J.K.A.U. Mar. Sci, , Vol.l,pp. 77-84(1410A,H./1990A.D.

Isozymal Relationships between Zoospores and VegetativeCells of Chlamydomonas euryale

Y AHIA A. MOHAMADDepartment of Biological Sciences, Faculty of Science,

United Arab Emirates, University.

ABSTRACT. Zoospores and vegetative cells of Chlamydomonas euryale(Lewin) have been compared through polyacrylamide gel electrophoreticpatterns of their total soluble proteins, a-Est., Alk. Ph., MDH, GDH,LAP, PER, TYR, GOT and AMY isozymal systems to check if any differ-ence might occur. Data have been treated according to a mathematical simi-

larityapproach.Though being two types of cells of the same species, vegetative cells and

zoospores showed 100% similarity in amylase isozyme pattern only.Similarities of T .S.P., a-Est., MDH, GDH, TYR, LAP and PER patternsranged from 50% to 80%. Still those of Alk. Ph. and GOT were quite dis-similar (0.0% similarity). The average similarity based on T .S.P. and nineisozyme systems used, was 50%. Possible reasons for the unexpected resultsare discussed and comments are made out.KEY WORDS: Chlamydomonas euryale, vegetative cells, zoospores,polyacrylamide gel ele'ctrop'horesis, total soluble proteins (T.S.P.), a-es-terases (a-Est.), alkaline phosphatases (Alk. Ph.), malate-dehydrogenases(MDH), glutamate dehydrogenases (GDH), leucine amino-peptidases(LAP), peroxidases (PER), tyrosinases (TYR), glutamate oxalacetatetransaminases (GOT), amylases (AMY), isozymal relationships.

Introduction

Isozymes can be products of different genetic sites, or may result from secondary alt-erations in the structure of a single polypeptide species. They may also be seen tofunction in different intracellular compartments, e.g., chloroplastic versus cytosolu-ble isozymes, and in different metabolic processes, e.g. glycolysis andgluconeogenesis. Taxonomists have exploited electrophoretic methods of isozyme

77

78 YahiaA. Mohamad

separation that in natural populations of sexually reproducing organisms to show: (a)numerous genetic loci are normally polymorphic; (b) some individual loci are ex-ceedingly variable; most species, even sibling species are genetically well differen-tiated from one another. Accordingly, isozyme patterns are very'useful charactersand particularly powerful tools at low taxonomic levels.

Isozyme analysis has been used to study the relation between Chlamydomonaseuryale and C. mowusii (Thomas and Delcarpio 1971). Mohamad (1981) made themost intensive work on isozymal chemotaxonomy of the genus Chlamydomonasusing 24 species including C. euryale. This work suggested grouping some speciesinto sub-genera and that two species are subject to split from the genusChlamydomonas. During his work, Mohamad noticed that C. euryale showed a veryweak growth in fresh water media, moderate growth in brackish water media (salin-ity 18%) and the best growth in enriched sea water media just as good as forDuna.iiella. Chlamydomonas euryale is then considered to be a euryhaline species to-lerant to big salinity change. Its zoospores are very small oblong in shape and very ac-tively motile.

The aim of the present work is to check if any difference could exist among theisozymal patterns of zoospores and vegetative cells of Chlamydomonas euryale.There is no report, so far, in the literature about any isozymal comparison of vegeta-tive cells and their reproductive units.

Material and Methods

The axenic culture of Chlamydomonas euryale has been obtained from Cambridgecollection. Culture has been grown in an enriched seawater medium at 18°C and 350footcandles supplied by cool flourescent lamps. It has been noticed that clouds offine green cells would appear concentrated at the liquid medium surface by the flaskwall. Microscopic examination showed that they were fine oblong very activelymotile reproductive units much smaller than the vegetative cells. Examination of thevegetative cells almost sedimenting near the bottom, showed that many of them in-cluded those small cells inside. The small cells have been pushing themselves rightand left inside the mother cell wall and escaped out one after another. Examinationof those reproductive units at successive time intervals showed no conjugation at all,so it was decided that they were zoospores rather than gametes. As these clouds weremaximum after three days of inoculation, probably as a response for the newmedium, big inocula were used (15 ml/100 ml medium). After three days of cultur-ing, green clouds were good enough at the surface. Cultures were centrifuged first at2500 g for 5 minutes. The pellet contained big vegetative cells only. The supernatantincluding the zoospores was recentrifuged at 5500 g for 10 minutes. Because of theiroblong shape and high active motility, zoospores used to stick by the surfa~e of theglass centrifuge tube and their sedimentation was much more slower than that of thevegetative cells. Pellets of both vegetative cells and zoospores were washed twiceusing phosphate buffer, pH 7.5, resulting a final cell suspension that was 0.5 M in tris.Cells were ruptured by passage through a French pressure cell (AMINCO) at 11,000

79lsozymal Relationships between Zoospores

pounds per square inch. Microscopic examination revealed a rupture yield in excessof 90%. All sample tubes and suspensions were kept in ice at about 5°C. The brokencell suspensions were centrifuged for 10 minutes at 7,800 g and the supernatant re-centrifuged for 15 minutes at 83,000 g in Beckman Ultracentrifuge (L3-40). Thesupernatants were concentrated in pretreated dialysis tubing over a sucrose bedunder vacuum. Concentrated samples were transferred into small sample tubes, towhich a few drops of bromophenol blue were added as electrophoretic marker. Allextracts were kept frozen for immediate isozymal analysis.

Electrophoretic gel preparations were the same as described by Scandalios (1969).All the chemicals used were supplied by sigma, BDH and J. T. Baker reagent grades.

For total soluble proteins, the recovered gels were fixed in 12.5% trichloroaceticacid for 10 minutes, and then stained overnight in 1.0% Coomassie blue inmethanol:acetic acid:water; 5: 1:5 v/v. Gels were then destained in fresh changes ofmethanol: acetic acid:water; 5:1:5 v/v for few minutes, one hour,S hours and a wholeday, respectively.

For isozymal analysis, the recovered gels were transferred directly to the approp-riate staining reaction mixture described in the original literature as follows:

1. a-esterase (Scandalios 1969).2. Alkaline phosphatase (Scandalios 1969).3. Malate dehydrogenase (Scandalios 1969).4. Glutamate dehydrogenase (Scandalios 1969).5. Leucine aminopeptidase (Smith and Van Frank 1975).6. Glutamate oxalacetate transaminase (Schwartz et al. 1963; modified by Shaw

and Prasad 1970).7. Peroxidase (Graham etal. 1965; modified by Shaw and Prasad 1970).8. Amylase (Scandalios 1969).9. Tyrosinase (Madhoshingh and Wood 1971).

Results

Individual gel runs are shown as line diagrams (Fig. 1) with migrations relative tobromophenol blue. Stainable bands of isozymes are considered both stacking and re-solving gels. Some workers ignore the staking gels, while some others (Rothman andLinden 1965, Murer 1971) do consider them.

The number of stained bands of total soluble proteins was much higher in case ofzoospores than that of the vegetative cells. Among the nine isozymal systemsstudied, those of a-esterases and malate dehydrogenases showed to be the most var-iable ones, while those of glutamate oxalacetate transaminases (GOT) and amylasesshowed to be least variable with quite a few bands. Like the case of total soluble pro-teins, the electrophoretic patterns of MDH, GDH and GOT had more bands forzoospores extracts than for those of vegetative cells. The reverse is true concerninga-esterases, alkaline phosphatases, peroxidases and leucine amino-peptidases. In

80 YahiaA. Mohamad

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case of tyrosinases and amylases, the zymograms of zoospores and vegetative cellsshowed equal number of bands..

Similarities between the patterns of zoospores and vegetative cells have been cal-culated for the total soluble proteins and the nine isozymal systems studied accordingto the similarity approach described by Sokal and Sneath (1963). Similarity percen-tages are showed in Table 1 and represented as individual and average dendrogramsin Fig. 2.

Similarity percentages between electrophoretic patterns of zoospores and vegetative cells.TABLE

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Discussion

Unexpectedly, the zymograms of nine isozymal systems as well as the total solubleprotein patterns of vegetative cells and zoospores of the same strain did not show ahigh percentage of similarity. Only one case of isozymal pattern identity (100% simi-larity) was found, that is amylase. Other cases of good similarity percentages werefound to be those of leucine aminopeptidase (80% ), malate dehydrogenase activity(73 %) and peroxidase activity (67%). a-esterase, glutamate dehydrogenase andtyrosinase patterns showed around 50% similarity, something that can not reflectstrong isozymal relations between zQPspores and vegetative cells in the culture sup-posed to be parent ones getting ready to divide. Still alkaline phosphatase and gluta-mateoxalacetate activity showed complete dissimilarity. These findings show that atleast some isozymal systems are different, and that the two types of cells could beidentical only in the starch metabolism. The idea that cells of the same species shouldhave the same and identical isozymal pattern should not be a straight rule all thetime. It should depend upon some conditions. For example, the physiological condi-tion of individuals, which would be certainly reflected on how operons work at diffe-rent genes coding for the isozymes associated with the physiological change. Thevegetative parent cells are big, old, many of them became non-motile and lost theirflagella and are ready to start several successive divisions. On the other hand, thezoospores are very small, new born and very actively motile cells. These, of course,are two cases that are physiologically quite different.

Zoospores were defined by Fritsch (1935) as naked flagellate protoplasts that showthe distinctive feature of their class. A single zoospore may be formed from the con-tents of each cell like in Oedogonium or, more commonly, division of the contentsinto two, four, eight or more parts (several hundreds in Cladophora) takes place andan equivalent number of zoospores is produced. He added that when zoospore for-mation is accompanied by division, the plasma membrane and the central vacuolemembrane are not utilized in the production of the swarmers. Pascher (1930) pointedout more differences that could exist between cells and their zoospores. He said "Ina considerable number of algae belonging to different classes the swarmers, eitherafter a brief period of flagellar activity or already at the time of liberation, assume anamoeboid state and may in this condition even show holozoic nutrition". Hartman,(1918) described some cytological difference that could exist between vegetativecells and zoospores in Chlamydomonas.

The above mentioned biochemical studies dealing with isozymal differences bet-ween the vegetative cells and zoospores of C. euryale do, support the morphologicaland physiological findings of earlier workers in this regard.

References

Fritsch, F .E. (1935) The Structure and Reproduction of the Algae, Cambridge University Press, Cam-

bridge.Graham, R.C., Lundholm, U. and Karnovsky, M.J. (1965) Cytochemical D~monstration of Peroxidase

Activity with 3-Amino- -Ethylcarbazole. J. Histochem. Cytochem. 13: 150.

83Isozymal Relationships between Zoospores.

Hartman, M. (1918) Ueber die Kem-und Zelletailung von Chlorogonium elongatum Dung, Arch. Protis-tenk. 39: 7 et seq.

Madhoshingh, C. and Wood, I.M. (1971) Dopa Stain for Precipitins in Gel, Anal. Biochem. 44: 523.Mohamad, Y.A. (1981) Biochemical Taxonomy of Chlamydomonas and Related Volvocalean Genera,

Ph.D. Thesis, Univ. of California, Los Angeles.Murer, R.R. (1971) Disc Electrophoresis and Related Techniques of Polyacrylamide Gel Electrophoresis,

Walter de Gruyter, Berlin, New York.Pascher, A. (1930) Neue Volvocalen (Polyblepharidinen-Chlanydomanadinen) Arch. F. Protistenk. 69:

103.Rothman, U. and Linden, S. (1965) Isolation of Lymphoid-Cell Protein with Relation to Delayed Hyper-

sensitivity, Nature 208: 389.Scandalios, J.G. (1969) Genetic Control of Multiple Molecular Forms of Enzymes in Plants, A review

Biochem. Genet. 3:37.Schwartz, M.K., Nesselbaum, J.S. and Bodansky, O. (1963) Procedure for Staining Zones of Activity of

Glutamic Oxalacetic Transaminase Following Starch Gel Electrophoresis, Am. J. Clin. Pathol-ogy. 40: 103-106.

Shaw, C.R. and Prasad, R. (1970) Starch Gel Electrophoresis of Enzymes, Biochem. Genetics. 4: 297-320.Smith, R.E. and Van Frank, R.M. (1975) The Use of Amino Acid Derivative of 4-methoxy-

naphthyl amine for the Assay and Subcellular Localization of Tissue Proteinases, in: Dingle, J. T.and Dean, R.I. (ed.), Lysosomes in Biology and Pathology, Elsevier North-Holland, Amsterdam,

pp.193-249.Sakal, R.R. and Sneath, P.H.A. (1963) Principals of Numerical Taxonomy, W.H. Freeman, New York.Thomas, D.L. and Delcarpio, J.B. (1971) Electrophoretic Analysis of Enzymes from Three Species of

Chlamydomonas, Amer. J. Bot. 58: 716-720.

YahiaA. Mohamad84

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