Plant Physiol. (1983) 73, 329-331
0032-0889/83/73/0329/03/$00.50/0
Changes in Photosystem II Account for the Circadian Rhythm in
Photosynthesis in Gonyaulax polyedra'
Received for publication May 3, 1983 and in revised form June 10, 1983
GORAN SAMUELSSON2, BEATRICE M. SWEENEY, H. ALLEN MATLICK, AND BARBARA B. PREZELIN
Department ofBiological Sciences, University ofCalifornia, Santa Barbara, California 93106
ABSTRACT
Cell-free extracts that show activity in photosynthetic electron flow
have been prepared from the unicellular dinoflagellate, Gonyawlax polyedra. Electron flow, as 02 uptake, was measured through both photosystem I and II from water to methyl viologen, through photosystem I
alone from reduced 2,6-dichlorophenol indophenol to methyl viologen
which does not include the plastoquinone pool or from duroquinol to
methyl viologen which includes the plastoquinone pool. Electron flow
principally through photosystem II was measured from water to diaminodurene and ferricyanide, as 02 evolution. Cultures of Gonyaulax were
grown on a 12-hour light:12 hour dark cycle to late log phase, then
transferred to constant light at the beginning of a light period. After 3
days, measurements of electron flow were made at the maximum and
minimum of the photosynthetic rhythm, as determined from measurements of the rhythm of bioluminescence. Photosynthesis was also measured in whole cells, either as 14C fixation or 02 evolution. Electron flow
through both photosystems and through photosystem II alone were
clearly rhythmic, while electron flow through photosystem I, including
or excluding the plastoquinone pool, was constant with time in the
circadian cycle. Thus, only changes in photosystem II account for the
photosynthesis rhythm in Gonyaulax.
The marine dinoflagellates are well known for their circadian
rhythmicity manifested in bioluminescence (16, 17, 19), cell
division (18, 20), and photosynthesis (5, 12, 13, 17). Both lightsaturated and light-limited rates of photosynthesis, measured
either by 02 evolution or 14C fixation, express rhythmicity in
whole cells of Gonyaulax (13). In addition, fluorescence emission
is about 2x as high during the day phase when photosynthesis
reaches a maximum than during the night phase when the rate
of photosynthesis is low (2, 21). The shape of the fluorescence
transient and delayed light emission curves also varies with the
phase of the circadian rhythm (21). Until now, however, it has
not been possible to dissect photosynthesis further, because cellfree preparations from dinoflagellates that were active in photosynthetic electron flow could not be prepared in spite of numerous attempts. Thus, substitute electron donors and acceptors
could not be used since they do not penetrate whole cells. Now,
however, one of us (G. S.) has developed a method for preparing
cell-free extracts of dinoflagellates which retain activity in electron flow through PSI and PSII. We have used this method to
assay cell-free extracts of Gonyaulax to determine the rates of
electron flow through the two photosystems separately at different times in a free-running circadian cycle in constant light. We
report here that rhythmicity is observed only in PSII.
MATERIALS AND METHODS
Cultures. Gonyaulaxpolyedra Stein, clone 70A isolated by one
of us (B. M. S.) was grown in unialgal batch cultures in f/2
medium (3) in alternating light (2.5 mw cm 2) and darkness, 12
h each at 19 to 200C. At late log phase of growth (5,000-10,000
cells ml-'), cultures were transferred to constant light at 0.9 mw
cm-2, at the same temperature at the beginning of a normal light
period. To insure uniformity between cultures, contents of all
flasks were mixed and redistributed. After 3 d, measurements of
electron flow were begun and were continued at different times
for several days. Circadian time (CT3) was determined from
frequent measurements of bioluminescence from the same cell
suspensions, as previously described (16). The midpoint of the
maximum in bioluminescence was taken as CT 1800 and the
interval between maxima in bioluminescence was divided by 24
to give circadian hours (CT 0000 is the time which corresponds
to the beginning of the light period in the previous light:dark
cycle [10]).
Whole Cell Photosynthesis. At each time point, the photosynthesis of whole cells was measured with an aliquot of the culture
concentrated 2x by gentle centrifugation (30 s, 100g, International Clinical Table-top Centrifuge), and resuspended in medium containing 10 mm NaHCO3. 02 evolution was measured
with a Clark-type 02 electrode (Rank Bros.) as described by
Delieu and Walker (1). Photosynthetic fixation of 14C NaHC03
was measured with illumination at 2 mw cm-2 for 15 min as
previously described (4) with the following modifications: l-ml
cell suspension containing 20 mm NaHCO3 was irradiated in a
scintillation vial in the presence of 0.5 yCi 14C; after 15 min, 1
ml 1 M HC1 was added, water was removed by drying, salts were
redissolved with a small amount of water, scintillation solvent
(ACS, Amersham Co.) was added, and the radioactivity was
counted with a Beckman model LS IOOC liquid scintillation
counter. Cell counts were made with a Sedgewich Rafter Counting Chamber. Chl was determined in acetone extracts by the
method of Jeffrey and Humphrey (7).
Preparation of Cell-Free Extracts. Cultures (1.5-2 L) were
harvested by centrifugation for 30 s at 100g as above. The pellet
was resuspended in 5 ml cold preparation medium (10 mM
Tricine [pH 7.2], 10 mM NaCl, 5 mM MgCl2, 10 mM KCI, and
0.2 M sucrose). Glass beads (0.45-0.5 mm diameter, B. Braun
Melsungen) were added to the suspension (10 parts to 3 parts
' Supported by grants from the National Science Foundation (NSF
3Abbreviations: CT, circadian time; MV, methyl viologen; DAD,
PCM80-01940 to B. M. S. and NSF OCE82-08187 to B. B. P.).
2Supported by a grant from Seth. M. Kempes Memorial Foundation, diaminodurene; FeCy, ferricyanide; DPIP, 2,6-dichlorophenol indoSweden. Present address: Department of Plant Physiology, University of phenol; asc, ascorbate; DQ, duroquinol; PQ, plastoquinone; LL, constant
light.
Umea, S-90187, Umea, Sweden.
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329 of Plant Biologists. All rights reserved.
Copyright © 1983 American Society
SAMUELSSON ET AL.
330
Plant Physiol. Vol. 73, 1983
[w/w] cell suspension) in a 40-ml conical centrifuge tube. After electrode and a Soltec chart recorder, model B18 1. For electron
cooling on ice for 5 min, the mixture was shaken on a Genie
Vortex Mixer at highest speed for 20 s to break the cells. The
suspension was separated from the beads by decanting, washing
the beads, and redecanting. The suspension was then centrifuged
for 2 min at 4°C at lOOg in a RC2B Sorval refrigerated centrifuge
to remove large cell debris. The supernatant was centrifuged at
12,000g for 5 min to pellet chloroplast fragments and other
membranes, the pellet was resuspended in 10 ml preparation
medium diluted 1/10 for 5 min and finally repelleted at 12,000g
for 5 min. The final pellet was resuspended in 5 ml preparation
medium and stored on ice until used.
Reaction Mixtures for Assay of Electron Transport. Whole
chain electron transport from water to MV as electron acceptor
was studied using a 4-ml assay mixture containing 10 mM
Tricine, 5 mM MgCl2, 1 mm NaN3, 1 mM MV, and 0.6-0.8 ml
cell-free extract containing 7 to 10 ug ml-' Chl a. 02 uptake was
recorded for 30 s on illuminating the mixture with saturating
light from a projection lamp filtered through CuSO4, using the
flow from water to DAD and FeCy (PSII), the reaction mixture
was 10 mm Tricine, 5 mM MgCl2, 1.5 mM FeCy, 0.5 mM DAD.
The DAD was dissolved in ice-cold HCI to a concentration of
15 mm of which 33.3 AI were added for 1 ml reaction mixture.
02 evolution was measured with the 02 electrode. PSI was
measured using two different electron donors to MV, DPIP
reduced with asc and DQ. The reaction mixture for the first
contained 10 mm Tricine, 5 mm Na ascorbate, 5 AM DPIP, 1 mm
NaN3, 1 mM MV, 2 ,uM DCMU, and 0.4 ml cell-free extract (47 zg Chl a ml-'). The reaction mixture for electron transport
from DQ to MV was 10 mM Tricine (pH 7.5), 1 mm NaN3, 1
mM MV, and 80 Al DQ solution. The DQ solution (16.4 mg
ml ') was prepared essentially as in Izawa (6). This solution was
prepared freshly for each experiment.
RESULTS
In whole cells of Gonyaulax, a clear circadian rhythm in
photosynthesis has been demonstrated in LL, with a maximum
400
at subjective midday and a minimum at night (13). The rhythm
in bioluminescence is clearly controlled by the same oscillator
(9, 15), but is 180° out of phase with the photosynthesis rhythm,
0
0
with maxima at subjective midnight. Bioluminescence, which
x
can be measured quickly with a minimum of material, was thus
300
7
x
0'
used as an indicator of circadian time so that the measurements
0'
0
-J
of photosynthesis could be made at the peak and trough of the
0
O
0
rhythm. Under the conditions used, the period of the rhythmicity
0og~~~~~~~~~~~~I
was
22.5 to 23 h.
z
E
E0'
0
10 O ou
The circadian rhythmicity is clearly preserved in cell-free
200
U,
extracts in electron flow from water to MV, which encompasses
E o~~~~~~~LL
UA
0
LLIL.
both PSII, PSI, and the intervening electron transport chain (6).
0
200
E
U)
Electron transport is high when bioluminescence is low, declines
z
IV
w
as bioluminescence increases during the night phase and inL)
E,
0
C')
creases again as the minimum in bioluminescence is reached
z
(Fig. 1). In general, the rate of electron flow through both
0
LI
0
photosystems as a function of time in a circadian cycle pallels
whole cell photosynthesis as measured by 02 evolution (Fig. 1).
0
O-aE
On the other hand, electron flow through PSI alone from DPIP/
14
18 22 02 06
asc to MV remains high and constant within experimental variHOURS CT
ation throughout the circadian cycle (Fig. 1). That electron flow
through the whole electron transport chain in photosynthesis
FIG. 1. Photosynthetic electron flow in cell-free extracts of Gonyaulax from water to MV shows circadian rhythmicity while that
polyedra at different times in a circadian cycle in constant light, compared through PSI alone from DPIP/asc to MV remains constant with
to 02 evolution by whole cells and bioluminescence. Abscissa, circadian time has been confirmed in two additional experiments
time determined as explained in text. Data from experiment I.
(Table I).
0
-
cy
Table I. Photosynthetic Electron Transport in Cell-Free Extracts of Gonyaulax polyedra with Different Electron Donors and Acceptors, Measured
in the Day and the Night Phases of the Circadian Cycle in Constant Light
Values for "C fixation in whole cells are included for comparison. Standard deviations and numbers of measurements averaged (in parentheses)
are given for each value. Chl a cell', 5.1-6.3 x 10-8 ,umol.
Cell-Free Extracts
Cells
Expt.
Expt.
~ ~CultureinWhlCes
to
WoeClsH20
H20 to MV
DAD/FeCy
ml1' x 10-3
jmol 14C mgl
DQ to MV
DPIP/ASC to MV
Mmol 02 mg-' Chi ah-'
Chlah-'
Day phase (03-06 CT)
II
10.9 ± 2.2
III
5.8 ± 0.2
IV
6.2 ± 0.9
226.0 ± 154.4 (6)
90.6 ± 3.5 (3)
-34.1 ± 0 (2)
-54.8 ± 21.4 (7)
-54.4 ± 8.9 (9)
34.1
54.4 ± 9.1 (6)
51.8 ± 16.8 (5)
-243 ± 10.6 (4)
-372.7 ± 51.7 (9)
-425.9 ± 40.9 (13)
-300.5 ± 27.6 (3)
-352.4 ± 50.4 (9)
-437.2 ± 40.1 (12)
Night phase (16-22 CT)
II
11.4 ± 0.1
III
6.1 ± 0.5
IV
5.8 ± 0.4
76.5 ± 16.2 (3)
72.3 ± 5.0 (3)
-20.3 ± 8.5 (6)
-39.0 ± 16.0 (15)
-36.4 ± 9.5 (4)
22.0 ± 4.6 (4)
35.8 ± 7.2 (8)
43.3 ± 1.3 (2)
-218.0 ± 29.4
-330.9 ± 57.4 (12)
-462.7 ± 21.2 (6)
-256 ± 31.9 (6)
-328.7 ± 54.6 (12)
-451.7 ± 58.6 (6)
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Copyright © 1983 American Society of Plant Biologists. All rights reserved.
A CIRCADIAN RHYTHM IN PSII IN GONYAUL4AX
331
To locate the control of the photosynthesis rhythm more electron transport are changing in response to signals from the
closely, we have used two other pairs of electron donors and circadian 'clock', and what the nature of these signals may be.
acceptors which interrupt the electron transport chain at different
authors wish to thank Dr. B. Martin, Urbana, IL, for
points. Electron flow was calculated on the basis of Chl a content theAcknowledgments-The
gift of DAD amd DQ. DAD was purified and converted to a HCI salt by D.
for purposes of comparison, inasmuch as the pigment content of Ort.
Gonyaulax in known not to change during the circadian cycle
(12). Electron flow through PSII alone was measured from water
LITERATURE CI'TED
to DAD/FeCy, an acceptor which intervenes close to Q, the
T, DA WALKER 1972 An improved cathode for the measurement of
primary acceptor for PSII (6). With this electron acceptor pair, 1. DELIEU
photosynthetic oxygen evolution by isolated chloroplasts. New Phytol 71:
values for day phase in both experiments were 1.5x those at
201-225
night (Table I). Furthermore, electron flow rates were almost 2. GOVINDJEE, D WONG, BB PREZELIN, BM SWEENEY 1979 Chlorophyll a fluorescence of Gonyaulaxpolyedra grown on a light-dark cycle and after transfer
identical to those for transport through the whole electron transto constant light. Photochem Photobiol 30: 405-41 1
port chain from water to MV. Thus, PSII alone can account for 3. GUILLARD
RRL, JH RYTHER 1962 Studies of marine diatoms. I. Cyclotella
the full circadian oscillation observed in cell-free extracts. The
nana Hustedt, and Detonula confervacea (Cleve) Gran. Can J Microbiol 8:
use of DQ as electron donor allowed the inclusion of the PQ
229-239
pool in measurements of electron flow through PSI, omitting 4. HARDING LW JR, BW MEESON, BB PREZELIN, BM SWEENEY 1981 Diel
periodicity of photosynthesis in marine phytoplankton. Mar Biol 61: 95PSII (6). This measurement of electron flow through only PSI
105
also was constant with time in the circadian cycle (Table I), and 5. HASTINGS
JW, L ASTRACHAN, BM SWEENEY 1961 A persistent daily rhythm
agreed in magnitude very closely with the values for electron
in photosynthesis. J Gen Physiol 45: 69-76
6. IZAWA S 1980 Acceptors and donors for chloroplast electron transport. Methflow from DPIP/asc to MV.
DISCUSSION
The findings reported here make it clear that the circadian
rhythm in photosynthesis reflects changes only in PSII, although
we cannot conclude whether these changes are on the oxidizing
or the reducing side of P68o. The similar arhythmicity obtained
when PSI was measured with DPIP/asc or DQ as an electron
donor provides evidence that rhythmicity does not arise from
changes in the PQ pool.
The amplitude of the rhythm in photosynthesis of both whole
cells and extracts was not as large as sometimes observed (Table
I). However, these cultures were in the stationary phase of growth
when electron flow was measured. This condition was chosen
deliberately in order to eliminate any changes in the photosynthetic machinery arising from the cell cycle which is known to
be under control of the circadian oscillator (18). However, such
stationary cultures have been observed to show rhythms of lower
amplitude than do cells growing logarithmically (1 1).
Our findings with Gonyaulax concerning the rhythm in electron flow in photosynthesis differ from those obtained with
Euglena, another organism with a circadian rhythm in photosynthesis (8). In the latter organism, electron flow through the whole
chain was observed to express rhythmicity as we found with
Gonyaulax. However, in Euglena, neither PSI nor PSII showed
a rhythm when measured separately. We can offer no explanation
for this discrepancy except to note that control of the circadian
cycle may well vary from organism to organism.
The findings reported here open the way to further investigation of the coupling between the circadian oscillator and photosynthesis. Such studies will be directed toward PSII and understanding what circadian changes in this part of the photosynthetic
ods Enzymol 69: 413-434
7. JEFFREY SW, GF HUMPHREY 1975 New spectrophotometric equations for
determining chlorophylls a, b, c, and c2 in higher plants, algae and natural
phytoplankton. Biochem Physiol Pflanzen 167: 191-194
8. LONERGAN TA, ML SARGENT 1979 Regulation of the photosynthesis rhythm
in Euglena gracilis. II. Involvement of electron flow through both photosystems. Plant Physiol 64: 99-103
9. MCMURRY L, JW HASTINGS 1972 No desynchronization among four circadian
rhythms in the unicellular alga, Gonyaulax polyedra. Science 175: 11371139
10. PMrTENDRIGH CS 1965 On the mechanism of the entrainment of a circadian
rhythm by light cycles. In J Aschoff, ed, Circadian Clocks. North Holland
Publishing Co., Amsterdam, pp 277-297
1 1. PREZELIN BB 1983 Photosynthetic physiology of dinoflagellates. In FJR Taylor,
ed, The Biology of Dinoflagellates, Blackwell Publishing Co, Oxford. In press
12. PREZELIN BB, BW MEESON, BM SWEENEY 1977 Characterization of photosynthetic rhythms in marine dinoflagellates. I. Pigmentation, photosynthetic
capacity and respiration. Plant Physiol 60: 384-387
13. PREZELIN BB, BM SWEENEY 1977 Characterization of photosynthetic rhythms
in marine dinoflagellates. II. Photosynthesis-irradiance curves and in vivo
chlorophyll a fluorescence. Plant Physiol 60: 388-392
14. Deleted in proof.
15. SWEENEY BM 1963 Resetting the biological clock in Gonyaulax with ultraviolet
light. Plant Physiol 38: 704-708
16. SWEENEY BM 1979 Bright light does not immediately stop the circadian clock
of Gonyaulax. Plant Physiol 64: 341-344
17. SWEENEY BM 1981 The circadian rhythms in bioluminescence, photosynthesis
and organellar movement in the large dinoflagellate, Pyrocystis fusiformnis.
In H-G Schweiger, ed, International Cell Biology 1980-1981. SpringerVerlag, Berlin, pp 807-814
18. SWEENEY BM 1982 Interaction of the circadian cycle with the cell cycle in
Pyrocystisfusiformis. Plant Physiol 70: 272-276
19. SWEENEY BM, JW HAsTINGS 1957 Characteristics of the diurnal rhythm of
luminescence in Gonyaulax polyedra. J Cell Comp Physiol 49: 115-128
20. SWEENEY BM, JW HASTINGS 1958 Rhythmic cell division in populations of
Gonyaulax polyedra. J Protozool 5: 217-224
21. SWEENEY BM, K SATOH, DC FORK 1982 A comparison of the fluorescence
and delayed light emission in Gonyaulax polyedra in day and night phases
of the circadian rhythm in constant light. Carnegie Inst Wash Year Book 81:
68-70
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