Studies of Chloroplast Development in Euglena. V. Pigment
Biosynthesis, Photosynthetic Oxygen Evolution and Carbon
Dioxide Fixation during Chloroplast Development 2,2
Arthur I. Stern,3 Jerome A. Schiff, and H. T. Epstein
Department of Biology, Brandeis University, Waltham, Massachusetts
Our studlies wvith Euglena (2, 6, 14, 19, 20) have
suggestedl that dark-grown cells containi approximately 30 proplastids whiclh develop into about 10
chloroplasts xvhen the cells are exposed to light. The
existence of these proplastids, originally inferred
from studlies on ultraviolet inactivation ain(l photoreactivation of chloroplast formiiation (14, 19). was
confirimie(I by fluorescence ani(I electron miiicroscopy
(2, 6). Other workers have stu(lie(l some of the initial steps invTolved wvhen dlark-groxvn cells are exposed
to light, suclh as the conversion of protochlorophyll to
chlorophyll (17). Other studies also exist of the related problemii of the physiology of chloroplast development in highler plants (3, 7, 23, 27).
Our earlier vork (20) also showed that chloroplast development in Euglena could be separated experimentally into 2 phases: replication of the system
which nmanufactures the chloroplast ancI development
of the proplastid into the mature chloroplast. In this
study x-e correlate the onset and kinetics of pigment
formlation., O. evolution and COi. fixation with the
developmilenit of the proplastid inito the mature chloroplast.
Materials and Methods
GroTl/t of Euglenia. Euigleina gracilis var. bacillaris Prinlgslheim (14) xv-as maintained aseptically in
the (lark at 250, for 1 year in 250-ml Erlenmeyer
flasks containiing 100 ml of Hutner's meclium pH 3.5
(10) xvith 5-ml transfers every 3 days. The starting
dark-grown stock maintainedl in
culture wN-as
laboratory for over 2 years (14). The imiiportance of
prolonged dark groxvtlh to entirely deplete chlorophyll
and chloroplast structures has been repeatedly emlphasized (14, 17); our dark-grown cells contain protochlorophyll ancI proplastids and lack chloroplasts and
chlorophyll completely. All manipulations xvere carried out under a green safelight in a (larkroolmi as
described I)reviously (14).
a
our
Received revised manuscript July 15, 1963.
This work was supported in part by research grant
RG-6344 from the Division of Research Graiits, Public
Health Service. The data are taken from a dissertation
submitted to the graduate faculty of Brandeis University
by A. I. Stern in partial fulfilment of the requirements for
the PhD degree.
3During the tenure of this xvork A. I. Stern wvas supported by a training grant in Developmenital Biology,
2G-883, from the National Institutes of Healtlh.
1
2
All light-grown cultures of wild type an(Ci mlutailt
cells with the exception of Y,BXD were grown as
lescribedI previously (14). Y,BXD is a slow growilng mlutant witlh a rather higlh reversioni rate wNhich
is grown under al)proximatelv 100 ft-c of lighlt for 3
to 4 weeks on the surface of pH 3.5 in3e(liumii solidifie(d
with agar containie(l in lhorizonltal Roux bottles.
Nor experimiienits with nondividing cells, a restillg
e(liumi was deevise(d containing (0.054-.t) mlannitol,
(0.01h[) MIgCl, an(d (0.01M\) KH,PO, per liter. It
lhas been shown that miiaininitol is not utilized as a carbon source for grow tli hv1 Euglenia (S. H. Hutner,
personial communication ) and(lwhile cells suspendle(d
in tlis miiediunm (loi)ot (livide, thev retain their viabilitv for long perio(ds of timie (75%,c after 30 days.)
FXor the (lescribe(d experinments 10 mlil of a 3-day ol(d
lark-grown culture were initro(luced inlto 1 liter of
growth minediunm or 200 nil -xvere ititro(luced into 500
ml of resting miie(liuml anlel these wvere placed on a
rotary shaker in tlle (lark for 70 lhours after whiclh
the cultures containe(d about 106 cells per ml, and the
cells in resting mie(diumii lhad ceasecl to divide. Tenml aliquots were tlhen transferre(d aseptically inlto
10-cmii sterile glass petri (lishes whiclh were then ex1ose(l to 100 ft-c, the optimal intensity for chloroplast
(levelopment (24), provided by a fixed light source
comp)osed of 20-w GE w-hite fluorescent lamps and(l
measure(I by meanis of a \Veston Illuminiation Meter
mo(lel 756. At appropriate tinmes, samlples were withdrawn for cell counlts, the cells wvere harvestedl by
centrifugation at 1000 X g for 10 imiinutes and xvere
x-aslhedI once with 0.1.m bicarbonate buffer (0.065.r
NaHCO3 an(l 0.035'm KHCO,) pH 8.2 (18). This
is referred to as "bicarbonate buffer! throughlout the
text.
02 Determiiixzatioaw. To mieasure °2 evolutioin, 10
to 20 X 106 cells vere suspend(led in a total of 3 nml of
0.1 a bicarbonate buffer in \Warburg vessels. 'Measuremlenits xvere carriedl out in a glass bottolmi Amiinco
Illuminiated \N'arburg apparatus at 260 xwith 150-\\x
Sylvania Projector Spotlamps as the light source.
I)uplicate flasks xvere used in all experimlents, one
l)eiig wrapped in alumiiinumii foil to serve as the (lark
control in order to correct for respiration. All flasks
were flushed withi 5%c/ CO2 in air for 15 mlilnutes in
the (lark and were theln subjected to illuminationl of
2500 ft-c xhich xvas found to be saturating for photosynithetic 0° evolution at all dexvelopmental stages.
220
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221
STERN ET AL.-PLASTID DEVELOPMENT, PHOTOSYNTHETIC PARAMETERS
In most experiments, measurements were taken over
the course of 2 hours but in computing the rates only
the initial slopes over 30 minutes were taken to minimize errors due to additional development of the
chloroplasts under the high light intensities.
CO2 Fixationt. In experiments where incorporation of C1402 was measured concomitantly with 02
evolution, the following modifications were introduced. After flushing the flasks containing 2.5 ml of
cell suspension in 0.1AO bicarbonate buffer, 0.1 ml of
radioactive bicarbonate solution was introduced into
the side-arnm of the flasks by attaching a short piece
of rubber tubing to the vent and drawing a negative
pressure on the manometer while the solution was
introduced from a pipette into the tubing (M. Gibbs,
personal conmmunication). In the same manner, the
radioactive solution was rinsed into the side arm with
a total of 0.4 ml of 0.1MA bicarbonate buffer. The
radioactive solution contained NaHC1403 (specific
activity 5 mc/nmmole) adjusted to give between 6 and
8 X 106 cpm per 0.1 ml of solution. After tipping-in
the contents of the side arm, the flasks were equilibrated for 10 nminutes before turning on the lights.
02 evolution was followed manometrically and after
30 minutes of light exposure, the cells were harvested
by centrifuging in the cold in a Servall angle head
centrifuge at 3500 X g for 10 minutes. The cells
were washed once with ice water and were brought to
a convenient volume. Aliquots containing about 5 X
106 cells were then taken for digestion by the wet oxidation method for Calvin et al. (4). The BaCO3
precipitate was counted with an end window gas flow
Atomic Instrument used in the proportional range
and corrected to infinite thickness by means of a
standard curve.
After all nmanomletry experiments aliquots were
taken and the cells harvested, covered with a pinch of
MgCO3, and the pigments were extracted with acetone until no color remaine(d in the cell pellet. The
extracts were brought to a known volume and their
absorbancy was measured in a Cary model 14 recording spectrophotometer. Chlorophyll concentrations
were determined by the method of Mackinney (15)
and carotenoid concentrations were estimated from
the absorbancy at 475 m,u using an average extinction
coefficient of Elerl = 2500 (N. I. Krinsky, personal
communication). In some experiments pigment concentrations were measured before the Warburg measurements but no significant difference was detected
between these and the post-Warburg measurements.
Aliquots for cell counts received a drop of saturated HgCl2 to fix the cells and were counted in a
Spencer Bright-Line hemocytometer.
Results
80 hours of development (2). The first parameters
have correlated with this developmental sequence
pigment concentrations.
Total chlorophyll and carotenoids per cell increase
markedly during development and the chlorophyllcarotenoid ratio increases steadily until a final stationary value of 2.5: 1 (mole/mole) is reached at
about 72 hours (fig 1, right). 02 evolution per cell
shows essentially similar kinetics (fig 1, left). Difficulties in measurement were encountered at very early
times of development since the rates of 02 evolution
in these cells were at or below the limit of resolution
of the manometric apparatus. Table I contains data
for early times of development. The earliest developmental stage for which reliable 02 evolution data
have been obtained here is about 10 hours, so we
conclude that the actual inception point for photosynevolution is somewhat earlier than this. On
thetic
the other hand, chlorophyll is formed immediately on
exposure to light (table I). The amount of chlorophyll formed after 2 hours of light exposure is not
stoichiometric with the protochlorophyll content of
dark-grown cells since about 3 X 10- 3 picograms
(pg) of protochlorophyll per dark-grown cell are
formed and the chlorophyll concentrations noted here
represent a 10-fold increase. This suggests an initial
synthesis of chlorophyll which is followed by resynthesis of more protochlorophyll and conversion to
chlorophyll. Chlorophyll synthesis then proceeds
slowly, if at all, until about 10 hours when further
significant synthesis begins. In contrast, the carotenoids do not appear to increase during these early
times of development. Krinsky (personal communication) has found, however, that there may be some
we
are
02
Table I
02
Evolution, C1402 Fixation, aild Pigmient Formation
Time
in hrs
0
02/cell
(pl)/hr
picoliters
0.60
0
0
....
....
+ 0.50
+ 1.20
0
0
- 0.50
+
2
0.05
....
+ 0.40
+ 0.40
0
0
- 0.19
....
-
4
C1402 fixed
cpm X 104
mgC/hr
6
8
+ 1.00
+ 1.00*
10
+ 0.76*
11
+ 1.00*
....
+ 1.66*
+ 0.31
Pigmnent SYnthesis and 0 Evolution. On exposure to light, proplastids in dark-grown cells begin to
increase in size (2, 6). By 15 hours each plastid
contains approximately 6 lamellae. Thereafter, lamella formation is linear with time until a fully mature plastid containing 13 lamellae is found at about
at
Early Stages of Chloroplast Development
These cells were suspended in the mannitol medium
and exposed to 100 to 150 ft-c.
* Significant (based on
Warburg per hour).
1,052
3,460
....
4,706
15,836
14,348
pg
0
0
0
0.02
0.07
0.03
0.02
0.09
0.07
0.10
0.14
0.19
0.22
0.25
0.33
0.21
0.16
0.19
0.20
0.15
0.15
....
....
0.23
0.47
change of 10 ,ul or
....
a
chl/cell carotenoid/
cell
pg
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Copyright © 1964 American Society of Plant Biologists. All rights reserved.
0.25
0.19
0.29
0.26
0.23
0.26
0.24
more
in the
222
PLANT PHYSIOLOGY
qualitative differences in the carotenoids of these cells
at this time. Nishimura and Huzisige (17) found a
lag period of about 15 hours in Euglena before net
synthesis of chlorophyll in dark-grown cells exposed
to light could be measured. The apparent differences
in the 2 laboratories nmay be due to culture conditions.
Besides growing their cells on a different mediunm
(butyric acid and glutamate as carbon sources), the
resting meedium Nishimura and Huzisige employed
containedl only phosphate buffer. High light intensities of about 880 ft-c were also used to induce chlorophyll synthesis and nmay have affected the cells' ability
to formii chlorophyll (24).
Photosynthetic 02 evolution on a chloropllyll basis
is shown on the left side of figure 1. 02 evolution is
high on a chlorophyll basis, initially, and later declines to a steady state value. This may reflect qualitative changes in the chlorophylls as the cells develop
photosynthetic capacity. Chlorophylls with varied
abilities to mediate 02 evolution may be produced
unequally at first but may reaclh equilibriumii values at
later stages of development.
Figure 1 also shows that the kinetics of pigment
formation and 02 evolution are unaffected by the
presence or absence of divisi'on. Nondividing cells
develop as well as dividing cells. This would suggest that most materials necessary for clhloroplast
synthesis alrea(ly exist in the (lark-growni cells.
Dark-grown cells exhibit pronminent paramylon bodies
which are composed of polymeric glucose (13) uponl
which they can probably draw as a carboni source.
Mutants. The photosynthetic comlpetence of various light-grown chloroplast mutanits of Euglena was
also measured in an effort to correlate the extent of
chloroplast development in these mlutants vith the
normal sequence. Figure 2 shows the (lata for:
P1BXL, Y1BXD, Y2BUL, Y3BUD and N3BUL and
a description of these mutants is found in table II.
For comparison, data are shown in figure 2 for wild
type cells in which chloroplast developnmenit has been
limited by low light intensities of 7 ft-c (24), and(
wild type cells at 24 and 48 lhours of dlevelopment at
normal intensities. Of the mutanits, P1BXL, which
forms normal-size(d chloroplasts, is the only onie Nvhich
21.MANN ITOL
I15
CM)
X
a
I°
IId
21EUGLENA MEDIUM
4
21
C.)
n~~~~~~~~~~~~
/
o
0 3
0 3
/
2
=o2~~~~~~~/CELLo
°-02/CHL.
O:
40
60
80
20
100
TIME AT 100-150 F.C. (HRS.)
20
40
60
80
100
TIME AT 100-150 F.C. (HRS.)
FIG. 1. Photosynthetic capacities and pigment contents of Euglena during chloroplast development. Dark-grown
cells were exposed to optimum light intensity at zero time and aliquots taken for measurement at the times indlicated.
Data are shown for nondividing cells (mannitol medium) and for dividing cells (Euglena medium).
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.~
STERN ET AL.-PLASTID DEVELOPMENT, PhIOTOSYNTHETIC PARANIETERS
exhibits significant photosynthetic 02 evolutionl.
This is not surprising since these plastids contain
patches of normal lamellae. The other mutants which
are blocked at early stages in development (Y1BXD
and Y3BUD) have not developed photosynthetic coImlpetence. Those mutants which lack chloroplast structures entirely even when growvn in the light (Y2BUL
and W3BUL), of course, never become photosynthetically active, nor do they form any chlorophyll.
The cells limited by low light intensity (7 ft-c) are
similar to cells which have developed at normal intensity (100 ft-c) for 48 hours as far as their pigments and photosynthetic competence is concerne(l,
reaching levels of about 50% of nornmal (24).
There is goodl correlation, then, between the presence of morphological structures necessary for photosynthesis and the photosynthetic competence of the
cells. It would seem that photosynthetic competence
is limlited to those cells which display at least a partial comlplenment of lamellae.
CO2 Fixationi. Another crucial inclicator of a
cell's photosynthetic competence is its ability to fix
CO, in the light. To correlate this with the morphological development of the chloroplast, C1402 fixation
-as nmeasured as a function of time for dark-grown
cells placed at the optimal intensity under nondividing
conditions (fig 3). Since the experiments were performied in \Varburg vessels, 02 evolution was mea-
-J
I
0
z GREEN
z
zo
0
lL
0
IC
0
0
GREENN
a80o
60
LUJ
A-J
0
80-
-j-_J -Joz
0 10 -i
0)
Zr.
40Q
0
223
Jul Ji
Joz
0QF40J
-)J
uJ uJoIzt
O
J
o
0
'
s
FIG. 2. Comparison of the photosynthetic capacity and pigment contents of mutants and other cells limited in chloroplast development. For a description of the mutants see table II. Development in wild type cells at 7 ft-c is limited
by light intensity (24). Wild type cells at 100 to 150 ft-c were measured at 24 and 48 hours by the same methods as
in figure 1. All values are expressed as a per cent of wild type cells grown at optimal light intensity. In all cases the
values for dark-grown cells (DK) have been taken as the zero poiIlts. Data for maximal 02 evolution cell/hour,
chlorophyll/cell, carotenoid/cell, chlorophyll/carotenoid (mole/mole) and 02 evolution on a chlorophyll basis (pl/pg)
are shown.
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224
PLANT PHYSIOLOGY
Table II
I)DsignationI of Mllnttants Derived fromii
Euglcnta gracilis var. bacillaris
Strain
Description of Light-grown cells
P1BXL
X-ray induced pale green, isolated in the light
and which by fluorescence and electron microscopy* appears to cointaini normal-sized chloro-
Y1BXD
X-ray induced yellow, isolated in the dark and
wlhich by fluorescence microscopy appears not
to develop beyond a very early (12 hr) stage
in chloroplast development.
Ultraviolet induced yellow, isolated in the light
and which by fluorescence and electron microscopy appears to lack chloroplasts com-
plasts which lack some or groups of lamellae.
Y2BUL
pletely.
Y 3BUD 'Ultraviolet induced yellow, isolated in the dark
and which is very similar morphologically to
Y ,BXD.
W3P,IL, 'Ultraviolet induced albino, isolated inl the light
and which by fluorescenice microscopy appears
to lack chloroplasts.
* Electron micrographs of these mutanits x-ill be publislhed
in a forthcom,niing paper.
sured concomitantly andl these (lata are also shown in
this figure. It was found that as little as 6 hours of
liglht exposure permitted the cells to fix significant
anmounts of C1402 as is seen in table I. The photosynthetic quotient was already approxinmately one by
15 hours of development.
The kinetics of CO2 fixation are sinilar to those
of 02 evolution and pigment formation (fig 1) and
in all cases, the kinetics are essentially linear after the
initial lag. The kinetics of lamiiella fornmation is also
linear from about 14 hours onwards, 1 lamella being
formed about every 11 hours (2). If lamella formation and the development of photosynthetic competence are linked, it should be possible to compute each
of the parameters measured on an 1 1 hour (or lamella) basis from the slopes in figures 1 and 3. Fronm
these values their plateau values can be predicted
( xvhich slhoul(d be 7.5 times any one rate since about
7.5 lamiellae are produced from the time at w hiclh
linear kinetics begins to the time at which the chloroplast is complete(d (2). When this calculation is
made, a close agreement is found with the experimental values obtained, as is seen (table III). The
Table III
Coiniparison1 of Predicted anid Experimtenttal Levels Attaimied for V"arionis Photosynithetic Paramietcrs
ditrinzg Chloroplast Developmlent
Theoretical* (expecte(d
at 72
Parameter
Experimental
hrs)
( 2 evolution (pl/cell/hr)
C1402 Fixation (cpm/mgC/hr)
Chlorophyll (pg/cell)
Carotenoid (pg/cell)
* Computed from the rate of
text).
21
370 X 103
16
3.7
20-22
300 x 10"
12-14
2.8-4.0
lamella formation (see
lanmella has been imlplicated as the site of photosyintlhesis on the basis of its structural presence in the
chloroplast, its association with pigments and the
fact that chloroplasts lacking lamellae are uinable to
photosynthesize (26, 28). These data, inclu(dinig
those for the mutaints, suggest that this interpretation
is valid for Euglenia.
Discussion
-
0
6
x
E
r
0
I-
E
I
x
'-
0
w
c0
0
0
20
40
60
80
100
TIME AT 100-150 F.C. (HRS.)
FIG. 3. Photosynthetic CO, fixation and
120
02
evolution1
in Euglena during cholorplast development. Dark-grown
cells were exposed to optimum light intensity at zero
time and aliquots taken for measurement at the times indicated. Data are shown for noindividinig cells (mannitol
medium).
Our findinigs in this study agree vell with those
foundl wvith other organisms, especially higher plants.
Phlotosynthesis in Euglena is correlated witlh the appearance an(l the development of the lamellae. After
a lag of about 6 hours, dark-grown cells exhibit photosynthetic capacity and electron micrographs (2, 6)
reveal that the proplastids are already undlergoing
structural chainges during this time. In higher
plants, etiolatedl leaves exposed to light also experience a lag perio(l, although slhorter (about 1-3 hours),
before photosynthetic activity is measurable. Mfaly
laboratories ( 1, 5 8, 11, 12, 16, 22, 23, 28) have repIorte(l the various changes in the proplasti(ds induce(d
witlh light and Ino doubt these are also relevent to
Euglena. Photosynthesis has been correlated wvitlI
the formation of chlorophyll a in higher plants (3,
23), in particular the C677 form of chlorophyll, thought
to be the chlorophyll-protein complex normally foundl
in green leaves (7). This form of chlorophyll does
not appear until 30 to 60 minutes after illunmination (21), so the C677 may be require(I before
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STERN ET AL.-PLASTID DEVELOPMENT, PHOTOSYNTHETIC PARAMETERS
photosynthesis is initiated and it may also be a limiting factor in the developmental sequence.
There is also evidence correlating the synthesis of
chlorophyll and the formation of grana in higher
plants. The latter process is light dependent but lamella discs have been observed to form in the dark
(11, 28, 29). Plants can accumulate some chlorophyll without the formation of grana and it is possible
that chlorophyll synthesis may control grana formation. These results are not inconsistent with our
findings for Euglena. Euglena forms no grana and
the mature chloroplast consists only of lamellae which
contain the photosynthetic pigments, and a pyrenoid
region. Because of this, the entire chloroplast in
Euglena has been compared to a single granum of
higher plant chloroplasts (9, 25). In higher plants
(3) the formation of pigments and development of
photosynthetic ability are nonlinear with time. In
Euglena our studies shoxv that formation of lamellae
appears to be linear with time and this is reflected in
the linear kinetics for the appearance of pigments and
photosynthetic capacity. This does not settle the
question of which parameter actually controls development. Chlorophyll synthesis may limit the formation of lamellae or the reverse may be equally true.
Indeed, some unknown third parameter may control
both. Until Euglena mu,tants are available which are
selectively blocked for either pigment or lamella formiiation, this will be difficult to answer since both
processes are light dependent and might have the sanme
action spectrum (17).
Summary
Measurements have been obtained of the photosynthetic capacity of dark-grown cells of Euiglena
gracilis var. bacillaris Pringsheim exposed to optimal
light intensities (100 ft-c). These include values for
oxygen evolution, carbon dioxide fixation, and pigment synthesis as a function of time in the light in
dividing and nondividing cells. There is an initial 6
to 8 hour lag in the rates of increase of all photosynthetic parameters when dark-grown cells are placed
in the light. This is followed by essentially linear
kinetics for all parameters until about 80 hours, when
these values are equivalent to those found for lightgrown cells. The kinetics of appearance for all
photosynthetic parameters are the same for dividing
and nondividing cells. The onset of linear kinetics
for all photosynthetic measurements is correlated
with the linear formation of the chloroplast lamellae
and the rates of all parameters are the same as the
rate of lamella formation. Various mutants limited
at different stages in chloroplast development were
also tested for their capacity to photosynthesize and a
correlation was found between photosynthetic ability
and the extent of chloroplast development.
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225
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Studies of Chloroplast Development in Euglena. VI. Light
Intensity as a Controlling Factor in Development 1, 2
Arthur I. Stern,3 H. T. Epstein, and Jerome A. Schiff
Department of Biology, Brandeis University, Waltham, Massachusetts
In a previous paper (13) we described the kinetics
of appearance of pigments and photosynthetic capacity
in dark-grown Euglena when the development of
proplastids into chloroplasts was induced with optimal
intensities of light. These processes were shown to
be strongly correlated with the development of lamella
structures within the chloroplasts. During the course
of this work it became apparent that the developmental process is strongly influenced by light intensity. The present paper describes the extent to
which light intensity controls the formation of chloroplast structure, pigments and photosynthetic capacity.
Materials and Methods
Euiglena gracilis var. bacillaris Pringsheimii was
grown as described previously (13) andl all techniques
for measuring 02 evolution, CO2 fixation, pigment
content, and cell numbers were also the same. Tennml aliquots of cells were transferred aseptically into
Received revised manuscript July 15, 1963.
This work was supported in part by research grant
RG-6344 from the Division of Research Grants, Public
Health Service. The data are taken from a dissertation
submitted to the graduate faculty of Brandeis University
by A. I. Stern in partial fulfilment of the requirements
for the PhD degree.
3 During the tenure of this work A. I. Stern was supported by a training grant in Developmental Biology,
2G-883, from the National Institutes of Health.
I
2
10-cm sterile glass petri dislhes which wxere then exposed to various light intensities. Intenisities of 4
to 700 ft-c (as measured with a \Veston illumination
meter) were obtained with Koclak neutral (lensity
filters or by varyiing the distance froml the fixe(d light
source compose(d of 20-w- GE white fluorescent lamllps.
All experimiienits were carrie(l out at 260.
Results and Discussion
Light Intensity and Photoswithetic Paramieters.
To define the optimial conditions for chloroplast developm-nentt. dark-grow-n cells rvere expose(d to intensities froml 7 to 700 ft-c for 100 hours. Since previous
experimenits had slhowx n that development proceeds
normally undler nondividinlg conditions (13), mannitol resting mle(liunm was used in all experinments. Figure 1 shows that 0., evolution anlld pigimlent formation
reach maximiial levels in the regioni of 100 ft-c all(n
higher intensities are inhibitory. At tile lowest intensity mleasured (7 ft-c) -, evolution andl pigimlent
production are only about 50% of tile mlaximlum, although 0 evolution on a chlorophyll basis andl the
chlorophyll-carotenoid ratios (mole/lmole) are equivalent to those in the cells developing at the optimal
intensity. A similar pattern w%as found for CO2 fixation. Dark-grown cells which had developed at 7 ft-c
for about 100 hours, fixed 165 and 180 X 103 cpml/
mg C/hour in separate experimlellts or app)roximllately
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