UDC 58
CODEN: ABCRA2
YU ISSN 0365—0588
ACTA BOT. CROAT. VOL. 40, 67—72, 1981.
UDC 582.251.72 : 575.24 = 20
P R O P E R T I E S AND S T R U C T U R E OF A
BLEACHED PIGM ENTLESS MUTANT
OF EU G L E N A G R A C I L I S *
ELENA MARCENKO
( R u d j e r B o s k o v ic I n s t i t u t e , Z a g r e b )
R e c e iv e d F e b r u a r y 10, 1981
Introduction
Diverse bleached m utants have been derived from the green flagellate
Euglena gracilis by physical and chemical agents (see e. g. K i r k 1967,
S c h i f f et al. 1971, P a r t h i e r and N e u m a n n 1977). These m u­
tan ts differ w ith regard to the presence or absence of a recognizable
plastid structure, as w ell as stigm a and carotenoid content ( L e f o r t 1963,
M o r i b e r et al. 1963, R o g e r s et al. 1972, B e n - S h a u l et al.
1972, M a r c e n k o 1973, K i v i c and V e s k 1974 a and 1974 b,
H e i z m a n n et al. 1976, K r o n e s t e d t and W a l e s 1976, P a r t h ­
i e r and N e u m a n n 1977). Most of the investigated m utants still con­
tain m ore or less expressed plastid structures resem bling proplastids of
th e etiolated w ild type Euglena. Some thylakoids, plastoglobuli and prolam ellar body-like structures have often been observed in such plastids.
In some cases very ab erran t structures, hardly recognizable as plastids, or
even no such stru ctu res w ere found.
The bleached m utants show, in m ost cases, m ore or less pronounced
carotenogenesis ( D o l p h i n 1970, M a r c e n k o 1973, G r o s s and
S t r o z 1975, P a r t h i e r and N e u m a n n 1977), lim ited photoinduced plastid developm ent ( S a l v a d o r et al. 1972, M a r c e n k o
1973, P a r t h i e r and N e u m a n n 1977) and photoinduced plastid en­
zyme synthesis ( P a r t h i e r and N e u m a n n 1977, R u s s e l l and
D r a f f a n 1978). M utants com pletely lacking plastids, stigm ata, and
pigm ents are com paratively rare.
The properties and u ltrastru c tu re of an obviously pigm entless, com­
pletely w hite Euglena m u tan t are examined.
* Dedicated to Professor Zvonimir D é v i d é on the occasion of his 60th
birthday.
A C T A B O T . C R O A T . V O L . 40, 1981.
67
ELENA
M ARCENKO
Materials
and
Methods
Two alm ost identical strains of a w hite Euglena m u tan t w ere de­
rived by chance from a carotenoid containing m utant Y -l ( M a r c e n k o
1973) by intense illum ination (over 10,000 lux) and subsequent grow th in
the d ark at room tem perature (the tem perature during illum ination was
not controlled).
Cells w ere grow n axenically in a rich organic m edium ( M a r c e n k o
1970), or in the synthetic m edium of C ram er and M yers m odified by P ad i l l a and J a m e s (1964).
G row th rates are expressed as K, the num ber of doublings per day,
according to H o o g e n h o u t and A m e s z (1965). Cell counts were
m ade in a haem ocytom eter.
P reparations for electron microscopy w ere done as described previ­
ously ( M a r c e n k o 1973).
E xtraction and analysis of pigm ents: Cells w ere harvested by centri­
fugation, washed w ith distilled w ater and extracted w ith acetone. The
ex tract was evaporated in vacuo and the residue tran sferred to n-hexan.
The spectrum was recorded w ith a P erk in Elm er Mod 137 spectrophoto­
m eter.
Results
Two com pletely w hite strains w ere obtained as the result of bleaching
of th e yellow m u tan t w ith light of high intensity. These m utants never
changed its w hite colour w h eth er grow ing in the d ark or in the light. They
never rev erted to th e yellow or green w ild type. According to the nom en­
clature of m utants of Euglena (S c h i f f et al. 1971) it could be called
WYL. The grow th of the tw o alm ost identical strains WYL-1 and WYL-2,
at d ifferen t tem peratures, com pared w ith the grow th of the wild type, is
show n in Table I. As shown by counting divisions of individual cells on
ag ar plates the slow grow th ra te of the w hite strains can, at least partially,
be attrib u ted to some cells dying a t the stage of cell division ( M a r c e n k o 1980). The grow th of the w hite strains is photoinhibited ( M a r c e n k o
1980). Photoinhibition of the grow th of some bleached strains of Euglena
has also been observed by C o o k (1968), M i t c h e l l (1971) and N i c o ­
l a s et al. (1980).
P igm ent analysis reveals no traces of chlorophyll or coloured caro­
tenoids. In th e short w ave region, however, some characteristic peaks
appear w hich are close, b u t not identical to the absorption m axim a of
phytoene indicating m ost probably, the presence of ergosterol ( G r o s s
and S t r o z 1975).
------------------------------------------------------------------------------------------------- ►
Figs. 1. and 2. Possible plastid remnants in a pigmentless Euglena mutant
WYL light-grown. Note the concentric lamellae and membrane-bound
non-osmiophilic vesicles (Fig. 2). 24,000 :1.
Fig. 3. Eyespot-region in WYL mutant, light-grown. Note the membrane-bound
vesicles of the stigma remnant in the vicinity of the flagellar region.
32,000 :1.
68
A C T A B O T . C R O A T . V O L . 40. 1981.
P R O P E R T IE S O F A B L E A C H E D P IG M E N T L E S S M U T A N T O F E U G L E N A G R A C I L I S
Figs. 1—3.
A C T A B O T . C R O A T . V O L . 40, 1981.
P R O P E R T IE S O F A B L E A C H E D P IG M E N T L E S S M U T A N T O F E Ü G L E N A G R A C IL IS
T a b l e I. Growth rates of Euglena strains at different temperatures in
the dark
Strain
Wild type
Wild type
Wild type
WYL-1
WYL-1
WYL-1
WYL-2
WYL-2
WYL-2
Temperature
Growth constant
K (number of
doblings per
day)
Generation time
20 *C
25 °C
30 “C
20 “C
25 °C
30 °C
20 “C
25 °C
30 °C
1,36
1,49
2,00
0,90
1,15
1,59
0,90
1,13
1,45
18h
16h
12h
27h
20h
15h
27h
21h
16’>
Light-m icroscopic exam ination reveals the absence of visible stigm a
in th e light- or dark-grow n m u tan t WYL, and no detectable plastids.
Electron-m icroscopic exam ination of light-grow n cells reveals, be­
sides num erous m itochondria, some structures of the size of proplastids
(ca 2 |xm) w ith v ery a b e rra n t lam ellae, often concentrically arranged (Fig.
1.). Sometim es these structures contain m em brane-bound em pty vesicles,
lacking osmiophily, of the size of plastoglobuli (Fig. 2). Such bodies rem inescent of plastid rem nants w ere not observed in the dark-grow n m utant.
Non-osmiophilic m em brane-bound vesicles of the size of stigm a-globules
in th e vicinity of the flagellar region w ere also present (Fig. 3). The p re­
sence of the p araflag ellar body in our m u tan t could not be confirm ed w ith
certainty.
L e f f and K r i n s k y (1967) claim ed to have obtained perm anently
bleached “white« cells of Euglena by th e m utagenic action of visible light
of high intensity, u n der aerobic conditions. B leached strains thus obtained
have been considered aplastidic, and lacking plastid DNA. To test w hether
these cells are possibly identical to the WYL m utant, wild type Euglena
was subjected to th e trea tm e n t described, b u t no com pletely pigm entless
or stigmaless strains w ere produced ( M a r c e n k o , unpublished). Only
chlorophyll was lacking, bu t stigma, carotenoids and some plastid struc­
tu res w ere present.
P r i n g s h e i m s ’ observation of stigm aless Euglena, produced by
heat bleaching and subsequent grow th in the dark (1951), could no t be
confirm ed in our laboratory. However, th e ir observations w ere based on
light-microscopic exam ination only, so th a t th e presence of stigm ata
m ight possibly have been overlooked. T here is also a difference in the
strains: th eirs was “bacillaris” and ours “Z ”.
Discussion
The yellow m u tan t Y -l containing distinct plastid structures and
carotenoids ( M a r c e n k o 1973), w hen grow n in the d ark and in the
light, can easily be bleached w ith th e sam e agents used for bleaching the
green Euglena ( M a r c e n k o 1974). The bleached cells can be called
“colourless" or ra th e r “pale-yellow ” as they also contain stigm ata and
exhibit light-induced carotenogenesis. The am ount of carotenoids in the
“pale-yellow ” m utants in th e d ark is negligible, and they appear alm ost
white, w hich m akes them easily distinguishable from th e yellow strain.
A C T A B O T . C R O A T . V O L . 40, 1981.
69
ELENA
M ARCENKO
Most of the bleached m utants derived from wild type green Euglena
also belong to the category of the “colourless” or “w hite” strains. The
term “w h ite” is ra th e r misleading, as m ost of these m utants exhibit some
plastid structures and some photoinduced carotenogenesis ( D o l p h i n
1970, K i v i c and V e s k 1974 a). Some of these “w hite” strains even
exhibit a lim ited chlorophyll synthesis (e. g. the strain “blanc” of S a l v a ­
d o r et al. 1972). D istinct plastid structures and carotenogenesis w ere
found in th e WSBUL m u tan t (P a 1 i s a n o and W a l n e 1976, P a r t h i e r and N e u m a n n 1977), earlier considered aplastidic. H e i z m a n n et al. (1976) observed in the sam e m utant some plastid structures
only after cycloheximide treatm ent.
T he WYL m utant described in the present paper is, however, com­
pletely pigm entless. Thus, it does not resem ble any of the bleached m u­
tan ts exam ined by P a r t h i e r and N e u m a n n (1977) which all con­
tain some carotenoids. In th eir strains these authors also observed some
prolam ellar bodies, osmiophilic globuli and even some stacked thylakoids,
all of which w ere absent from our strain. It is m ore closely like the alm ost
aplastidic strains described, e. g. by K i v i c and V e s k (1974 a), and the
em pty “eye-spot” vesicles resem ble th a t of Euglena derived Astasia longa
( K i v i c and V e s k 1974 b). Astasia longa is a natu rally occurring pig­
m entless Euglena which lacks any plastid stru ctu re ( R o g e r s et al.
1972, K i v i c and V e s k 1974 a).
I t is proposed th a t the term “w hite” should be reserved for strains
which under no condition develop any visible pigm ent. The proper charac­
terization of strains becomes p articu larly im portant in studies on the cor­
relation of plastid stru ctu re and function.
Summary
The stru ctu re and grow th w ere exam ined in a white, com pletely
pigm entless, m u tan t of Euglena gracilis. A lthough it lacks any pigments,
coloured stigm a and defined plastids, some non-osm iophilic structures
resem bling stigm a vesicles and, possibly, very ab erran t rem nants of
plastids appear in this strain. The value of the exact characterization of
bleached m utants is discussed.
*
I am indebted to Dr. N. L j u b e s i c for electron microscopy of the spe­
cimens.
References
Ben-Shaul, Y., R. Silman, I. Ophir, 1972: Effects of streptomycin on the ultra­
structure of plastids in Euglena. Physiol, vég. 10, 255—268.
Cook, J. R., 1968: Photo-inhibition of cell division and growth in euglenoid
flagellates. J. Cell. Physiol. 71, 177—184.
Dolphin, W. D., 1970: Photoinduced carotenogenesis in chlorotic Euglena gracilis.
Plant Physiol. 46, 685—691.
Gross, J. A., R. J. Stroz, 1975: The carotenoid hydrocarbons of Euglena gracilis
and derived mutants. Plant Physiol. 55, 175—177.
70
A C T A B O T . C R O A T . V O L . 40, 1981.
P R O P E R T IE S O P A B L E A C H E D P IG M E N T L E S S M U T A N T O F E U C L E N A G R A C I L I S
Heizmann, P., G. F. Salvador, V. Nigon, 1976: Occurrence of plastidial rRNAs
and plastidial structures in bleached mutants of Euglena gracilis. Exp.
Cell Res. 99, 253—260.
Hoogenhout, H., J. Amesz, 1965: Growth rates of photosynthetic microorganisms
in laboratory cultures. Arch. Mikrobiol. 50, 10—24.
Kirk, J. T. O., 1967: Genetic control and plastid biochemistry. In: Kirk J. T. O.,
R. A. E. Tilney-Basset: The Plastids. Freeman, London, San Francisco.
Kivic, P. A., M. Vesk, 1974a: An electron microscope search for plastids in ble­
ached Euglena gracilis and in Astasia longa. Can. J. Bot. 52, 695—699.
Kivic, P. A., M. Vesk, 1974b: The structure of the eyespot apparatus in bleached
strains of Euglena gracilis. Cytobiologie 10, 88—101.
Kronestedt, E., B. Wales, 1976: On the presence of plastids and eyespot appa­
ratus in a porfiromycin-bleached strain of Euglena gracilis. Protoplasma
84, 75—82.
Leff, J., N. I. Krinsky, 1967: A mutagenic effect of visible light mediated by
endogenous pigments in Euglena gracilis. Science 152, 1332—1334.
Lefort, M., 1963: Étude comparative de certains mutants dépigmentés chez
1’Euglena gracilis et chez d’autres algues. C. R. Acad. Sci. (Paris) 256,
5190—5192.
Marčenko, E., 1970: The effect of illumination regimen on temperature-induced
and spontaneous bleaching in Euglena gracilis. Acta Bot. Croat. 29, 27—32.
Marčenko, E., 1973: Plastids of the yellow Y -l strain of Euglena gracilis. Proto­
plasma 76, 417—433.
Marčenko, E., 1974: Effect of light and temperature on the bleaching of the
yellow mutant Y -l of Euglena gracilis. Protoplasma 82, 119—123.
Marčenko, E., 1980: Effect of isopropyl N-phenylcarbamate on Euglena. Acta
Bot. Croat. 39, 71—78.
Mitchell, J. L. A., 1971: Photoinduced division synchrony in permanently ble­
ached Euglena gracilis. Planta 100, 244—257.
Moriber, L. G., B. Hershenov, S. Aaronson, B. Bensky, 1963: Teratological chloroplast structures in Euglena gracilis permanently bleached by exo­
genous physical and chemical agents. J. Protozool. 10, 80—86.
Nicolas, P., G. Freyssinet, V. Nigon, 1980: Effect of light on glucose utilization
by Euglena gracilis. Plant Physiol. 65, 631—634.
Padilla, G. M., T. W. James, 1964: Continuous culture of Protozoa. In: Methods
in Cell Physiology. Vol. I (Prescott, D. M. ed.) Academic Press, New
York, London.
Palisano, J. R., P. L. Walne, 1976: Light and electron microscopy of two per­
manently bleached cell lines of Euglena gracilis (Euglenophyceae) Nova
Hedwigia 27, 455—478.
Parthier, B., D. Neumann, 1977: Structural and functional analysis of some
plastid mutants of Euglena gracilis. Biochem. Pflanzen 171, 547—562.
Pringsheim, E., O. Pringsheim, 1951: Experimental elimination of chromatophores and eye-spot in Euglena gracilis. New Phytologist 51, 5—76.
Russell, G. K., A. G. Draff an, 1978: Light-induced enzyme formation in a chlo­
rophyll-less mutant of Euglena gracilis. Plant Physiol. 62, 678—682.
Rogers, T. D., V. E. Scholes, H. E. Schlichting, 1972: An ultrastructural compa­
rison of Euglena gracilis Klebs, bleached Euglena and Astasia longa
Pringsheim. J. Protozool. 19, 133—139.
Salvador, G., V. Nigon, F. Richard, P. Nicolas, 1972: Structure et propriété d’un
nouveau mutant blanc d’Euglena gracilis. Protistologia 8, 533—540.
Schiff, J. A., H. Lyman, G. K. Russel, 1971: Isolation of mutants from Euglena
gracilis. In: Methods in Enzymology. Vol. 23A (San Pietro A. ed.) Acade­
mic Press, New York, London.
A C T A B O T . C R O A T . V O L . 40, 1981.
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ELENA M ARCENKO
SADRŽAJ
S V O JST V A I S T R U K T U R A B E Z B O JN E M U T A N T E V R STE KU GLE N A G R A C I LI S
Elena Marčenko
( I n s t i t u t » R u đ e r B o šk o v ić « , Z a g re b )
Istraživani su ra st i u ltra stru k tu ra bijele bezbojne m utante vrste
Euglcna gracilis. Iako bez pigm enta, u stanicam a postoje ostaci stigme i
plastida, koji sadržavaju samo koncentrične lam ele i ostatke globula.
Ukazano je na potrebu točnije karakterizacije izblijeđenih m utanti
euglena.
D r . K le n a M a r č e n k o
I n s t i t u t » R u đ e r B o šk o v ić «
B i j e n i č k a 54
YU-410Q1 Z a g r e b ( J u g o s l a v ij a )
72
A C T A B O T . C R O A T . V O L . 40, 1981.