Abstract
A factor exists in the tissues of Elysia hedgepethi that stimulates
the release of photosynthetically fixed carbon from isolated Codium
fragile chloroplasts. The factor is labile at high and low temperatures.
non-particular, and predominantly present in the chloroplast-bearing
parapodia of E. hedgepethi. It may have a permanent effect on the release
of photosynthates without damaging the chloroplasts' photosynthetic
capabilities.
Introduction
E. hedgepethi is a saccoglossan mollusc which bores into
siphonaceous algae, extracts its chloroplasts, and incorporates them into
the cells lining its digestive diverticulum. These chloroplasts have been
shown by Trench et al (1973a) to remain photosynthetically active in the
slug. Hinde and Smith (1972) have shown that the chloroplasts may remain
functional more than two months after acquisition. Isolated chloroplasts
of C. fragile, one food source of E. hedgepethi, have been shown to remain
photosynthetically active five days following isolation, although the
activity shows a marked decrease (Trench et al., 1973a).
Trench et al. (1973a) showed that the chloroplasts in E. hedgepethi
release a number of photosyntetic products, primarily galactose, to their
host cells. They estimated that at least 36% of the total carbon fixed by
the chloroplasts is released to the animal. Although different figures have
been reported, up to 10% of photosynthetic products are released from
isolated chloroplasts in a simple mannitol plus salts solution.
It was hypothesized that a factor exists in the homogenate of Elysja
viridis which stimulates this leakage of photosynthetic products (Gallop.
1974). Gallop found that the factor primarily exists in the
chloroplast-bearing tissues of the digestive diverticulum of E. viridis.
The factor is present in the clear supernatent of the homogenate and was
found to be thermolabile, non-particulate, and water soluble. No such
factor is present in a homogenate of C. fragile (Gallop, 1974; Milkowski.
personal communication).
My experiments investigated the properties of this factor which
seems to cause a permanent change in the activity of the chloroplasts
(Trench, 1973). Its thermolability, kinetics of action, reactivity with
various enzymes, and permanent effects on isolated C. fragile
chloroplasts were investigated.
Materials and Methods
Collection and maintenance of material
Elysia hedgepethi were obtained feeding on Bryopsis corticulans at
the Moss Landing Harbor (California), and on C. fragile off the coast of
Santa Barbara, California. The animals were maintained in 9 degree
Celcius running sea-water tanks under normal daily illumination. They
were fed a mixed diet of B. corticulans and C. fragile,
Experimental and dietary C. fragile was obtained daily from Hopkins
Marine Station, Pacific Grove, California.
Isolation of C. fragile chloroplasts.
Chloroplasts were obtained from C. fragile fronds by the procedure
of Shephard et al. (1968), as modified by Trench et al. (1973a).
Determination of the chloroplast concentration
A portion of the chloroplast suspension was added to 90% cold
acetone and centrifuged at high speed 12,000 x g for 10 minutes. Optical
densities were read at 663 nm and the following formula was used:
o.d. 775.05 = schlorophyll al (mMol) (Dawson et al, 1986)
Preparation of E. hedgepethi homogenates
Parapodial tissue of E. hedgepethi was gently blotted, weighed, and
disrupted using a Dounce homogenizer in the chloroplast suspension
mediumf of Shephard et al. (1968). The crude homogenate was
ultracentrifuged at 41000 x g for 60 minutes at 4 degrees Celcjus.
Approximately 70-80 mg fresh weight animal/mi was used in the crude
homogenate.
Experimental conditions and procedure
Chloroplast suspensions containing approximately 40mg
chlorophylliml were incubated with 10 microCuries Nah14co,/mi for 2
+ hereafter referred to as "assay medium"
hours at 16 degrees Celcius. Two flourescent light bulbs at 10 cm were
judged to be sufficient light to stimulate photosynthesis (R. Zimmerman.
personal communication), and a preliminary experiment showed that this
system increased photosynthesis 6.8 x over isolated chloroplasts
incubated in the dark. The suspensions were centrifuged 12,000 x g for 1
minute and washed 2 times.
The various incubations (60 minutes at 16 degrees Celcius) were
under indirect room light. At the end of the incubation, the suspension
was centrifuged, the supernatent removed, and the pellet washed in fresh
medium. Fixed "C was assessed by resuspending the pellet in absulute
methanol and incubating this suspension at 50-60 degrees Celcius for 10
minutes; solids remaining in the extract were removed by centrifugation
before counting radioactivity.
Sample analysis
Excess H'“COg was driven from the supernatents, washings, and
pellet extractions by adding 10% TCA and shaking the samples for 15
minutes. This was found to drive off 99.8% of unfixed 140. Acidified
samples were transferred to scintillation vials containing 5 ml Ecolume
scintillation cocktail and counted on a Beckmann liquid scintillation
counter.
Results
Stimulation of photosynthate release by E. hedgepethi homogenate
A homogenate made of the parapodial tissues of fed specimens of
E. hedgepethi stimulates an increase in the release of photosynthate from
isolated pre-loaded chloroplasts. (Table 1) The time course of release of
photosynthate in the presence of animal homogenate varies from that of
normal assay solution. (Figure 1) A fairly logarithmic curve reflects this
release. However, the release of a chloroplast suspension in assay medium
alone reaches a maximum in 15 minutes and remains constant up to 5
hours. (Milkowski, personal comunication)
lo insure that this result is not an artifact of an increase in the
osmolarity of the solution, l incubated the chloroplasts in 1.0 oSM
solutions. The solutions used in the isolation and resuspension of the
chloroplasts are normally 0.65-0.7 osM. (Shephard et al, 1968) There was
no significant difference between the 0.6 osMand 1.0 osM incubations.
(Table 2)
lo insure that the additional counts in the supernatent were not an
artifact of damage to the chloroplasts due to the animal homogenate.
pre-incubated the suspension in the homogenate. Following this the
suspension was spun down, wached, and incubated in H'4CO»“. (Table 3)
Although the percentage release results varied in the two experiments, the
total fixed "C following the incubation were nearly the same, indicating
ono permanent damage to the photosynthetic capacity of the chloroplasts,
This was also reflected in the findings that no chlorophyll had leaked into
the assay medium of animal homogenate solution following incubations.
(Milkowski, personal communication)
Stability of the translocating factor
As reported by Gallop (1974), the effect of the E. hedgepethi
homogenate on the chloroplast suspension is greatly diminished by a 10
minute, 100 degree Celcius incubation in a boiling water bath. In fact.
found that the releasing activity of the homogenate was reduced to the
level of assay solution alone. (Table 4)
1o fürther examine the stability of the translocating factor,
incubated the homogenate at room temperature for 1.5 hours. This
produced a marked decrease in the translocation-enhancing ability of the
homogenate. (Table 5) In addition, an 8.5 hour, 4 degree Celcius incubation
produced similar results. (Table 6)
In an attempt to characterize this releasing factor, I incubated the
E. hedgepethi homogenate for one hour at room temperature in 0.1 ma/ml
pronase to determine whether the factor is a protein. As a control,
incubated the tissue homogenate and pronase against tissue homogenate,
assay soution, and assay solution and pronase. (Table 7) All four groups
showed greater than normal counts. In fact, assay solution containing
pronase showed greater release than assay solution.
Discussion
My experiments have shown interesting characteristics of the
translocating factor described by Gallop (1974). This factor is
water-soluble, present in the high-speed supernatent of E. hedgepethi
homogenates, and exctremely thermolabile. It causes a reproducable
increase in photosynthate release from pre-loaded chloroplasts. It does
not reduce the photosynthetic capacity of the chloroplasts, yet it may have
a permanent effect on the permeability of their membrane for certain
forms of fixed carbon, as was evidenced in the pre-incubation
experiments.
The time course of photosynthate release is interesting in its
comparison to the findings of Milkowski (personal communication) that
release in normal asay solution reached a maximum within 15 minutes,
The curve l obtained may reflect two different hypotheses. First, it may
indicate that an upper level of release exists, past which the homogenate
has no effect. Ötherwise, it may be yet another reflection of the
thermolability of the factor. In my 1.5 hour, room temperature incubation.
the reactivity of the factor was greatly diminished. My logarithmic curve
could indicate that as the factor breaks down, the rate of reactivity
decreases. The 100 degree Celcius incubation of the E. hedgpethi
homogenate also supports this hypothesis. The factor appears
thermolabile at high and low temperatures in very short periods of time.
Although it is not conclusive whether pronase has an effect on the
stability of this factor, it is clear that pronase reduces the selective
permeability of the envelope of the extremely hearty C. fragile
chloroplast. This reason, in addition to its thermolability, it was difficult
to characterize the factor. The factor may be a proteolytic enzyme itself.
lo investigate this possibility, addition of a variety of enxyme inhibitors
to the homogenization medium may be informative,
The hypothesis that this factor may be an enzyme was stressed in
my series of experiments. This is because the parapodial tissue used to
obtain these data contains a large amount of the digestive diverticulum of
the animal. The factor may be a variation of an enzyme used by other
saccoglossums to digest chloroplasts. That the factor is present in the
digestive tissues may explain its thermolability. When the lysosomes
ruptüre during homogination, the digestive enzymes they release may
destroy the translocating factor.
In order to expand upon and improve these experiments, a few points
should be considered. Increasing the number of times that a chloroplast
suspension is pelleted and resuspended seems to lower the activity of the
chloroplasts. The chloroplasts should be harvested from C. fragile
gathered the day of the experiment in order to maximize the retention of
their photosynthetic capacity. Also, E. hedgepethi homogenate should be
stored in a -80 degree Celcius freezer because the mucus and solutes tend
to lower the freezing point of the homogenate, leaving the homogenate
partially active. Finally, the time that the homogenate spends out of the
freezer should be minimized. Further experiments, including the addition
of protease inhibitors, could include forcing the homogenate through a
dialysis membrane to determine the size of the factor. This may be
difficult due to the presence of mucus, which may clog the dialysis pores
or make the factor stick to the membrane.
Acknowlegements
Iwould like to thank Rob Swezey and Denis Larochelle for their time and
advice, without which this project would not have been possible.
References
Dawson, M. C., Daphne C. Elliot, William H. Elliot, and Kenneth M. Jones,
1986. Data for Biochemical Beasearch. Oxford University Press, New
York.
Gallop, Angella. 1974. Evidence for the presence of a ’factor in Elysia
viridis which stimulates photosynthate release from its symbjotic
chloroplasts. New Phytol. 83: 445-450.
Hinde, R. and D. C. Smith. 1972. Persistence of functional chloroplasts in
Elysia viridis (Opisthobranchia, Saccoglossa). Nature New Biology
239: 30-31.
Shephard, D. C., W. B. Levin, andR. G. S. Bidwell. 1968. Normal
photosynthesis by isolated chloroplast. Biochim. biophys. Acta, 32;
413-420.
Trench, R. K., J. Elizabeth Boyle, and D. C. Smith. 1973a. The association
between chloroplasts of Codium fragile and the Mollusc Elysia
viridis: I. Characteristics of isolated Codium chloroplasts. Proc. R.
Soc. Lond. A. 184: 51-61.
Trench, R. K., J. Elizabeth Boyle, and D. C. Smith. 1973b. The association
between chloroplasts of Codium fragile and the Mollusc Elysja
viridis: II. Chloroplast ultrastructure and photosynthetic carbon
fixation in E. viridis. Proc. R. Soc. Lond. A. 184: 63-81
TABLE 1: Effect of E. hedgepethi homogenate on pre-loaded chloroplasts
% of total fixed 170
Incubation Medium (n)
released into medium
Parapodial tissue extract (3)
25.7 f 1.5
Assay medium (3)
12.3 + 1.5
TABLE 2: Effect of osmotic changes of assay solution on photosynthate
translocation
Incubation Medium (n)
% of total fixed
released into medium
Assay medium (0.6 oSM), (2)
6.6 + 0.0
Assay medium (1.0 OSM), (2)
9.7 + 0.1
TABLE 3: Effect of pre-incubation with E. hedgepethi homogenate on
photosynthate translocation
% of total fixed 4
Incubation Medium (n)
Total cpm
released into medium
released
Expt 1 Expt 2
Expt 1 Expt 2
Parapodial tissue extract (2)
78.4 83.2
2.5e4 1.8e4
+4.3 + 1.9 + 2.0e3 + 2.0e3
12.0e3
Assay medium (2)
86.911.2 68.940.7 2.7e4 2.3e411.0e3
TABLE 4: Effect of 100 degree Celcius, 10 minute incubation on the
stability of E. hedgepethi homogenate
% of total fixed 140
Incubation Medium (n)
released into medium
Parapodial tissue extract, incubated (2)
6.6 + 0.8
Parapodial tissue, not incubated (2)
28.0 + 1.3
TABLE 5: Effect of pronase on the stability of E. hedgepethi homogenate
% of total fixed 140
Incubation Medium (n)
released into medium
Parapodial tissue extract (2)
42.0+ 2.0
Parapodial tissue extract + pronase (2)
50.7 + 0.7
Assay medium (2)
30.9 + 2.8
Assay medium + pronase (2)
46.0 + 0.5
TABLE 6: Effect of 20 degree Celcius, 90 minute incubation on the
stability of E. hedgepethi homogenate
% of total fixed 4
Incubation Medium (n)
released into medium
Parapodial tissue extract (4)
12.0 + 0.9
Assay medium (4)
11.9 + 2.2
TABLE7; Effect of 4 degree Celcius, 8.5 hour incubation on the stability of
E. hedgepethi homogenate
% of total fixed 170
Incubation Medium (n)
released into medium
9.2+ 0.7
Parapodial tissue extract (4)
8.1+1.2
Assay medium (2)
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