THE EFFECTS OF STARVATION ON THE SYMBIOTIC CHLOROPLASTS IN ELYS IA VIRIDIS: A FINE STRUCTURAL STUDY

Transcription

1 New Phytol. (1980) 84, THE EFFECTS OF STARVATION ON THE SYMBIOTIC CHLOROPLASTS IN ELYS IA VIRIDIS: A FINE STRUCTURAL STUDY BY C. R. HAWES* AND A. H. COBBf Department of Botany, University of Bristol, Bristol, U.K. {Received 14 May 1979) SUMMARY An ultrastructural approach was used to study the eflfects of light and dark starvation on chloroplasts symbiotic in the mollusc Elysia viridis. Starvation for 27 days in a 16 h light/8 h dark photoperiod resulted in loss of starch grains and a gradual disintegration of the chloroplasts by a progressive increase in the inter-thylakoid spaces, resulting in the separation and degeneration of the photosynthetic membranes. To account for these observations a process of photodestruction is suggested. Chloroplasts in animals starved in total darkness for a corresponding period remained relatively intact, and showed a marked increase in the number of plastoglobuli per chloroplast. This increase in plastoglobuli may be a result of the normal ageing process of chloroplasts with a redundant photosynthetic apparatus. INTRODUCTION Freshly collected specimens of the sacoglossan mollusc Elysia viridis, Montague, show rates of photosynthesis comparable to their food plant, the siphonaceous marine alga Codium fragile, sur. Hariot (Trench, 1975). However, once the animals are removed from the seaweed their photosynthetic performance slowly declines, which suggests a turnover of the symbiotic chloroplasts (Hinde and Smith, 1975). Gallop (1975) attempted to determine this rate of turnover by measuring the rate of aquisition of chloroplasts from I'^C-labelled Codium fronds, followed by their subsequent loss from the animal. However, her results were unclear due to the lag period necessary to permit defecation of the chloroplasts from the gut. In this study an ultrastructural approach was used to estimate chloroplast age in animals previously starved for up to 40 days in either light or total darkness, and evidence for the likely fate of the chloroplasts is presented. MATERIALS AND METHODS Specimens of Elysia viridis were collected from intertidal rock pools at Bembridge, Isle of Wight and starved in aerated sea water at 9 C either in the dark or under a photoperiod of 16 h light/8 h dark, provided by an overhead bank of flourescent lights (Atlas Gro-lux) which delivered 64 W m-^ at the water surface. Specimens were taken at intervals and fixed for electron microscopy as described by Hawes (1979). * Present address: Department of Botany, Universit>' of Oxford, South Parks Road, Oxford. t Present address: Department of Life Sciences, Trent Polytechnic, Burton Street, Nottingham X/80/ $02.00/ The New Phytologist

2 376 C. R. HAWES AND A. H. COBB RESULTS Chloroplasts in the digestive cells of freshly collected Elysia are subcircular to ovoid in cross-section, and the double envelope is bounded by a host membrane. The stroma contains a few plastoglobuli, ribosomes and up to three starch grains. Thylakoid lamellae traverse the stroma in pairs or triplets (Plate 1, No. 1; Hawes, 1979). Starvation in a 16 h light/8 h dark photoperiod After 6 days' starvation in this light regime few starch grains are observed in the symbiotic chloroplasts (Plate 1, Nos 2, 3), but otherwise they generally exhibit the same ultrastructure as chloroplasts in fresh animals (Plate 1, No. 2). Some chloroplasts, however, show signs of disruption caused by an increase in the intra-thylakoid spaces, Table 1. The effect of light and dark starvation on chloroplast diameter, plastoglobuli size and plastoglobuli number in Elysia viridis Chloroplast Plastoglobuli Plastoglobuli Treatment diameter (/im) diameter (/im) per chloroplast Fresh Elysia 2 71 (80) 0134 (108) 5-12 (50) 27 d light-starved 3-5O**» (36) 5 79 N.S. (48) 28 d dark-starved 2 08*** (56) 0158 N.s. (159) 9 91»* (49) Figures in parentheses refer to the size of the sample from which the mean values were calculated. Chloroplast and plastoglobuli diameters represent the mean cross-section measurements from electron micrographs. The significance of the mean values was determined using Student's t test. N.s. - not significant. Significant at P = 0 01 level. *** Significant at P = level. with a separation of the membranes (Plate 1, No. 3) and an overall swelling of the chloroplast (Table 1). After 27 days' starvation all the symbiotic chloroplasts are affected in this manner. The host membrane which surrounds individual chloroplasts is, however, still present (Plate 2, No. 1, single arrows), but may be ruptured in places (Plate 2, No. 1, double arrows). Other plant cell organelles, such as mitochondria ingested with the chloroplasts as cytoplasmic droplets (Cobb, 1977; Hawes, 1979), are no longer apparent after prolonged starvation (Plate 2, No. 1). After 40 days' starvation a complete disintegration of many of the photosynthetic membranes has taken place, resulting in electron transparent areas in the digestive cells, which presumably represent the sites of degenerated chloroplasts (Plate 2, Nos 2, 3). However, chloroplasts in various stages of this degenerative process are commonly seen in fresh Elysia (Plate 3, No. 1). Vacuoles are present in the digestive cell of both fresh and starved Elysia, which apparently contain the membranous remains of chloroplasts (Plate 3, Nos 2, 3). Similar vacuoles, with the remains of up to three chloroplasts, are also observed in the digestive cells of naturally pale animals (Plate 4, no. 3). Starvation in the dark When the animals are starved for 28 days in total darkness the symbiotic chloroplasts show no symptoms of thylakoid degeneration (Plate 4, Nos. 1, 2). There is, however, a total loss of starch grains, a marked increase in the number of plastoglobuli

3 Effects of starvation on symbiotic chloroplasts 377 in the stroma, and a 25 % reduction in chloroplast cross-sectional diameter (Plate 4, No. 2; Table 1). Table 1 summarizes the eflect of light and dark starvation on symbiotic chloroplast diameter, plastoglobuli size and number in Elysia. Chloroplasts swell during light starvation and shrink in the dark, during which there is a significant increase in plastoglobuli number per chloroplast. DISCUSSION The results show that with prolonged starvation in a 16 h light/8 h dark regime there is a progressive degeneration of the chloroplasts in the digestive cells of Elysia. This is seen as a swelling of the chloroplasts coupled with a disintegration of the thylakoid lamellae system caused by the separation of the thylakoid membranes, and a final breakdown of the membranes. A proposed explanation for this breakdown of the photosynthetic membranes is given later. The separation of the thylakoid membranes is a similar phenomenon to that described by Kulandaivelu and Hall (1975) in isolated spinach chloroplasts, but it is not possible to quantify the present results as it is likely that the chloroplasts in Elysia are not of even age. This would explain the presence of both intact and degenerating chloroplasts in animals starved for 6 days (Plate 1, Nos 2, 3). Indeed, degenerating chloroplasts were also found in fresh Elysia (Plate 3,' No. 3), which supports the suggestion of Gallop (1975) that there is a turnover of chloroplasts in the digestive cells. These observations, however, are further complicated by the report that siphonaceous algal chloroplasts may exist as a heterogenous group, and that the different subpopulations may exhibit different biochemical functions within the algal cell (Luttke, Rahmsdorf and Schmid, 1976). If this is the case, then the different chloroplast populations in Elysia may be expected to age at diflferent rates. Thus, although a distinction can be made, at the ultrastructural level, hetween freshly ingested and older chloroplasts, an accurate age of the chloroplasts cannot be determined. When Elysia are starved in the dark for 28 days the chloroplasts appear relatively intact, but there is a marked increase in the number of plastoglobuli per chloroplast (Plate 4, Nos 1,2; Table 1). In higher plants platoglobuli are believed to function as lipid storage sites outside the thylakoids, and mainly contain the lipophilic quinones that function as the biological oxidation-reduction catalysts in the photochemically active thylakoids. The synthesis of these quinones continues after that of the thylakoids and chlorophylls are finished, so that the plastoglobuli represent reservoirs for excess lipids (Lichtenthaler, 1968). Consequently, according to Lichtenthaler (1968), there is a close correlation between plastoglobuli and chloroplast development. If, for some reason, thylakoid synthesis is prevented, or the thylakoids break down, there is an increase in the number and/or volume of the plastoglobuli. Indeed, the plastoglobuli shown in Plate 4, Nos 1, 2, appear typical of 'old' chloroplasts by this classification. It remains unclear, however, whether this abundance of pbstoglobuli in Elysia chloroplasts has resulted from a prevention of thylakoid synthesis, thylakoid breakdown, or perhaps more likely, by the normal ageing process of chloroplasts with a redundant photosynthetic apparatus. In contrast, when animals are starved in the light for 27 days there is an almost complete degeneration of the chloroplast membranes (Plate 1, No. 3 to Plate 2, No. 4). However, as the chloroplasts of animals starved in the dark remain relatively intact

4 378 C. R. HAWES AND A. H. COBB over this period, the destruction is most probably light-induced, and not as a result of digestive enzyme action. To account for this destruction of chloroplasts in the light rather than in the dark we suggest the process of photodestruction rather than digestion. One possible mechanism for photodestruction is that in the animal cell environment the chloroplasts are less well protected against the toxic effect of photochemically produced superoxide, singlet oxygen and/or hydroxyl-free radicals. One characteristic of C. fragile chloroplasts in their algal environment is their ability to accumulate large quantities of storage starch (Cobb, 1977; Hawes, 1979), and long chain polyphosphate (Cobb, 1978). After ingestion into the hepatopancreas of the animal these storage products are metabolized during the first 14 days of starvation (Plate 1, Nos 1 to 3; Cobb, 1978). Only after depletion of their storage products do the symbiotic chloroplasts begin to show signs of photodestruction (when starved in the light), or plastoglobuli accumulation (when starved in the dark). A significant rate of carbon turnover is required to dissipate destructive photochemical energy (Krause et al., 1977), but, with symbiotic chloroplasts in starved animals, the rates of carbon fixation slowly decline (Hinde and Smith, 1975). Thus a declining rate of carbon assimilation, combined to a depletion of chloroplast storage products, may slowly render the symbiotic organelles less able to dissipate excess photochemical energy, so that net free oxygen radical production, and hence lipid peroxidation eventually ensue (Halliwell, 1978). We suggest therefore that animals may survive on their symbiotic chloroplasts for at least 14 days (i.e. the estimated time for the total depletionof chloroplast storage products), depending on the prevailing light intensities. Trench, Boyle and Smith (1973) suggested that the ultimate fate of symbiotic C. fragile chloroplasts was digestion by the host digestive cells in a phagocytotic vacuole which formed around the chloroplasts. This was supported by electron micrographs of apparently digested chloroplasts in starved animals. Digested chloroplasts are reported here in both starved and fresh animals (Plate 3, Nos 2, 3), but this method of chloroplast destruction is totally different to, and far less common than the photodestruction proposed for the majority of chloroplasts in the light/dark starved animals. The phagocytotic membrane is derived from the host membrane that surrounds the chloroplasts in the digestive cells. ACKNOWLEDGEMENTS We would like to thank Professor D. C. Smith for critically reading the manuscript, and the S.R.C. for financial support. REFERENCES COBB, A. H. (1977). The relationship of purity to photosynthetic activity in preparations of Codium fragile chloroplasts. Protoplasma, 92, 137. COBB, A. H. (1978). Inorganic polyphosphate involved in the symbiosis between chloroplasts of alga Codium fragile and mollusc Elysia viridis. Nature, Lond., 272, 554. GALLOP, A. (1975). Chloroplast symbiosis. D.Phil. Thesis, Oxford. HALLIWELL, B. (1978). The chloroplast at work. A review of modern developments in our understanding of chloroplast metabolism. Progress in Biophysics and Molecular Biology, 33, 1. HAWES, C. R. (1979). Ultrastructural aspects of the symbiosis between algal chloroplasts and ElysK' viridis. New Phytologist 83, 445. HINDE, R. & SMITH, D. C. (1975). The role of photosynthesis in the nutrition of the mollusc Elysia viridis. Biological Journal of the Linnaean Society, 7, 161.

5 The New Phytologist, Vol. 84, No. 2 I. HAWES AND A. H. COBB Plate 1 (Facing p. 378)

6 The Nezv Phytologist, Vol. 84, No. 2 Plate 2 C. R. HAWES AND A. H. COBB

7 The New Phytologist, Vol. 84, No. 2 Plate 3 ^C'--, L HAWES AND A. H. COBB

8 The New Phytolgisto, Vol. 84, No. 2 C. R. HAWES AND A. H. COBB Plate 4

9 Effects of starvation on symbiotic chloroplasts 379 KRAUSE, G. H., LORIMER, G. H., HEBER, U. & KIRK, M. R. (1977). Photorespiratory energy dissipation in leaves and chloroplasts. Proceedings of the 4th International Congress on Photosynthesis, 299. KULANDAIVELU, G. & HALL, D. O. (1976). Ultrastructural changes in in vitro ageing spinach chloroplasts. Zeitschrift fiir Naturforschung, 31, 82. LiCHTENTHALER, H. K. (1968). Plastoglobuli and the fine structure of plastids. Endeavour, 102, 144. LiJTTKE, A., RAHMSDORF, U. & SCHMID, R. (1976). Heterogeneity in chloroplasts of siphonaceous algae as compared with higher plant chloroplasts. Zeitschrift fur Naturforschung, 31c, 108. TRENCH, R. K. (1975). Of 'leaves that crawl': functional chloroplasts in animal cells. Symposia for the Society of experimental Biology, 29, 229. TRENCH, R. K., BOYLE, J. E. & SMITH, D. C. (1973). The association between chloroplasts of Codium fragile and the mollusc Elysia viridis. II. Chloroplast ultrastructure and photosynthetic carbon fixation in E. viridis. Proceedings of the Royal Society {London), B 184, 63. EXPLANATION OF PLATES PLATE 1 No 1 Chloroplasts in the digestive cells of freshly collected specimens of E. viridis. x No. 2. Intact chloroplasts in a digestive cell of E. viridis after 6 days' light/dark starvation, x No. 3. Partially disrupted chloroplasts after 6 days' light/dark starvation, x PLATE 2 No 1 Chloroplasts after 27 days' light/dark starvation. All the chloroplasts show signs of disruption. The host membrane is still present (single arrows), but may be ruptured in places (double arrows). X10400.,. r No. 2. Chloroplasts after 40 days' light/dark starvation. Electron transparent areas represent the sites of degenerated chloroplasts. x r,. No. 3. Disrupted chloroplast after 40 days' light/dark starvation. Very little remains of the photosynthetic membranes, x PLATE 3 No 1 Disrupted chloroplasts in a freshly collected specimen of E. viridis. x No. 2. Remains of a chloroplast in a digestive vacuole of an animal after 40 days' light/dark starvation. X17100 No. 3. Remains of a chloroplast in a digestive vacuole in a fresh specimen of E. viridis. x PLATE 4 No. 1. Chloroplasts in digestive cells after 28 days' starvation of the host in total darkness. A marked increase in the number of plastoglobuli per chloroplast is evident, x No. 2. Chloroplasts after 28 days' starvation, x r 77 j- No. 3. Remains of chloroplasts within digestive vacuoles in naturally pale specimens ot t. vtridis. X

10