Evidence that cryptomonad evolved from photosynthetic eukaryotic endosymbionts

GEOFFREY IAN McFADDEN

Plant Cell Biology Research Centre, School of Botany, University of Melbourne, Parkville, Victoria 3052, Australia

Summary

Unicellular of the division Cryptophyta pos- the rRNAs may be transcribed from genes in this sess an unusual subcellular compartment of un- nucleus. This identification of a second, nucleus- known derivation. This compartment, which is containing eukaryotic compartment within these partitioned off from the main cytoplasm by two cells supports the hypothesis that cryptomonads membranes, contains a and a small contain a reduced photosynthetic eukaryotic endo- nucleus-like organelle surrounded by - symbiont. like particles. Electron-microscopic in situ hybrid- ization has been used to show that the in this subcellular compartment are eukaryotic. In addition, eukaryotic rRNA has been localised Key words: chloroplasts, cryptomonads, endosymbiosis, /;; within the nucleus-like organelle, suggesting that situ hybridization, ribosomes, ribosomal RNA.

Introduction main supporting evidence for this hypothesis is that chloroplasts in these algae are surrounded by four mem- Photosynthesis evolved in prokaryotes (Krishna Rao et branes (Gibbs, 19816). The inner pair of membranes is al. 1985) and was acquired by through endo- considered homologous to the green chloroplast symbiosis of a prokaryote (Margulis, 1981; Taylor, 1987; envelopes (Whatley, 1981; Whatley et al. 1979; Cavalier- Gray, 1988; Giovannonieia/. 1988). The photosynthetic Smith, 1986). The two outer membranes are termed the prokaryotic endosymbiont in green has been chloroplast (CER) (Gibbs, reduced to a semi-autonomous organelle bound by two 19816). The outermost CER membrane, which is usually membranes, the chloroplast. The inner chloroplast mem- continuous with the nuclear envelope/rough endoplas- brane probably represents the endosymbiont's plasma mic reticulum and bears eukaryotic ribosomes, is putati- membrane, while the outer membrane could be derived vely derived from the endomembrane system of the host from the outer membrane of the endosymbiont (Cavalier- cell, while the inner CER membrane could represent the Smith, 1987) or the phagocytotic of the host cell plasma membrane of the eukaryotic endosymbiont (Whatley and Whatley, 1981; Whatley et al. 1979; (Whatley and Whatley, 1981; Dodge, 1979; Cavalier- Dodge, 1979). It is envisaged that many of the endosym- Smith, 1986). biont's genes were transferred to the host One group from the , the crypto- (Harrington and Thornley, 1982; Weeden, 1981), monads, may be an intermediate stage in the reduction of thereby stabilizing the symbiotic relationship and con- a photosynthetic eukaryotic endosymbiont to a chloro- verting the endosymbiont to a true organelle (Cavalier- plast with four limiting membranes. Cryptomonads Smith and Lee, 1985). (Division Cryptophyta) are common marine or fresh- Photosynthetic algae of the Kingdom Chromista water biflagellates (Gantt, 1980). Between the inner and (Cavalier-Smith, 1986) (e.g. , brown algae, gol- outer pairs of membranes surrounding the chloroplast in den , cryptomonads) are thought to have cryptomonad cells is a small compartment termed the acquired their chloroplasts 'third hand'. According to this periplastidal space, which contains ribosome-like par- hypothesis (illustrated in Fig. 1), a photosynthetic pro- ticles and a small organelle resembling a nucleus, known karyote became a chloroplast in an unknown eukaryotic as the (Greenwood, 1974; see Fig. 2, this intermediary, which in turn became an endosymbiont paper). It has been proposed that the nucleomorph is a within a second phagotroph (Greenwood et al. 1977; vestigial nucleus from a eukaryotic endosymbiont and Whatley and Whatley, 1981; Whatley et al. 1979; Dodge, that the ribosome-like particles in the periplastidal space 1979; Cavalier-Smith, 1982, 1986; Gibbs, 1981a). The are ribosomes from the endosymbiont's cytoplasm Journal of Cell Science 95, 303-308 (1990) Printed in Great Britain © The Company of Biologists Limited 1990 303 Fig. 1. Hypothetical sequence of endosymbioses in evolution of cryptomonads (see text for references). A photosynthetic prokaryote (Pr) was engulfed by an unknown (Eul) and became a chloroplast in an unidentified photosynthetic eukaryote. The photosynthetic eukaryote was in turn engulfed by a second phagotroph (Eu2) and became an endosymbiont to create autotrophs like cryptomonads. Vestiges of the nucleus and cytoplasm of the photosynthetic eukaryotic endosymbiont (Eul) would be retained in a subcellular compartment of the second host. In situ hybridization studies reported here identify this compartment as containing eukaryotic ribosomes. Preliminary evidence suggests that rRNA for these ribosomes may be encoded by a second nucleus derived from the eukaryotic endosymbiont (Eul).

(i.e. those in the chloroplast) or eukaryotic origin (nu- clear-encoded cytoplasmic ribosomes) by electron mi- croscopy (McFadden et al. 1988; McFadden, 1989). I have examined cryptomonads by high-resolution in situ hybridization using two probes, one specific for eukary- otic ribosomal RNA (rRNA) and the other specific for prokaryotic rRNA. The ribosomes in the periplastidal space are shown to be eukaryotic and are quite probably transcribed from genes in the nucleomorph.

Materials and methods

Cultures The cryptomonad ( caudata Geitler, Melbourne University Culture Collection no. CrlO) was grown under a fluorescent illumination in WARIS-H media as described Periplastidal \~ // Chloro space (McFadden and Melkonian, 1986). The (Bacillus sp.) Cytoplasm were contaminants in one of the algal sub-cultures.

Standard electron microscopy Cells were fixed in 2% glutaraldehyde in 50mM-piperazine- Ar,A''-bis[2]-ethanesulphonic acid (pH7) at 4°C for one hour. Cells were then post-fixed in 1 % OsCj and embedded in Spurr's resin.

In situ hybridization fixation Fig. 2. Diagram showing subcellular compartments in a For in situ hybridization the cells were fixed in glutaraldehyde cryptomonad. At the anterior is a gullet with two emergent as above, washed in the buffer, dehydrated to 70% ethanol and flagella. The main nucleus with its nucleolus (No) is at the embedded in LR Gold resin (McFadden et al. 1988). Fixation posterior. Four membranes surround the chloroplast. An with OsO4 is not compatible with in situ hybridization (McFad- extension of the nuclear membranes, the chloroplast den, 1989). endoplasmic reticulum (CER), envelopes the chloroplast and associated compartment. Between the CER and the Probes chloroplast envelopes is the periplastidal space, which The eukaryotic rRNA probe was a 1000 bp (base pair) BamHl/ contains ribosome-like particles and the nucleomorph. Bar, EcoRl nuclear genomic fragment of the 18 S rRNA gene from 1 /tm. Pisum sativum (Jorgensen et al. 1987). The prokaryotic rRNA probe was an 800 bp EcoRl fragment partially encoding 16 S rRNA from the chloroplast genome of Chlamydomonas rein- (Greenwood et al. 1977; Whatley and Whatley, 1981; hardtii (Eco 36/37, according to Grant et al. 1980). Whatley

304 G. I. McFadden Hybrids were detected with anti-biotin antibody and a second- Cryptomonads ary antibody conjugated to colloidal gold as previously de- The general morphology and physiology of cryptomo- scribed (McFadden, 1989). nads is described in detail elsewhere (Gantt, 1980). Briefly, cryptomonads are small, bean-shaped biflagel- Results lates containing a and Cz plus phycobilipro- teins. Fig. 2 illustrates the compartmentation in a crypto- Specificity of the probes monad cell. The chloroplast is bounded by four membranes (Fig. 2). Between the inner and outer pairs At the stringency used, the prokaryotic probe hybridizes of membranes is the periplastidal compartment (Figs 2 only with prokaryotic rRNA and the eukaryotic probe and 5), which contains ribosome-like particles and the hybridizes only with eukaryotic rRNA. Fig. 3 shows the nucleomorph (Figs 2 and 5). The nucleomorph is two probes hybridized to bacteria. The prokaryotic probe bounded by a double membrane with pores and has an labels ribosomes in the cytoplasm of bacterial cells electron-opaque zone resembling a nucleolus plus several (Fig. 3A) but the eukaryotic probe does not label bac- smaller electron-dense globules (Fig. 5D). terial ribosomes (Fig. 3B). Fig. 5 shows the periplastidal compartment of crypto- Fig. 4 shows the two probes hybridized to cryptomo- monad cell and labelling of the compartment with the nad cells. The prokaryotic probe labels only the ribo- probes for eukaryotic and prokaryotic rRNA. The peri- somes in the chloroplast stroma (Fig. 4A). The eukary- plastidal compartment is heavily labelled with the eukary- otic rRNA probe labels the ribosomes in the cytoplasm otic probe (Fig. 5B) but is not labelled with the prokary- (Fig. 4B). In addition, the eukaryotic probe also labels otic probe (Fig. 5C). Fig. 5D,E shows the nucleomorph the nucleolus in the nucleus of the cryptomonad cell of cryptomonads and the labelling of the nucleomorph (Fig. 4B). Nucleoli were labelled at a mean density of 339 2 with the probe for eukaryotic rRNA. The electron- gold particles per jj,m (standard deviation =190, w=20). opaque nucleolus-like region of the nucleomorph is The prokaryotic probe does not hybridize to rRNA in the labelled with the eukaryotic probe (Fig. 5E). Nucleolus- nucleolus (Fig. 4A). The sense probes do not label any like regions were labelled at a mean density of 406 gold structures (not illustrated). particles per /mi2 (standard deviation=260, ;i=10). Other structures in the nucleomorph including the dense globules and the surrounding matrix are not labelled with the eukaryotic probe (Fig. 5E). The prokaryotic probe does not label any structures in the nucleomorph (Fig. 5C).

Discussion

High-resolution in situ hybridization shows that a probe specific for eukaryotic rRNA hybridizes to the periplasti- dal space of cryptomonads. A probe specific for prokary- otic rRNA labelled the chloroplast ribosomes in crypto- monads but did not label the periplastidal space. Previous studies employing standard electron microscopy showed ribosome-like particles in the periplastidal space (Green- wood, 1974). These particles are larger than the prokar- yotic ribosomes in the chloroplast and are similar in size to eukaryotic cytoplasmic ribosomes (Sepenswol, 1973; Gillott and Gibbs, 1980; Gibbs, 1981a). In addition, cytochemical studies have localized RNA within the * 4 periplastidal compartment (Hansmann, 1988). The pres- ent results demonstrate the presence of eukaryotic rRNA in the periplastidal space and provide additional proof that the periplastidal compartment of cryptomonads contains eukaryotic ribosomes. 3A B The eukaryotic ribosomes in the periplastidal compart- ment of cryptomonads are isolated from the main cyto- Fig. 3. Bacterial cells (Bacillus sp.) labelled with the plasm by the two membranes of the CER (Gibbs, 19816; prokaryotic probe and the eukaryotic probe. A. Longitudinal and see Fig. 2 of this paper). Since no mechanism for section of bacterium labelled with prokaryotic probe. The ribosomes in the peripheral cytoplasm are labelled. Very little transport of rRNA across membranes is known, the label is present on the central nucleoid. Bar, 0.5 ^m. rRNA genes are presumably located in the periplastidal B. Bacterium labelled with the eukaryotic probe. No compartment. The nucleomorph contains DNA (Ludwig structures are labelled. Only a single gold particle and Gibbs, 1985, 1987; Hansmann et al. 1986) and has (arrowhead) is present. Bar, 0.5 f«m. been proposed to contain genes for periplastidal ribo-

Evolution of algal chloroplasts 305 chl •/••

no-

:< *

chl

4A cy B

Fig. 4. Sections of a cryptomonad (Chroomonas caudala) cell labelled with the prokaryotic probe or the eukaryotic probe. A. Longitudinal section of posterior end labelled with prokaryotic probe. The ribosomes in the stroma of the two chloroplast lobes (chl) are heavily labelled. Ribosomes in the cytoplasm (cy) and rRNA transcripts in the nucleolus (no) are not labelled. Bar, 0.25 ,um. B. Parallel section to A, labelled with eukaryotic probe. Ribosomes in the cytoplasm (cy) and transcripts in the nucleolus (no) are heavily labelled. The chloroplast ribosomes (chl) are not labelled. Bar, 0.25 jum. somes (McKerracher and Gibbs, 1981; Cavalier-Smith, experiments demonstrate that the nucleolus-like area 1986; Hansmann et al. 1986; Hansmann, 1988; Ludwig contains eukaryotic rRNAs and strongly suggest that and Gibbs, 1985, 1987). I have shown that a probe for these are transcribed from an aggregation of rRNA genes eukaryotic rRNA, which labels nascent ribosomes at the in the nucleomorph. Presumably, the rRNAs exit the site of transcription in eukaryotic nucleoli (McFadden et nucleomorph via the small pores in the surrounding al. 1988; McFadden, 1989;' and this paper), labels an double envelope and become ribosomes in the periplasti- electron-opaque region within the nucleomorph at com- dal compartment. The cryptomonad nucleomorph thus parable intensity. Previous workers have localized RNA possesses four characteristics typical of a eukaryotic within this electron-opaque region and have suggested nucleus: a double membrane envelope with pores that it is a nucleolus (Gillott and Gibbs, 1980; Hans- (Greenwood, 1974; Gillott and Gibbs, 1980), DNA mann, 1988; Ludwig and Gibbs, 1987). The present (Ludwig and Gibbs, 1985, 1987; Hansmann et al. 1986),

Fig. 5. Sections of cryptomonad showing labelling of labelled. Bar, 0.5 fim. D. Standard electron micrograph of periplastidal compartment with eukaryotic probe and cryptomonad nucleomorph region showing cytoplasm (cy) on prokaryotic probe. A. Standard electron micrograph showing either side of the chloroplast (chl), chloroplast endoplasmic nucleus (mi), chloroplast (chl) with (py), reticulum (cer), chloroplast envelopes (ce), periplastidal space nucleomorph (nin) and the periplastidal space (five arrows). ("&), and nucleomorph (nm) with pores (arrowheads), Bar, 0.5//m. B. Similar section to A, labelled with eukaryotic electron-dense globules (small arrows) and nucleolus-like probe. The nucleolus in the nucleus (nu) is labelled, as are region (open arrow). Bar, 0.25 fan. E. Nucleomorph region of the ribosomes in the main cytoplasm. The periplastidal cryptomonad labelled with eukaryotic rRNA probe. The compartment (five arrows) is heavily labelled. The chloroplast (chl) is not labelled. The ribosomes of the chloroplast stroma (chl) is not labelled. Bar, 0.5 fan. cytoplasm (cy) on either side of the chloroplast are labelled. C. Similar section to A and B, but labelled with the Within the nucleomorph (nm), the darker nucleolus-like prokaryotic probe. The stromal region of the chloroplast (chl) region (large open arrow) is labelled. The electron-dense is heavily labelled. The main nucleus (nu) and cytoplasm are globules (small arrows) and surrounding matrix of the not labelled. The periplastidal space (five arrows) and nucleomorph are not labelled. Bar, 0.25 fan. nucleomorph (nm) are not labelled. The pyrenoid (py) is not

306 C. I. McFadden self-replication (McKerracher and Gibbs, 1981; Morrall space by the CER) is considered unlikely (Cavalier- and Greenwood, 1982), and a nucleolus-like zone con- Smith, 1986) but cannot be discounted at present. If taining eukaryotic rRNA (this paper). cryptomonads contain a eukaryotic endosymbiont (ex- There are two possible origins of the nucleomorph and ogenous origin of nucleomorph and periplastidal ribo- periplastidal ribosomes: autogenous or exogenous. An somes), then the nucleotide sequence of rRNAs from the autogenous origin (where the nucleomorph is a portion of periplastidal compartment and the main cryptomonad the main nucleus partitioned off into the periplastidal cytoplasm should be divergent. While the in vita

A X

chl

Evolution of algal chloroplasts 307 ization results presented here show that the ribosomes in GILLOTT, M. A. AND GIBBS, S. P. (1980). The cryptomonad both the periplastidal space and main cytoplasm are nucleomorph: its ultrastructure and evolutionary significance. jf. Phycol. 16, 558-568. eukaryotic, they do not give any further measure of their GIOVANNONI, S. J., TURNER, S., OLSEN, G. J., BARNS, S., LANE, relatedness. To address this question I am sequencing D. J. AND PACE, N. R. (1988). Evolutionary relationships among clones of rRNA genes from the cryptomonad. and green chloroplasts. J. Bad. 170, 3584-3592. The in situ hybridization studies suggest that the GRANT, D. M., GILLHAM, N. W. AND BOYNTON, J. E. (1980). Inheritance of chloroplast DNA in Chlamvdomonas reinhardtii. nucleomorph of cryptomonads is a transcriptionally ac- Proc. natn. Acad. Sci. U.S.A. 77, 6067-6071. tive eukaryotic nucleus isolated within a subcellular GRAY, M. W. (1988). Organelle origins and ribosomal RNA. compartment that also houses eukaryotic translation Biochem. Cell Biol. 66, 325-348. machinery and a chloroplast. The power of this tech- GREENWOOD, A. D. (1974). The Cryptophyta in relation to nique for identifying the evolutionary affinities of differ- phylogeny and photosynthesis. In Electron Micwscopy 1974 (ed. J. V. Sanders and D. J. Goodchild), pp. 566-567, Canberra: ent compartments in complex chimaeric cells with mul- Australian Academy of Sciences. tiple genomes is apparent. Why cryptomonads have this GREENWOOD, A. D., GRIFFITHS, H. B. AND SANTORE, U. J. (1977). extra nucleus and eukaryotic compartment remains an Chloroplasts and cell compartments in . Br. Phvcol. open question. More advanced members of the Kingdom J. 12, 119. Chromista such as diatoms, brown algae and golden HANSMANN, P. (1988). Ultrastructural localization of RNA in cryptomonads. Pmtoplasma 146, 81-88. flagellates have no nucleomorph or ribosomes in their HANSMANN, P., FALK, H., SCHEER, U. AND SITTE, P. (1986). periplastidal space (Cavalier-Smith, 1986). If these or- Ultrastructural localization of DNA in two species by ganisms are derived from cryptomonads by a loss of the use of a monoclonal DNA antibody. Eur.J. Cell Biol. 42, 152-160. nucleomorph and periplastidal ribosomes, then the nuc- HARRINGTON, A. AND THORNLEY, A. L. (1982). Biochemical and leomorph function must have either been assumed by genetic consequences of gene transfer from endosymbiont to host genome. J. molec. Evol. 18, 287-292. another compartment or become redundant. JORGENSEN, R. A., CUELLER, R. E., THOMPSON, W. F. AND The in situ hybridization results reported here strongly KAVANAGH, T. A. (1987). Structure and variation in rDNA of support the hypothesis that cryptomonads converted pea. Characterization of a cloned repeat and chromosomal rDNA from phagotrophy to autotrophy by permanently har- variants. PI. molec. Biol. 8, 3-12. KRISHNA RAO, K., CAMMACK, R. AND HALL, D. O. (1985). bouring a photosynthetic eukaryotic endosymbiont Evolution of light energy conversion. In Evolution of Prokaryotes (Greenwood et al. 1977; Whatley and Whatley, 1981; (ed. K. H. Schleifer and E. Stackenbrandt), pp. 143-173, New Whatley ef a/. 1979; Dodge, 1979; Cavalier-Smith, 1982, York: Academic Press. 1986; Gibbs, 1981a,6; Ludwig and Gibbs, 1985, 1987; LUDWIG, M. AND GIBBS, S. P. (1985). DNA is present in the nucleomorph of cryptomonads: further evidence that the Hansmann et al. 1986; Hansmann, 1988). chloroplast evolved from a eukaryotic endosymbiont. Pmtoplasma 127, 9-20. I am grateful for a Queen Elizabeth II Research Fellowship. LUDWIG, M. AND GIBBS, S. P. (1987). Are the of Dr D. Hill kindly provided the cryptomonad culture and cryptomonads and Chlorarachnion the vestigial nuclei of eukaryotic Fig. SD, and Dr W. Thompson and Dr N. Gillham donated the endosymbionts? Ann. N.Y. Acad. Sci. 503, 198-211. clones from which the probes were derived. I am particularly MARGULIS, L. (1981). Symbiosis in Cell Evolution, San Francisco: indebted to Professor A. E. Clarke and members of the Plant Freeman and Co. Cell Biology Research Centre for their assistance in recombi- MCFADDEN, G. I. (1989). In situ hybridization in plants: from macroscopic to ultrastructural resolution. Cell Biol. Int. Rep. 13, nant DNA manipulations and the use of many resources. Ms I. 3-21. Bonig provided invaluable assistance with the microscopy. MCFADDEN, G. I., CORNISH, E. C, BONIG, I. AND CLARKE, A. E. (1988). A simple fixation and embedding method for use in hybridization histochemistry of plants. Histochem. jf. 20, 575-586. References MCFADDEN, G. I. AND MELKONIAN, M. (1986). Use of Hepes buffer for algal culture media and electron microscopy. Phvcologia 25, 551-557. CAVALIER-SMITH, T. (1982). The origin of . Biol.J. Linn. MCKERRACHER, L. AND GIBBS, S. P. (1981). Cell and nucleomorph Soc. 17, 289-306. division in the alga Cryptomonas. Can.jf. Bot. 60, 2440-2452. CAVALIER-SMITH, T. (1986). The kingdom Chromista: Origin and MORRALL, S. AND GREENWOOD, A. D. (1982). Ultrastructure of systematics. In Progress in Phycological Research, vol. 4 (ed. F. nucleomorph division in species of Cryptophyceae and its Round and D. J. Chapman), pp. 309-347, Bristol: Biopress Ltd. evolutionary implication. J. Cell Sci. 54, 311-328. CAVALIER-SMITH, T. (1987). The simultaneous symbiotic origin of SEPENSWOL, S. (1973). of the cryptomonad mitochondria, chloroplasts, and microbodies. Ann. N.Y. Acad. Sci. paramecium. Expl Cell Res. 76, 395-409. 53, 55-71. TAYLOR, F. J. R. (1987). An overview of the status of evolutionary CAVALIER-SMITH, T. AND LEE, J. J. 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308 C. /. McFadden