The Pyrenoid Ultrastructure in Oocystis Lacustris

Fottea 9(1): 149–154, 2009 149

Dedicated to Dr. Pe t r Ma r v a n on the occasion of his 80th birthday

The pyrenoid ultrastructure in Oocystis lacustris Ch o d a t (Chlorophyta, Trebouxiophyceae)

Maya P. St o y n e v a 1, Elisabeth In g o l i ć 2, Georg Gä r t n e r 3* & Wim Vy v e r m a n 4

1 Sofia University “St Kliment Ohridski”, Faculty of Biology, Department of Botany, 8 bld. Dragan Zankov, BG– 1164 Sofia, Bulgaria 2 Forschungsinstitut für Elektronenmikroskopie und Feinstrukturforschung, Steyrergasse 17, A–8010 Graz, Austria 3* Leopold-Franzens-Universität Innsbruck, Institut für Botanik, Sternwartestr. 15, A–6020 Innsbruck, Austria; corresponding author: e-mail: [email protected] 4 Ghent University, Department Biology, Laboratory of Protistology and Aquatic Ecology, Krijgslaan 281–S8, B–9000 Gent, Belgium

Abstract: The fine structure of vegetative cells of Oocystis lacustris has been studied with special attention to the ultrastructure of the pyrenoid and its starch sheath. The TEM-investigation showed that the pyrenoid matrix is homogenous, not traversed by thylakoids and the surrounding starch sheath is continuous, horseshoe-shaped or fragmented in 2 starch plates. This starch sheath structure is regarded as a common feature within Oocystis and closely related genera Eremosphaera and Neglectella.

Key words: Oocystis lacustris, ultrastructure, pyrenoids, starch sheath

Introduction light microscopе (Et t l 1980) especially when stained with reagents such as Lugol’s iodine Photosynthetic pigments, storage products and solution. The main structure of the pyrenoid structure of plastids are some of the important matrix (homogenous, perforated, lamellate or features in the taxonomy of eukaryotic algae. In traversed by thylakoids) is also visible in LM with many types of algae within the chloroplast occurs extraordinary optical equipment and when stained a dense proteinaceous body, visible with light with reagents such as azocarmine-G solution microscope and designated as a pyrenoid. The (Et t l 1976, 1983, Gä r t n e r 1985). The electron term “pyrenoid” was created by Sc h m i t z (1882) microscopy only cleared characteristic internal who was the first to associate this structure with an details of the pyrenoid matrix (Gi bbs 1962, Do dg e effect on the accumulation of starch grains in the 1973, Pi c k e t t -He a p s 1975, Fr i e d l 1989, In g o l i ć & chloroplasts of green algae. Nowadays it is well Gä r t n e r 2003). Among the members of the genus known that pyrenoids contain the carbon fixing Oocystis A. Br. the ultrastructure of a pyrenoid enzyme Rubisco and are commonly associated with horseshoe-shaped starch sheath was first with formation of storage products (e.g. Gr a h a m shown by Sc h n e pf , Ko c h & De i c h g r ä b e r (1966, & Wi l c o x 2000, Le e 2008). A remarkable number p. 165, fig. 33) in a schematic graph of Oocystis of morphological types of pyrenoids exists (e.g. solitaria Wi t t r o c k f. maior Wi l l e . Later on, Do dg e 1973, Et t l 1980, Wh a t l e y 1993). The Ro b i n s o n & Wh i t e (1972, p. 112, fig. 5) presented absence or presence of pyrenoids in vegetative a pyrenoid in one TEM-micrograph of Oocystis cells was already used as a taxonomic criterion apiculata W. We s t , and recently So l d o et al. on the generic level of algae (St a r r 1955, Hi n d a k (2005, p. 314, fig. 2 A) documented the pyrenoid 1977-1990) whereas the morphology of the starch of Oocystis nephrocytioides Fo t t et Ča d o in a sheath itself, its structure, and location can assist micrograph of an ultra thin cell section. in the identification of green algal species (Br o w n In this paper the ultrastructural details of & McLe a n 1969, Et t l 1976, Ko m á r e k & Fo t t the pyrenoid in cells of Oocystis lacustris Ch o d a t 1983, Et t l & Gä r t n e r 1988a). The starch sheath are described for first time and comparison with of green algal pyrenoids is normally visible with the related genera Neglectella Vo d e n ič a r o v 150 St o y n e v a et al.: The pyrenoid ultrastructure

et Be n d e r l i e v , Eremosphaera De Ba r y and Siderocelis Fo t t is done.

Material and methods

Oocystis lacustris material was obtained from selected phytoplankton samples from Lake Tanganyika dated June–July 2003 when it formed dense populations (St o y n e v a et al. 2007) and fixed in acid Lugol’s solution. For detailed description of localities, sampling and methods refer to St o y n e v a et al. (2007). For TEM study cells were fixed a) in 3% glutaraldehyd in 0,1 M cacodylate buffer and b) in 1% aqueous Os O4 in 0,1 M cacodylatbuffer, dehydrated in acetone and embedded in Spurr’s resine¸ ultrathin sections were stained with uranyl acetate and lead citrate (Re y n o l ds 1963). Electron micrographs were taken with a Tecnai 12 (FEI) microscope equipped with a Gatan ccd camera.

Results

In ultra thin sections most of the vegetative cells of Oocystis lacustris contain one parietal chloroplast filling more than half of the cell size (Figs 1c, 3c). However, sometimes also two chloroplasts, and, occasionally, four or more of them have been observed. Their thylakoids occur in pairs. In each chloroplast one pyrenoid with a homogenous matrix is situated and surrounded by a thick starch sheath (Figs 1p, 2p, 3p). The diameter of the pyrenoid body is between 1 and 1.5 µm; the thickness of the starch sheath is about 0.25 µm. Thylakoids are not traversing the pyrenoid matrix. The starch sheath around the pyrenoid appears like a closed ring (Fig. 2) or as a horseshoe-shaped starch plate (Fig. 3). There can also be a sheath consisting of two starch plates, more or less regular in thickness (Fig. 1 st). Additionally, single lenticular starch grains, which are not in close association with the pyrenoid, are visible inside the chloroplast (Fig. 2s). These stroma starch grains may reach

Fig. 1 Vegetative cell of Oocystis lacustris with 1 chloroplast (c) and a starch sheath (st) consisting of two starch plates around the homogenous matrix of the pyrenoid (p). n = nucleus. Scale bar 1 µm.

Fig. 2 Pyrenoid (p) with homogenous starch sheath and additional stroma starch grains (s) in the chloroplast. n = nucleus. Scale bar 1µm.

Fig. 3 Chloroplast (c) with pyrenoid (p) and homogenous horseshoe-shaped starch sheath. Scale bar 1µm. Fottea 9(1): 149–154, 2009 151 considerable dimensions (up to 0.25–0.75 µm). A ultrastructure and the pyrenoid construction would single nucleus is embedded in the cell lumen (Figs be necessary. 1n, 2n). The cell wall is multilayered (Figs 1–3). The genus Siderocelis Fo t t was also Its appearance in wavy structure, most probably, placed into the Oocystoideae (Fo t t 1976) based is a result of fixation and dehydration during on detailed light microscopy (Fo t t 1976, Hi n d á k preparation. 1977-1990) but yet the fine structure of its cells in most of the species is unknown. Identical cell wall structures of Amphikrikos nanus (Fo t t Discussion et He y n i g ) Hi n d á k = Siderocelis nana Fo t t et He y n i g to Oocystis species were documented by The finding of the multilayered cell wall during Cr a wf o r d & He a p (1978). Recent TEM-studies this study is in conformity with all previous data, of Siderocelis irregularis Hi n d á k from Lake which showed that the cell walls in Oocystaceae Tanganyika revealed its pyrenoid organization: Bo h l i n are composed of several layers and this matrix traversed by single undulating thylakoids diacritic criterion is in accordance with the and starch sheath consisting of 2–10 plates molecular data (Ko m á r e k 1979, He pp e r l e et al. (St o y n e v a et al. 2008, p. 798, figs. 21, 22). This 2000). structure is clearly different from the pyrenoids of The pyrenoids and their starch components Oocystis and Eremosphaera, as they are discussed are of great value among the main diagnostic below. This could be accepted as additional prove features for identifying coccal green algae with for the exclusion of Siderocelis from Oocystaceae light microscope. Their structure can be cleared (Et t l & Ko m á r e k 1982, Ko m á r e k & Fo t t 1983). up by using staining procedures and squashing- Nevertheless, the degree of relationship of method (Et t l & Gä r t n e r 1988b, 1995). For Siderocelis to the Oocystaceae and its inclusion further taxonomic investigations of unicellular in the trebouxiophycean lineage (Ts a r e n k o et al. green algae on species level TEM studies of 2006) need support of molecular investigations, ultrastructural details of cell components are which still are lacking. important. Among them the pyrenoid matrix In the genus Neglectella Vo d e n ič a r o v (homogenous, with invaginations or traversing et Be n d e r l i e v , generally regarded as close thylakoids) combined with details of the starch to Oocystis, a pyrenoid with massive, thick sheath composition are significant.B r o w n & Bo l d continuous and homogenous starch sheath is (1964) and Br o w n & McLe a n (1969) were the described (Be n d e r l i e v 1971, Vo d e n ič a r o v & first who used the ultrastructure of the pyrenoid Be n d e r l i e v 1971). According to the comparative and number and position of starch grains to studies of Be n d e r l i e v (1971) and the text in classify various species of the green algal genera Vo d e n ič a r o v & Be n d e r l i e v (1971) the pyrenoid Chlorococcum Me n e g h i n i and Tetracystis Br o w n of Neglectella is of the same type as the pyrenoid et Bo l d . In the genus Trebouxia Pu y m a l y tubular of Eremosphaera viridis De Ba r y . This coincides or ramified invaginations into the pyrenoid matrix extensively with the descriptions given by Fo t t and thylakoids traversing through the matrix were & Ka l i n a (1962) but is in opposition to Sm i t h shown to be species-specific (Fr i e d l 1989, In g o l i ć & Bo l d (1966, p. 25) where in E. viridis “a & Gä r t n e r 2003). number of polygonal starch grains often surround Profound light microscopical investigations the pyrenoids, especially in aging or nitrogen- of some genera of the Oocystaceae family have deficient cells”. Such discrepancies could be been published previously (e.g. Pl a y f a i r 1916, based on different cultivation conditions. Recently Sk u j a 1956, Fo t t & Ka l i n a 1962, Fo t t & in the description of Eremosphaera tanganyikae Ře h á k o v á 1963, Sm i t h & Bo l d 1966, Ře h á k o v á St o y n e v a , Gä r t n e r , Co c q u y t et Vy v e r m a n 1969, Hi n d á k 1977–1990, Ko m á r e k & Fo t t (St o y n e v a et al. 2006) some diagnostic features 1983) and recently LM observations on Oocystis of pyrenoid and starch sheath - visible with light lacustris from tropical Lake Tanganyika have been microscope - were included. The starch sheath was documented by St o y n e v a et al. (2007). However, shown to contain two plates (St o y n e v a et al. 2006, for a comprehensive cytomorphological and figs. 37, 39). These results generally coincide with taxonomic study of the whole group (subfamilies our LM observations on Oocystis lacustris, where Oocystoideae and Eremosphaeroideae in Ko m a r e k continuous starch sheath was detected (St o y n e v a & Fo t t 1983) still more investigations of cell et al. 2007, p. 587) and with our recent TEM 152 St o y n e v a et al.: The pyrenoid ultrastructure investigations, showing that starch plates do not References exceed two in number. The presence or absence of a pyrenoid is Be n d e r l i e v , K. (1971): Bau und Entwicklung des documented for most species of Oocystis (e.g. Pyrenoids bei Eremosphaera viridis und Neglectella eremosphaerophila Vo d . et Be n - Sk u j a 1956, Bo u r r e l l y 1966, Ph i l i p o s e 1967, d e r l . – Nova Hedwigia 21: 789–800. Hi n d á k 1977–1990, Ko m á r e k 1983, Ko m á r e k Bo u r r e l l y , P. (1966): Les algues d’eau douce. I. – & Fo t t 1983, Jo h n & Ts a r e n k o 2002), but 511pp., Boubée & Cie., Paris. the organization of the starch sheath was often Br o w n , R. M., Jr. & McLe a n , R. J. (1969): New taxo- neglected. A special note about the continuous nomic criteria in classifications of Chloro- starch sheath was provided by Fo t t & Ča d o (1966) coccum species. II. Pyrenoid fine structure. – J. in their LM diagnosis of Oocystis nephrocytioides. Phycol. 4: 114–118. The TEM photo in So l d o et al. (2005) reveals Br o w n , R. M. & Bo l d , H. C. (1964): Phycological a pyrenoid with homogenous matrix and bipartite Studies 5. Comparative studies of the algal starch sheath. Observations with TEM of Oocystis genera Tetracystis and Chlorococcum. – Univ. apiculata showed a “bilenticular” pyrenoid type Texas Publ. 6417: 1–213. Ch a d e f a u d , M. (1941): Les pyrénoïdes des algues et (Ch a d e f a u d 1941, Ho r i & Ue d a 1967) traversed l’ existence chez ces végétaux d’un appareil by thylakoids and enclosed by a starch sheath cinétique intraplastidial. – Ann. Sci. Nat. Bot. fragmented in two parts (Ro b i n s o n & Wh i t e 11: 1–44. 1972). Cr a wf o r d , R. M. & He a p , P. F. (1978) : Transmission Our results on Oocystis lacustris coincide electron microscopy X-ray microanalysis of partially with the observations on O. apiculata. two algae of the genera Scenedesmus and In O. apiculata, as well as in O. lacustris, the Siderocelis. – Protoplasma 96: 361–367. starch envelope around the pyrenoid appears Do dg e , J. D. (1973): The Fine Structure of Algal Cells. continuous or fragmented in two parts. But O. – 261 pp., Academic Press, London, New York. apiculata pyrenoids are traversed by a simple Et t l , H. (1976): Die Gattung Chlamydomonas Eh r e n b e r g . – Beih. Nova Hedwigia 49: (single) tubular thylakoid system as it is shown by 1–1122. o b i n s o n h i t e R & W (1972, p.112, fig. 5). This was Et t l , H. (1980): Grundriß der allgemeinen Algologie. never observed in cells of O. lacustris where the – 549 pp., VEB Gustav Fischer, Jena. pyrenoid matrix appeared homogenous and was Et t l , H. (1983): Chlorophyta I. Phytomonadina. not traversed by thylakoids (Figs 1–3). Therefore – In: Et t l , H., Ge r l o ff , J., He y n i g , H. & it is clear that the type of pyrenoid matrix Mo l l e n h a u e r , D. (eds): Süßwasserflora von (homogenous or traversed by thylakoids) still Mitteleuropa 9. – 807 pp., Gustav Fischer, needs further studies and, most probably, is not a Stuttgart, New York. diacritic feature in Oocystis and in Oocystaceae. Et t l , H. & Gä r t n e r , G. (1988a): Chlorophyta II. However, it seems that continuous or slightly Tetrasporales, Chlorococcales, Gloeodendrales. – In: Et t l , H., Ge r l o ff , J., He y n i g , H. & fragmented starch sheath with at most two starch Mo l l e n h a u e r , D. (eds): Süßwasserflora von plates is a common diagnostic feature in members Mitteleuropa 10. – 436 pp., Gustav Fischer, of the genus Oocystis and its closely related genera Stuttgart, New York. Neglectella and Eremosphaera, but this still has to Et t l , H. & Gä r t n e r , G. (1988b): Eine einfache Methode be verified by further observations with TEM on zur Darstellung der Struktur der Stärkehüllen more species. von Pyrenoiden bei Grünalgen (Chlorophyta). – Arch. Protistenk. 135: 179–181. Et t l , H. & Gä r t n e r , G. (1995): Syllabus der Boden-, Acknowledgements Luft- und Flechtenalgen. – 721 pp., Gustav The work was partially supported in the frame of a Fischer, Stuttgart, Jena, New York. research fellowship for the first author (M. P. St.) by the Et t l , H. & Ko m á r e k , J. (1982): Was versteht man Belgian Science Policy Office (CLIMLAKE project) unter dem Begriff “coccale Grünalgen”? – and by a research fellowship for the third author (G. Arch. Hydrobiol. Suppl. 60, 4, Algol. Stud. 29: Gä.) financed by the Austrian Research Association 345–374. (MOEL-project nr. 118). Fo t t , B. (1976): Oocystis und verwandte Gattungen aus der Unterfamilie der Oocystoideae; Namensänderungen, taxonomische Notizen und Bestimmungsschlüssel. – Preslia 48: 193–206. Fo t t , B. & Ča d o , I. (1966): Oocystis nephrocytioides Fottea 9(1): 149–154, 2009 153

sp. nov. – Phycologia 6: 47–50. Pl a y f a i r , G. I. (1916): Oocystis and Eremosphaera. Fo t t , B. & Ka l i n a , T. (1962): Über die Gattung – Proceedings of the Linnean Society of New Eremosphaera De Ba r y und deren South Wales 41: 107–147. taxonomische Gliederung. – Preslia 34: 348– Pr i n t z , H. (1913): Eine systematische Übersicht der 358. Gattung Oocystis Nägeli. – Nyt Magazin for Fo t t , B. & Ře h á k o v á , T. (1963): Infraspecific Naturvidenskaberne 51: 165–203. variability in Oocystis coronata Le m m e r m a n n . Ře h á k o v á , H. (1969): Die Variabilität der Arten der – Nova Hedwigia 6: 239–243. Gattung Oocystis A. Br a u n . – In: Fo t t , B. (ed.): Fr i e d l , T. (1989): Comparative ultrastructure of Studies in Phycology. – pp. 145–196, Academia, pyrenoids in Trebouxia (Microthamniales, Praha. Chlorophyta). – Pl. Syst. Evol. 164: 145–159. Re y n o l ds , E. S. (1963): The use of lead citrate at Gä r t n e r , G. (1985): Die Gattung Trebouxia Pu y m a l y high pH as an electron opaque stain in electron (Chlorellales, Chlorophyceae). – Arch. microscopy. – J. Cell. Biol. 17: 208–212. Hydrobiol. Suppl. 71, Algol. Studies 41: 495– Ro b i n s o n , D. G. & Wh i t e , R. K. (1972): The fine 548. structure of Oocystis apiculata W. We s t with Gi bbs , S. P. (1962): The ultrastructure of the pyrenoids particular reference to the wall. – Br. Phycol. J. of the green algae. – J. Ultrastruct. Res. 7: 262– 7: 109–118. 272. Sc h m i t z , F. (1882): Die Chromatophoren der Algen. – Gr a h a m , L. E. & Wi l c o x , L. W. (2000): Algae. – 640 180 pp., Max Cohen & Sohn, Bonn. pp., Prentice-Hall, Upper Saddle River, NJ. Sc h n e pf , E., Ko c h , W. & De i c h g r ä b e r , G. (1966): Zur He pp e r l e , D., He g e w a l d , E. & Kr i e n i t z , L. (2000): Cytologie und taxonomischen Einordnung von Phylogenetic position of the Oocystaceae Glaucocystis. – Arch. Mikrobiol. 55: 149–174. (Chlorophyta). – J. Phycol. 36: 590–595. Sk u j a , H. (1956): Taxonomische und Biologische Hi n d á k , F. (1977–1990): Studies on the Chlorococcal Studien über das Phytoplankton Schwedischer algae (Chlorophyceae), I-V. – Biologické práce, Binnengewässer. – Nova Acta Reg. Soc. Scient. Veda, Bratislava. Upsaliensis, ser. IV, 16: 1–404. Ho r i , T. & Ue d a , R. (1967): Electron microscope Sm i t h , R. L. & Bo l d , H. C. (1966): Phycological studies on the fine structure of plastids in studies.VI. Investigation of the algal genera siphonous green algae with special reference to Eremosphaera and Oocystis. – Univ.Texas their phylogenetic relationships. – The Science Publ. 6612: 7–121. Rep., Tokyo Kyoiku Daigaku, Sec. B, 12/187: So l d o , D., Ha r i , R., Si gg , L. & Be h r a , R. (2005): 225–244. Tolerance of Oocystis nephrocytioides to copper: In g o l i ć , E. & Gä r t n e r , G. (2003): Ultrastructure intracellular distribution and extracellular of pyrenoids in green algal taxonomy. – complexation of copper. – Aquatic Toxicology Performance Report 2001/2002. – pp. 90–91, 71: 307–317. Forschungsinstitut für Elektronenmikroskopie, St a r r , R. C. (1955): A comparative study of Techn. Universität Graz, Austria. Chlorococcum Me n e g h i n i and other Jo h n , D. M. & Ts a r e n k o , P. M. (2002): Chlorococcales. spherical, zoospore-producing genera of the – In: Jo h n , D. M., Wh i t t o n , B. A. & Br o o k , A. J. Chlorococcales. – Indiana Univ. Publ., Sci. Ser. (eds): The Freshwater Algal Flora of the British 20: 1–111. Isles. An Identification Guide to Freshwater and St o y n e v a , M. P., Co c q u y t , C., Gä r t n e r , G. & Terrestrial Algae. – pp. 327–408, Cambridge, Vy v e r m a n , W. (2007): Oocystis lacustris Ch o d . Cambridge University Press. (Chlorophyta, Trebouxiophyceae) in Lake Ko m á r e k , J. (1979): Änderungen in der Taxonomie der Tanganyika (Africa). – Linzer. Biol. Beitr. 39: Chlorococcalalgen. – Arch. Hydrobiol./Algol. 571–632. Stud. Suppl. 56, 24: 239–263. St o y n e v a , M. P., Gä r t n e r , G., Co c q u y t , C. & Vy v e r - Ko m á r e k , J. (1983): Contribution to the chlorococcal m a n , W. (2006): Eremosphaera tanganyikae sp. algae of Cuba. – Nova Hedwigia 37: 65–180. nov. (Trebouxiophyceae), a new species from Ko m á r e k , J. & Fo t t , B. (1983): Chlorococcales. - In: Lake Tanganyika. – Belg. J. Bot. 139: 3–13. Hu b e r -Pe s t a l o z z i , G. (ed.): Das Phytoplankton St o y n e v a , M. P., In g o l i ć , G., Ko f l e r , W. & Vy v e r m a n , des Süßwassers 7. – 1044 pp., Schweizerbart, W. (2008): Siderocelis irregularis. (Chloro- Stuttgart. phyta, Trebouxiophyceae) in Lake Tanganyika. Le e , R. E. (2008): Phycology. – 547 pp., Cambridge – Biologia 63: 795–801. University Press, Cambridge. Ts a r e n k o , P. M., Wa ss e r , S. P. & Ne v o , E. (eds) (2006): Pi c k e t t -He a ps , J. D. (1975): Green Algae. – 606 pp., Algae of Ukraine: Diversity, Nomenclature, Sinauer Ass. Inc., Sunderland. Taxonomy, Ecology and Geography, Vol. Ph i l i p o s e , M. T. (1967): Chlorococcales. – 365 pp., 1. – 713 pp., A. R. G. Gantner, Ruggell, ICAR, New Delhi. Liechtenstein. 154 St o y n e v a et al.: The pyrenoid ultrastructure

Vo d e n ič a r o v , D. & Be n d e r l i e v , K. (1971): Neglectella gen. nov. (Chlorococcales). – Nova Hedwigia 21: 801–812. Wh a t l e y , J. M. (1993): Chloroplast ultrastructure. – In: Be r n e r , T. (ed.): Ultrastructure of Microalgae. – pp. 135–204, CRC Press, Boca Raton, Ann Arbor, London, Tokyo.