of
Egregia are similar to those occurring in the same
organelle of other organisms described by Nass
and her coworkers (12-14) and Kislev et al. (10).
F r o m the present work we can conclude, however,
that DNA-containing areas of both chloroplasts
and mitochondria of the same organism appear to
be of similar nature. The dimensions of D N A areas
of both organelles compare favorably well and the
sizes of the D N A fibrils within the containing areas
are similar (15-25 A thick). These fibrils are
smaller than nuclear D N A fibrils, which, in this
instance, may reflect the absence of histones (12,
14, 16). It is well known, however, that there are
considerable differences in the molecular weight,
density, and the composition of base pairs between
chloroplast and mitochondrial DNA, on one hand,
and nuclear DNA, on the other. For instance, in
higher plants chloroplast D N A is much richer in
guanine and cytosine than is nuclear I ) N A (10, 20),
but in Euglena and other alga the reverse is true
(5, 19). Suyama and Bonner (21) have reported
the difference in the density of D N A from mitochondria and nuclei in several plant species. They
also demonstrated that the mitochondrial D N A of
these plants is identical but that the nuclear D N A
shows some variation. Recently, it was shown that
certain mitochondrial D N A has a ring structure
(22). In general, evidence from qualitative analysis
and electron microscopy seems to indicate, therefore, that chloroplast D N A and mitochondrial
D N A tend to resemble bacterial and, perhaps,
blue-green algal D N A more than they do nuclear
DNA.
This work was supported in part by National Research Council of Canada (Grant A-2288).
Received for publication 4 October 1966.
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T H E JOURNAL OF CELL BIOLOGY • VOLUME 33, 1967