Download Extremely rapid effects of polyunsaturated fatty acids and N

Lipids and Signalling: Oxylipins 3
Extremely rapid effects of polyunsaturated fatty acids and N-acetylglucosamine
on free-radical metabolism in cultured potato plant cells
P. A. Kashulin’, M. N. Merzlyak, P. M. Zhiboedov and V. K. Zhirov
Polar-Alpine Botanical Garden, Kola Science Centre, I 84230 Apatity, Faculty of Biology Moscow State University,
I 19899 Moscow, Russia
Abstract
Materials and methods
T h e effects of arachidonic and linoleic acids,
separately and in co-operation with N-acetylglucosamine oligomers, on Solanum tuberosum
plant suspension cell cultures were investigated in
terms of the fluorescent oxygen-activated-speciessensitive dye 2‘,7‘-dichlorofluorescin diacetate.
T h e inductors used triggered extremely rapid
(within 2-10 min) generation of H,O, in the cells;
the majority was expressed in cultures treated
with combined polyunsaturated fatty acid and Nacetylglucosamine oligomers. T h e stimulation of
free-radical generation may be related to defensive mechanisms modulating a plant-pathogenicmicro-organism interaction.
In experiments,
14-day-old cell cultures of
S. tuberosum L. var. T e m p in the steady-state
phase of culture growth underwent 3 min of centrifugation at 2000 g and were resuspended in fresh
Murashige and Scoog-based medium (pH 7.2)
up to a density 2 x lo4 cell/ml. T h e native carbohydrate derivatives of surface glycoproteins were
modelled by the N-acetyl-D-glucosamine trimer
[(GlcNAc),] synthesized at the A. N. Bakh
Institute of Biochemistry (Moscow, Russia).
Linoleic and arachidonic acids (Sigma), either separately or in co-operation with (GlcNAc),, were
considered as race-non-specific inductors for cells.
Intracellular free-radical production was quantified in terms of the intracellular probe 2‘,7‘dichlorofluorescin diacetate (DCFscinDA). T h e
quantity of fluorescent 2‘,7‘-dichlorofluoresceine
(DCFscein), formed when DCFscinDA is oxidized in cells by hydrogen peroxide, depends
directly on the size of the free-radical population
in the dye microenvironment [2,3]. All fluorescent
dyes were from Serva. T h e concentration of the
acetone dye stock solution used in suspension
cultures was 0.3 yo (v/v). Cell-free incubation
medium was considered as a base fluorescence
control. Accumulation of the fluorescent product
DCFscein in cells was monitored with a Jasco-550
spectrofluorimeter at excitation and emission
wavelengths of 495 and 523 nm, respectively. Data
are given as means& S.E.M.
Introduction
T h e polyunsaturated fatty acids (PUFAs) and N acetylglucosamine polymers that are transported
to the contact surfaces or constitute the surface
structures of penetrating fungus hyphae are considered as potential elicitors of defensive responses in plants. Despite the accepted concept
that different mechanisms underlie induction of
defence reactions in plants mediated by specific
carbohydrate and race-non-specific lipid derivatives, in many cases the early stages of both
scenarios may be accompanied by cellular generation of active oxygen forms [l]. This work
aimed to model the action of hydrocarbon and
lipid derivatives on plants in suspension cultures
of potato (Solanum tuberosum) cells. T h e system
based on this species in combination with various
races of potato blight fungus, Phytophthora infestans (Mont.) de Bari, or their components, is
widely accepted as a model to study general plantpathogen relationships.
Results and discussion
T h e vitality of the cells, by analysis of intracellular
esterase activity, was carried out using 2’,7’dichlorofluoresceine diacetate (DCFsceinDA) [2].
Addition of micromolar DCFsceinDA concentrations to the cell culture was followed by a green
emission, qualitatively analogous to enzymic formation of DCFscein in incubation medium. This
fluorescence showed the vitality of cells used, as
the emission of derivatives of fluorescein is used
routinely to mark living plant cells. T h e cells were
capable of keeping the dye for no less then 2-3 h.
Addition of DCFscinDA to the cell culture resulted in a slow elevation of fluorescence near 523 nm
Key words: oxygen activated species, phytoimmunity, PUFA.
Abbreviations used: PUFA. polyunsaturatedfatty acid; (GlcNAc),,
N-acetyl-o-glucosamine trimer; DCFscein, 2’,7’-dichlorofluoresceine; DCFsceinDA, 2’,7’-dichlorofluoresceine diacetate;
DCFscinDA, 2’,7’-dichlorofluorescin diacetate.
‘To whom correspondence should be addressed (e-mail
[email protected]).
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Biochemical Society Transactions (2000) Volume 28, part 6
Table I
Comparative effects of arachidonic and linoleic acids on 5. tuberosum suspension cell cultures to produce DCFscein
The fluorescence intensity at 523 nm reached in 4 min after addition of inductor, relative to the fluorescence of 0 0 I m M DCFscein in cellfree incubation medium is presented The cell density used was 2 x I O4 c e l l s h Values in parentheses are the numben of repeated
expenments
Fluorescence of DCFscein in cells (relative units)
Inductor
Inductor concentration (mM) . . .
Arachidonic acid
Linoleic acid
2.0
4.0
6.0
8.0
8. I +2.0 (5)
4.8f I .2 (5)
8.8f2.7 (5)
6.9f I .8 (5)
9.6f3.I (4)
7.8f2.3 (4)
9.853.2 (4)
8.2f2.8 (4)
Table 2
The synergetic effects of N-acetylglucosamine and lipid inductors on production of DCFscein in S. tuberosum
suspension cell culture
Experimental details were as for Table I Values in parentheses are the numben of repeated expenments
Fluorescence (relative units)
____
Fluorescence
-
-
~
Inductor-free cells
(GlcNAc), ( I mM)
(GlcNAc), ( I mM)
followed by linoleic
acid (2 mM)
5k0.4 (4)
4.2f I .6 (4)
20.6f2.8 (4)
and was evidence of DCFscein formation from
DCFscinDA. This relatively weak fluorescence
(see Table 2) presumably reflects the basal level
of free-radical generation, which was not limited
by deacylation activity of intracellular esterases.
Preincubation for 10 min of DCFscinDA-treated
cell culture with (GlcNAc), or P U F A promoted
the rapid rise of intracellular oxygen free radicals
up to saturation levels. There were no significant
differences found between the effects of arachidonic and linoleic acids (Table 1). At low concentration the effectiveness of arachidonic acid
seemed higher, but the difference vanished with
the rise in inductor concentration.
T h e first recognition stages in plant-pathogen
interaction may be followed by the involvement of
PUFAs in this process. T o model this scenario the
addition of (GlcNAc), and, consequently, PUFAs,
was tested. T h e addition of arachidonic acid to
cell cultures after 10 min of preincubation with
DCFscinDA and a few minutes with (GlcNAc),
resulted in a drastic and rapid ( < 2 min) increase
in fluorescence near 523 nm, which amounted to
5-fold enhancement of the effect of (GlcNAc),
alone (Table 2). Analogous action was induced by
linoleic acid.
T h e results presented show arachidonic
0 2000 Biochemical Society
~~
(GlcNAc), ( I mM)
followed by
arachidonic acid (2 mM)
23.6f4.1 (4)
acid ' s capability of activating natural free-radical
metabolism in suspension cell culture and reveal
the synergism of PUFAs and (GlcNAc):, in the
stimulation of H,O, generation. T h e generation of
H,O, took place at the very onset of elicitor
addition. This supports the available data that the
activation of free-radical metabolism is an early
stimulus-dependent defence reaction in plant
cells. In such a short time it is hardly, if at all,
possible for de novo protein synthesis to be responsible for the free-radical production. According to theoretical work on the question, H,O,
formed under elicitor stimulation may be involved
in the realization of oxidative o r second-messenger
functions as an alternative receptor-linked plasmamembrane redox component in host plant cells
[4]. T h e synergetic enhancement of free-radical
metabolism by mutual effects of elicitors of differing chemical natures supports the evidence that
the activity of arachidonic acid in induction of
defence reactions in potato plants may be increased
by p-glucan pretreatment [S-71. T h e co-operative
and abrupt activation of free-radical metabolism
by different inductors may underlie the mechanism of the hypersensitive immune reaction of
potato plants infected with Phytophthora infestans
fungus.
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Lipids and Signalling: Oxylipins 3
5 Kurantr, M. J, and Zacharius, R. M. (198 I ) Physiol. Plant
Pathol. 18, 69 -7 I
6 Maniara, G., Laine, R. and Kuc, J. A. ( 1984) Physiol. Plant
Pathol. 24, 177- I 8 6
7 Preisig, C. L. and Kuc. J. A. ( 1985) Arch. Biochem. Biophys
236,379-389
References
I Doke. N. and Chai. H. B. (I 987) in Molecular Determinants
o f Plant Disease, pp. 235-25 I , Springer-Verlag,Tokyo
2 Ferrer, A. S.. Santema, j. S., Hilhont. R. and Viser, J, W. G.
( 1990) Anal. Biochem. 187, I 29- I 32
3 Hauglald. R. P. (1992) in Molecular Probes. Handbook of
Fluorescent Probes and Research Chemicals, 5th edn,
Molecular Probes, Eugene
4 Apostol, I., Heinstein, P. F. and Low, P. S.( I 989)
Plant Physiol. 90, 109- I I 6
Received I 5 June 2000
o-Hydroxylation of epoxy- and hydroxy-fatty acids by CYP94A I :
possible involvement in plant defence
F. Pinot"', M. Skrabst, V. Compagnon", J.-P. Salaun*, I. Benveniste", L. Schreibert and F. Durst"
*IBMP-CNRS UPR406 Dept. d'Enzymologie Cellulaire et Moleculaire, 28 rue Goethe 67083 Strasbourg, France,
and TLehrstuhl fur Botanik II, Julius Von Platz 3, D-97082 Wurzburg, Germany
Abstract
Introduction
T h e C,, fatty acid derivatives 9,lO-epoxystearic
acid and 9,lO-dihydroxystearic acid were hydroxylated on the terminal methyl by microsomes of
yeast expressing CYP94Al cloned from Vicia
sativa. 'The reactions did not occur in incubations
of microsomes from yeast transformed with a void
plasmid or in the absence o f N A D P H . After
incubation of a synthetic raceniic mixture of 9,lOepoxystearic acid, the chirality of the residual
epoxide was shifted to 66:34 in favour of the
9S,10R enantiomer. Both the 9S,10R and 911,lOS
enantiomers were incubated separately. We
determined respective K,,, and YniLxvalues
of 1.2 & 0.1 pM and 19.2 0.3 nmol/min per nmol
of cytochrome P450 for the 9R,lOS enantiomer
and of 5.9 0.1 p M and 20.2 & 1.O nmol/min per
nmol of cytochrome P450 for the 9S,10R enantiomer. This demonstrated that CYP94A1 is enantioselective for the 9R, 10S, which is preferentially
formed in V . sativa microsomes. Cutin analysis
of V . sativa seedlings revealed that it is mainly
constituted of derivatives of palmitic acid, a C,,
fatty acid. Our results suggest that CYP94A1
might play a minor role in cutin synthesis and
could be involved in plant defence. Indeed,
18-hydroxy-9,10-epoxystearic acid and 9,10,18trihydroxystearic acid have been described as
potential messengers in plant-pathogen interactions.
We previously showed that, in Vicia sativa microsomes, oleic acid is subjected to a cascade of
reaction involving three different enzymes : an
epoxygenase, an epoxide hydrolase and a cytochrome P45O-dependent o-hydroxylase [I].
Using oleic acid as a starting material, these
enzymes are able to produce in nitro the major C,,
cutin monomers. Inhibition studies suggested the
presence of distinct fatty acid o-hydroxylases [2].
This was confirmed recently by the cloning of
CYP94A1 [3] and CYP94A2 [4]. When expressed
in yeast, CYP94A1 catalyses the w-hydroxylation
of saturated and unsaturated fatty acids with chain
lengths ranging from C,, to CIS.[3]. Treatment of
etiolated V . satizm seedlings by the plant hormone
methyl jasmonate led to an accumulation of transcripts coding for CYP94A1 and, concomitantly,
to a stimulation of microsomal fatty acid whydroxylase activity [ S ] .
Experimental
CYP94A1 was expressed in Saccharomyces cerevisiae as described in [3]. Enzymic activities
were determined by following the rate of metabolite formation by T L C [l]. Chiral analyses were
performed by using pure synthetic 9R, 1 OSepoxystearate methyl ester as a standard. Briefly,
radiolabelled enantiomers of 9,lO-epoxystearic
acid were separated on H P L C (Waters, equipped
with two 510 pumps, and a U6K injector from
Waters) using a chiral column (Column Chiracel
OB, 4.6 x 2 5 0 m m ; J. J . Baker Chemical Co.).
Key words, cytochrome P450. epoxide. messenger.
'To whom correspondence should be addressed (e-mall
franck.pinot(a)bota-u1p.u-strasbg.fr).
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