Photosynthetic Rhythms in I. flaccida
Harris
Page 2.
Abstract
Endogenous circadian rhythms in photosynthesis are
described for the first time in the red alga Iridaea
flaccida (S. & G.)Silva. These rhythms persist under
constant conditions for three days showing a 36% difference
between maximum photosynthetic rates at midday and minimum
rates at night. Respiration is constant over the circadian
cycle. Changes in the initial slopes of photosynthesis¬
irradiance curves at different circadian times suggest
the involvement of the light reactions in this rhythmicity.
Photosynthetic rates of I. flaccida are comparable in air
and in water when the thallus is fully hydrated. A small
amount of desiccation enhances the photosynthetic rates,
while continued desiccation results in a marked decrease.
Photosynthetic Rhythms in I. flaccida
Harris
Page 3
Introduction
Though circadian rhythms in algae have been described,
only a few algal species have been examined. The green
alga Acetabularia (Terborgh, 1967), the dinoflagellate
Gonyaulax polyedra (Hastings et al., 1961), and the diatom
Phaeodactylum tricornutum (Livingston, 1964) all have
circadian photosynthetic rhythms. These rhythms have
been well documented and many interesting techniques for
their measurement were used. For example, Prezelin and
Sweeney (1977) described diurnal periodicity in the photo¬
synthetic capacity of G. polyedra in terms of variations
in the initial slopes of photosynthesis-irradiance (P-I)
curves. The absence of changes in either pigment concen¬
trations or in cell numbers throughout the time-course of
the rhythmic changes in photosynthesis indicated a photo¬
synthetic rhythm.
Despite the documentation of such rhythms in some
algae, there has been no investigation to date of circadian
photosynthetic rhythms in red algae, although the effects
of environmental factors such as light, temperature, and
exposure to air have been studied (Kanwisher, 1966; Hodgson,
in prep; Johnson et al., 1974). Johnson and his coworkers
reported that Iridaea flaccida has a higher photosynthetic
rate in air than in water, and that the photosynthetic rate
increases after some degree of desiccation. However, the
potential effects of endogenous circadian rhythmicity in
Photosynthetic Rhythms in I. flaccida
Page 4
Harris
photosynthesis and respiration were not adequately considered
in that study. The studies presented here examine endogenous
rhythms in photosynthesis of Iridaea flaccida. In addition
desiccation as a consequence of periodic exposure during the
tidal cycle was studied to determine its effects on the
rates of photosynthesis.
Materials and Methods
Iridaea flaccida was collected from a relatively
protected location in Monterey Bay at Mussel Point, Pacific
Grove, California. Three vertical zones were designated as
High, +3 to +4 ft, Middle, +2 to +3 ft, and Low, +1 to +2 ft
above mean lower low water. The algae were collected at low
tide, then kept in clear plastic bins filled with fresh sea
water. These bins were placed in a constant temperature
room at 15°C, and maintained under dim constant light at
irradiances from 25 to 30 uE/m/s as provided by cool white
fluorescent lamps. The algae were incubated for at least
24 hours under these conditions before being used for ex¬
periments. Preliminary studies showed that different portions
of the thallus have different photosynthetic capabilities.
Only middle portions of the thallus were used, and I. flaccida
which had been grazed upon or which had reproductive struc¬
tures were avoided.
Photosynthesis and respiration of algae submerged in
sea water were measured with a Gilson constant pressure
differential respirometer. This procedure has been documented
Photosynthetic Rhythms in I. flaccida
Harris
Page 5
by Umbreit et al. (1972). The sidearm of each Gilson
flask contained a standard bicarbonate buffer solution
to keep carbon dioxide levels at normal levels throughout
the duration of each experiment (Pratt, 1943). One gram
fresh weight samples were placed into 10 ml fresh sea water
and allowed to equilibrate for 30 min., a period found to
be necessary before a maximum photosynthetic rate was
achieved. Readings were taken every 15 min. for one hour.
Constant illumination of 195 pE/m/s was supplied by flood¬
lights in the respirometer. Experiments requiring a range
of irradiances used layers of neutral density screens over
individual Gilson flasks. The algal samples were sub¬
sequently dried to a constant weight in an 80°C drying
oven. Similar procedures were used for respiration meas¬
urements made in darkness except the sidearm contained
0.4 ml of 30% KOH to absorb carbon dioxide.
Photosynthesis in air was measured using a Beckman
Model 215 Infared Gas Analyzer. I. flaccida was kept
under the same preincubation conditions as described above.
Before placing 10.0 g fresh weight samples in the flow¬
through sample chamber, they were shaken ten times to re¬
move excess water. Carbon dioxide changes were recorded
on a Beckman stripchart recorder. The temperature inside
the flow-through chamber was kept at 15°C with refrigerated
water baths. The light source was an overhead 500 watt
tungsten-iodide lamp combined with neutral density screens
to achieve a range of irradiances from 17.5 to 1400 uE/m2/s.
After photosynthetic measurements, these algal samples
Photosynthetic Rhythms in I. flaccida
Harris
Page 6
were dried to constant weight.
Different levels of desiccation were achieved by placing
samples on strips of aluminum foil and allowing them to
desiccate on the laboratory bench at 22°C. The extent
of desiccation is expressed as a ratio of the desiccated
weight to the initial fully hydrated weight, and reported
as per cent.
Results
A preliminary 24 h study of the photosynthetic rates
of Iridaea flaccida revealed a well-defined endogenous
circadian rhythm in algae collected from the Low and Middle
zones. I. flaccida from the High zone, however, did not
show this photosynthetic rhythmicity. A 72 h experiment
supported the early observation and indicated that this
rhythm persists with only slight damping. See Figures 1A,
1B, and 1C. A 36% difference in photosynthetic rates
between maximum values in the subjective day and minimum
values in the subjective night was measured during the
first 48 h for the Middle and Low zones. Maximum photo¬
synthesis was reached between 1100 and 1300 h, while
photosynthetic rates were lowest between 0200 and 0500 h.
The respiratory rate did not show an endogenous rhythm
nor great variability throughout the day. See Figure 2.
Photosynthetic rates in air and in water were measured
over a range of irradiances from 17.5 to 290 pE/m/s.
Photosynthesis-irradiance (P-I) curves were constructed
Photosynthetic Rhythms in I. flaccida
Harris
Page 7
from the data at distinct circadian times over a 24 h
period. See Figures 3 and 4. Photosynthetic rates at
light saturation (Pmax) were estimated by taking the mean
of the last five data points. Table 1 presents these
values determined at different circadian times (ct) where
ct O is local dawn (0600). Pmax values at ct 6 were con¬
sistently higher than at other circadian times, corresponding
to the diurnal changes in Pmax reported in the 72 h study.
Initial slopes were determined by linear regression analyses
which assumed a zero intercept and utilized photosynthetic
values between O and 130 uE/m2/s. Table 1 gives values for
the initial slopes of these P-I curves with the corresponding
standard errors and correlation coefficients. The initial
slopes of the P-I curves, as calculated by least squares
linear regression (Sokal & Rohlf, 1969), were directly
proportional to the circadian time and the magnitude of
Pmax values.
A desiccation study revealed that photosynthetic rates
of I. flaccida increased during desiccation to 80% of its
fully hydrated weight. See Figure 5. After this degree
of desiccation was reached, the rates of photosynthesis
decreased precipitously.
Discussion
An endogenous photosynthetic rhythm in the red alga
Iridaea flaccida has been demonstrated. Samples from the
Low intertidal zone provided the clearest evidence of this
Photosynthetic Rhythms in I. flaccida
Harris
Page 8
rhythm. These fluctuations in photosynthetic rate coincide
with the natural light-dark regime. There is clearly
anticipation of cyclic changes in irradiance throughout
the 24 h day as indicated by the persistence of these
rhythms in the absence of external cues. I. flaccida
from the Middle zone also exhibited an endogenous circadian
rhythm with a lesser degree of persistence. I. flaccida from
the High intertidal zone did not have a clear rhythm in the
preliminary 24 h experiment nor in the 72 h experiment. The
photosynthetic rates seemed to increase and decrease with¬
out regard to either the natural light-dark regime or the
tidal cycle, although superimposition of the two would be
extremely difficult to interpret. The High zone is exposed
approximately 16 h/day as compared to an average 11.3 h/day
for the Middle, and 6.8 h/day for the Low. This longer
period of exposure may have some deleterious effects on
photosynthetic rates and may result in fluctuating Pmax
values. Further investigation of the effects of exposure
will be necessary before any firm conclusions can be drawn
about the photosynthetic fluctuations of I. flaccida in the
High intertidal.
Rates of respiration over a 24 h period indicated that
the respiration was relatively constant for all three zones.
Therefore, variations in respiratory rates cannot account
for the diurnal periodicity in photosynthesis.
Photosynthesis-irradiance experiments were conducted to
further elucidate aspects of the photosynthetic rhythm. These
experiments gave estimates for maximum photosynthesis (Pmax)
Photosynthetic Rhythms in I. flaccida
Harris
Page 9
at different circadian times. Both in air and in water
values
photosynthetic measurements gave increased P,
at midday and decreased Pmay
values in the late afternoon
and evening. These changes in the values of Pyax in the
P-I studies are consistent with the endogenous photosynthetic
rhythmicity observed in the 72 h experiment.
The changes in the initial slopes of the P-I curves
suggest involvement of the light reactions in photosynthetic
rhythmicity as was found for the dinoflagellate Gonyaulax
polyedra (Prezelin and Sweeney, 1972 a, b). They hypothe¬
sized that the change they observed in initial P-I slopes is
due to some conformational change in the thylakoid membranes
with which the light reactions are associated. Steeper
initial slopes indicate more efficient photosynthetic
abilities at particular circadian times. The photosynthetic
measurements in air reported here clearly show this rela¬
tionship. The initial slopes of the P-I curves are pro¬
gressively steeper from ct O to ct 6, and then decrease
by ct 12. Thus, the photosynthetic efficiency is greater
at ct 6 than at other circadian times.
Photosynthetic
measurements in water yielded similar results. This dif¬
ference in initial slopes suggests that the mechanism
controlling the endogenous photosynthetic rhythm in Iridaea
flaccida resides in components of the photosynthetic light
reactions. Differences in initial slopes would not be
anticipated if the rhythm was due to some circadian change
in the dark reaction components since initial photosynthesis
Photosynthetic Rhythms in I. flaccida
Harris
Page 10
in the light-limiting irradiances would proceed at a rate
independent of circadian times.
These P-I curves also give some indication as to
whether photosynthetic rates in air are comparable to
those in water. A conversion from mg Co/g dry wt./h to
ul O/g dry wt./h was calculated so that direct comparisons
could be made. Photosynthetic rates in air are only
slightly less than those in water. This is in direct
contrast to the findings by Johnson et al. (1974), who
found photosynthetic rates in air 2.94 times greater than
photosynthetic rates in water for I. flaccida.
The effects of desiccation on photosynthetic rates
were measured for I. flaccida from the Middle zone. Results
indicate that a small amount of drying enhances net photo¬
synthesis. This effect was prominent when the algal
samples were desiccated to approximately 80% of their
fully hydrated weights. Desiccation in excess of this
amount caused marked reductions in photosynthetic rates.
I. flaccida is exposed many hours each day and the increased
photosynthetic rates during exposure are of interest.
Extreme desiccation is not a problem for I. flaccida in
the Middle and Low zones because it retains sea water on
the underside of the thallus under natural conditions. The
High zone is exposed for longer periods, however, and
damage due to desiccation may be of greater significance
for algae in this zone. Further studies will be directed
toward measuring the relationship between photosynthetic
Photosynthetic Rhythms in I. flaccida
Harris
Page 11
rates and desiccation under natural conditions.
Acknowledgments
I wish to thank Dr. Lawrence Harding, Dr. I.A. Abbott,
and Dr. John Phillips for their encouraging support and
occasional pat on the back. I am also grateful to Lyn
Hodgson for her help and to Robin Burnett for assistance
in the statistical analyses of the P-I data.
Photosynthetic Rhythms in I. flaccida
Page 12
Harris
Literature Cited
Hastings J.W., L. Astrachan, B.M. Sweeney. 1961. A persistent
daily rhythm in photosynthesis. J. Cell
Physiol. 45: 69-76.
Johnson W.S., A. Gigon, S.L. Gulmon, H.A. Mooney. 1974.
Comparitive photosynthetic capacities of
intertidal algae under exposed and submerged
conditions. Ecology 55: 450-453.
J.W. 1966. Photosynthesis and respiration in some
Kanwisher,
seaweeds. p. 407-420. In H. Barnes (ed.). Some
contemporary studies in marine science. Allen &
Unwin, London.
Livingston L., Palmer J.D.,F.D. Zusy. 1964. A persistent
diurnal rhythm in photosynthetic capacity.
Nature 203: 1087-1088.
Prezelin B.B.,B.W. Meeson, and B.M. Sweeney. 1977. Charac¬
terization of photosynthetic rhythms in marine
dinoflagellates. Plant Physiol. 60: 384-387.
Prezelin B.B., B.M. Sweeney. 1977. Characterization of
photosynthetic rhythms in marine dinoflagellates.
Plant Physiol. 60: 388-392.
Sokal R.R., F.J. Rohlf. 1969. Regression. pp.407-417. In
R. Emerson, D. Kennedy, R. Park (ed.). Biometry
W.H. Freeman & Co., San Francisco, 776p.
Photosyntheti
flaccida
Page 13
Harris
Terborgh J., G.C. Mcleod. 1967. The photosynthetic rhythm
of Acetabularia crenulata. I. Continuous
measurements of oxygen exchange in alternating
light dark regimes and in constant light at
different intensities. Biol. Bull. 133: 659-669.
R.H. Burris, and J.F. Stauffer. 1972.
Umbreit, w.w.,
Constant pressure manometry. pp. 100-110.
Manometric & Biochemical Techniques, Burgess
Publishing Co., Minneapolis. 387p.
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Photosynthetic Rhythms in I. flaccida
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Figure Legends
Figures 1A
Photosynthesis (Pmax) of Iridaea flaccida
1B
measured at light saturation for algae pre-
10
incubated and maintained in dim constant light.
Alternate dark and light bands represent sub¬
jective night and day, respectively. Values
are the means of triplicate samples with vertical
bars representing standard errors. Samples
were collected from the A) Low, B) Middle, and
C) High intertidal zones.
Figure 2
Respiration of Iridaea flaccida from Low, Middle,
and High intertidal zones. Algal samples were
preincubated and maintained in dim constant light
but measurements were taken under total dark
conditions. Alternate light and dark bands
represent subjective day and night, respectively.
Values are the means of triplicate samples with
vertical bars representing standard errors.
Figure 3
Photosynthesis-irradiance (P-I) curves for
Iridaea flaccida in water. Photosynthetic
measurements were made at distinct circadian
times beginning at ct O or subjective dawn (0600).
Photosynthetic Rhythms in I. flaccida
Harris
Page 16
Photosynthesis-irradiance (P-I) curves for
Figure 4
Iridaea flaccida in air. Photosynthetic
measurements were made at distinct circadian
times beginning at ct O or subjective dawn
(0600 h). A factor of 509 was calculated
assuming a photosynthetic quotient of 1.0,
for conversion from mg Co/g dry wt./h to
ul O/g dry wt./h for direct comparisons of
in air and in water photosynthesis.
Figure 5
The relationship between extent of desiccation,
as measured by per cent wet weight, and photo¬
synthesis for Iridaea flaccida.
.
PHOTOSYNTHESIS (uI O9/g dry wt./h)
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