ABSIRACI
After 15 minutes, 1 12 of the photosynthate fixed by isolated
chloroplasts of Codium fragile was released to the control medium
The percentage released remained constant over a five hour period.
Chloroplasts incubated in homogenates of Cfragile did not release a
greater percentage of fixed carbon over chloroplasts incubated in
the control medium. Extracts from Elysia hedgpethi that were
starved for 16 days also did not induce the release of photosynthate
from isolated chloroplasts. Further experiments were done using
E hedgpethi that were starved for 22 days and then re-fed Cfragile
until their green color was restored (7 days). Homogenates of the
re-fed slugs again showed no increase in the percentage of fixed
carbon released. In one experiment using extracts from Aplysiopsis
smithi, 52.3% of the photosynthate was released from isolated
chloroplasts, over 2.5 times the percentage released to the control
medium.
INTRODUCTION
The sacoglossan Elysia hedgpethi is known to incorporate
chloroplasts from the siphonaceous alga Codium fragile within the
cells of its digestive diverticula (Greene, 1970b). It has been shown
that the chloroplasts taken up by the animal remain
photosynthetically active after incorporation into the animal
(Greene, 1970a: Greene and Muscatine, 1972). Studies done on Elysia
viridis, another sacoglossan, demonstrate that the chloroplasts of
Cfragile are likewise incorporated into the digestive cells and
continue to photosynthesize at rates comparable to those in the
intact alga (Trench et al., 1973b). Further investigations by Trench,
Boyle and Smith (1973b) indicate that 368 of the photosynthate
fixed by the ingested chloroplasts is released to the animal tissue.
Trench et al. (1973b) found that a greater percentage of fixed carbon
was released from chloroplasts incubated in Eviridis homogenates
when compared with the percentage released from chloroplasts
incubated in simple mineral solution. This suggests that the animal
homogenate contains a factor which modifies the permeability of
the chloroplast envelope and hence, stimulates the release of fixed
carbon.
Gallop (1974) reported that the activity of the photosynthate
release factor was reduced significantly when chloroplasts were
incubated in homogenates from the non-chloroplast bearing tissues
of Eviridis. It has therefore been proposed that the presence of
chloroplasts within the digestive cells of symblotic sacoglossans is
necessary for the synthesis of the release factor (Gallop, 1974).
The studies presented here examine the activity of the
photosynthate release factor in Ehedgpethi when the digestive
cells are lacking chloroplasts versus its activity once chloroplasts
have been restored. To further investigate the relationship
between the photosynthate release factor in sacoglossan
homogenates and symbiotic chloroplasts, a comparative study was
done using the homogenate from Aplysiopsis smithi, a sacoglossan
unable to retain chloroplasts in its tissues.
MATERIALS AND METHODS
Collection and Maintenance of Animals:
E hedgpethi were collected on Cfragile in Santa Barbara. he
animals were maintained without food in a running sea water
aquarium illuminated with a battery of fluorescent lamps. Several
animals were starved for 22 days then transferred to an aerated
beaker containing filtered sea water and fresh Cfragile where they
were allowed to feed for 7 days.
Aplysiopsis smithi were collected from the mud flats at Noss
Landing State Beach (California). These animals were maintained in
a running sea water aquarium on a diet of Cfragile and Bryopsis
corticulans
Collection of C fragile:
Fresh C fragile was collected for use in each experiment from
the rocky intertidal zone at Hopkins Marine Station, Pacific Grove.
Chloroplast Isolation:
Chloroplasts were isolated from intact Cfragile using the
procedure developed by Shephard et al. (1968) with the following
modifications. After resuspending the initial pellet in 10 ml
washing medium, the suspension was spun at approximately 800 g
for 4 minutes. The supernatant was then spun at 1000 g for
10 minutes (Shephard et al., 1968). Äfter discarding the new
supernatant fraction, the pellet was resuspended in 10 ml washing
medium and spun at approximately 800 g for 4 minutes as before.
The chloroplast suspension was again spun at 1000 g for
10 minutes, the supernatant discarded and the pellet resuspended in
the final chloroplast isolation medium containing: mannitol 0.6 M;
potassium dihydrogen orthophosphate 10°9 M; hepes 5 x 10-3M
potassium chloride 10°2 M; magnesium chloride 5 x 10°9 M; adjusted
to ph 7.2.
Preparation of Sacoglossan Homogenate
Whole animals were homogenized with a Dounce homogenizer at
a final concentration of 75 mg wet weight animal/ mi chloroplast
isolation medium (see above). Crude homogenates were then
centrifuged at 41,000 g for 60 minutes (4°) and the supernatant
used for the experimental incubation medium (Gallop, 1974).
Preparation of C fragile Homogenate:
C fragile fronds were minced with a razor blade, homogenized
in a Potter- Elueham homogenizer, then centrifuged at 1700 g for
15 minutes (4°C). The supernatant was the homogenate fraction
tested.
Assay of Photosynthate Release Eactor Actwity
Chloroplasts suspended in isolation medium were incubated
with Nah'4coz (10 uC1/m1) for 2 hours at saturating photosynthetic
irradiance (R. Zimmerman, personal communication). The amount of
radjoactive carbon fixed by chloroplasts incubated in the light was
greater than the amount of radioactive carbon fixed by chloroplasts
incubated in the dark by a factor of 13.7. Chloroplasts were then
collected by centrifugation (12,000 g, 2 min), washed in fresh
isolation medium, resuspended in the desired experimental
homogenate and incubated for 60 minutes at room temperature. In
control experiments, chloroplasts were incubated in isolation
medium at the same final chlorophyll concentration. After 60
minutes, the suspensions were pelleted down (12,000 g, 2 min), the
supernatants acidified and counts of radioactive carbon measured
(see below). The pellets were resuspended in 1 m1 95% hot methano
to extract the remaining photosynthate.
Chlorophyll Estimation:
Ope volume of chloroplast suspension was added to 9 volumes
cold acetone then centrifuged at 3900 g for 10 minutes. The amount
of extracted chlorophyll was calculated by measuring the
absorbance of the supernatant at 663 nm (effrey, 1968). The
reading was then standardized by the following equation:
OD reading (mMolar)/ 75.05/ 1000 x MW Chlorophylla
(893.53 g/mol) x 10 (dilution factor) = (chlorophylD mg/ml.
Estimation of Eixed Carbon:
Supernatant fractions from the incubation media, washings and
extracted pellets were transferred to a 24-well plastic culture
dish. To drive off residual radioactive bicarbonate the solutions
were made 102 in trichloroacetic acid (TCA) and shaken vigorously
for 15 minutes. The acidif ied fractions were mixed with 5 ml
scintillation fluid and the radioactivity analyzed with a Beckman
scintillation counter.
RESULIS
Analysis of Photosynthate Released Over Iime:
Chloroplasts containing radioactive photosynthate accumulated
over 2 hours were resuspended in fresh isolation medium and
incubated at room temperature. At times 15, 30, 45, 60, 90, 120,
180, and 289 (in minutes af ter resuspension) aliquots of the
suspension were centrifuged and the supernatant and pellet
fractions analyzed for radioactivity (figure 1). The results indicate
that the amount of fixed carbon leaked to the suspension medium
reaches a maximum value by 15 minutes with no further increase
over 5 hours.
Effect of C fragile Homogenate on the Release of Photosynthate
from Isolated Chloroplasts.
When chloroplasts containing radioactive photosynthate were
incubated in Cfragile homogenate they did not produce a greater
release of fixed carbon in comparison to chloroplasts incubated in
the control medium (Table 1 ).
Effect of Starved E hedgpethi Homogenate on the Belease of
Photosynthate from Isolated Chloroplasts:
Chloroplasts incubated in the homogenate from E hedgpethi
that were starved for 16 days show no significant difference in the
percentage of fixed carbon released when compared with control
medium incubation (Table 1).
Effect of Re-fed E hedgpethi Homogenate on the Release of
Photosynthate from Isolated Chloroplasts:
The homogenate from animals that were starved of
chloroplasts and then re-fed Cfragile does not enhance the
percentage of fixed carbon released to the incubation medium
(Table 1).
Effect of A smithi Homogenate on the Belease of Photosynthate
from Isolated Chloroplasts.
These results demonstrate a marked increase over control
values in the amount of fixed carbon released when chloroplasts
were incubated in A smithi homogenate (Table 1).
DISCUSSION
When labeled, isolated chloroplasts are incubated in the
supernatant of Cfragile homogenate there is no greater release of
fixed carbon than occurs in control medium incubation. This could
suggest that the mechanism for release of photosynthate in intact
Cfragile is lost when the alga is homogenized. Alternatively, it is
conceivable that no such release factor is synthesized by C fragile
Le., the intrinsic release rate of photosynthate is sufficient to meet
the plant's needs. An informative experiment would be to determine
whether the time course of release of photosynthate from
chloroplasts incubated in Cfragile homogenates is similar to that
in control medium.
The experiments with Ehedgpethi show no evidence for the
presence of a photosynthate release factor in the homogenates of
the starved animals nor in the homogenates of the re-fed animals.
However, previous reports indicate that such a factor is found in the
homogenates of E viridis (Trench et al. 1973b). Evidence for the
presence of such a release factor in Ehedgpethi is presented in a
recent study done by Francis Glaser (unpublished) who reports an
increase in the amount of photosynthate released from chloroplasts
incubated in E hedgpethi homogenates over the values demonstrated
by control medium incubation. From these experiments ! conclude
that the release factor is synthesized only when chloroplasts are
present in the digestive diverticulum of Ehedgpethi and fürther,
that this release factor is broken down when chloroplasts are lost
from E hedgpethi upon starvation.
It is possible that more than 7 days are required for the
release factor to achieve a quantity sufficient to yield à
measureable effect on the release of photosynthate. Yet, in a
presentation by Mart in Kozdrowicki on June 3, 1988 (Symposium of
the China Point Academy of Sciences - XXIV Annual) it was
announced that the chlorophyll content of Ehedgpethi starved free
of chloroplasts and then re-fed on Cfragile for 7 days was
equivalent to the chlorophyll content of animals that had not been
starved. Perhaps there is both a minimum chlorophyll concentration
and a minimum time period required for the activity of the
photosynthate release factor to be regained.
More likely, the release factor was destroyed during incubation.
Glaser (personal communication) reported that the activity of the
release factor was severely diminished after Ehedgpethi
homogenates were incubated for one hour at room temperature, the
temperature at which! conducted my incubations.
Data collected from A smithi homogenates strongly suggest
the presence of a photosynthate release factor. Unlike that of
E hedgpethi homogenates, the release factor of A smithi
homogenates, if such a factor exists, is not destroyed by incubation
at room temperature. However, since Asmithi are incapable of
incorporating chloroplasts (R. Swezey, personal communication) it
may be that this sacoglossan homogenate contains damaging
digestive enzymes which lyse chloroplast envelopes. To test this
the supernatant from chloroplasts incubated in A smithi extracts
should be measured for absorbance at 663 nm. A strong absorbance
would be indicative of chlorophyll in the supernatant and can be
interpreted as lysis of the chloroplasts (Denis Larochelle, personal
communication). In addition, the functionality of chloroplasts
should be tested after incubation in A smithihomogenates i.e., the
amount of labeled carbon fixed by chloroplasts incubated in A
smithi homogenates should be compared to the amount of
radioactive carbon fixed by chloroplasts of the control medium
incubation.
when ! tested the control medium and the medium from re-fed
E. hedgpethi homogenate for chlorophyll the results were negative.
Furthermore, chloroplasts incubated in homogenates of E hedgpethl
showed the same photosynthetic ability as chloroplasts incubated in
control medium (F.Glaser, personal communication). Thus, it is
evident that the release of photosynthate is not due to severe lysis
of the chloroplast envelope when chloroplasts are incubated in
E hedgpethi homogenates.
The incubations of chloroplasts in E hedgpethi extracts should
be repeated at 17 °C to compare with data collected by Glaser and
others. Finally, to help clarify the limitations of using tissue
extracts to study living systems, further investigations should
ipvolve invivo studies on the release of photosynthate from the
symbiotic chloroplasts of E hedgpethi.
SUMARY
The amount of photosynthate released from chloroplasts to the
isolation medium reaches a maximum value in 15 minutes.
Homogenates from starvedE hedgpethi and re-fed Ebedgpethi did
pot demonstrate the ability to release fixed carbon from isolated
chloroplasts, whereas chloroplasts incubated in the homogenate of
A.smithireleased 52.3% of the total labeled photosynthate, over 2.5
times the percentage released in the control incubation.
Acknowledgements:
Iwould like to thank Dr. Robert R. Swezey for his support and
enthusiasm and for the many hours he devoted to this scientific
endeavor. Special thanks to Denis Larochelle for all of his help and
to Dr. David Epel for granting the use of his laboratory, making this
research possible.
12
REEERENCES
Gallop, A. (1974). Evidence for the presence of a' factor' in Elysia
viridis which stimulates photosynthate release from its
symbiotic chloroplasts. New Phytol, 73, 1111.
Greene, R. W. (1970 a). Symbjosis in sacoglossan opisthobranchs:
Translocation of photosynthetic products from chloroplast to
host tissue. Halacologia, 10, 369.
Greene, R. W. (1970 b). Symbiosis in sacoglossan opisthobranchs:
Symbiosis with algal chloroplasts. Malacologia, 10, 357.
Greene, R. W. & Muscatine, L. (1972). Symbiosis in sacoglossan
opisthobranchs: Photosynthetic products of the chloroplasts.
Mar Biol, 14, 253.
Jeffrey, S. W. (1968). Pigment composition of siphonales algae in
the brain coral Eavia. BiolBulL, 135, 141.
Shephard, D. C., Levin, W. B. & Bidwell, R. G. S. (1968). Normal
photosynthesis by isolated chloroplasts. Biochim biophys Acta,
32,413.
Trench, R. K., Boyle, J. E. & Smith, D.C. (1973 b). The association
between chloroplasts of Codium fragile and the mollusc
Elysia viridis: II. Chloroplast ultrastructure and photosynthetic
carbon fixation in Eviridis. Proc R. Soc Lond. B., 184, 63.
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FIGURE 1. RELEASE OF PHOTOSYNTHATE FROM CHLOROPLASTS IN
ISOLATION MEDIUM
Chloroplasts containing photosynthate accumulated over
2 hours were resuspended in fresh isolation medium then incubated
at room temperature. At times 15, 30, 45, 60, 90, 120, 180, and
289 (in minutes after resuspension) aliquots of the suspension
were centrifuged and the supernatant and pellet fractions analyzed
for radioactivity. Figure 1. is a plot of the percentage of fixed
carbon released to the isolation medium at each time point tested.
0-
o I
81
% FIXED CARBON RELEASED
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