BIOGENESIS OF CHLOROPLAST MEMBRANES I. Plastid Dedifferentiation in a Dark-Grown Algal ...

32 Pages·2003·2.76 MB·English

BIOGENESIS OF CHLOROPLAST MEMBRANES
I. Plastid Dedifferentiation in a Dark-Grown
Algal Mutant (Chlamydomonas reinhardi)
I. OHAD, P. SIEKEVITZ, and G. E. PALADE
From The Rockefeller University, New York 1001. Dr. Ohad's present address is the Department D
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ABSTRACT m h
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This paper describes the morphology and photosynthetic activity of a mutant of Chlamy- ://ru
domonas reinhardi (y-l) which is unable to synthesize chlorophyll in the dark. When grown pre
heterotrophically in the light, the mutant is indistinguishable from the wild type Chlamy- ss.o
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domonas. When grown in the dark, chlorophyll is diluted through cell division and the /jcb
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zymes associated with the photosynthetic process (alkaline FDPase, NADP-linked G-3-P df/3
dehydrogenase, RuDP carboxylase), as well as cytochrome f and ferredoxin, continue to 5/3
be present in relatively high concentrations. The enzymes involved in the synthesis of the /52
1
characteristic lipids of the chloroplast (including mono- and digalactoside glycerides, /12
6
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phosphatidyl glycerol, and sulfolipid) are still detectable in dark-grown cells. Such cells 11
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accumulate large amounts of starch granules in their plastids. On onset of illumination, /5
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dark-grown cells synthesize chlorophyll rapidly, utilizing their starch reserve in the process. .p
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At the morphological level, it was observed that during growth in the dark the chloroplast f b
lamellar system is gradually disorganized and drastically decreased in extent, while other y gu
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subchloroplast components are either unaffected (pyrenoid and its tubular system, matrix) st o
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or much less affected (eyespot, ribosomes). It is concluded that the dark-grown mutant 02
possesses a partially differentiated plastid and the enzymic apparatus necessary for the Jan
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INTRODUCTION
Since membranes are currently recognized as 15), but these organs represent already complex
"elementary structures" extensively used in the structures in which membranes are only one of a
construction of a large number of cell organs, series of components (16-19). Moreover, much of
membrane biogenesis should be considered a basic the recent literature (cf. (20, 21) for review) deals
process that underlies the differentiation, growth, with local (satellite) DNA's and their possible
and replication of many cell organs and of the cell function as autonomous or auxiliary genomes. Rel-
itself. Some information is already available on the evant to this paper is the discovery of satellite
biogenesis-and its regulation-of whole cell DNA's in Chlamvdomonas (22-25). Information
organs, such as mitochondria and chloroplasts (I- directly concerning operative and regulatory proc-
521
esses in membrane biogenesis is now needed in crease the membrane content of their chloroplasts;
order to understand a basic step in biological or- all this happens without cell division. The struc-
ganization: the transition from molecular to sub- tural and enzymic modulations that occur in the
cellular and cellular structures. chloroplast of the mutant during "degreening,"
The study of membrane biogenesis would be i.e. transition from the light-grown photosynthetic,
facilitated by the availability of a system with the to the dark-grown nonphotosynthetic state, form
following characteristics: (1) synthesis of mem- the subject of this paper. The analysis was carried
branes at such rates as to allow adequate sampling out at the level of the whole cell with the intent to
time, and in quantities and configurations such as continue it at the chloroplast and chloroplast disc
to permit isolation for biochemical analysis during level in subsequent steps.
the formative stages; (2) easy identification of the The results of experiments dealing with the re-
new cellular structures; (3) the membranes should verse process ("greening"), i.e. the formation of
have a distinct biochemical activity easily recog- the chloroplast disc system and reappearance of
nizable against the background of the metabolic photosynthetic activity, will be reported in the
activity of the cell and stable enough to permit accompanying paper.
assay in vitro after isolation; (4) the machinery D
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necessary for the synthesis of membrane compo- MATERIALS AND METHODS wn
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nents should be present in a latent state and be Description of Mutant ade
aucptoivna taecdt ivbayti oan ,s imthpel es ypshteymsi oslohgoiucladl pstriemfeurleunst;i al(l5y) Chlamydomonas reinhardi wild type and y-1 mutant d from
synthesize membrane components rather than in- were kindly supplied by Dr. R. Sager, Department of http
crease all cellular synthetic activities. Biological Sciences, Hunter College, New York. ://ru
degTrheeesse byid eeatilo lraeteqdu irpelmanetn tles avaerse omr aeltg ateo ivna wriahbiclhe mis uTltoahwtei,o ntmh. eu Atraelntvhte orusgigeohnn e ,t rhaeyt e- lm,b uaatcarkoti soetno ayrsa- lt+ea ysis-p l+ohni gttahon, eyoa-unlsd- press.org
the formation of chloroplast membranes is induced stocks must be cloned regularly to maintain the /jcb/a
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absence of cell division, of photosynthetic mem- segregation in crosses, as expected for a unit factor f/3
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brane components and their assembly into mor- (26). However, it is not yet established whether the /3/5
phoTlhoeg icaaimlly oaf ntdh isb iwocohrkem isi catoll yc hdairsatcintecrti zuen istsu.ch a loorc antoionnch roofm thoes ogmeanle. wSoitmhien gtehnee ticce lld aist a chsurogmgeosst oimt atol 21/126
be nonchromosomal (27, 28), such as its response to 31
system using a dark-grown mutant of Chlamv- 10
streptomycin as a mutagen. Nonetheless, its pattern of /5
domonas reinhardi, a unicellular alga with a single 2
large p]astid, as a generator of chloroplast mem- unit-factor inheritance is different from the maternal 1.pd
inheritance found in the more than 40 different f b
branes when exposed to light. In wild type Chlamy- nonchromosomal genes so far identified in Chlamy- y g
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dcohmloornoapsh, ytlhle inc ethlles dhaarvke ast hwe elal baisl iitny thtoe lsigyhntth. eIst izise, dcohmemoniacsa l( 2l9e)s.i oTnh ienrveo lavreed oinnl yth fee ws indgaltea- gaesn et om thueta tbioion- est on
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therefore, assumed that they, unlike Angiosperms of y-l; that available suggest a block between proto- 2 Ja
among the higher plants and certain species of chlorophyll(ide) and chlorophyll(ide) (30), but a nu
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Euglena and Ochromonas, contain the entire enzy- block at an earlier point is not excluded. ry 2
Mutant cells grown in the dark or light will be 0
matic equipment necessary for this synthesis. The 23
referred to hereafter as chyd cells and chyl cells, re-
mutant in question apparently lacks part of this
spectively.
equipment, for if placed in the dark it will continue
to grow and multiply, so that each cell will progres- Culture of Algae
sively lose its initial chlorophyll content. As will be The cells were grown in a medium which is similar
shown below, this occurs by simple dilution to that described by Sager and Granick (31) and has
through cell division of the chlorophyll originally the following composition: 1000-ml solution A (Na
synthesized in the light. Concomitantly, the discs citrate, 1.7 X 10-3 M; Na acetate, 7.5 X 10-3 M;
KHPO , 5.7 X 10-4 M; KH PO , 7.4 X 10-4 M;
of the chloroplasts disappear, though the chloro- 2 4 2 4
NH NO , 3.7 X 10- 3 M; MgSO4, 1.2 X 10-3 M;
plast itself persists as a distinctive structure. Upon CaC41 , 33.6 X 10- 4M; FeCI3, 3.7 X 10-5M) supple-
2
reexposure to light, these yellow cells will synthe- mented with 1-ml solution B (a trace-metal solution
size chlorophyll, by a light-activated step, and in- consisting of (mg/100 ml): H3B03, 100; ZnSO4.7
522 THE JOURNAL OF CELL BIOLOGY -VOLUME 35, 1967
H 0, 100; MnSO .H 0, 40;CoC12-6 H 0, 20; described above, so as to bring the cell concentration,
2 4 2 2
Na2MO4.2 H20, 20; CuSO45 H20, 6.25). A five- after replenishing with fresh medium, to the original
times concentrated stock solution of medium A could value of -0.8 X 106 cells/mil. Under these conditions,
be kept at 4 for 1-2 wk, the trace metals B being the generation time in the dark was approximately 22
added only before autoclaving, which was carried out hr as compared with 18-20 hr in the logarithmic
at 15 psi for 20 min for volumes up to 2 1, and for 40 phase of growth. Three culture flasks were used for
min for volumes of 2-15 1. each experiment. Since variation in chlorophyll con-
Stock cultures were kept on agar slants prepared by tent and cell count did not exceed 10% from one
adding 2% agar (Difco) to the above medium. These flask to another, the cells harvested from all three
stock cultures were maintained by transferring to flasks were pooled for enzymic and morphological
fresh agar medium every 4-6 days. analysis.
All liquid culture incubations were at 250 either in
The Greening Process
the dark or light (-700 ftc) in a photosynthetic
incubator (New Brunswick incubator shaker, model This was followed by incubating chyd cells in the
G-27) operated at one shaking cycle/second and culture medium at final concentration ranging from
provided with white fluorescent lamps. 1 X 106 to 2 X 107 cells/mi. The ratio, surface-to-
Mass liquid cultures of chyd cells were initiated by volume, in the incubation vessel was about 0.5-0.7 D
inoculating a loop of cells from a 2- to 4-day old cmrl. Illumination was provided by white fluorescent ow
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agar-slant culture into an Erlenmeyer flask containing lamps supplying about 700 ftc at the level of the cell loa
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100 ml of liquid medium and allowing growth in the suspension. ed
light for two to three generations (usually overnight). Bacterial contamination was checked by examining fro
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Fernbach flasks containing 2 1 of medium were at various time intervals in the phase-contrast micro- h
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through a sterilized cotton filter, and the flasks were for 2 min in the cold, washed with fresh medium /jcb
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(chyl cells), a sterile semicontinuous culture apparatus The number of cells in the sample was estimated by df/3
was used consisting of a reservoir (15 1) and a culture counting in a hemocytometer. 5/3
flask equipped with a ground-glass inlet to allow During the preparation steps, chyd cells were oc- /52
1
replenishing with fresh medium and a ground-glass casionally exposed to dim artificial or natural light /12
6
outlet at the bottom to drain periodically the contents for periods of <Z15 min in the cold. Special care to 31
1
of the vessel (Fig. 1). The initial inoculation was with avoid such exposure was unnecessary, since chilled 0/5
two to three loops of cells grown in the light on agar cells kept in the light for up to 1 hr showed neither 21
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slants. For logarithmic growth, the culture was increase in the amount of chlorophyll, nor changes in df b
harvested every 24 hr by leaving in the growing flask their morphology as compared with nonexposed cells. y g
only 100-200 ml of the old culture and replenishing ue
the volume with 2 1 of fresh medium. By this method, Preparationo f Homogenates st o
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tion of 2 X 10scells/ml in a French press (32) fitted 3
The Degreening Process
with an oil pump and accessory valve (Blackhawk
This was followed by growing the cells in the Industrial Products Co., Butler, Wis.) designed to
semicontinuous culture apparatus described above. insure a constant pressure (5000 psi) throughout the
The cells were first grown in continuous light (about process. Under these conditions, all the cells and
700 ftc) up to a concentration of - 1.3 X 106 cells/mil practically all the chloroplasts were completely frag-
then half the volume was drained (1.25 1) and used mented, as ascertained by light and electron micros-
for enzyme assays and morphological studies. The copy.
volume of the culture was brought up to 2.5 1 with
Enzyme Assays
fresh medium (final cell concentration -0.8 X 106
cells/ml), and the incubation continued in the dark All the activities or concentrations are expressed on
for the rest of the experiment. Every 22-24 hr, an a per cell basis, rather than on a protein basis, since
adequate volume of the culture was withdrawn, as the protein concentrations vary during the greening
OHAD, SIEKEVITZ, AND PALADE Biogenesis of Chloroplast Membranes. 1 523
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FIGURE 1 Apparatus for semicontinuous culture of algal cells. The reservoir (R) was connected through Ja
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the ground-glass sterile joints (sj) with the inlet (I) on the culture flask (CF). This was done by removing a
cover cl from plug pi, inserting plug pi into inlet c2 and then opening the clamp (ell). Bubbling of C0 -air ry 2
2 02
mixture, sterilized by passage through the cotton filter F and dispersed by the sintered glass filter (g), 3
1
provided positive pressure in the culture flask preventing contamination when inlet (I) was open. Air
escapes through filter F2. Drainage of culture was carried out through outlet c3 and clamp C12. An enlarged
view of the ground-glass connection tube is shown in Fig. 1 a. The long neck (n) of plugs pl and p3 allow
passage of liquid without wetting the ground joints, thus ensuring sterility. The rubber connections (rl and
r2) were of latex surgical tubing which can withstand autoclaving three to five times. All the ground-glass
joints were of pyrex glass. The joints were sterilized by glowing over a Bunsen flame each time the covers
were removed.
process, but cell number remains invariant. Respira- The cells were resuspended in the growing medium at
tion and oxygen evolution were measured in a a concentration of 108 cells/ml, and 02 uptake was
Warburg apparatus with the use of incandescent followed in the dark and light in the presence of 10 N
lamps providing 500 ftc at the level of the vessels. KOH in the center well. For oxygen evolution
524 THE JOURNAL OF CELL BIOLOGY VOLUME 35, 1967
measurements, the cells were resuspended in a 1:1 deep waterbath; illumination was for 30-sec intervals,
mixture of 0.07 M KHCO3 and 0.11 M NaHCO3, with readings in the dark at 340 myp; a dark-blank of
pH 8.4, and saturated with CO bybubbling a mixture similar composition but covered with aluminum foil
2
of 5% CO in air (33). The specific rate of oxygen was always run, and the readings subtracted from
2
evolution (mole/min/108 cells) was dependent on those of the light-activated samples. There was no
cell concentration in the range of 2 X 108 to 2 X 107 reaction in the light without the added ferredoxin.
cells per ml (or 6 X 107 cells per flask). This was The source of the ferredoxin was the dialyzed extract
probably because of insufficient illumination at high of the acetone precipitate prepared from spinach as
cell concentrations. Measurements were, therefore, described by San Pietro and Lang (39). In measuring
performed at those concentrations at which the the appearance of NADP photoreduction during
response was proportional to cell concentration, and greening, the above assay conditions were used with
extrapolated to 108 cells per flask. equal amounts of spinach ferredoxin extract added at
Succinic dehydrogenase was measured in homoge- all time points. In the assay for ferredoxin activity in
nates, as described by Bernath and Singer (34). Chlamydomonas, a similar extraction (39) was per-
The alkaline FDPasej was assayed as described by formed on packed green or yellow cells and the
Racker and Schroeder (35) and Smillie (36) in a extract assayed as described above, with a dilute
system containing in 1.0 ml final volume: 10 /moles homogenate of green cells as a source of the light-
D
fructose 1-6 diphosphate; 10 pUmoles MgC12; 3 activated reaction. In this manner, a quantitative ow
moles EDTA; 100 moles Tris buffer, pH 8.8; and measure of ferredoxin activity was made by measuring nlo
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the requisite amount of homogenate. The reaction the rate of NADP photoreduction catalyzed by an de
was carried out at room temperature (26-28°) for extract from 109 chyl or chyd cells. Rates in both d fro
30 min and stopped by adding TCA to a final con- assays were calculated from the linear parts of the m h
centration of 10%. Inorganic phosphate in the curves, and all activities were expressed as moles ttp
acid-soluble supernatant was determined by the NADP reduced. ://ru
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sayed as described by Smillie and Fuller (38) in 3.0 volume containing 12 moles of Tris buffer, pH 8.0, /jcb
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of homogenate. The reaction was run at room tem- were used in the comparison of light-grown and /52
perature in a Beckman DU spectrophotometer with dark-grown cells, in order to overcome somewhat the 1/12
a Gilford recording attachment by first preincubating radioactivity dilution effect by different amounts of 631
aaldl diinnggr etdhiee nstsu besxtrcaetpet. sOubpstitcraatle dfeonr si1t0y mchina,n gaensd wtheerne nonradioactive CO2 in the two cases. Reactions were 10/52
read at 340 m/; the rate was calculated from the run at room temperature for 5 and 10 min, with 1.pd
laisn ethaer pexatritn cotfi otnh ec oceufrfviec,i enwt itfho r 6N.2A2 DXP H1.06/cm2/mole vlaacrkyiinngg saumbsotruanttes. oAft hthoem oegnedn aotfe thaen di nwcuitbha tiao nb,l a1n.k5 f by gu
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The photoreduction of NADP (39) was carried out ml of boiling 70% alcohol were added, along with st o
by incubating at room temperature, in a Beckman two drops of glacial acetic acid; the reaction vessels n 0
2
DU spectrophotometer cuvette: 30 moless of Tris were heated for I min in a boiling water bath, cooled, Ja
buffer, pH 7.5, 1 mole of MgC12, 0.2 mole of and aliquots were plated for counting. Linear rates nua
NADP, a spinach extract (1I mg protein) containing were obtained with time and tissue concentration ry 2
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ferredoxin, and the requisite amount of homogenate after correcting for zero time control and a blank 23
in a final volume of 3.0 ml. The light source was an without substrate.
incandescent lamp (700 ftc) placed before a 10-cm The Hill reaction was measured according to James
and Das (41) in a system containing 30 pumoles of
1The following abbreviations were used in this
phosphate buffer, pH 6.6, 17 Mmoles of KCI, 0.1
paper: FDPase, fructose- 1,6-diphosphatase; RuDP,
molee of DCI, and different amounts of homogenate
ribulose-l ,5-diphosphate; RuDP carboxylase, ribu-
in a final volume of 3.0 ml. Light was shown as above
lose-l,5-diphosphate carboxylase; DCI, dichloro-
in 30-sec intervals and the change in OD at 600 mg
phenol indophenol; ftc, foot candle; ER, endoplasmic
reticulum; PPN reductase, photosynthetic pyridine measured with a Zeiss spectrophotometer; a dark
nucleotide reductase; EDTA, ethylenediaininetetra- control and a control with o-phenanthroline (0.4
acetate; G-3-P, glyceraldehyde-3-phosphate; TCA, /mole) were always run. The rates were linear with
trichloroacetic acid; NADP, nicotinamide adenine time and homogenate concentration after subtracting
dinucleotide phosphate. the values of the dark control.
OHAD, SIEKEVITZ, AND PALADE Biogenesis of Chloroplast Membranes. I 525
Chemical Determinations chromatography of nondeacylated lipids was carried
out on commercial silicic acid-impregnated paper
Total chlorophyll was measured in 80% acetone
(Whatman, No. SG 81) with the use of the solvent
extracts of the harvested cells or homogenates as
systems described by Marinetti (47). The nondeacyl-
described by Arnon (42).
ated phospholipids and sulfolipids were located by
Cytochrome f was measured by the method of
using extracts of cells preincubated in the light either
Chance (43) as described by Hill and Bonner (44, with 32po or 35SO4 and comparing the radio-
45), using a split beam or double beam spectropho-
autograms with those of lipids extracted from 4C-
tometer, and recording in liquid nitrogen or at room
labeled cells. There was only one spot on the 5S-
temperature the difference spectra (ox/red) of 80%
labeled radioautogram, presumably the sulfolipid
acetone-extracted cells suspended in 0.1 M Na-
described by Benson et al. (48). The individual
phosphate buffer, pH 7.4.2
phospholipids were tentatively identified by their
Total lipids were extracted from the whole cells
location (46, 47). Monogalactosylglyceride distearate
and chromatographed after deacylation as described
(kindly supplied by Dr. M. Kates, National Research
by Wintermans (46). The resolved deacylated mono-
Council, Ottawa, Canada) served as a marker for the
and digalactosylglycerides were identified with the
monogalactosyl lipid. There were only two spots
aid of purified radioactive markers (kindly donated by
staining with the Schiff reagent, one identified with D
Dr. A. A. Benson, Scripps Institute, La Jolla, Calif.) o
the monogalactosyl marker, and the other corre- w
on radioautograms of the chromatograms, and by sponding to the position of the digalactosyl lipid (46). nloa
staining with the Schiff reagent.3 Two-dimensional The pattern of the chromatograms was highly re- ded
producible and corresponded well with that of plant fro
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2 We are very thankful to Dr. Walter D. Bonner, lipids shown by Marinetti (47). h
Jpoehrfnosromni nFgo utnhdeaseti ond,e teUrnmivinearstiiotyn so, f Panedn nsiynltvearnpirae,t infogr by Inthceo raplograaet iionn st heo fd a3r2kp o0o,r lig3h5tS Ow4e, re ocra rr1ie4dC -aocuett abtye ttp://rup
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t3h Ae rmesoudltisf.ication of Schiff reaction was kindly sup- tThhee sarmadei oparcotciveed urme ataesr ifaolr t(haem goruenetn inngo teedx peinri mfeignutsre. ss.org
plied by Dr. M. Kates, National Research Council, legends) was added to the incubation medium at the /jcb
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Ottawa, Canada. zero time of the experiments. Analyses for protein and rticle
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All micrographs show sections of Chlamydomonas reinhardi cells fixed in 2% 0s04 in 263
0.1 M phosphate buffer (pH 7.2), embedded in Epon, and sectioned at Z 500 A with 11
0
diamond knives. The sections were doubly stained with U02 acetate and Pb citrate and /52
1
micrographed in a Siemens Elmiskop I. .p
d
f b
General Abbreviations y g
u
e
ce, chloroplast envelope; cm, chloroplast matrix; cr, chloroplast ribosomes; d, disc; db, st o
n
bent or folded over disc; dc, dictyosome; m, mitochondrion; mb, cell membrane; n, nucleus; 0
2
ne, nuclear envelope; no, nucleolus; o, osmiophilic globule; p, pyrenoid; r, cytoplasmic Ja
n
ribosomes; rer, rough-surfaced endoplasmic reticulum; sg, starch granules; sp, starch plates; ua
t, pyrenoid tubules; v, vacuole; w, cell wall. ry 2
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2
3
FIGURIE 2 General morphology of a mutant cell grown in the light (chyl) in batch type
culture. The figure shows the profile of a medially sectioned cell whose cell wall is marked
w and cell membrane mb. The cup shape of the chloroplast and the location of the pyren-
oid (p) within the thickened posterior part of the plastid are clearly shown. The chloro-
plast is bounded by chloroplast envelope (ce) (see Fig. 5 for further detail) and contains
many grana (g), i.e., stacks of fused discs connected by one or more free or fused discs.
Osmiophilic globules (o) and a few starch granules (sg) are scattered among the grana.
The pyrenoid (p) appears as a large, finely granular mass of polygonal profile surrounded
by discontinuous shell of starch plates (sp) and penetrated by a system of tubules (t).
Within the chloroplast cup are located the nucleus (n), dictyosomes (de) and their associ-
ated vacuoles (v), and endoplasmic reticuluml cisternae of transitional type (tc). Mitochon-
dlia (m) are concentrated at the anterior pole of the cell and between the chloroplast and
the cell membrane. A contractile vacuole is Ilalrked cv and a flagellum fl. X 20,000.
526 THE JOURNAL OF CELL BIOLOGY VOLUME 5, 1967
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OlRAD, SIEKEVITZ, AND PALADE Biogenesis of Chloroplast Membranes. 527
lipid were carried out as described in the following Preparation of Specimens
paper (49).
for Electron Microscopy
Insoluble starch was measured in the residue after
acetone extraction of whole cells. The residue was Whole cells were fixed in 2% OsO04 in 0.1 M
washed twice with 2% Na2CO3, and recovered each phosphate buffer, pH 7.4, or in 2% glutaraldehyde in
time by centrifuging at 3000 g, 5 min. Since the cell 0.2 M cacodylate buffer, pH 7.4, containing 2 X 10-3
wall was not disrupted by this procedure, all starch M CaCI or MgCI .Fixation was at 40 for overnight.
2 2
granules were retained inside the sac formed by the Specimens fixed in the last fixative were washed and
cell wall, thereby allowing good recovery at low-speed postfixed overnight in 2% Os04 in the same buffer.
centrifugation. The sediment was hydrolyzed in N Some of the specimens were stained in block with
H2SO4 at 100° for 10 min and the amount of total 0.5% uranyl acetate in acetate Veronal buffer, pH
sugar was determined in the 3000-g, 5-min supernate 6.0, before dehydration.
of the hydrolysate by using the phenol-sulfuric acid After fixation, specimens were dehydrated in a
reagent of Dubois et al. (50). The same values were series of graded alcohols and finally embedded in
obtained if hydrolysis was carried out at pH 2, for Epon as in Luft's procedure (52). Sections were cut
20 min. Since the cellulosic cell wall is not signifi- with a Porter-Blum Servall MT2 microtome using a
cantly affected by N H2SO4 even after more prolonged diamond knife (DuPont de Nemours, Wilmington, Do
exposure, H2SO4 was consistently used to ensure Del.), and were mounted on 200-mesh grids coated wn
complete dissolution of the starch. Starch granules with a formvar film or on naked 400-mesh grids. The load
wpreerpea riesdo lainte d0 .0f4o rM i Tderinst ifbiucfafteior,n pHfro m7. 4, haotm 5o0g0e0n aptesis. sceictrtaioten,s aws erdee ssctraiibneedd wbyi thR euyrannoyldl s ac(e5t3a)t,e aanndd tlheeand ed from
The homogenate, centrifuged at 15,000 g for 20 min, covered with a thin film of carbon. A Siemens http
yielded a hard white pellet overlaid by a fluffy Elmiskop I was used at magnifications of 6,000 to ://ru
pigment-rich layer. The latter was easily removed by p
60,000. re
gentle pipetting with 2% NaCO3. The white pellet The amount of membranes forming the lamellar ss.o
wasa sa bthovene, reasnuds pefinndaelldy ienx 2tr%ac teNda 2CwOith3 , 8w0%as headc ettwonicee. system of the chloroplast, and the incidence of fusion rg/jcb
lines of grana were estimated from electron micro- /a
Trimedhfeirnena ,tc ritef1iisl0eei0dd °u g)ea r swa wnhsautisalc erhacs hwy oihebf ilytdve ea darpyc oiigdnwl gud hceoysrsid zecre oo nlay(ns0sidsi.s3 t i-tn2raga t c oepfs)H .s ot Irfto2 mn wg(a1laly0s- gdmeraiscpcrrhoisbg eradwp ihtbhsy th(Lmeo auugdsne i f(io5cf4a )t.ai o npRsra,o ncde3od0mu,0rl0ey0 stitamok iel4na0r ,e0tl0oe0 c)tt rhoaontf rticle-pdf/35/3
tose and higher oligosaccharides, or by a-amylase sections including comparable amounts of the chloro- /52
1
(hog pancreas, Worthington) hydrolysis which gave plant area were scanned with a grid having a spacing /12
maltose and small amounts of glucose. The identifica- of 1 cm. The scanning was done on prints enlarged 631
1
tion of the hydrolysis products was made by descend- to X 100,000. The membrane content index is 0/5
ing paper chromatography in N-propanol-ethylace- defined as the number of intersections of membrane 21
.p
tate-water (7/3/1 by volume) with a benzidine profiles or grana fusion lines with the grid lines df b
reagent for detection (51). divided by the surface of the chloroplast area scanned. y g
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FIGURE 3 Mutant cell grown in the light in batch type culture. Section cutting through ry 2
the center of the bottom of the chloroplast cup. The chloroplast is bounded by a chloro- 02
3
plast envelope (ce) and is occupied by numerous grana, each formed hy a pile of fused discs.
Most grana are perpendicularly sectioned (g); a few appear in oblique (gl) and grazing
(92) sections. The latter show a full-faced view of the large festooned discs and show clearly
the irregularly thickened disc rims (arrows). Such thickened rims appear as dense dots at
the end of many normally sectioned discs throughout the chloroplast. In addition to those
fused into grana, a few free or intergranar discs are present. The space among the grana is
occupied by a relatively tight matrix (stroma) which contains osmiophilic globules (o),
starch granules (sg), and numerous ribosomes. (The latter are hardly visible at this magni-
fication; see, however, Figs. 10 and 11.)
The pyrenoid (p) occupies the center of the chloroplast bottom and consists of a finely
granular core surrounded by a discontinuous shell of starch plates (sp) and penetrated by
a network of tubules (t). The profile marked cl is part of the irregularly deeply cut rim of
the chloroplast cup. X 27,000.
528 THE JOURNAL OF CELL BIOLOGY ·VOLUME 35, 1967
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OIIAD, SIEKEVITZ, AND) ALADE Biogenesis of Chloroplast Membrane. I 529
An average of 1700 cm2 was scanned at each time The cell is contained within a cell wall of vary-
point. ing thickness (0.1-0.2 ,) and fibrillar text ure (Figs.
2 and 3) which in normal sections can be resolved
RESULTS into as many as six to ten layers of fibrils. In each
layer, the fibrils are oriented parallel to one
Growth Characteristics of Light-Grown and
another, but from one layer to the next their ori-
Dark-Grown Cells
entation changes by a large angle. The thickness
The generation time of both wild type and mu- of the fibrils is -30 A, as expected for single cellu-
tant cells was found to be 18-20 hr when grown in lose elementary fibrils (55). The space between
the dark and 6 hr when grown in continuous adjacent fibril layers appears to be "empty" (i.e.,
light. Logarithmic growth was maintained in all occupied only by embedding plastic). Whether
cultures up to concentrations of -2 X 106 cells/ this free space is present also in vivo, or is the result
ml. The concentration of chlorophyll in chyl cells of extraction of soluble components during speci-
in logarithmic growth was 18-20 pg/107 cells, in- men preparation, is not known.
creasing in the stationary phase to 30-50 MUg/017 The cell membrane consists of a single unit mem-
cells. The chlorophyll content of chyd cells was brane ~100 A in thickness. Its outer dense leaflet Do
<0.3 pug/107 cells after seven to nine generations is usually thicker and more intensely stained than wnlo
of growth in the dark. the inner dense leaflet (Figs. 3 and 7). The cell ade
d
When attempts were made to grow chyd cells fro
m
in liquid semicontinuous culture, it was found that 5 h
ocfatmene tahpe pacruelntutr ea fdteerte r1io4 rattoe d 18a ngde nceerlalt iodnesa.t h Tbhee- 2 ttp://rup
- Short axis re
cells of the original culture were sometimes slowly ----- Long axis ss.o
irse palbalcee dto bsyy wnthhaets izsee emchs lotor obpeh ya lbl aicnk tmheu tdaanrtk ,w hthicuhs 2 rg/jcb/a
being similar to the wild type. Green colonies Chloroplast rticle
were observed also in cultures grown in the dark %It -pd
on agar plates whenever these cultures were not f/35
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transferred to new medium within 2-3 wk. The /5
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1
back mutant, however, does not accumulate in I - /1
2
light-grown liquid cultures during as many as 50 63
1
1
to 60 generations, as tested by inoculating cells 0/5
from such cultures on agar slants or liquid cultures I_ 21
and allowing growth in the dark. i .pdf b
Cytoplasm y g
Morphology of the Mutant Grown in Light ', I ue
and Dark st on
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Mutant cell populations during the logarithmic Jan
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2
microscopes. When grown in the light, the cells I: 3
were ellipsoidal with a short axis of 5-8 pu and a I
long axis of 8-12 pt; cells grown in the dark were 150 200 250 300 350 400 A
more rounded. Both chyl and chyd cells were FIGURE 4 Size distribution of cytoplasmic ribosomes
motile and did not form clumps or aggregates. and of ribosome-like particles in chloroplasts of light-
grown mutants. The particles are slightly elongated.
At the electron microscopic level, the morphol-
Full line, short axis; dotted line, long axis. A, chloro-
ogy of chyl cells was, in general, similar to that of
plast particles; B, cytoplasmic ribosomes. Measure-
chyd cells except for the lamellar system of the
ments were made with a magnifier (X 10) on micro-
chloroplast and its starch content. In the following
graphs taken at 40,000 electron magnification, enlarged
description, the structure of both light- and dark- photographically to 100,000. The same cell sections
grown cells would be considered together except were used for measurement of both kinds of particles.
for the intrachloroplast membranes. The data were collected from a total of ten micrographs.
530 THE JOURNAL OF CELL BIOLOGY VOLUME 35, 1967