Endosymbiotic Chloroplasts in Molluscan Cells Contain Proteins Synthesized After Plastid Capture

The Journal of Experimental Biology 199, 2323–2330 (1996) 2323 Printed in Great Britain © The Company of Biologists Limited 1996 JEB0480 ENDOSYMBIOTIC CHLOROPLASTS IN MOLLUSCAN CELLS CONTAIN PROTEINS SYNTHESIZED AFTER PLASTID CAPTURE SIDNEY K. PIERCE1,*, RACHEL W. BIRON1,† AND MARY E. RUMPHO2 1Department of Zoology, University of Maryland, College Park, MD 20742, USA and 2Department of Horticultural Sciences, Texas A & M University, College Station, TX 77843, USA Accepted 11 July 1996 Summary Endosymbiotic chloroplasts within the cells of the and subsequently translocated into the chloroplasts. Thus, ascoglossan slug Elysia chlorotica synthesize a variety of the plastids not only synthesize proteins during this long- proteins including the large subunit of ribulose-1,5- lived association, but the host cell seems to play a role in bisphosphate-carboxylase oxygenase (RuBisCO) and the plastid protein turnover. photosystem II protein D1. In addition, the effects of protein synthesis inhibitors suggest that some chloroplast- Key words: endosymbiosis, chloroplast, slug, Elysia chlorotica, associated proteins are synthesized in the animal cytosol protein synthesis. Introduction Most plant–animal symbioses are cellular associations, with selectively take up and retain the algal chloroplasts (West, either entire algal cells residing between animal cells 1979). The long-term survival of the chloroplasts within the (Muscatine et al. 1975; Trench, 1979; Pardy et al. 1983) or a animal cells suggests that these plastids possess an extremely complete plant cell existing within a vacuole produced by the high degree of genetic and biochemical autonomy, or that the animal cell (Muscatine et al. 1975; Anderson, 1983). However, host cells of E. chlorotica play a significant role in promoting in the marine ascoglossan slug Elysia chlorotica, a single the survival of the chloroplasts, or perhaps both. organelle, the chloroplast from the filamentous marine alga Although associations of this type occur in other Vaucheria litorea, subsists within the animal cell cytosol in the ascoglossan species (Taylor, 1970; Greene, 1970), including complete absence of the rest of the plant cell (Graves et al. the closely related European ascoglossan Elysia viridis, which 1979; West, 1979). The slug has earned the nickname ‘leaves forms a symbiosis with chloroplasts from the alga Codium that crawl’ (Trench, 1975) because, when unfurled and fragile (Trench, 1975), none is capable of maintaining the crawling along the sandy bottoms of tidal marshes, the slug, endosymbiotic chloroplasts for as long as E. chlorotica. The which is a deep shade of green owing to the high density of longevity of the E. chlorotica/V. litorea symbiosis suggested chloroplasts within its tissues, bears a close resemblance to a that a study of this relationship might provide information small green leaf. As a result of this remarkable symbiosis, E. about the degree of genetic autonomy of the endosymbiotic chlorotica survives starvation conditions in the laboratory for chloroplasts as well as the contribution that the slug cells may as long as 9 months when provided with adequate light for be making to the longevity of the chloroplasts. photosynthesis, continuing to display a net positive The ability of the plant organelle to survive within an animal incorporation of carbon from photoautotrophic CO2 fixation cytoplasm suggests that more fundamental biochemical (West, 1979; S. K. Pierce, unpublished observations). interactions exist between the cell and the foreign organelle The E. chlorotica/V. litorea symbiosis is not inherited, but than would occur in an extracellular symbiosis. In plant cells, must be re-established with each generation of slugs (West, chloroplast function requires both the integration of two 1979; Trench, 1975; Taylor, 1970). Adult E. chlorotica feed separate and distinct genomes, that of the plant nucleus and only upon two species of alga, V. litorea and V. compacta that of the chloroplast itself (Kowallik, 1989; Berry-Lowe and (West, 1979). At metamorphosis from planktotrophic veliger Schmidt, 1991; Gounaris et al. 1986), and a coordinated to its adult form, E. chlorotica forms an intracellular symbiotic transduction of light signals between the cytosol and the association with chloroplasts from V. litorea. After juvenile chloroplast (Mullet, 1993). Several of the chloroplast proteins slugs have ingested the contents of V. litorea filaments, specific necessary for continued photosynthesis, including several that cells lining the tubules of the extensive digestive system suffer photo-oxidative damage during the light reactions of *e-mail: [email protected]. †Present address: Department of Biology, Dartmouth College, Hanover, NH 03784, USA. 2324 S. K. PIERCE, R. W. BIRON AND M. E. RUMPHO photosynthesis, must be continuously replaced (Schuster et al. marsh near Menemsha Pond on Martha’s Vineyard Island, 1988; Orwitz, 1990) and are encoded by the plant nuclear Massachusetts, USA. The animals were maintained in 18.9 or genome (Keegstra, 1989; Soll and Alefsen, 1993). The 37.8 l aquaria at 10 °C in aerated artificial sea water (ASW) chloroplast genome itself also encodes several light-labile (Instant Ocean, 925 mosmol kg−1) on a 16 h:8 h light:dark cycle proteins required for photosynthesis (Greenburg et al. 1989; (GE cool-white fluorescent tubes, 15 W). Animals were Mattoo et al. 1989; Barber and Anderson, 1992). In addition, collected either in the early spring (animals that had the synthesis, maintenance and degradation of some of these overwintered in the marsh) or in the autumn (progeny from the chloroplast proteins are often light-sensitive, mediated through overwintered animals) (see West et al. 1984, for a description cytosolic photoreceptors (Gamble and Mullet, 1989a,b; of the E. chlorotica life cycle). Christopher and Mullet, 1994). Thus, the persistence of Vaucheria litorea (C. Agardh) were collected from the same photosynthetic function of the V. litorea chloroplasts within E. marsh and were maintained in sterile culture in an incubation chlorotica cells suggests the existence of a mechanism for chamber at 22 °C in aerated enriched ASW (250 mosmol kg−1) either long-term maintenance or de novo synthesis of these [modified from f/2 medium (Bidwell and Spotte, 1985); all proteins in the absence of both the plant nucleus and nutrient concentrations were reduced to 40 % of the cytoplasm. Unless some unidentified mechanism exists within recommended concentrations, and Na2SiO3 was omitted], with the V. litorea chloroplast to repair the damage that occurs 24 h illumination (GE fluorescent white tubes, 40 W). The during light exposure, those proteins that are usually encoded decreased osmolality and nutrient concentrations discourage by the plant nuclear genome and targeted to the plastid must the growth of marine diatoms and other contaminants, while be synthesized for the endosymbiotic chloroplasts, either on allowing the euryhaline algae to thrive. The medium was filter- the plastid ribosomes or on the animal cytosolic ribosomes. In sterilized through a 0.2 µm mesh filter prior to use. Algal addition, the regulatory signals that normally originate from filaments collected from the field were thoroughly washed with the plant cytosolic photoreceptors must be entirely absent, ASW and then transferred to the sterile medium. After 2 days, somehow taken over by the symbiotic plastid, or somehow dark green growing tips (1–3 cm in length) were trimmed from accomplished in the molluscan cytosol. the algae with dissecting scissors, rinsed with sterile ASW Although some earlier attempts were made to determine the (250 mosmol kg−1) and transferred to fresh medium. Every biochemical interactions between the cells of E. viridis and its 2 days for several weeks, small (<1 cm in length) growing tips endosymbiotic chloroplasts, very little is known about protein were cut from the rapidly growing algae and transferred to new synthesis and turnover within the endosymbiotic chloroplasts. medium, until a clean culture was obtained. Thereafter, Autoradiography of thin sections of 3H-leucine labeled E. cultures were transferred to fresh medium every 7–10 days. viridis indicated small amounts of radioactivity over a small New cultures were inoculated as needed by transferring tips to proportion of the chloroplasts, and specific tests for the large new medium. subunit of ribulose-1,5-bisphosphate-carboxylase oxygenase (RuBisCO) failed to demonstrate any synthesis (Trench, 1975). Chloroplast isolation Additional research on the synthesis of other chloroplast Slugs proteins within E. viridis has not been forthcoming, perhaps We discovered that a mucolytic agent, N-acetyl-L-cysteine stymied by the insurmountable technical difficulty of (Mucomyst) (Reas, 1963; Grassi and Morandini, 1976), producing an isolated chloroplast fraction from the huge eliminates most of the copious mucus that may have foiled amount of mucus produced by the slugs (Trench et al. 1973). other attempts to study this symbiosis, while leaving the Thus, until now, almost no information is available on the chloroplasts intact. The concentration of Mucomyst required nature of the biochemical mechanisms that permit the to disperse mucus effectively in homogenates of slugs sustenance and maintenance of the plant organelle in the (500 mmol l−1) was determined in preliminary experiments by foreign environment of the molluscan cytoplasm. observing the disappearance of mucus strands in homogenates We have recently developed a protocol that produces

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