FEMS MicrobiologyLetters 68 (1990) 77-82
Published by Elsevier
77
FEMSLE 03924
The natural herbicide, cyanobacterin, specifically disrupts
thylakoid membrane structure in Euglena gracilis strain Z
F l o r e n c e K. G l e a s o n
Departmentof Plant Biology. Unit'ersio"of Minnesota. St. Paul MN. U.S.A.
Received20 November 1989
Accepted 21 November 1989
Key words: Cyanobacterin, Euglena graciiis; Thylakoid membrane disruption
1. S U M M A R Y
Cyanobacterin is a natural product produced
by the c y a n o b a c t e r i u m (blue-green alga),
Seytonema hofmanni. The compound has been
chemically characterized and shown to inhibit
electron transport in photosystem !1. Although the
herbicide is lethal to photoautotrophs, photoheterotrophically-grown organisms such as Euglena
gracilis can survive and grow in saturating concentrations of cyanobacterin. Electron micrographs of treated E. gracilis cells show extensive
damage to the thylakoid membranes of the chloroplasts, similar to the effects observed with 3-(3. 4dichlorophenylFl, l-dimethyl urea (DCMU). Unlike the synthetic herbicide, cyanobacterin ,~pecifically disrupts thylakoid membranes and does not
affect other cellular membranes or heterotrophic
growth.
Correspondenceto: Florence K. Gleason, Department of Plant
Biology, 220 BioScienceCenter. University of Minnesota, St.
Paul, Mlq 55108, U.S.A.
2. I N T R O D U C T I O N
Cyanobacterin is a natural product excreted by
the filamentous cyanobacterium. Scytonema
hofmanni, UTEX 2349. The compound has been
chemically characterized as a diaryl-substituted
y-lactone with a chlorine substituent on one of the
aromatic rings [1]. it is a highly effective algicide
[2] and may be produced as an allelopathic substance substance by S. hofmanni, which is found in
benthic or terrestrial habitats.
Like many synthetic herbicides, such as 3(3, 4-dichlorophenyi)-l, l-dimethyl urea ( D C M U )
and atrazine, the natural product inhibits photosynthetic electron in photosystem !I. However,
cyanobacterin appears to have a unique binding
site. Analysis of photosynthetic electron transport
in the presence of various Hill acceptors suggests
that it acts on the oxidizing side of the quinone-B
electron acceptor [3]. it had previously been observed that the natural product disrupts thylakoid
membrane structure [4] and perhaps even the outer
cell membranes in cyanobacteria [2]. To eurther
characterize the in vivo activity of cyanobacterin,
its effects on ultrastructure were determined in the
eukaryotic also, Euglena gracilis.
0378-1097/90/$03.50 © 1990 Federation of European MicrobiologicalSocieties
78
3. M A T E R I A L S A N D M E T H O D S
Axenic cultures of Euglena gracilis strain Z
(University of Texas Collection, strain 753) were
maintained at 2 5 ° C on semisolid medium at a
constant illumination of 30/~mol m -2 s -1 photos y n t h e t i c a l l y active r a d i a t i o n ( P A R ) . T h e
organisms were grown photoheterotrophically o n
medium enriched with yeast and beef extract as
described by Starr [4]. All cultures were grown at
2 5 ° C and 60 ~mol m -2 s "i PAR. G r o w t h was
monitored by measuring the increase in turbidity
at 420 nm.
Cyanobacterin was extracted from Scytonema
hofmanni ( U T E X 2349) as previously described
[1]. The c o m p o u n d was dissolved in ethyl ether.
Fig. 2. UItrastructure of photoheterotroplm~iiy-grown £ug/ena
gracilis 24 h after addition of 4.6 tiM cyanobacterin. C.
chloroplast; M, mitochondrion; N, nucleus; S, storage granule;
v, vesicles. Bar = I/tin.
3.1. Electron microscopy
Exponentially growing cells of E. gracilis were
removed from cultures approximately 24 h after
addition of cyanobacterin or solvent to controls
culture. Cells were centrifuged at 2000 x g for 5
rain a n d fixed in 4% 81utaraldehyde in cacodylate
buffer, p H 7,5. T h e cells were then post-fixed,
e m b e d d e d a n d stained with lead citrate-uranyi
acetate as previously described [6]. Sections were
examined in a Hitachi H-600 transmission electron microscope.
4. R E S U L T S A N D D I S C U S S I O N
Fig, 1. Uhrastructure of photoheterotrophicaily.grown Eus/ena
gracilis taken from control culture treated with ethyl ether. C,
chloroplast; G, Golgi apparatus; M, mitochondrion; N,
nucleus; S, slorage granule; t, thylakoids, Bar = !ttm,
As previously shown, cyanobacterin inhibits
growth of autotrophieally grown E. gracilis at a
concentration of 4.6 p M ; photoheterotrophically
grown cells remain viable at this concentration b u t
chlorophyll content declines to negligible values in
the presence of the algicide [7]. Cyanobacterin, at
nanomolar concentrations also inhibits photosynthetic electron transport in isolated chloroplasts
from heterotrophically grown E. gracilis [8].
Cyanobacterin was found to specifically disrupt
chloroplast ultrastructure in both autotrophically
and photoheterotrophically grown E. gracilis.
However, the fact that photoheterotrophically
growing cells survive and even resume growth in
Fig. 3. Ultrastructure of photoheterotrophically-grown Euglenagracilis 24 h after addition of 46 pM cyanobacterin. C. chloroplast:
M. mitochondrion: N, nucleus: S, storagegranule; t, remnants of tbylakoid membranes. Bar = 1 ~m.
the presence of the herbicide made it possible to
study the effect in otherwise healthy cells. Fig. 1
shows the ultrastructure of an untreated cell.
Chloroplasts exhibit stacks of thylakoid membranes arranged in parallel arrays typical of E.
gracilis. Also visible in the micrograph are a
nucleus and mitochondria. After 24 h in the presence of 4.6 /~M cyanobacterin, the orderly
arrangement of thylakoids is disrupted as seen in
Fig. 2. Although the chloroplast membranes remain intact, the thylakoids appear vesiculated.
There is no obvious damage to other membranes
or organelles. Treatment for longer periods of
time or with higher doses (up to 46/tM) leads to
complete loss of thylakoid structure as shown in
Fig. 3. The effect of the algicide is apparently
highly specific for thylakoid membranes.
The effects of cyanobacterin on cell ultrastructure were previously described in the prokaryote,
Synechococcus sp. Subtle changes in structure and
staining intensity were seen after a short exposure
of these photoautotrophic cells to the natural
product [4] More profound changes, especially in
cell membranes, may be secondary damage due to
the lethal effect of cyanobacterin on photoautotrophs. However, unlike most cyanobacteria, the
eukaryotic alga Euglena gracilis can grow in the
light but also utilize an external carbon source. In
the presence of cyanobacterin, these cells remain
viable and will resume growth. In contrast, the
algicide is lethal to E. gracilis grown under photoautotrophic conditions.
Effects similar to those shown in Figs. 2 and 3
on thylakoid membrane morphology after treatment of Euglena gracilis with the photosystem II
inhibitor, DCMU, have been reported. The morphological changes required high doses of herbicide and took 6-10 days to develop in cells grown
on lactate [9,10]. The ultrastructure of other
organelles such as mitochondria was also affected
since DCMU at high concentrations is known to
inhibit respiratory electron transport. Apparently
this is not true for cyanobacterin. Both cyanobacterin and DCMU disrupt the pH gradient
across thylakoid membranes which develops during photosynthetic electron transport [11], However, the natural product is highly specific and
does not affect other cellular processes even at
saturation. The site and mode of action of cyanobacterin seem to be different from classical photosystem II inhibitors as shown in herbicide-resistant mutants of the cyanobacterium, Anacystis
nidulans [12]. Nevertheless, it has a similar effect
on thylakoid membrane ultrastructure. DCMU has
been shown to secondarily inhibit membrane lipid
biosynthesis in Euglena [13]. This is apparently
not true of cyanobacterin since only thylakoid
membranes are damaged in the presence of the
natural product. It may be that the thylakoid
membrane disruption seen in the presence of photosynthetic electron transport inhibitors is a general phenomenon resulting from inhibitor binding
and disruption of electron transport which leads
to a loss of the normal membrane potential and
subsequent degradation.
ACKNOWLEDGEMENTS
Research contribution No. 230. This work is
the result of research sponsored by the Minnesota
Sea Grant Program, supported by NOAA Office
of Sea Grant, Department of Commerce, under
Grant No. NA86AA-D-SGl12 R/NP-7. The U.S.
Government is authorized to reproduce and distribute reprints for government purposes,
notwithstanding any copyright notation that may
appear hereon. The expert assistance of Dr. Joy
Paulson is gratefully acknowledged.
REFERENCES
Ill Pignatello, J,J., Porwoll, J,, Carlson, R.E., Xavier, A,,
Gleason, F,K, and Wood,J,M, (1983) J. Org, Chem. 48,
4035-4037,
[2] Mason,C.P., Edwards,K.R,,Carlson,R.E., Pignatello,J.,
Gleaso~l, F.K, and Wood, J.M, (1982) Science 215,
400-4~2,
[3] Gleasoa, F.K, and Case, D.E. (1986) Plant Physiol.80,
834-837,
[4] Gleason,F,K, and Paulson,J,L (1984) Arch. Microbiol,
138, 273-277.
[5l Start, R,C, (1964)Amer,J, Bot, 51,1013-1044.
16] Gleason,F,K, and Ooka, M.P. (1978) Cytobiologie(Enr.
J. Cell Biol. ) 16, 224-234.
[71 Gleason, F,K. and Baxa, C,A. (1986) FEMS Microbiol.
Leu. 33, 85-88,
[8] Baxa, C.A. and Gleason. F,K. (1985) Abst. Am. SOC.
Microbiol. p. 191.
[9] Calvayrac, R. and Ledoigt, G. (1976) Plant Sci. Lett. 7,
249-263.
[10| Calvayrac, R.. Bomsel, J-L. and LavaI-Martin, D. (1979)
Plant Physiol. 63. 857-865.
[11] Thoma, WJ. and Gleason, F.K. (1987) Biochemistry 26,
2510-2515.
[12] Mallipudi, L.R. and Gleason, F.K. (1989) Plant Sci. 60,
149-154.
[13] Troton. D., Calvayra¢, R. and LavaI-Martin, D. (1986)
Biochim. Biophys. Acta 878. 71-78.