Download The metabolism of photosynthetically fixed

Biological Journal of the Linnean Society, 10: 329-342. With 2 plates
September 1 9 7 8
The metabolism of photosynthetically fixed
carbon bv isolated chloroplasts from Codium
fragile (Chlorophyta: Sipionales) and by
Elysia viridis (Mollusca: Sacoglossa)
ROSALIND HINDE
Department of'Agrictiltirru1Science, University o j Oxjbrd"
Accepted f o r publication August I977
The metabolism of photosynthetically fixed 14C by isolated chloroplasts from Codiurn fmgile is
compared with that by Elysia viridis (which contains C. fragile chloroplasts). There are marked
differences between t h e two in the formation and subsequent metabolism of both soluble and
insoluble products. Less than 6% of the carbon fixed by the isolated chloroplasts during a
1 5 min pulse of l4C-bicarbonare in the light is released into the medium over t h e succeeding
2 4 h. During photosynthesis glycollate and glucose monophosphate are the only labelled
compounds released; after the pulse very little glycollate is released and over 2 4 h only glucose
monophosphate and an unidentified compound are found in t h e medium. In E. viridis
photosynthetically fixed carbon can b e recovered from compounds of all major classes found in
animals. Soon after the pulse, hexoses are the most heavily labelled compounds, b u t two hours
later amino acids are more heavily labelled than hexoses. The unidentified compound is not
found in t h e animals. E. viridis can absorb and metabolize exogenous glycollate and glucose.
Earlier authors' suggestions that glucose is the compound which moves from the chloroplast t o
t h e animal cell are discussed, and it is proposed that both glucose monophosphate and glucose
are formed outside the chloroplast from triose phosphate exported from it.
KEY WORDS: - Mollusca - Sacoglossa - Chlorophyta - Siphonales - chloroplast symbiosis
- carbon metabolism - isolated chloroplasts.
CONTENTS
. . . . . . . . . . . . . . . . . . .
Introduction
Materials and methods
. . . . . . . . . . . . . . . .
General methods
. . . . . . . . . . . . . . .
Experiments with isolated chloroplasts
. . . . . . . . .
Experiments with Elysia viridis
. . . . . . . . . . .
Metabolism of a pulse of fixed 14Cb y E. viridb
. . . . .
Metabolism of exogenous glucose and glycoUic acid b y E. viridb
Results
. . . . . . . . . . . . . . . . . . . .
Metabolism of a pulse of fixed "C by isolated chloroplasts of C.fragi7e
Metabolism of a pulse of Fixed ''C by E. viridis
. . . . . . .
Clycollic acid
. . . . . . . . . . . . . .
Glucose
. . . . . . . . . . . . . . . .
Discussion
. . . . . . . . . . . . . . . . . . .
Acknowledgements
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References
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Present address: School of Biological Sciences. Botany Building, University of Sydney, Australia 2006.
329
3 30
R HINDE
INTRODUCTION
Photosynthetically fixed carbon plays an important role in the nutrition of
the marine mollusc Elysia uiridis (Montagu). Hinde & Smith (1975) showed
that when E. uiridis are starved in the light they lose weight much more slowly
than when they are starved in the dark, and that the rates of photosynthesis
under the conditions of these experiments are of the right order to account for
the observed weight differences.
This paper describes a preliminary study of how the animal metabolizes
photosynthetically fixed carbon released from its “symbiotic” chloroplasts.
Previous experimental investigations of E. uiridis have been described by
Trench & Smith (1970), Trench, Boyle & Smith (1973a, b, 1974), Gallop
(1974) and Hinde & Smith (1972, 1975). These show that the animal contains
undamaged chloroplasts from its food plant (Codium fragile (Sur.) Hariot) in
the cells which line its digestive diverticula. These chloroplasts are able to fix
carbon photosynthetically at rates comparable to those of the intact alga; at
least 36% (and probably a good deal more) of the carbon they fix in
photosynthesis leaves the chloroplasts and enters the cells of the animal. The
chloroplasts can remain active in the animal for three months at least, but their
photosynthetic activity does decline during this time. Their apparent inability
to synthesize chlorophyll, ribulose biphosphate carboxylase and chloroplast
membrane proteins in the absence of the plant cell nucleus (Trench et al.,
1973b; Trench & Ohlhorst, 1976), probably accounts for this loss of activity.
Three types of experiment are described in this paper.
(1) Experiments concerning the metabolism of a pulse of fixed 14C by
isolated chloroplasts of C. fragile: the chloroplasts were exposed to a 15 min
‘pulse’ of NaH14C03, and the fate of the photosynthetically fixed 14C was
followed for a ‘chase’ period of 24 h. The objects of these experiments were:
(i) t o gain further information about the compounds released from the
chloroplasts, during and after a period of photosynthetic activity and (ii) to
provide a control for comparison with the experiments in which the
metabolism of a pulse of fixed 14C was followed in intact animals over a period
of 96 h. (It was not possible to study the isolated chloroplasts past 24 h, since
although they can still photosynthesize after 4 days in isolation, preliminary
experiments showed that after about two days some of the plastids lysed,
making interpretation of results very difficult. 1
(2) Experiments concerning the metabolism of a pulse of fixed 14C by
Elysia uiridis: the animals were exposed to a 15 min pulse of NaH14C03 in the
light and then kept in unlabelled sea-water for chase periods of up t o 96 h. At
intervals samples were taken and extracted in one of two ways. One method
(extraction with methanol followed b ammonium sulphate and then potassium hydroxide) separated the fixed YC into soluble and insoluble fractions,
and allowed direct comparison with the experiments on isolated chloroplasts.
The other (extraction with trichloracetic acid and ethanol) separated the
components of the tissues into chemical classes, as indicated in Table 3.
(3) Experiments concerning the metabolism of exogenous glucose and
glycollic acid by E. uiridis: glycollic acid is the principal radioactive compound
released to the medium when chloroplasts isolated from C. fragile are allowed
to fix 14C-bicarbonate photosynthetically in a simple suspension medium.
METABOLISM OF PHOTOSYNTHETICALLY FIXED CARBON
331
When the chloroplasts are suspended in a homogenate of E. viridis movement
of fixed carbon out of the chloroplasts is greatly increased, and both glucose
and glycollic acid are released (Trench et al., 1973b; Gallop, 1974). If these
compounds are the ones which pass from the chloroplasts to the cells of the
animal in the intact association, then they must be readily metabolized by the
animal, as movement of fixed carbon between the partners takes place rapidly
and on a large scale. Glucose is readily metabolized by all animals, including
molluscs; however, the role of glycollic acid and ;he importance of the
glyoxyllate cycle in molluscan metabolism do not appear to have been
investigated. Glucose and glycollic acid were therefore supplied to the intact
animals via the external medium, to confirm that the animals can metabolize
them. In this way the metabolism of single labelled compounds could be
studied; during photosynthesis a large number of labelled compounds are
formed, and it is not possible to follow the fate of one of these at a time.
MATERIALS AND METHODS
General methods
C. fragile and t'. viridis were collected at Bembridge, Isle of Wight, and
maintained in the laboratory in aquaria of aerated sea-water a t 7"-8"C.
Illumination of approximately 2700 lux at bench level was provided for 12 h in
each 24 h period by banks of fluorescent tubes. Incubations with NaHI4CO3
(supplied by the Radiochemical Centre, Amersham) were carried out in
McCartney bottles in a 'Perspex' water bath lit from below by fluorescent tubes
and kept at 19"C. The light intensity at the base of the bottles was 23,680 lux.
Experiments with isolated chloroplasts
Chloroplasts were isolated from C. fragile by the method of Trench et al.
(1973a). The isolated chloroplasts were taken up into the suspension medium
without added carrier bicarbonate or carbonate (Trench et al., 1973a); aliquots
(2 ml each) were pipetted into McCartney bottles and 'pre-incubated' in the
water bath for 5 to 1 5 min. Then NaH14C03 (final concentration 10 pCi.ml-')
was added and after 1 5 min incubation each sample was centrifuged. The
medium (medium 1) was removed and stored in a deep freeze. The pellet was
resuspended in 2 ml of suspension medium (with no NaHI4CO3) and kept at
room temperature under normal room lighting. Thus the samples which were to
have a chase period of 2 or 6 hours were kept in the light throughout the chase;
those with a chase period of 24 hours were left in darkness for approximately
13 hours overnight (the chase periods all began between 11.00 and 11.30 h).
After 0, 2, 6 and 24 hours, samples were centrifuged and the medium
(medium 2) was frozen. The chloroplasts were resuspended in 2 ml cold
absolute methanol and the suspension was kept at 4°C until the chloroplasts
were quite white. After centrifugation, 0.5 ml of the methanol extract was
diluted with acetone and used for determination of chlorophyll (Jeffrey,
1968). The pellet was then extracted successively for 15 min each with 80%
methanol (at 45" C), with water, with 5% (NH,), SO4 and with KOH (each at
100°C). Radioactivity of aliquots of medium 1, medium 2 and each of the
3 32
R HlNDE
extracts was assayed as outlined above. To identify the soluble products of
photosynthetic carbon fixation, and the compounds which had left the
chloroplasts, the absolute methanol extract (or in some cases the bulked
absolute methanol, 80% methanol and water extracts) and samples of medium
1 and medium 2 were chromatographed in two dimensions by the method of
Bassham & Calvin (1957). Radioactive compounds were localized by autoradiography on 'Kodirex' no screen X-ray film. Radioactivity in the spots was
determined using an end-window Geiger-Muller counter. Single spots were
eluted from the two-dimensional chromatograms and identified by running
them against known compounds in one or more of the following solvents: (1)
picric acid : tert-butanol : water (2 : 80 : 20, w/v/v) for phosphate esters
(Loughman & Martin, 1957); (2) ethanol : 0.880 ammonia : water
(160 : 5.6 : 34.4 v/v/v) and (3) phenol : water ( 3 : 1 w/v) in an atmosphere of
ammonia (both modified from I. Smith, 1960) for organic and amino acids; (4)
ethyl methyl ketone : acetic acid : water : pyridine (70 : 2 : 15 : 15) (von Holt
& von Holt, 1968) for carbohydrates and organic acids.
Experiments with E. viridis
Metabolism of a pulse of fixed 14C by E. viridis
E. viridis were pre-incubated in sea-water for 15 min, then incubated for
1 5 min in sea-water containing NaH14C03 (20 pCi.ml-'). The slugs were then
transferred to unlabelled sea-water and kept at 7"-8"C with illumination of
approximately 2700 lux (for 12 h per 24 h day) until the end of the chase
period. Immediately after the pulse of NaH14C03, and 2, 6, 24, 48 and 96 h
later two slugs were removed and extracted with the series of solvents used for
the isolated chloroplasts; the tissues were treated with each solvent for 1 h.
Scintillation counting and chromatography were carried out as described for
the experiments with isolated chloroplasts. This made it possible to carry out
chlorophyll determinations and allowed comparisons to be made with the
results obtained with isolated chloroplasts. As well, after 96 h (expt. 1) and
after 0, 24, and 96 h (expt. 2) another two slugs were extracted by the method
of Lenhoff & Roffman (1971, method 11) (modified by using centrifugation to
separate soluble from insoluble material at each stage of the fractionation of
the tissues). Briefly, this method involves homogenizing the tissue in cold 5%
trichloracetic acid (TCA), and treating both the resulting solution and the
insoluble residue with hot absolute ethanol. The material which is insoluble in
both cold TCA and hot ethanol is then treated in hot TCA (5%). Aliquots of
each extract were assayed for radioactivity as described under General
Methods.
Metabolism of' exogenous glucose and glycollic acid by E. viridis
Slugs were incubated for 6 h in 2 ml sea-water to which 14C-glucose or
''C-glycollate (Radiochemical Centre, Amersham) had been added. The
incubations were carried out in the dark at room temperature (18.5" to 21°C).
In the first experiment one slug was used for each treatment; 2.0pCi of
glucose-U-14C were used for the glucose treatment, and 1.4 pCi of sodium
glycollate for the glycollic acid treatment. In the second experiment two slugs
were used for each treatment, and the amount of the labelled compound was
METABOLISM O F PHOTOSYNTHETICALLY FIXED CARBON
333
doubled in each case. In both experiments the medium consisted of 2 ml
sea-water plus the compound to be tested. The stated specific activities were
2-4mCi.mM-' for the glucose and 16.1mCi.mM-' for the glycollate. In each
experiment samples of the media were taken for scintillation counting
immediately before and immediately after incubation; after incubation, the
slugs were washed well in fresh sea-water, killed in hot absolute methanol and
extracted in methanol, (NH,), SO4 and KOH as described above. Counting and
chromatography were carried out as in the other experiments.
RESULTS
Metabolism of a pulse of fixed I4C by isolated chloroplasts from C. fragile
The results of these experiments are shown in Fig. 1 and Table 1. The
chloroplasts released relatively little fixed 14C during the pulse. During the
15 min incubation with NaH14C03 the chloroplasts released 5.4% (kO.80) of
the total 14C fixed (mean and standard deviation for eight observations) (see
Figure 1. Autoradiographs of two-dimensional chromatograms of methanol-soluble compounds
from media and pellets of isolated chloroplasts from Codium fragile, immediately after a
1 5 min pulse of NaHI4CO, in the light, and after a chase period. A. Labelled compounds in
medium 1, immediately after pulse. B. Labelled compounds in medium 2 (which did n o t
contain NaHI4CO,) 6 h after end of pulse. C. Labelled compounds in chloroplast pellet
immediately after pulse D. Labelled compounds in chloroplast pellet 24 h after end of pulse. I,
giycollic acid; 11, unknown compound 1; 111, glucose monophosphate; IV, phosphoglyceric acid;
V. danine t unidentified amino acid; 0, origin.
R HINDE
334
Table 1. Results of experiments with isolated chloroplasts of Codium fragile.
Chloroplasts were exposed to a 15 min ulse of NaH14C03 in the light, then left
in medium without NaHP4C03 for the chase period
Time after end of
pulse (h)
Total fixed carbon
( 1 0 3 counts/min/pg
chlorophyll)
fived C in:
medium during pulse
Expt
Expt 2
% total
Expt 1
}mean
Expt 2
medium during chase
methanol and water
soluble
insoluble
+ KOH[(NH,),SO,
extracts]
Expt 1
}mean
Expt 2
Expt 1,
Jmean
Expt
Expt l} mean
Expt 2
0
2
6
24
81.5
5.9
83.9
7.2
52.1
8.1
58.3
6.9
"3
5.1
4.7
-1-
'"}
4.6} 5.8
5.0
7.0
5.2
12.0
9*7}io.9
'"}
8.9
'"j}
11.5
45.3k8. 39.6j110
10.8
13.3
u-6}47.7
50.7
57.6
"'356.6
W};
43'3}37.7
32.0
::::}26.2
50.8
48.4
46.8
""}40.2
34.5
Table 1). Trench et al. (1973a) obtained a value of 2% for release of fixed I4C
by chloroplasts of C fragile, but their experiments were carried out under
slightly different conditions, and the chloroplasts were exposed to NaH14C03
for 1 h. Table 1 also shows that during the first 2 h of the chase period 11%
more of the total fixed carbon was released to the medium; over the succeeding
24 h there was no further loss of 14C to the medium. Although a large number
of labelled compounds was found in the chloroplasts in all stages of the chase
period, only three were found in the medium in significant amounts, even after
24 h. This indicates that there was very little lysis of chloroplasts over the 24 h
in isolation. Even in the first 2 h after isolation the chloroplast membranes are
not very "leaky" to organic compounds; the fact that after 2 h there was very
little further loss of carbon from the chloroplasts may indicate that the
permeability of their membranes to organic compounds changed slowly in
response to removal from the cytoplasm. Similar decreases in permeability to
metabolites occur in the plasmalemma of algae which are symbiotic with fungi
(D. C.Smith et al., 1969) and with animals (Trench, 1971).
The labelled compounds released during the pulse were clearly different
from those released during the chase. Autoradiographs of twodimensional
chromatograms showed that glycollic acid was the major radioactive compound
released to the medium during the pulse but glucose monophosphate was also
released. In these experiments about 17% of the radioactivity on the
chromatograms was in glucose monophosphate. Only slight traces of other
compounds were found (Fig. 1A). Six hours after the end of the pulse hardly
any radioactive glycollate had been released into the chase medium (Fig. 1B).
There were 2 distinct spots-a glucose monophosphate and an unidentified
compound ('unknown 1'). The amount of unknown 1 varied between
experiments; in some there was very little present and the glucose monophosphate was the only labelled compound released, while in others unknown
METABOLISM OF PHOTOSYNTHETICALLY FIXED CARBON
335
1 was as obvious as in Fig. 1B. Unknown 1 did not react with ninhydrin. It is
still possible that it is an amino acid, but if so it must have been present on the
chromatograms in very low concentrations, but with a high specific activity. It
was run on one-dimensional chromatograms in solvents 2 and 4 with a number
of substances, most of which are expected to run in the same area of the
two-dimensional chromatogram as unknown 1 (glucose, ribose, tyrosine, valine,
phenylalanine, leucine, alanine, serine, glycine, glycollic, glyoxyllic, lactic,
glyceric, malic, citric, succinic, a-ketoglutaric and fumaric acids, glutamine and
asparagine). Unknown 1 did not run with any of these markers; it ran as a
single spot in both solvents.
During the chase period there were some striking changes in the distribution
of 14C among compounds inside the chloroplasts (Table 1, Fig. l C , D). Since
there was only a very slight decline in the total 14C content of the chloroplasts
over this period, these changes must represent movements of carbon between
different pools within the chloroplast rather than loss of carbon preferentially
from certain pools. The main movement shown in Table 1 was from the
KOH-soluble fraction into water- and methanol-soluble compounds. This
suggests that part, at least, of the pool of insoluble compounds within the
chloroplasts was turning over fairly fast.
The most striking changes in the pattern of fixed 14C in methanol- and
water-soluble compounds in the chloroplasts involved glycollic acid and
unknown 1. Immediately after the end of the pulse 25% of the soluble fixed
14C was in glycollic acid (Fig. 1C). Twenty-four hours later the major labelled
compound was unknown 1, which now contained 40% of the soluble 14C while
glycollic acid accounted for no more than 1% (Fig. 1D). Since little or no
glycollate was lost from the chloroplasts after the end of the pulse, its
disappearance from the soluble fraction clearly indicates that it had been
metabolized. I t was probably not broken down to COz since if the glycollic
acid had been respired, the 14C02 would have to have been refixed with very
great efficiency to account for the results. The appearance of unknown 1 in the
plastids and the medium at the time when glycollate disappeared naturally
suggests that unknown 1 was formed (directly or indirectly) from glycollate,
but the timing of these changes may well have been coincidental. In most
plants glycollic acid is metabolized via glyoxyllic acid, serine and glycine
(Tolbert, 1974), but chromatography of unknown 1 shows that it is not any of
these compounds. The amount of 14C appearing in unknown 1 in 24 h was
almost exactly the same as the amount lost from the KOH fraction. The nature
of the insoluble material in the chloroplasts of C.fragile is not known, but at
least a part of it has the peculiar property of being acid labile (Trench e t al.,
1973a).
Metabolism o f a pulse of fixed 14Cb y E. viridis
The results of these experiments are shown in Tables 2 and 3 and Fig. 2A, B.
During the chase period there was a progressive loss of fixed 14C from the
animals. The loss over 96 h was ap roximately 50% in one experiment and 30%
in another (measured as fixed C per pg chlorophyll). This loss does not
necessarily indicate loss of 14C in respiratory C 0 2 ; much of it may have been
due to the secretion into the medium of mucus synthesized from labelled
r
R. HINDE
336
Table 2. Percentage of total fixed counts in soluble (methanol + water extracts)
fractions of Elysia uiridis after a 1 5 min pulse of NaH14C0, in the light.
Ammonium sulphate + potassium hydroxide extracts (‘insoluble material’) make
up the residue of the fixed counts
Time after end of
pula (h)
0
2
6
24
48
96
Expt 1
Expt 2
83.1
80.0
60.5
65.7
44.6
56.6
44.8
49.5
35.1
37.8
33.3
43.0
Mean
81.6
63.1
50.6
47.2
36.5
38.2
Table 3. Percentage of total fixed counts in compounds of various classes in E.
viridis after a 15 min period of photo-synthesis in NaH14C0,, as shown by
fractionation with trichloracetic acid (TCA) and ethanol
Time after end of pulse (h)
0
24
Class of compound
Expt 2
Expt 2
Expt 2
Expt 1
soluble in cold TCA and
in ethanol
small molecules (amino
acids, monosaccharides,
etc.)
76
50
25
17
TCA soluble and ethanol
insoluble
oligosaccharides,
oligonucleotides
7
15
25
56
1
4
4
4
10
9
14
10
2
5
9
4
Fraction
TCA insoluble, ethanol lipids, lipid soluble
soluble
compounds, small
proteins
cold TCA insoluble,
ethanol insoluble, hot
TCA soluble
nucleic acid
components
insoluble in cold TCA, protein
ethanol, and hot TCA
96
(Totals are less than loo%,as not all labelled material was recovered during fractionation; the results are
presented to demonstrate that compounds of all classes did become labelled in the 4 days following the
pulse. )
sugars (Trench et al., 1972). The proportion of
in methanol-insoluble
compounds rose over the first 48 h, and there was a corresponding fall in
soluble material (Table 2). This movement is in direct contrast with that seen in
the isolated chloroplasts, where there was movement out of the insoluble
fractions. The proportion of fixed 14C incorporated into insoluble material
during the pulse was higher in the isolated chloroplasts than in intact animals;
this may be related to the fact that the amount of fixed carbon released from
the chloroplasts to the cells of the animals is much greater than the amount
released from the chloroplasts to the isolation medium (Trench et al., 1973b).
Table 3 shows that there was a marked rise in the proportion of 14C in the
TCA-soluble, ethanol-insoluble fraction during the 96 h chase. Oligosaccharides
and oligonucleotides make up most of this fraction (Lenhoff & Roffman,
1971). Presumably the oligosaccharides, which are precursors of mucus,
contain most of the label in this fraction in E. uiridis. There was definite
incorporation into lipid, protein and nucleic acid, but only at a low level, and
METABOLISM O F PHOTOSYWHETICALLY FIXED CARBON
337
Figure 2. Autoradiographs of two-dimensional chromatograms of methanol soluble compounds
from Elysia viridis A. At end of 1 5 min pulse of NaHI4CO, in the light. B. Two hours after end
of 1 5 min pulse of NaH14C0, in the light. C. After 6 h in medium containing “C-glycollic acid.
D. After 6 h in medium containing ‘‘C-glucose. I, glycollic acid; VI, hexose(s), tail of spot
probably setine; VII, glutamic acid; VIII, aspartic acid; IX, gIycine; X, serine; XI, hexose; XII,
unknown compound 2; XIII, alanine; 0, origin
there was little change in the I4C content of these fractions during the four
days of the chase. Table 3 also illustrates the variability in metabolism of the
slugs-in both experiments the slugs were near the end of their life cycle and
had started t o spawn (cf. Hinde & Smith, 1975).
As in the isolated chloroplasts, there were marked changes in the distribution
of fixed I4C among methanol-soluble compounds in the slugs during the chase
period. Immediately after the pulse, most fixed 14C was in hexoses (Fig. 2A) as
previously described by Trench et al. (197313). Within 2 h the 14C in hexoses
had decreased considerably, and it remained at a low level for the rest of the
chase. From 2 h after the end of the pulse until the end of the chase a large
proportion of the methanol-soluble fixed 14C was in amino acids, particularly
aspartic and glutamic acids (Fig. 2B). Labelled alanine, on the other hand, was
most abundant at the end of the pulse, and its abundance decreased during the
chase. Labelling of glycollic acid was much less prominent in the intact animds
than in the isolated chloroplasts, and there was no labelled glycollate in the
animals after 6 h. Unknown 1, so important during the chase in the isolated
plastids, was not detected at all in the animals.
R HINDE
338
Metabolism of exogenous glycollic acid and glucose by E. viridis
Glycollic Acid
The results in Table 4 show that only a small proportion of the glycollate in
the medium was taken up by the animals; however radioactivity derived from it
was found in both soluble and insoluble compounds in the tissues. Figure 2C
shows that a large proportion of the 14C in the soluble fraction in these slugs
was in glycollic acid, but that there was also appreciable activity in hexose and
in several amino acids, including serine, glycine, alanine, and aspartic and
glutamic acids. The discrepancy between the amount of 14C lost from the
medium and the amount recovered from the animals presumably represents
respiratory losses. Some of the hexoses formed from the glycollate may have
been incorporated into mucus and secreted back into the medium, so that the
actual uptake may have been higher than the apparent uptake.
Table 4. Uptake and incorporation of 14C-glycollicacid and 14Cglucose by
E. viridis
Glycollic acid
Expt2
Expt 1
Glucose
Expt 1
Expt2
~~
original radioactivity’ lost from medium in 6 h
5.9
6.4
64.3
78.4
radioactivity lost from medium which was
recovered from animals
60.2
50.0
50.0
53.7
% recovered radioactivity in methanol and water
soluble compounds
84.4
88.4
69.7
81.0
%
%
Radioactivity measured as counts per minute by scintillation counting.
Glucose
Much of the 14C was lost from the medium, indicating a marked uptake of
exogenous glucose. Again, since some absorbed 14C-glucose may have been
released back t o the medium in mucus, uptake may have been even greater than
indicated in Table 4. The discrepancy between 14C lost from the medium and
that recovered from the tissues may again indicate respiratory loss. Within the
tissues a higher proportion of 14C was incorporated into insoluble compounds
than was the case with glycollic acid. Chromatograms of the labelled
compounds in the methanol-soluble extracts (Fig. 2D) showed a general
similarity to those from animals two or more hours after the pulse of
NaH14C03 (e.g. Fig. 2B), except that rather more 14C had been incorporated
into hexoses, and there was an extra, unidentified compound (‘unknown 2’).
Some labelled glycollic acid was formed in one experiment, but not in the
other.
DISCUSSION
The results of these experiments confirm that photosynthetically fixed
carbon is actively metabolized by E. viridis by a number of pathways. Most of
the fixed carbon is incorporated first into small molecules, but there is
increasing incorporation into the fraction containing oligosaccharides during
METABOLISM OF PHOTOSYNTHETICALLY FIXED CARBON
3 39
the chase period. This result is in agreement with the earlier reports that a
substantial part of the photosynthetically fixed carbon is incorporated into
mucus in sacoglossans with symbiotic chloroplasts (Trench et al., 1972).
The experiments on the uptake of 14C-glucose confirm that glucose is
actively metabolized by the animals; indeed the distribution of 14Cin methanol
soluble compounds after the animals have been incubated in ''C-glucose in the
dark is remarkably similar to that after exposure to a pulse of NaH14C03 in the
light. Trench et al. (1973b) have suggested that glucose may be the main
compound released from the chloroplasts when they are in the cells of
E. vindis. In these experiments isolated chloroplasts of C'.fragile have not been
observed to release significant amounts of free glucose, but they do release
glucose monophosphate.
The chloroplasts of higher plants are only slightly permeable to glucose
monophosphates, and almost all the fixed carbon which is translocated t o the
cytoplasm leaves the chloroplasts as triose phosphate (Walker, 1976). Codium
chloroplasts differ from those of higher plants in several ways (e.g. in their
ability to continue to carry out photosynthesis for long periods in isolation,
and to withstand osmotic shocks which would rupture higher plant chloroplasts
(Trench et al., 1973a)), and it is possible that they also differ in their transport
systems for organic compounds. This may account for the apparently specific
transfer of glucose monophosphate to the medium. If this is the case, the
appearance of large amounts of glucose in homogenates of E. viridis (Trench et
al., 1973b; Gallop, 1974) would be due to dephosphorylation of glucose
monophosphate by enzymes from the animal cells. If, on the other hand, the
membranes of C. fragile chloroplasts are similar t o those of higher plant
chloroplasts in their permeability, then the apparent transport of large amounts
of glucose monophosphate must be an artifact. In this case the simplest
assumption is that triose phosphate is the compound exported by the
chloroplasts, both in Codium and in Elysia. Attempts to isolate the chloroplasts
of Codium lucasii by a method almost identical with the one used in the work
described in this paper (Hinde, unpublished) resulted in preparations containing
a high proportion of intact chloroplasts. However, electron micrographs
showed that many of these chloroplasts were surrounded by a thin layer of
cytoplasm which was bounded by a membrane. Ben-Shaul et al. (1975)
obtained similar preparations when they attempted t o isolate the chloroplasts
of Codium vermilara by a gentler method. If the chloroplasts used in the
experiments reported here were contaminated with cytoplasm, the glucose
monophosphate may well have been formed in this cytoplasmic layer from
triose phosphate exported by the chloroplasts in the usual way.
There is no evidence that the chloroplasts in the cells of E. viridis are
associated with cytoplasm from C fragile. Electron micrographs of E. viridis
(Trench et al., 1973b) showed that most of the chloroplasts were enclosed only
by the two-layered chloroplast envelope. Some chloroplasts were surrounded
by an extra membrane outside the chloroplast envelope, but this appeared to
have been produced by the animal cell, and there was no cytoplasm between
this membrane and the chloroplast. I t was suggested that the extra membranes
may have been those of phagocytic vacuoles, via which the chloroplasts were
being incorporated into the digestive cells, or of autophagic vacuoles, in which
defunct chloroplasts are apparently digested by the host cell (Trench et al..
14
340
R HINDE
1973b). However, sacoglossans which contain chloroplasts have not been
assayed for enzymes of algal origin (other than those found normally in
chloroplasts), and the possibility that components of the alga, other than
chloroplasts, remain in the digestive cells has thus not been entirely ruled out.
As in other symbiotic associations (Smith e t al., 1969) rapid removal of the
compounds which move between the chloroplasts and the animal cells is likely
to be very important in maintaining the flow of photosynthate from the
chloroplasts, by preventing their building up t o very high concentrations close
to the chloroplast envelope. In many symbioses removal of the mobile
compounds is achieved by their conversion t o compounds which cannot
re-enter the exporting partner. Thus, if triose phosphates were being exported
by the chloroplasts in E. viridis their rapid conversion to glucose phosphate
would serve this purpose (assuming the chloroplasts of C.fragile are relatively
impermeable to glucose phosphate). Enzymes from the animal cells are
presumably capable of synthesizing glucose phosphate from triose phosphate,
so that it is not necessary to postulate the presence of alp1 cytoplasm or
mitochondria in the association. Assuming that this mechanism operates in
intact E. viridis, the stimulatory effect of homogenates of E. viridis on
“leakiness” of C frugile chloroplasts may simply be due t o the presence in the
homogenate of enzymes which can synthesize glucose from triose phosphate
exported by the chloroplasts.
I t is not clear whether the digestive cells of sacoglossans such as E. viridis are
specially adapted for exploitation of chloroplasts, or whether any animal cell
could stimulate the flow of photosynthate from chloroplasts if it could
incorporate them into its cytoplasm. Nass (1969) showed that cultured mouse
fibroblast cells could take up isolated spinach chloroplasts and that these
chloroplasts retained their structural integrity, Hill activity and ability t o fix
CO, for at least 5 days, 2 days and 1 day respectively. However, Nass did not
investigate the possibility that photosynthetically fixed carbon might pass from
the chloroplasts to the “host” cells. Since only those tissues of E. viridis which
contain chloroplasts are able to stimulate the export of photosynthate from
isolated C fkagile chloroplasts (Gallop, 1974) the presence of chloroplasts may
itself stimulate synthesis of the necessary enzymes in the cytoplasm of the
mollusc cell. Glycollic acid was the main compound released by isolated
chloroplasts during photosynthesis. The amount of glycollic acid produced in
the light in higher plants and in algae depends on a number of factors.
Glycollate is formed by the reaction of oxygen with ribulose biphosphate,
which is also the primary acceptor for CO, in photosynthesis, so the
availability of carbon dioxide and of oxygen are the most important factors
controlling the rate of production of glycollic acid (Tolbert, 1974). The
experiments described here show that glycollare is a less important product in
the animal than iF the isolated chloroplasts. Perhaps respiration in the animals’
tissues keeps the concentration of oxygen low (and the CO, concentration
high) in the cells which contain the chloroplasts, thus decreasing the
production of glycollic acid. If this is the case, glycollate may not be
particularly important in the transfer of fixed carbon from the chloroplast to
the animal cell. The experiments in which I4C-glycollate was supplied t o the
animals show that the association can metabolize glycollate. Of course, it is still
possible that glycollate is an important transfer compound: if it were rapidly
METABOLISM OF PHOTOSYNTHETICALLY FIXED CARBON
341
used by the animals in the synthesis of other compounds (for example, amino
acids) its concentration in the tissues would remain low throughout the
experimental period because of its high turnover rate. The experiments on the
metabolism of exogenous glycollic acid show that it is used in the synthesis of a
number of amino acids, and the pulsechase experiments show that amino acids
form a major part of the pools of methanol soluble compounds in E. viridis from
two hours after the end of the pulse of photosynthesis. These experiments were
carried out during the spawning season for E. uiridis from Bembridge, and it is
possible that the animals had a particularly high demand for amino acids at this
time, for the production of eggs and sperm.
The results illustrate the major differences between the metabolism of the
chloroplasts when they are in isolation and when they are inside animal cells.
These differences can be summarized as follows:
% lqC released
incorporation into glycollate
IqC incorporation into ‘unknown 1’
‘‘C incorporation into insoluble material
lqc
In isolation
In animal
5.8%
substantial
substantial
higher
40%(minimum)
moderate
nil
lower
These differences provide a fresh example of the dangers of assuming that the
physiology of an organelle in isolation is the same as its physiology when it is
under the controlling influence of a cell. However, given this caveat (which
applies to almost all studies of isolated organelles) the experiments clearly
demonstrate the great potential of C fragile for the study of isolated
chloroplasts, provided the problem of whether or not these preparations are
contaminated t o an unacceptable level with cytoplasm from the alga is solved.
The fact that there was no detectable loss of I4C from the chloroplasts t o the
medium during the last 22 h of the chase period is a remarkable demonstration
of their resistance to lysis. The metabolism of glycollic acid, the turnover of
insoluble carbon and the identity and origin of unknown 1 are problems which
should be relatively easy to study.
ACKNOWLEDGEMENTS
I wish to thank Professor D. C. Smith, firstly for his help through many
valuable discussions during this work, and secondly, for his immensely useful
critical reading of the manuscript. Miss Stella Dahlin provided excellent
technical assistance. I would also like to thank Mrs C. Northover, of Bembridge,
who collected Elysia and Codium for this work, and Mr B. Lester and Mr J.
Fairburn, for their help in preparing the plates. The work was supported by the
Science Research Council.
Note added in pro0 f
Cobb (1977) has confirmed that C. fragile chloroplasts isolated by the
method used here are contaminated by algal cytoplasm.
COBB, A. H., 1977. The relationship of purity to photosynthetic activity in preparations of Codium
fmgile chloroplasts. Protoplasma, 92: 1 37-146.
342
R HINDE
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