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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 . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330 331 331 331 332 332 332 333 333 335 338 338 338 341 342 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. 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