Download Enzymes of the biosynthesis of octadecanoid

Journal of Experimental Botany, Vol. 52, No. 354, pp. 11±23, January 2001
REVIEW ARTICLE
Enzymes of the biosynthesis of octadecanoid-derived
signalling molecules
Florian Schaller1
Lehrstuhl fuÈr Pflanzenphysiologie, Ruhr-UniversitaÈt Bochum, D-44780 Bochum, Germany
Received 14 June 2000; Accepted 16 August 2000
Abstract
It is known that octadecanoids, i.e. jasmonic acid
and related compounds are involved in plant defence
reactions against (1) microbial pathogens, (2) herbivores and (3) damage by UV-B or UV-C light as
well as (4) senescence and (5) mechanotransduction.
Jasmonic acid is likely to occur ubiquitously in the
plant kingdom, and it has also been found in some
fungi. The pathway of octadecanoid biosynthesis was
elucidated in the early 80s by Vick and Zimmerman.
This review summarizes recent progress in the
identification and characterization of octadecanoid
biosynthetic enzymes and in the understanding of the
regulation of octadecanoid biosynthesis.
Key words: Lipase, lipoxygenase, allene oxide synthase,
allene oxide cyclase, 12-oxo-phytodienoic acid reductase.
Introduction
More than 100 years ago, Charles Darwin and Julius von
Sachs postulated the existence of chemical messengers
that are responsible for the regulation of plant development. The ®rst such messenger, the growth hormone
indole-3-acetic acid, was identi®ed in the 1930s. Since
then, the phytohormones have been and still are an area
of active research, but also one of major ignorance. Some
analogies to animal hormones have become apparent,
since over recent years brassinosteroids and jasmonates,
resembling in structure the animal steroids or prostaglandins, respectively, have been identi®ed as plant
signalling compounds.
In 1962, the methyl ester of jasmonic acid was isolated
by Demole and coworkers as a major fragrance in the
etheral oil of jasmine (Demole et al., 1962), and the free
1
Fax : q49 234 32 14187. E-mail: [email protected]
ß Society for Experimental Biology 2001
acid was identi®ed at the same time in culture ®ltrates
of the fungus Botryodiplodia theobromae (Aldridge
et al., 1971). Jasmonic acid is known to be involved
as a signalling compound in multiple aspects of plant
responses to their biotic and abiotic environment. Today
it is known that jasmonic acid, in planta levels of which
range from 0.01±3.0 mg g 1 FW (Mueller et al., 1993;
Mueller and Brodschelm, 1994), is only one of several
bioactive compounds within the class of octadecanoids
which regulate a broad spectrum of plant responses (for
review see Sembdner and Parthier, 1993).
The pathway of jasmonic acid (JA) biosynthesis is
shown in Fig. 1. Biosynthesis is believed to start with
the oxygenation of free a-linolenic acid (LA), which
is converted to (9Z,11E,15Z,13S)-13-hydroperoxy-9,11,
15-octadecatrienoic acid (13(S)-HPOT) in a reaction
catalysed by a 13-lipoxygenase (LOX). The presumed
release of LA from membrane lipids through the action
of a lipase may be triggered by local or systemic signals
like oligogalacturonides, chitosan, systemin (NarvaÂezVaÂsquez et al., 1999; Farmer and Ryan, 1992; Mueller
et al., 1993) or wounding (NarvaÂez-VaÂsquez et al., 1999;
Conconi et al., 1996a). The 13(S)-hydroperoxide serves
as a substrate for several enzymes like divinylether
synthase, peroxygenase, hydroperoxide lyase, and hydroperoxide reductase (Fig. 1) (BleÂe and Joyard, 1996;
MareÂchal et al., 1997) or allene oxide synthase (AOS)
(Vick and Zimmerman, 1981, 1987). AOS converts
13(S)-HPOT to an unstable epoxide (12,13(S)-epoxy9(Z),11,15(Z)-octadecatrienoic acid, 12,13-EOT) which
is cyclized by allene oxide cyclase (AOC) to the ®rst
cyclic and biologically active compound of the pathway,
12-oxo-10,15(Z)-phytodienoic acid (OPDA). Reduction
of the 10,11-double bond by a NADPH-dependent
OPDA-reductase then yields 3-oxo-2(29(Z)-pentenyl)cyclopentane-1-octanoic acid (OPC-8 :0) which is believed
to undergo three cycles of b-oxidation to yield the end
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Schaller
product of the pathway, i.e. JA. Evidence for this
b-oxidation process has emerged from radiotracer experiments, which have allowed the detection of 3-oxo-2(29-pentenyl)cyclopentane hexanoic acid (OPC-6 :0) and
3-oxo-2-(29-pentenyl)cyclopentane butanoic acid (OPC4:0) as intermediates in the conversion of OPDA to JA
(Vick and Zimmerman, 1983). While this biosynthetic
pathway results in the production of JA with (3R,7S)con®guration (i.e. (q)-7-iso-JA) (Vick and Zimmerman,
1984), JA extracted from plant tissue is predominantly
in the thermodynamically more favourable (3R,7R)trans-con®guration (i.e. ( )-JA) (Quinkert et al., 1982).
The biosynthesis of JA appears to involve three different
compartments (Fig. 4): the conversion of LA to OPDA
is localized in the chloroplasts (Vick and Zimmerman,
1987; Song et al., 1993; Laudert et al., 1997), while the
reduction of racemic OPDA to OPC-8 :0 (OPR1 and
OPR2) occurs in the cytoplasm (Schaller and Weiler,
1997a, b). Finally, the postulated steps of b-oxidation,
i.e. conversion of OPC-8 :0 to JA, are believed to occur
in peroxisomes (Gerhardt, 1983; Vick and Zimmerman,
1984). Like JA, OPDA may accumulate to substantial
amounts in plant tissue (Stelmach et al., 1998). In
contrast, OPC-8 :0 occurs only in trace amounts.
The release of a-linolenic acid
By analogy to mammalian eicosanoid biosynthesis,
a phospholipase A (PLA) activity has been postulated
to be involved in JA biosynthesis (Figs 1, 3). An increase
of PLA activity could be detected in tobacco cells 2 h
after elicitation (Roy et al., 1995) and in soybean after
treatment with extracts of a pathogenic fungus
(Chandra et al., 1996). A rapid release of LA preceding
the accumulation of JA upon elicitation (Mueller et al.,
1993) or wounding (Conconi et al., 1996a) supported
evidence for a regulated lipase (Fig. 4) which releases
LA for octadecanoid biosynthesis from membrane lipids
(NarvaÂez-VaÂsquez et al., 1999). Considering the localization of the early enzymes of JA biosynthesis, i.e.
LOX, AOS and AOC in the chloroplasts, it is not the
amount of LA within whole cells (which should be
generally suf®cient for JA biosynthesis and even more
so after induction by stress; Conconi et al., 1996a;
Moreau and Preisig, 1993), but the amount of LA in
the chloroplasts that needs to be determined. Since
biosynthesis of OPDA occurs in the chloroplasts, the
major sources of LA for JA biosynthesis may be
monogalactosyl diacylglycerol and digalactosyl diacylglycerol as previously suggested (Simpson and Gardner,
1995). Galactosyl lipids are predominantly located in
chloroplasts and are degraded after wounding (Conconi
et al., 1996a) or mechanical stimulation (Weiler et al.,
1993).
Lipoxygenase
Lipoxygenases (LOXs) are non-haem iron-containing
dioxygenases that catalyse the oxygenation of fatty acids
to their corresponding hydroperoxy derivatives (Figs 1, 3)
and are widely distributed in animals, in man and higher
plants (Siedow, 1991). Plant LOX proteins consist of
a single polypeptide chain with a molecular mass of
about 94±104 kDa. They have been correlated with plant
growth and development, maturation and senescence
(for review see Siedow, 1991), with responses to pathogen
attack (Rance et al., 1998; VeÂroneÂsi et al., 1996) and
wounding (Farmer and Ryan, 1992; Saravitz and
Siedow, 1995, 1996) and the biosynthesis of signalling
molecules, such as JA and OPDA (Fig. 3) (Creelman
and Mullet, 1997; Vick and Zimmerman, 1987). Higher
plants contain multiple LOXs (Fig. 2) comprising at
least eight identi®ed isoforms in soybean (for review
see Brash, 1999). It has been shown that many plant
LOXs are soluble, cytoplasmatic enzymes (Siedow, 1991;
Hildebrand, 1989; Gardner et al., 1991). Nevertheless,
the presence of LOXs has been demonstrated in other
cellular compartments, such as the stroma of chloroplasts
(Douillard and Bergeron, 1981), lipid bodies (Feussner
and Kindl, 1994; Feussner and KuÈhn, 1995), vacuoles
(Tranbarger et al., 1991), and mitochondrial fractions
(Braidot et al., 1993).
A stroma-localized plastidial LOX (LOX2) appears
to be responsible for the wound-induced biosynthesis
of JA in Arabidopsis (Bell et al., 1995). Charge modi®cations of soluble LOXs, however, may result in their
association with membranes (Droillard et al., 1993).
Alternatively, soluble LOXs may be transferred via
vesicles to target membranes where they attack polyunsaturated fatty acids of glycerolipids or free fatty
acids liberated by membrane-bound phospholipases
(Fornaroli et al., 1999). Since linolenic acid dissolves
poorly in aqueous media at typical physiological pH
values of 7.0±7.4 it is not likely to be available as
a substrate for soluble LOXs (Brash, 1999). Therefore,
membrane lipids attacked by membrane-associated LOXs
are likely to be the initial substrate of octadecanoid
biosynthesis (Siedow, 1991).
Allene oxide synthase
According to the biosynthetic pathway of Vick and
Zimmerman, jasmonates originate from LA via the
reaction of a 13-LOX. The resulting product (9Z,11E,
15Z,13S)-13-hydroperoxy-9,11,15-octadecatrienoic acid
(13(S)-HPOT) serves as a substrate for several enzymes
(BleÂe, 1998) like hydroperoxide lyase, hydroperoxide
reductase, divinylether synthase, and peroxygenase
(Fig. 1). The dehydration of 13(S)-HPOT to the unstable
epoxide (9Z,11E,15Z,13S,12R-12,13-epoxy-9,11,15-octadecatrienoic acid) by allene oxide synthase (AOS,
Enzymes of octadecanoid biosynthesis
13
oligogalcturonides
wounding
O
COOH
chitosan
m
em
br
an
e
systemin
13-keto-9(Z),11(E),15(Z)octadecatrienoic acid
Lipase
OH
COOH
COOH
13-OH-9(Z),11(E),15(Z)octadecatrienoic acid
Lipoxygenase
linolenic acid
Lipoxygenase
Hydroperoxidereductase
O
OH
OOH
COOH
COOH
COOH
O
etheroleic acid
13(S)-HPOT
Divinylethersynthase
15,16-epoxy-13-OH-9(Z),
11(E)-octadecadienoic acid
Peroxygenase
AOS
Hydroperoxidelyase
HO O
COOH
O
n-hexenal
COOH
COOH
12,13-EOT
AOC
O
OH
COOH
COOH
O
OHC
12-oxo-9(Z)dodecenoic acid
OPDA
OPR
COOH
O
COOH
Isomerization Faktor
COOH
OHC
12-oxo-10(E)dodecenoic acid
OPC-8:0
-oxidation
COOH
COOH
HOOC
traumatic acid
O
jasmonic acid
Fig. 1. a-Linolenic acid: a central substrate for the production of defence-related metabolites with biological activity. AOS: allene oxide synthase;
AOC: allene oxide cyclase; OPR: 12-oxo-phytodienoic acid reductase; 13(S)-HPOT: (9Z,11E,15Z,13S)-13-hydroperoxy-9,11,15-octadecatrienoic acid;
12,13-EOT: (9Z,11E,15Z,13S,12R-12,13-epoxy-9,11,15-octadecatrienoic acid; OPDA: 12-oxo-10,15(Z)-octadecatrienoic acid; OPC-8:0: 3-oxo-2(29(Z)pentenyl)-cyclopentane-1-octanoic acid.
formerly hydroperoxide isomerase and hydroperoxide
dehydrase) (Vick and Zimmerman, 1981, 1987) represents
the ®rst speci®c step in octadecanoid biosynthesis. The
unstable allene oxide does either spontaneously hydrolyse
to a mixture of a- and c-ketols (80% and 10%,
respectively) and racemic cis-12-oxophytodienoic acid
(OPDA, 10%) or, in a concerted action with allene oxide
cyclase (AOC), it cyclizes to enantiomerically pure
9S,13S-OPDA (i.e. cis(q)-OPDA) (Hamberg and
Fahlstadius, 1990; Laudert et al., 1997), the substrate
for jasmonic acid biosynthesis (Fig. 1).
AOS was ®rst puri®ed and subsequently cloned from
¯ax seeds (Song and Brash, 1991; Song et al., 1993),
guayule (Pan et al., 1995), Arabidopsis (Laudert et al.,
1996), tomato (Sivasankar et al., 2000; Howe et al., 2000),
and barley (Maucher et al., 2000) and was also puri®ed
from corn (Utsunomiya et al., 2000). AOS is a cytochrome P450 of the CYP74 family (Laudert et al., 1996;
Song et al., 1993; Pan et al., 1995) which is independent
of oxygen, NADPH and P450 reductases for enzymatic
activity and is characterized by a lack of transmembrane
segments and low af®nities for CO. According to
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Schaller
Fig. 2. Phylogenetic tree of plant lipoxygenases. The amino acid sequences were compiled into the phylogenetic tree using `clustalW' and `TreeView'.
Arabidopsis thaliana, LOX 2 accession JQ2391; Arabidopsis thaliana, LOX accession JQ2267; Cucumis sativus, LOX (lipid body) accession X92890;
Cucumis sativus, LOX accession U36339; Glycine max, LOX 1 accession AAA33986; Glycine max, LOX 2 accession J03211; Glycine max, LOX 3
accession X13302; Glycine max, LOX 4 (VSP 94) accession P38417; Glycine max, LOX 5 (vlxB) accession U50075; Glycine max, LOX 7 accession
U36191; Glycine max, LOX accession X56139; Glycine max, LOX vlxC accession U26457; Hordeum vulgare, LOX A accession L35931; Hordeum
vulgare, LOX B accession AAB60715; Lens culinaris, LOX 1 accession P38414; Lycopersicon esculentum, LOX (loxC, chloroplast) accession U37839;
Lycopersicon esculentum, LOX (chloroplast) accession AAB65767; Lycopersicon esculentum, LOX A accession P38415; Lycopersicon esculentum, LOX
B accession P38416; Nicotiana tabacum, LOX accession S57964; Oryza sativa, LOX 2 accession A53054; Oryza sativa, LOX 2 accession S23454;
Phaseolus vulgaris, LOX accession S22153; Pisum sativum, LOX (cytoplasmic, seed) accession X07807; Pisum sativum, LOX (seed) accession
CAA34906; Pisum sativum, LOX G accession X76124; Solanum tuberosum, LOX 2 accession U60201; Solanum tuberosum, LOX 3 accession U60202;
Solanum tuberosum, LOX accession U24232; Solanum tuberosum, LOX H1 accession X96405; Solanum tuberosum, LOX H3 accession X96406;
Solanum tubersosum, LOX 1 accession U60200; Vicia faba, LOX 1 accession CAA97845.
differences in the haem-binding domain which is distinct
from that of most other P450 enzymes, the CYP74 family
is subdivided in two classes. The CYP74A subfamily
contains all AOSs, whereas the pepper hydroperoxide
lyase and the tomato hydroperoxide lyase are the sole
member of the CYP74B subfamily thus far identi®ed
(Matsui et al., 1996; Howe et al., 2000). AOS activity has
been localized in the chloroplasts of spinach leaves (Vick
and Zimmerman, 1987) and barley (Maucher et al.,
2000).The AOS cDNAs from ¯ax (Song et al., 1993),
tomato (Sivasankar et al., 2000; Howe et al., 2000) and
Arabidopsis (Laudert et al., 1996) encode transit peptides
characteristic for chloroplast targeting. Similar transit
peptides could not be found, however, in the AOS from
rubber particles of guayule (Pan et al., 1995) and in the
two isoforms of the barley AOS (Maucher et al., 2000).
Catalytic activity
While AOS from ¯ax and Arabidopsis will convert the
13(S)-hydroperoxides of linoleic and linolenic acids to the
respective allene oxides (Song and Brash, 1991; Laudert
et al., 1996) at comparable rates, the Km for 13(S)-
octadecadienoic acid of barley AOS1 is approximately
5-fold higher compared to that for 13(S)-octadecatrienoic
acid. The 15(S)-hydroperoxides of C20 fatty acids as
well as the 9(S)-hydroperoxides of linoleic and linolenic
acid are also substrates of barley AOS (Maucher et al.,
2000). The physiological relevance of the latter activity,
however, remains obscure.
Physiology
Wounding, OPDA and JA induce the expression of both
¯ax and Arabidopsis AOS (Harms et al., 1998; Laudert
and Weiler, 1998) (Fig. 4). Arabidopsis AOS is also
induced by ethylene, an indication that these two phytohormones might interact in some way (O'Donnell et al.,
1996; Laudert and Weiler, 1998). Salicylic acid, an
activator of pathogenesis-related gene expression, induces
Arabidopsis AOS but represses ¯ax AOS, indicating different regulation of AOS gene expression among plant
species (Harms et al., 1998; Laudert and Weiler, 1998).
Overexpression of ¯ax AOS in transgenic potato plants
led to a 6±12-fold increase in the endogenous JA levels,
but despite the fact that the transgenic plants had `resting'
Enzymes of octadecanoid biosynthesis
15
Fig. 3. Plant lipoxygenases metabolize a-linolenic acid to produce
signalling compounds, induce defence mechanisms and produce energy.
JA levels similar to those found in wounded wild-type
plants, pin2 genes were not constitutively expressed
(Harms et al., 1995). In contrast, the overexpression of
Arabidopsis AOS in Arabidopsis and tobacco did not
alter basal levels of JA, but in these transgenic plants,
peak jasmonate levels were 2±3-fold higher after wounding as compared to wounded, untransformed plants and
the transgenic plants reached the maximum JA-level
signi®cantly earlier than wounded control plants. These
®ndings suggest that overexpression of AOS might be a way
of controlling defence dynamics at least in Arabidopsis and
tobacco (Laudert et al., 2000). Considering the results of
both Harms et al. and Laudert et al., it could be argued
that it is not the absolute, constitutive level of octadecanoids that determines the induction of defence mechanisms, but the relative increase of signalling molecules after,
for example, wounding (Harms et al., 1998; Laudert and
Weiler, 1998). The ®ndings that the expression of the
AOS gene is upregulated by JA and OPDA as well as
their methyl esters (Laudert and Weiler, 1998) demonstrate the autoinduction of the ®rst speci®c enzyme of
the jasmonate pathway by its end products. Treatments
which do not lead to endogenous accumulation of
jasmonates in barley (e.g. SA, ABA, NaCl, and pathogen
infection) failed to give upregulation of AOS expression
(Maucher et al., 2000).
Fig. 4. The biosynthesis of jasmonic acid takes place in three different
compartments and is highly regulated. LA: a-linolenic acid; LOX:
lipoxygenase; AOS: allene oxide synthase; AOC: allene oxide cyclase;
OPR: 12-oxo-phytodienoic acid reductase; 13(S)-HPOT: (9Z,11E, 15Z,
13S)-13-hydroperoxy-9,11,15-octadecatrienoic acid; 12,13-EOT: (9Z,
11E,15Z,13S,12R-12,13-epoxy-9,11,15-octadecatrienoic acid; OPDA: 12oxo-10,15(Z)-octadecatrienoic acid; OPC-8:0: 3-oxo-2(29(Z)-pentenyl)cyclopentane-1-octanoic acid; JA, jasmonic acid; SA, salicylic acid; BR,
brassinolide; Cor, coronatine.
Regulation of the Arabidopsis AOS gene
In order to gain a better understanding of the mechanisms
involved in the control of AOS gene expression, the AOS
gene of Arabidopsis was isolated and characterized
(Kubigsteltig et al., 1999). The promoter region contains
cis-elements such as CAAT boxes and ACGT elements
(Foster et al., 1994) found in many regulated genes. These
elements are almost identical to a stress response element
described previously (Goldsbrough et al., 1993) as being
salicylate-reponsive and a putative ethylene-reponse
element (Montgomery et al., 1993; Itzhaki et al., 1994),
respectively. In young transgenic plants (up to 21 d
(tobacco) and 11 d (Arabidopsis) post-germination)
expressing the pAOS :: uidA reporter gene, no GUS
16
Schaller
activity was observed. In older plants, however, increasing GUS activity could be detected and was correlated
with increasing leaf age. Generally, GUS activity was
higher in Arabidopsis compared to tobacco. Furthermore,
Arabidopsis pAOS::uidA reporter gene plants showed
expression of the AOS gene in pollen, which is consistent with the observation that jasmonates are required
for male fertility in Arabidopsis, as both the coronatinand jasmonate-insensitive mutant coi1 (Feys et al., 1994)
and the triple mutant fad3-2 fad7-2 fad8, incapable of
producing normal levels of JA, are male-sterile (McConn
and Browse, 1996). Furthermore, jasmonates have been
shown to occur in pollen (Yamane et al., 1982; Miersch
et al., 1998). After wounding, both local and systemic
activation of the AOS promoter could be detected in
Arabidopsis and tobacco. Two hours after wounding,
GUS activity was observed in the main veins of
Arabidopsis and tobacco leaves as well as the petioles
and 24 h after wounding the whole shoot had reacted
uniformly. In contrast to the systemic induction after
wounding, after the application of jasmonates, such as
OPDA, and JA as well as the octadecanoid analogue,
coronatin, only a local induction of AOS promoter
activity was observed in the treated leaves. JA anduor
OPDA are thus unlikely to serve as systemic inducers
of defence reactions in Arabidopsis (Kubigsteltig et al.,
1999). Rather, octadecanoids act locally at the site of
application, that is, they behave like short distance
messengers in planta. Deletion analyses of the Arabidopsis
AOS promoter localized a putative cis-element for
wound inducibility to a domain of only 50 base pairs.
The experiments to clarify if this element is both
necessary and suf®cient to confer wound-inducibility are
in progress (I Kubigsteltig, personal communication).
Allene oxide cyclase
In 1981, Vick and Zimmerman (Vick and Zimmerman,
1981) postulated the existence of a hydroperoxide cyclase
for the conversion of 13(S)-HPOT into 12-oxo-phytodienoic acid and the allene oxide, 12,13(S)-epoxylinolenic
acid, has been shown to serve as the immediate precursor of
OPDA in plants (Fig. 1; Hamberg and Hughes, 1988;
Baertschi et al., 1988; Crombie and Morgan, 1988). In
1990, an allene oxide cyclase (AOC) was identi®ed as
a soluble enzyme in corn with a molecular mass of about
45 kDa (Hamberg and Fahlstadius, 1990). Recently, AOC
was puri®ed from maize kernels (Ziegler et al., 1997),
cloned from tomato (Ziegler et al., 2000) and the substrate speci®city of the enzyme has been analysed (Ziegler
et al., 1999).
Catalytic activity
Allene oxide cyclase (AOC, EC 5.3.99.6) catalyses the
cyclization of the unstable allene oxide 12,13(S)-
epoxy-9(Z),11,15(Z)-octadecatrienoic acid (12,13-EOT)
yielding 12-oxo-10,15(Z)-phytodienoic acid (OPDA) the
®rst octadecanoid bearing the characteristic cyclopentenone structure (Figs 1, 4). Potato AOC in combination
with recombinant Arabidopsis AOS expressed in E.coli
resulted in the production, in vitro, of highly asymmetrical cis-OPDA consisting nearly exclusively of the
9(S),13(S)-form (i.e.the cis(q)-enantiomer) (Laudert
et al., 1997), which is the enantiomer occurring in plant
tissues (Stelmach et al., 1998; Laudert et al., 1997) and
the precursor of JA biosynthesis. The maize enzyme is
a dimer of 47 kDa apparent molecular mass which accepts
12,13(S)-epoxy-9(Z),11,15(Z)-octadecatrienoic acid, but
not 12,13(S)-epoxy-9(Z),11-octadecadienoic acid, as a substrate (Ziegler et al., 1997, 1999). This is in contrast to
AOS which produces both allene oxides from the
respective substrates, 13(S)-hydroperoxylinolenic acid
or 13(S)-hydroperoxylinoleic acid. Thus, the speci®city
in the octadecanoid biosynthetic pathway results from
AOC, rather than LOX or AOS.
The extremely short half-life of allene oxides (t1u2 less
than 30 s in water), the optical purity of natural OPDA,
and the absence of a- and c-ketols in plant tissues in vivo
(Weiler et al., 1998), suggest a tight coupling of the AOS
and AOC reactions possibly in a synthase-cyclase complex.
12-oxo-phytodienoic acid reductase
Vick and Zimmerman (1984), analysing the metabolism
of 18O-labelled OPDA in several plant tissues, identi®ed
3-oxo-2-(29(Z)-pentenyl)-cyclopentane-1-octanoic
acid
(OPC-8 :0) as one of the labelled metabolites. The enzyme
catalysing the reduction of the CˆˆC double bond of the
conjugated enone moiety (Fig. 1) was called 12-oxophytodienoic acid reductase (OPR). OPR was identi®ed
as a protein of a molecular mass of about 54 kDa, with
a KM of 15 mM for its substrate OPDA and a preference
for NADPH as the reducing agent. Molecular cloning
of the reductase from Arabidopsis (Schaller and Weiler,
1997b) revealed a close relationship to Otto Warburg's
Old Yellow Enzyme (OYE), a ¯avoprotein enone reductase (Warburg and Christian, 1932, 1933), to morphinone
reductase of Pseudomonas putida, an enzyme which
reduces morphinone to hydromorphone or codeinone to
hydrocodone (French and Bruce, 1995) and other OYErelated proteins (Fig. 5). OYE (EC 1.6.99.1) was initially
isolated from brewer's bottom yeast and was the ®rst
enzyme shown to possess a ¯avin cofactor (Warburg and
Christian, 1932, 1933). Despite extensive biochemical and
spectroscopic characterization, the physiological function
of the enzyme has remained obscure. OYE has been
described as a diaphorase catalysing the oxidation of
NADPH in the presence of molecular oxgen, but a physiological oxidant has not been found (Massey and Schopfer,
1986). A number of arti®cial electron acceptors for
Enzymes of octadecanoid biosynthesis
17
Fig. 5. Phylogenetic tree of the family of Old Yellow Enzymes. The amino acid sequences were compiled into the phylogenetic tree using `clustalW'
and `TreeView'. Agrobacterium radiobacter, GTN-reductase accession CAA74280; Arabidopsis thaliana, OPR 1 accession AAC78440; Arabidopsis
thaliana, OPR 2 accession AAC78441; Arabidopsis thaliana, OPR 3 accessions AAF67635, AAD38925, CAB66143, AAD19764; Arabidopsis thaliana,
OPR-like protein accession AAC33200; Azotobacter vinelandii, 2-cyclohexen-1-one reductase accession BAA88211; Candida albicans, estrogen-binding
protein accession P43084; Catharanthus roseus, OPR-like accession T09943; Enterobacter cloacae, pentaerythritol tetranitrate reductase accession
AAB38683; Escherichia coli, N-ethylmaleimide reductase accession P77258; Kluyveromyces lactis, KYE 1 (OYE 1) accession P40952; Lithospermum
erythrorhizon, LEDI-5a protein accession BAA83083; Lithospermum erythrorhizon, LEDI-5b protein accession BAA83084; Lycopersicon esculentum,
OPR 1 accession CAB43506; Lycopersicon esculentum, OPR 2 accession AJ278331; Lycopersicon esculentum, OPR 3 accession AJ278332; Pseudomonas
putida, morphinone reductase accession S64687; Saccharomyces cerevisiae, OYE 1 accession Q02899; Saccharomyces cerevisiae, OYE 2 accession
NP_012049; Saccharomyces cerevisiae, OYE 3 accession NP_015154; Saccharomyces cerevisiae, OYE-like accession CAA65573; Schizosaccharomyces
pombe, OYE A accession Q09670; Schizosaccharomyces pombe, OYE B accession Q09671; Vigna unguiculata, OPR-like accession T11580.
reduced OYE has been identi®ed including the ole®nic
double-bond of a,b-unsaturated ketones and aldehydes
(Stott et al., 1993; Vaz et al., 1995). Several OYE homologues have been reported in prokaryotic and eukaryotic
organisms (Stott et al., 1993; French and Bruce, 1994;
Franklund et al., 1993; Miura et al., 1997; Buckman and
Miller, 1998). The puri®cation (Schaller and Weiler,
1997a) and molecular cloning of a plant OPR (Schaller
and Weiler, 1997b) led to the identi®cation of the ®rst
member of the family of OYEs in higher plants.
Up to now, two OPRs from Rock Harlequin (Corydalis
sempervirens, Schaller and Weiler, 1997a; Schaller et al.,
1998), three OPR isoforms from Arabidopsis (Schaller and
Weiler, 1997b; Biesgen and Weiler, 1999; MuÈssig et al.,
2000; Schaller et al., 2000) and three OPR isoforms from
tomato (Straûner et al., 1999a; A Schaller, personal
communication) have been described. A phylogenetic tree
of a sequence alignment of the amino acid sequences of
the three Arabidopsis and tomato OPRs as well as OYEs
1±3 from Saccharomyces and other plant OYE-related
sequences is shown in (Fig. 5). Sequence similarities were
observed over the entire lengths of the protein sequences,
furthermore two of the three amino acids involved in
substrate binding are conserved throughout the plant and
the yeast sequences (tyr376 and his192), whereas asp195 is
replaced by his in most of the plant sequences.
Catalytic activity
Looking at the phylogenetic tree of OYEs and their
related enzymes and the OPRs (Fig. 5) it becomes
obvious that enzymes of the OPR3-type from
Arabidopsis and tomato are more closely related to the
OYEs than OPR1 and OPR2. This re¯ects the enzymatic
properties of the enzymes. While OPRI from Corydalis
sempervirens (Schaller et al., 1997a), LeOPR1 from
tomato (Straûner et al., 1999b) and OPR1 and OPR2
from Arabidopsis (Schaller et al., 2000) preferentially
catalyse the reduction of cis( )-OPDA, the isomer which
may result from a partial uncoupling of the AOSuAOC
reactions, OPRII from Corydalis sempervirens (Schaller
et al., 1998), LeOPR3 from tomato (A Schaller, F Schaller
and P Macheroux, unpublished results) and OPR3 from
Arabidopsis (Schaller et al., 2000) are the enzymes which
18
Schaller
prefer cis(q)-OPDA, the naturally occurring precursor
of JA biosynthesis, as their respective substrate. OPR3
rather than its relatives OPR1 and OPR2, is thus the
enzyme involved in JA biosynthesis in Arabidopsis and
tomato. The functions of OPR1 and OPR2 are not yet
understood, but it seems likely that they are not involved
in JA biosynthesis. However, looking at the broad
substrate speci®city of Le OPR1 and Arbidopsis OPR1
(Straûner et al., 1999b; F Schaller, unpublished results)
it is tempting to speculate that other physiologically
relevant compounds may be reduced by this enzyme.
Yeast OYE forms charge transfer (CT) complexes with
a variety of aromatic and heteroaromatic compounds
carrying an ionizable hydroxyl group. Formation of the
CT complex results in a long wavelength transition in the
absorbance spectra. A positive correlation was observed
between the absorbance maximum of the newly formed
long wavelength transition and the Hammett paraconstant of the p-substituted phenolic compounds, which
can be considered as a measure of the phenolate
ionization potential (Massey et al., 1984). This ®nding
and the pH dependence of binding suggest that in the
CT complex, the bound phenolate and the oxidized ¯avin
act as the electron donor and acceptor, respectively.
It could be demonstrated that a series of phenolic
ligands form CT complexes with LeOPR1 (Straûner et al.,
1999b) and LeOPR3 as well as OPR3 from Arabidopsis
(A Schaller, F Schaller and P Macheroux, unpublished
results). This ®rst demonstration of such complexes in
plant ¯avoproteins provides further biochemical evidence
for the relationship between yeast OYEs and their plant
homologues. It could be concluded from these experiments that the ¯avin moiety acts as the acceptor and the
phenolic ligand as the donor in the LeOPR1 CT complex,
as was concluded earlier for yeast OYE (Abramovitz and
Massey, 1976). Work towards the elucidation of LeOPR
structure by X-ray diffraction is in progress (A Schaller,
personal communication) and will yield an even better
understanding of the relationship between yeast and plant
OYEs.
Physiology
The analysis of the regulation of OPR activity is of
fundamental interest because JA is not the only biologically active compound of the jasmonate pathway. It
could be demonstrated that the ®rst cyclic intermediate,
OPDA, functions as an endogenous signal transducer
in mechanotransduction (Stelmach et al., 1998; Weiler
et al., 1993, 1994) and, furthermore, it could be demonstrated that OPDA may be more relevant in vivo than
JA for signal transduction in elicited cell-suspension
cultures from several plant species (Blechert et al., 1995;
Parchmann et al., 1997). In tendrils of Bryonia dioica,
there is a strong and transient increase in OPDA levels
after mechanical stimulation while JA levels remain
unaffected (Blechert et al., 1999). Thus the OPRs and
OPR3 in particular may play a decisive role in octadecanoid signalling in that the enzyme appears to control
the metabolite ¯ow from the biologically active C18compound OPDA to the C12-metabolite JA (Fig. 1).
OPRs may thus regulate the level of OPDA independently
from that of JA. Changes in the relative concentration of
octadecanoid compounds (i.e. the oxylipin signature)
rather than the concentration of any single compound,
may be relevant for the activation of diverse physiological
responses (Weber et al., 1997).
The expression of OPRs was analysed on the mRNA
and protein levels as well as in transgenic plants expressing the GUS reporter gene under the control of the
Arabidopsis OPR promoters. Transient changes in steadystate OPR1 and OPR2 mRNA levels were observed in
response to wounding, UV-C illumination and heatucold
stress (Fig. 4). Likewise, the expression of GUS under
the control of the OPR1 and OPR2 promoters was stimulated after touch- and wind-stimulation, as well as after
wounding and UV-irradiation (Biesgen and Weiler,
1999). The very same conditions, which are known to
stimulate octadecanoid signalling transduction (Farmer
and Ryan, 1990; Falkenstein et al., 1991; Conconi et al.,
1996b; Stelmach et al., 1998), also resulted in a rapid
and transient increase of OPR3 mRNA (MuÈssig et al.,
2000). The signi®cance of transcriptional activation of
OPR genes remains unclear, however, since no induction
was observed for OPR1 and 2 at the levels of protein or
enzymatic activity (Biesgen and Weiler, 1999).
b-Oxidation
12-(18O)-oxo-PDA,
10,11-dihydro-12-(18O)-oxo-PDA
as well as the fully saturated 10,11,15,16-tetrahydro12-(18O)-oxo-PDA are converted to JA or 9,10-dihydroJA, respectively and, in addition, two labelled C16 and
C14 intermediates with the carboxyl chain shortened by
two and four carbons, respectively, have been isolated,
suggesting that three cycles of b-oxidation are the
terminal steps in JA biosynthesis (Figs 1, 4) (Vick and
Zimmerman, 1983, 1984). The compartmentalization of
these b-oxidation steps has not been investigated yet.
Presumably, they are localized in the peroxisomes anduor
glyoxysomes, since these are the only sites where
b-oxidation is known to occur in plants (Gerhardt,
1983; Vick and Zimmerman, 1984). Alternatives should
be considered, since b-oxidation of unsaturated fatty
acids, such as oleic acid, terminates after only two cycles,
leaving a D5,6-enoyl-CoA which cannot be b-oxidized
further unless the double bond is reduced (Tserng and Jin,
1990). The in¯uence of a cyclohexenone ring attached to
an acyl chain on the ef®ciency of the b-oxidation enzymes
Enzymes of octadecanoid biosynthesis
is unknown, nor has it been shown whether or not OPC8:0 can be converted to its coenzyme A thioester required
to initiate b-oxidation. Likewise, the question of whether
the transformation of the C18-compound OPDA to the
C12-compound JA should be considered a biosynthetic,
or rather, a catabolic reaction is still open. It has been
reported that OPDA is active per se in the induction of
benzophenanthridine alkaloids after elicitation of plant
suspension cultures (Blechert et al., 1995). Therefore,
these authors assumed that OPDA and OPC-8 :0 are
primary signal transducers in the elicitation process,
notwithstanding the biological activity of JA and its
possible interaction with the same hypothetical receptor.
Furthermore, OPDA is 50±100 times more active as
compared to JA in eliciting tendril coiling in Bryonia
dioica (Weiler et al., 1994). Thus, just like in mammalian
eicosanoid metabolism where b-oxidation is a characteristic degradation route and usually associated with
complete loss of the biological activity, it is possible that
JA represents a degradation product of biologically more
active C18-compounds, which is then further inactivated
in the cell by conjugation (Sembdner et al., 1988).
Discussionuperspectives
Although all enzymes of JA-biosynthesis are present in
low levels in uninduced plants, it is now certain that key
steps in the pathway are under inductive control (Fig. 4).
This induction may lead to an autoinduction of the
biosynthetic pathway or may help to regulate the metabolite pools of the biologically active C18-compounds
versus those of the C12-compounds. OPR operates at
such a decisive point in the pathway. If one bears in
mind that the OPDA content in plants varies by more
than one order of magnitude between different species
(Stelmach et al., 1998), it becomes obvious that (1) the
19
release of OPDA from its biosynthetic compartment, the
chloroplast, anduor (2) the reduction of released OPDA
by OPR may be rate-limiting steps in JA biosynthesis.
Since the localization of OPR3 from Arabidopsis and
tomato is not yet known the reduction of cis(q)-OPDA
may be localized in the peroxisomes.
The observation that OPR3 mRNA levels are upregulated by 24-epi-brassinolide (MuÈssig et al., 2000) suggests
a functional relationship between octadecanoid and
brassinolide signalling. Some further observations which
point to this connection are: (1) brassinosteroids are present in pollen at very high concentrations, and jasmonic
acid is required for pollen fertility (Xie et al., 1998) and
(2)OPR3 gene expression by membrane damage after
detergent treatment is impaired in the brassinosteroidde®cient mutant cbb1 (MuÈssig et al., 2000).
Because of the fact that neither JA nor OPDA led to
a systemic induction of the AOS promoter like, for
example, wounding (Kubigsteltig et al., 1999), it seems
clear that (1) these octadecanoids are not involved in
systemic defence signalling (Fig. 6), rather they behave
like local response regulators and (2) the systemic
activation of the AOS promoter after wounding of a leaf
or a root does involve other wound factor(s) released
by these organs. On the other hand a crucial role for
octadecanoids in herbivore defence could be demonstrated: (1) the LA-de®cient fad 3-2 fad 7-2 fad 8 mutant of
Arabidopsis (McConn et al., 1997) lacks the ability to
accumulate JA after wounding and is more susceptible
to attacks of chewing insects than the wild-type, (2) the
defenceless mutant (def 1) of tomato does not raise the
JA-level after wounding, produces less proteinase
inhibitor and is more susceptible to herbivore attack than
the wild-type (Howe et al., 1996), (3) the JA-insensitive
mutant (coi1) of Arabidopsis is more susceptible to herbivore attack than wild-type plants (Rojo et al., 1998).
Therefore it may be possible that there exist different
Fig. 6. There exist three wound-induced signalling pathways in higher plants (adapted from Weiler et al., 1999).
20
Schaller
signal transduction pathways and defence mechanisms
for herbivore attack and wounding, respectively. For
the described systemic accumulation of octadecanoids
after wounding, chemical and physical messengers are
discussed: (1) the octadecapeptide systemin in tomato (for
review see Schaller and Ryan, 1996), (2) hydraulic (for
review see Malone and Alarcon, 1995) and (3) electrical
(Wildon et al., 1992) signals. Aspects of wound signalling
will be discussed in detail in Jose J SaÂnchez-Serrano's
review in this issue.
Acknowledgements
The author is grateful to Professor Dr EW Weiler for his
encouraging interest in and critical reading of the manuscript,
and the Deutsche Forschungsgemeinschaft, Bonn for ®nancial
support.
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