Planta 1 ml - s-1). This resulted in an equilibrium between the physiological gas exchange at the surface of the thalli and the concentrations in the medium. Therefore, the recordings represent a relative measure of the rates of net photosynthesis or respiration. The continuous flow through the chamber was generated by the head of water between the reservoir and the outlet from the chamber. Water from the outlet was pumped back into the reservoir. All connections were made with silicone tubing. The Plexiglas chamber was kept in a translucent water bath at 18~ C and irradiated from below via a mirror. Red light was supplied from a projector (Prado universal; Leitz, Wetzlar, FRG) whose light was filtered through 3 mm of red Plexiglas (Nr.501 ; R6hm & Haas, Darmstadt, FRG). Blue light was provided from a cold lightmicroscope lamp with a bifurcated light pipe (KL 1500; Leitz) using
a blue glass filter (% max: 410 nm; halfband width: 1l0 nm; Leitz). The whole setup was protected from other irradiation by a lighttight black wooden box.
Results Initial results showed that the degree o f e n h a n c e m e n t o f red-light-saturated photosynthesis by blue light was very variable, relative to the rates in red light, and so the measurements were extended over longer periods o f time. This lead to the discovery that photosynthesis in red light was rhythmic (Fig. 2). The r h y t h m was circadian, as it continued under c o n s t a n t environmental conditions and displayed a period that was n o t exactly 24 h. In continuous red light, the period was usually a b o u t 23 h. [The circadian times (CT) which are referred to in this paper are measured f r o m the beginning o f the light phase in the culture conditions immediately preceding the experiment.] Pulses o f blue light enhanced the rates o f p h o t o s y n thesis, but the degree o f stimulation depended on the time in the circadian r h y t h m at which blue light was given (Fig. 2). W h e n blue light was given at the peaks o f the r h y t h m the response was small, whereas blue light given in the troughs resulted in a m u c h stronger response. D a r k respiration was n o t rhythmic and was n o t affected by blue-light pulses. Immediately after periods o f photosynthesis, the rate o f d a r k respiration was high, but declined rapidly during the next 30 min and m o r e slowly after that (see Figs. 7 and 8 for examples). The effects o f blue light on CO2 c o n s u m p t i o n were essentially the same as those on O2 evolution. However,
2 min blue light
..
. .......... \.
9
,--5h m
d[02]
black box
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Q,I >
reservoir [1 []
o
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Fig. 1. Experimental setup for continuously recording rates of 02 evolution and CO2 consumption in an open system
Fig. 2. Circadian 0 2 production and CO2 consumption of Ectocarpus in continuous red light and after blue-light pulses of 2 min duration, given at various times of the day. Rates of photosynthetic 02 evolution and CO2 consumption were measured under continuous red light at an irradiance (46 W ' m - E ) that saturated photosynthesis. Arrowheads indicate start and end of red-light irradiation. Pulses of blue light (2 min at 3.2 W 9 m -2) were given at the times indicated by the arrows. Note that the rhythm of CO2 uptake was delayed by about 5 h relative to the rhythm of 02 evolution
R. Schmid and M.J. Dring: Fast blue-light response of photosynthesis in Ectocarpus
E
55
blue light pulse duration: l m i n
,7" CT 17 t
o= o
/
(:u
,v"
/
o"
F~
I=:
A,.'"""
,V" >
.... 9 .......... t...
o
J/
10"6
~"CT 6
. . ..""''"
10"5
photon fLuence
10"4
10.3
[mol-m "2 ]
Fig. 4. Effect of photon fluence of blue light on the magnitude of stimulation of photosynthesis in Ectoearpus. Photosynthetic rates were measured under saturating irradiances of red light (50 W 9m-Z). Blue-light pulses of l-rain duration at various irradiances were given at the peaks (CT 6) and at the troughs (CT 17) of the photosynthetic rhythm. The difference between the maximum of 02 production after blue-light stimulation and the rate in red light before the stimulus was plotted versus the blue-light fluence
QJ
o~ ~a o
0
5 10 time [mini Fig. 3A, B. Kinetics of stimulation of Oz evolution after induction by blue light (BL) in Eetoearpus (A). Insert (13) shows another experiment at a different time scale. Horizontal dottedline shows the rate of 02 evolution before the start of the blue-light stimulus. Blue-light induction was for 1 min at different irradiances (fluences are indicated at the curves). Blue light was given at either the peak (CT 6) or the trough (CT 17) of the photosynthetic rhythm. The increase of 02 evolution in continuous blue light at the highest irradiance is shown for comparison (broken line) the kinetics o f these effects could not be resolved, because o f the slow response of the CO2-electrode system used. Nevertheless, the rhythm of CO2 consumption appeared to be delayed (by approx. 5 h in the experiment shown in Fig. 2) relative to that o f O2 production. However, the delay was variable and could be as little as 1 h. Thus phase differences could be a consequence o f the time the electrode requires to approach an equilibrium recording. Using a higher time resolution (Fig. 3), it was apparent that photosynthesis began to rise 15 to 20 s after the start o f a 1-min pulse o f high-irradiance blue light. After 3 to 5 rain, a maximum was reached, followed by a gradual decline of photosynthetic rate. A difference between the kinetics of stimulation after a blue-light pulse and that in continuous blue light was first visible after 3 rain (Fig. 3, hatched area). The decline after reaching the maximum was not continuous, and a second lower peak was often observed about 20 min after the blue-light pulse (Fig. 3, insert B). This indicated that the enhancement of photosynthesis by blue light consisted of at least two components. When lower irradiances of blue light were used, the onset o f photosynthetic stimulation was progressively delayed (Fig. 3). Irradiances below the blue-light threshold (see next paragraph) were effective only as long as the blue irradiation was applied (not shown). The time taken to respond at different irradi-
ances was the same at the maxima (CT 6) and minima (CT 17) of the rhythm.
Sensitivity to blue light. The dependence of the degree of stimulation of photosynthesis on the blue-light fluence was measured in the troughs (CT 17) and the peaks (CT 6) of the rhythm. Using a 1-min pulse, the irradiance of blue light was changed by means of neutral-density filters. F o r both o f the circadian times, the magnitude o f the response of Oz production was proportional to the logarithm of the blue-light fluence, but the slope o f the line for pulses at CT 17 was greater than that for CT 6 (Fig. 4). The two lines extrapolated towards the same threshold value of fluence (approx. 10-6 mol 9 m-2). The maximum photosynthetic rates after the highest bluelight fluences used were only about two-thirds of those under the influence o f continuous blue light. Therefore, the response was probably not saturated by blue light in these pulse experiments. Circadian rhythm of photosynthesis. The photosynthetic rhythm was very stable, at least under the conditions used in these experiments. N o conditions have yet been found that caused the rhythm to run out or disappear. Even after three weeks of measurement, there was no change of period or amplitude. Short pulses of blue light had no effect on the characteristics o f the circadian rhythm (Fig. 2), even when these treatments were close to saturation for the stimulation of photosynthesis. Irradiation with prolonged blue light, if added to continuous red light, caused at least three distinct effects (Fig. 5): (i) the amplitude of the rhythm was reduced relative to that in red light alone, because of the stronger response of photosynthesis to blue light in the troughs of the rhythm; (ii) the maxima of the rhythm o f 02 evolution in blue + red were at different phase positions, compared with the maxima in red light alone; (iii) after the end of
R. Schmid and M.J. Dring: Fast blue-light response of photosynthesis in Ectocarpus
56 blue tight
on:A] off: V
T
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a blue glass filter (% max: 410 nm; halfband width: 1l0 nm; Leitz). The whole setup was protected from other irradiation by a lighttight black wooden box.
Results Initial results showed that the degree o f e n h a n c e m e n t o f red-light-saturated photosynthesis by blue light was very variable, relative to the rates in red light, and so the measurements were extended over longer periods o f time. This lead to the discovery that photosynthesis in red light was rhythmic (Fig. 2). The r h y t h m was circadian, as it continued under c o n s t a n t environmental conditions and displayed a period that was n o t exactly 24 h. In continuous red light, the period was usually a b o u t 23 h. [The circadian times (CT) which are referred to in this paper are measured f r o m the beginning o f the light phase in the culture conditions immediately preceding the experiment.] Pulses o f blue light enhanced the rates o f p h o t o s y n thesis, but the degree o f stimulation depended on the time in the circadian r h y t h m at which blue light was given (Fig. 2). W h e n blue light was given at the peaks o f the r h y t h m the response was small, whereas blue light given in the troughs resulted in a m u c h stronger response. D a r k respiration was n o t rhythmic and was n o t affected by blue-light pulses. Immediately after periods o f photosynthesis, the rate o f d a r k respiration was high, but declined rapidly during the next 30 min and m o r e slowly after that (see Figs. 7 and 8 for examples). The effects o f blue light on CO2 c o n s u m p t i o n were essentially the same as those on O2 evolution. However,
2 min blue light
..
. .......... \.
9
,--5h m
d[02]
black box
!ll I
~\"-..
Q,I >
reservoir [1 []
o
d[O~]/dt ~u
recorder
[e t e c f r ~ ~ , die021/eL red
I
~
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~
I
0oo
I
ooo
Local lime
blue .
Light"TL f i t t e r ' ~ guide
0oo
coLd u Light source
Fig. 1. Experimental setup for continuously recording rates of 02 evolution and CO2 consumption in an open system
Fig. 2. Circadian 0 2 production and CO2 consumption of Ectocarpus in continuous red light and after blue-light pulses of 2 min duration, given at various times of the day. Rates of photosynthetic 02 evolution and CO2 consumption were measured under continuous red light at an irradiance (46 W ' m - E ) that saturated photosynthesis. Arrowheads indicate start and end of red-light irradiation. Pulses of blue light (2 min at 3.2 W 9 m -2) were given at the times indicated by the arrows. Note that the rhythm of CO2 uptake was delayed by about 5 h relative to the rhythm of 02 evolution
R. Schmid and M.J. Dring: Fast blue-light response of photosynthesis in Ectocarpus
E
55
blue light pulse duration: l m i n
,7" CT 17 t
o= o
/
(:u
,v"
/
o"
F~
I=:
A,.'"""
,V" >
.... 9 .......... t...
o
J/
10"6
~"CT 6
. . ..""''"
10"5
photon fLuence
10"4
10.3
[mol-m "2 ]
Fig. 4. Effect of photon fluence of blue light on the magnitude of stimulation of photosynthesis in Ectoearpus. Photosynthetic rates were measured under saturating irradiances of red light (50 W 9m-Z). Blue-light pulses of l-rain duration at various irradiances were given at the peaks (CT 6) and at the troughs (CT 17) of the photosynthetic rhythm. The difference between the maximum of 02 production after blue-light stimulation and the rate in red light before the stimulus was plotted versus the blue-light fluence
QJ
o~ ~a o
0
5 10 time [mini Fig. 3A, B. Kinetics of stimulation of Oz evolution after induction by blue light (BL) in Eetoearpus (A). Insert (13) shows another experiment at a different time scale. Horizontal dottedline shows the rate of 02 evolution before the start of the blue-light stimulus. Blue-light induction was for 1 min at different irradiances (fluences are indicated at the curves). Blue light was given at either the peak (CT 6) or the trough (CT 17) of the photosynthetic rhythm. The increase of 02 evolution in continuous blue light at the highest irradiance is shown for comparison (broken line) the kinetics o f these effects could not be resolved, because o f the slow response of the CO2-electrode system used. Nevertheless, the rhythm of CO2 consumption appeared to be delayed (by approx. 5 h in the experiment shown in Fig. 2) relative to that o f O2 production. However, the delay was variable and could be as little as 1 h. Thus phase differences could be a consequence o f the time the electrode requires to approach an equilibrium recording. Using a higher time resolution (Fig. 3), it was apparent that photosynthesis began to rise 15 to 20 s after the start o f a 1-min pulse o f high-irradiance blue light. After 3 to 5 rain, a maximum was reached, followed by a gradual decline of photosynthetic rate. A difference between the kinetics of stimulation after a blue-light pulse and that in continuous blue light was first visible after 3 rain (Fig. 3, hatched area). The decline after reaching the maximum was not continuous, and a second lower peak was often observed about 20 min after the blue-light pulse (Fig. 3, insert B). This indicated that the enhancement of photosynthesis by blue light consisted of at least two components. When lower irradiances of blue light were used, the onset o f photosynthetic stimulation was progressively delayed (Fig. 3). Irradiances below the blue-light threshold (see next paragraph) were effective only as long as the blue irradiation was applied (not shown). The time taken to respond at different irradi-
ances was the same at the maxima (CT 6) and minima (CT 17) of the rhythm.
Sensitivity to blue light. The dependence of the degree of stimulation of photosynthesis on the blue-light fluence was measured in the troughs (CT 17) and the peaks (CT 6) of the rhythm. Using a 1-min pulse, the irradiance of blue light was changed by means of neutral-density filters. F o r both o f the circadian times, the magnitude o f the response of Oz production was proportional to the logarithm of the blue-light fluence, but the slope o f the line for pulses at CT 17 was greater than that for CT 6 (Fig. 4). The two lines extrapolated towards the same threshold value of fluence (approx. 10-6 mol 9 m-2). The maximum photosynthetic rates after the highest bluelight fluences used were only about two-thirds of those under the influence o f continuous blue light. Therefore, the response was probably not saturated by blue light in these pulse experiments. Circadian rhythm of photosynthesis. The photosynthetic rhythm was very stable, at least under the conditions used in these experiments. N o conditions have yet been found that caused the rhythm to run out or disappear. Even after three weeks of measurement, there was no change of period or amplitude. Short pulses of blue light had no effect on the characteristics o f the circadian rhythm (Fig. 2), even when these treatments were close to saturation for the stimulation of photosynthesis. Irradiation with prolonged blue light, if added to continuous red light, caused at least three distinct effects (Fig. 5): (i) the amplitude of the rhythm was reduced relative to that in red light alone, because of the stronger response of photosynthesis to blue light in the troughs of the rhythm; (ii) the maxima of the rhythm o f 02 evolution in blue + red were at different phase positions, compared with the maxima in red light alone; (iii) after the end of
R. Schmid and M.J. Dring: Fast blue-light response of photosynthesis in Ectocarpus
56 blue tight
on:A] off: V
T
>_
o~ o ,5' "G il~ '....
