Báo cáo khoa học: Cytochrome P450 metabolizing fatty acids in plants: characterization and physiological roles pptx

MINIREVIEW
Cytochrome P450 metabolizing fatty acids in plants:
characterization and physiological roles
Franck Pinot
1
and Fred Beisson
2
1 Institut de Biologie Mole
´
culaire des Plantes. CNRS – Universite
´
de Strasbourg, Strasbourg, France
2 Department of Plant Biology and Environmental Microbiology, CEA ⁄ CNRS ⁄ Aix-Marseille University, Cadarache, France
Introduction
Plants produce a wide variety of oxygenated deriva-
tives of fatty acids (FAs) which are involved in vari-
ous important biological functions ranging from
waterproofing to signalling and plant defence. Most
oxygenated FAs are the products of enzyme-cataly-
sed reactions. Hydroperoxides of lipid signalling
result mainly from the action of lipoxygenases. The
hydroxy FAs of some seed oils (e.g. ricinoleic acid
of castor bean) and membrane lipids (2-hydroxy FA
of sphingolipids) are synthesized by homologues of
Keywords
catabolism; cutin; cytochrome P450;
defense; epoxyacids; fatty acid; plant
envelope; reproduction; sporopollenin;
suberin
Correspondence
F. Pinot, IBMP-CNRS UPR 2357, Institut de

Botanique, 28 rue Goethe, F-67083
Strasbourg Cedex, France
Fax: +33 (0)368851921
Tel: +33 3 68 85 19 99
E-mail:
(Received 22 June 2010, revised 15
September 2010, accepted 22 October
2010)
doi:10.1111/j.1742-4658.2010.07948.x
In plants, fatty acids (FA) are subjected to various types of oxygenation
reactions. Products include hydroxyacids, as well as hydroperoxides, epox-
ides, aldehydes, ketones and a,x-diacids. Many of these reactions are cata-
lysed by cytochrome P450s (P450s), which represent one of the largest
superfamilies of proteins in plants. The existence of P450-type metabolizing
FA enzymes in plants was established approximately four decades ago in
studies on the biosynthesis of lipid polyesters. Biochemical investigations
have highlighted two major characteristics of P450s acting on FAs: (a) they
can be inhibited by FA analogues carrying an acetylenic function, and
(b) they can be enhanced by biotic and abiotic stress at the transcriptional
level. Based on these properties, P450s capable of producing oxidized FA
have been identified and characterized from various plant species. Until
recently, the vast majority of characterized P450s acting on FAs belonged
to the CYP86 and CYP94 families. In the past five years, rapid progress in
the characterization of mutants in the model plant Arabidopsis thaliana has
allowed the identification of such enzymes in many other P450 families (i.e.
CYP703, CYP704, CYP709, CYP77, CYP74). The presence in a single spe-
cies of distinct enzymes characterized by their own regulation and catalytic
properties raised the question of their physiological meaning. Functional
studies in A. thaliana have demonstrated the involvement of FA hydroxy-
lases in the synthesis of the protective biopolymers cutin, suberin and

sporopollenin. In addition, several lines of evidence discussed in this
minireview are consistent with P450s metabolizing FAs in many aspects of
plant biology, such as defence against pathogens and herbivores, develop-
ment, catabolism or reproduction.
Abbreviations
FA, fatty acid; x, omega position; P450, cytochrome P450.
FEBS Journal 278 (2011) 195–205 ª 2010 The Authors Journal compilation ª 2010 FEBS 195
FA desaturases. But for the vast majority of other
oxygenated FA derivatives, the insertion of oxygen
atoms in the carbon chain is dependent on cyto-
chrome P450s (P450s).
The involvement of P450s in FA metabolism in
plants was first described in the context of cutin and
suberin studies [1,2]. These protective biopolymers are
made mostly of hydroxyfatty acids and a,x-dicarbox-
ylic acids esterified to each other and to glycerol. The
hydroxyl groups of the FAs can be located at the ter-
minal methyl (x position) or on various internal posi-
tions [3]. Enzymes capable of hydroxylation of FA
thus have a key role to play in cutin and suberin syn-
thesis by allowing the production of bifunctional FA
derivatives which can serve as monomers for polymeri-
zation. The pioneering studies on the biosynthesis of
polymers of oxygenated FAs have shed light on the
capacity of P450s to catalyse in-chain and x-hydroxyl-
ation of FAs, but also to catalyse the introduction of
other functional groups in FAs such as epoxy groups,
which are common substituents in cutin or suberin
monomers of some plant species [1,2]. Incubation of
lauric acid (C12:0) and its unsaturated analogues with

microsomal fractions of various plant species con-
firmed the existence in plants of distinct P450s able to
catalyse different reactions [4–6]. More recent studies
involving the characterization of plant mutants allowed
the identification of additional P450s catalysing FA
oxidation (Table 1). The results of the studies pre-
sented in this minireview demonstrate the involvement
of FAs in the synthesis of plant hydrophobic barriers
and suggest their implication in several other biological
processes.
Identification of the first fatty acid
hydroxylases
Typically, FA hydroxylation activities are barely detect-
able in whole-plant extracts even when using purified
microsomal fractions as the enzyme source and radiola-
belled FAs as substrates. This is because plant FA
hydroxylases are either low in abundance or expressed
in specific tissues and ⁄ or in response to stress [7]. This
has represented a major difficulty to their identification
and biochemical analysis. To circumvent this problem,
researchers have taken advantage of two major features
of P450s acting on FAs: (a) they can be regulated at the
transcriptional level by biotic and abiotic stresses, and
(b) they can be inactivated by FA analogues carrying an
acetylenic function.
In a pioneering work on plant FA hydroxylases [8],
abiotic stresses were used to compensate for the low
level of enzyme present in plant tissue. Exposure of
Vicia sativa seedlings to clofibrate, a hypolipidaemic
drug, strongly induced x-hydroxylase activities [8] and

enabled biochemical investigations [9,10]. In micro-
somes of clofibrate-treated seedlings, inhibition of lau-
ric and oleic acid hydroxylation by a C18 FA carrying
a terminal acetylenic function followed different kinet-
ics. This was the first demonstration of the existence of
Table 1. In vitro activity and physiological roles of characterized plant cytochrome P450s metabolizing fatty acids.
Cytochrome P450 Plant Enzymatic activity Physiological role Ref
94A1 Vicia sativa x-hydroxylation Signalling? catabolism? [13]
94A2 Vicia sativa x- and in-chain hydroxylations ? [15]
94A5 Nicitiana tabacum x-hydroxylation, diacid formation Signalling? catabolism? [22]
94C1 Arabidopsis thaliana x-hydroxylation, diacid formation Signalling? catabolism? [18]
86A1 Arabidopsis thaliana x-hydroxylation Suberin synthesis [14]
86A2 Arabidopsis thaliana x-hydroxylation Cutin synthesis, defence [24]
86A8 Arabidopsis thaliana x-hydroxylation Cutin synthesis, pleiotropic role [23]
86A4 Arabidopsis thaliana x-hydroxylation Cutin synthesis [34]
86A22 Petunia hybrida x-hydroxylation Estolide synthesis [30]
86A33 Solanum tuberosum x-hydroxylation Suberin synthesis [29]
86B1 Arabidopsis thaliana x-hydroxylation Suberin synthesis [26]
81B1 Helianthus tuberosus in-chain hydroxylation Signalling? defence? [20]
703A2 Arabidopsis thaliana in-chain hydroxylation Sporopollenin synthesis [37]
704B1 Arabidopsis thaliana x-hydroxylation Sporopollenin synthesis [38]
704B2 Oryza sativa x-hydroxylation Sporopollenin synthesis [39]
709C1 Triticum aestivum in-chain hydroxylation Signalling? defence? [19]
726 A1 Euphorbia lagascae Epoxidation Seed oil synthesis [31]
77A4 Arabidopsis thaliana Epoxidation in-chain hydroxylation Signalling? defence? [21]
77A6 Arabidopsis thaliana in-chain hydroxylation Cutin synthesis [34]
92B1 Petunia hybrida x-hydroxylation Flowering? [54]
78A1 Zea mays x-hydroxylation Flowering? [55]
Cytochrome P450 metabolizing fatty acids in plants F. Pinot and F. Beisson
196 FEBS Journal 278 (2011) 195–205 ª 2010 The Authors Journal compilation ª 2010 FEBS

distinct FA x-hydroxylases in a single species. Weiss-
bart et al. [10] demonstrated that 11-dodecynoic acid
irreversibly inhibited x-hydroxylation of lauric acid in
V. sativa microsomes. The mechanism of inhibition
has been explored using FA-metabolizing P450s from
animals. In these P450s, inactivation results from heme
[11] or protein alkylation [12]. Postulating that inhibi-
tion of plant P450s followed the same mechanism,
inhibition by a FA with an acetylenic function was
used to clone the first plant FA x-hydroxylase
(CYP94A1). Incubation of [1-1
4
C]-11-dodecynoic acid
with V. sativa microsomes allowed covalent tagging of
a protein responsible for x-hydroxylase activity on lau-
ric acid. An internal peptide sequence from the radio-
labelled protein was determined and a full-length
cDNA was isolated [13].
Another strategy based on conserved motifs led to the
identification in Arabidopsis thaliana of a second plant
FA hydroxylase. Using the similarity of an Arabidopsis
expressed sequence tag (EST) sequence and the con-
sensus sequence of the fungal CYP52 family of alkane
hydroxylases and the mammalian CYP4A family of
alkane hydroxylases, a cDNA was cloned and shown to
encode a member (CYP86A1) of a new plant P450
family. When expressed in yeast, CYP86A1 displayed
x-hydroxylase activity on a range of saturated and unsat-
urated C12-C18 fatty acids but not on C16 alkane [14].
Catalytic properties and active site

Plants P450s can catalyse different types of reactions
using FAs as substrates. The most typical products of
reactions are x- and in-chain hydroxy fatty acids, but
FAs can also be epoxidized, transformed to dicarbox-
ylic FA (Fig. 1) or in the case of hydroperoxides,
cleaved to shorter aliphatic compounds (see below).
Few studies have addressed what determines substrate
specificity and regioselectivity. One has to keep in
mind that most of the knowledge concerning these two
aspects of FA hydroxylase enzymology comes from
biochemical studies performed after heterologous
expression. Also, activity measurements were per-
formed with free FA, but natural substrates are very
often not known and might be different (acyl-CoAs,
glycerolipids, etc.).
From a thermodynamic point of view, oxidation of
the terminal methyl of a fatty acid is disfavoured in
comparison with oxidation of a secondary carbon.
This implies that x-hydroxylases possess a highly
structured active site. Using site-directed mutagenesis,
Kahn et al. [15] demonstrated that a conservative sub-
stitution of Phe494 in CYP94A2 cloned from V. sativa
led to a shift in the regiospecificity of lauric acid
hydroxylation from the x- position to the x-1 position.
It was concluded that Phe494 supplies constraints that
maintain the terminal methyl of lauric acid near the
ferryl oxo species. The aliphatic nature of FAs makes
it very likely that hydrophobic interactions are impor-
tant for the positioning of the substrate in the active
site. This was confirmed by Rupashinghe et al. [16]

who studied five members of the CYP86 family from
Arabidopsis. Using modelling based on the known
crystal structure and 3D models of FA-metabolizing
P450s in bacteria and animals, the authors confirmed
HOOC
HOOC
OH
HOOC
O
H
HOOC
COOH
HOOC
OH
HOOC
O
-hydroxylation
Epoxidation
In chain hydroxylation
Aldehyde formation
Dicarboxylic acid formation
A
E
B
C
D
F
CYP94C1
CYP94C1
CYP94C1

CYP77A4
CYP709C1
Fig. 1. Metabolization of oleic acid and its derivatives by plant P450s. Reactions presented in this scheme have been described after incuba-
tion of oleic acid with microsomes of yeast expressing distinct P450s [18,19,21]. (A) Oleic acid, (B) 18-hydroxyoleic acid, (C) octadec-9-
en-18-al-oic acid, (D) octadec-9-en-1,18-dioic acid; (E) 9,10-epoxystearic acid; (F) 17-hydroxyoleic acid.
F. Pinot and F. Beisson Cytochrome P450 metabolizing fatty acids in plants
FEBS Journal 278 (2011) 195–205 ª 2010 The Authors Journal compilation ª 2010 FEBS 197
that CYP86 models have a binding site packed with
hydrophobic residues (Fig. 2). However, in some cases,
the results also suggest the presence of polar residues
in the binding site. This is the case for CYP94A1 [17],
CYP94C1 [18] and CYP709C1 [19] which metabolize
in vitro 9,10-epoxystearic acid with a higher efficiency
than C18:1, C18:2 or C18:3. This suggests a strong
interaction between the oxiran and a polar residue of
the active site. The enantioselectivity of CYP94A1 for
9R,10S-epoxystearic acid supports this hypothesis. The
high constraints on the substrate in the active site are
also illustrated by the example of CYP77A4, which
epoxidizes C18:2 to 12,13-epoxyoctadeca-9-enoic acid
presenting a strong enantiomeric excess in favour of
the 12S ⁄ 13R enantiomer representing 90% of the
epoxide. Some hydroxylases, e.g. CYP81B1 [20] or
CYP77A4 [21], exhibit a regioselectivity depending on
the aliphatic chain length. This is likely because of the
anchoring of different substrates via interaction of
the carboxyl group with the same polar residue of the
enzyme. By contrast, some hydroxylases show strict
regioselectivity: whatever the chain length, CYP94A1
[13] and CYP709C1 [19] exclusively attack the x- and

x-1 position, respectively. This clearly indicates that in
these cases, the carboxyl group does not interact with
a specific polar residue of the active site.
The structure of metabolites also depends on the
chemical motif present on the substrate. CYP94A5
from tobacco [22] and CYP94C1 from Arabidopsis [18]
catalyse the x-hydroxylation of fatty acids, but oxida-
tion of primary alcohol by these enzymes leads to the
formation of dicarboxylic FAs. Using microsomal frac-
tion of V. sativa, Weissbart et al. [10] showed that
x-hydroxylases form epoxides when the terminal car-
bon is engaged in an unsaturation. The same observa-
tion was made with in-chain hydroxylase from
microsomes of Heliantus tuberosus [20]. These results
were obtained with unsaturated analogues of lauric
acid, but recently CYP77A4, an in-chain hydroxylase
cloned from Arabidopsis, was shown to be able to
epoxidize physiological C18:1, C18:2 and C18:3 [21].
Physiological role
Characterization of the first FA-metabolizing P450
enzymes showed that they exhibited different regiose-
lectivities and substrate specificities and were differ-
ently regulated. This suggested that many P450s acting
on FAs existed within the same species and acted on
distinct substrates in a variety of biochemical path-
ways. EST and genome sequencing projects demon-
strated that P450s acting on FAs belonged to
multigenic families and analysis of mutants confirm
their involvement in several biological processes
(Table 1).

Cutin and suberin biosynthesis
FA x-hydroxylation
The first plant gene encoding a FA x-hydroxylase,
CYP86A1, was identified in A. thaliana based on
sequence homology with x-hydroxylases from mam-
mals and yeast. The encoded protein was characterized
after heterologous expression [14]. A protein from the
same subfamily, CYP86A8, was the first FA x-hydrox-
ylase for which a mutant (lacerata) was isolated [23].
The presence of a maize transposon in the coding
sequence of CYP86A8 led to a pleiotropic mutant phe-
notype with organ fusion, altered cell differentiation,
reduced apical dominance and delayed senescence. The
organ fusion phenotype observed in lacerata was simi-
lar to that of Arabidopsis overexpressing a cutinase.
This was consistent with implication of this enzyme in
Arabidopsis cutin synthesis. TEM of the epidermis
showed that the structure of the cuticle was altered,
which suggested that some of these phenotypes were
due to a defect in the epidermal cuticle. It was thus
proposed that a major role of lacerata was the produc-
tion of the omega-hydroxy FA constitutive of the cutin
polymer matrix of the cuticle. Reverse genetics
approaches in Arabidopsis and potato enabled investi-
gation of the putative involvement of other members
of the CYP86 family in the synthesis of cutin and the
Fig. 2. Predicted substrate contact residues in all five CYP86A
models with predicted conformation for oleic acid binding. Con-
served residues are coloured in green, similar residues are coloured
in gold, nonconserved residues are in elemental colours. The heme

within the catalytic site and oleic acid (C18:0) are shown in space-
filling format. From Rapusinghe et al. [16].
Cytochrome P450 metabolizing fatty acids in plants F. Pinot and F. Beisson
198 FEBS Journal 278 (2011) 195–205 ª 2010 The Authors Journal compilation ª 2010 FEBS
other major FA-based polyester of plants (suberin).
The att1 mutant disrupted in CYP86A2 [24] displayed
increased sensitivity to the bacteria Pseudomanas syrin-
gae, and water loss and a disorganized cuticle struc-
ture, which was consistent with a role in cutin
biosynthesis. The Arabidopsis horst mutant, impaired
in the coding sequence of CYP86A1, showed a total
aliphatic root suberin content that was reduced to
60% compared with wild-type [25]. This reduction was
because of the strong decrease of C16 and C18
x-hydroxyacids and corresponding diacids. This obser-
vation corroborates in vitro studies performed by Ben-
veniste et al. [14] who showed that microsomal
preparations of yeast expressing CYP86A1 metabo-
lized C16 and C18 with high efficiency compared with
other FAs tested. The fact that the content in the satu-
rated very long-chain (C22–C24) omega-hydroxy FA
of suberin was not affected by in CYP86A1 knockouts
indicated that synthesis of these monomers was due
mostly or completely to proteins other than CYP86A1.
Coexpression of a second member of the CYP86
family, CYP86B1, using suberin biosynthetic genes and
in silico gene expression analysis of its tissue specificity,
suggested its possible implication in the syntesis of
suberin monomers. This was confirmed by study of the
ralph mutant possessing an alteration in the coding

sequence of CYP86B1 [26]. This mutation resulted in a
strong reduction of C22 and C24 x-hydroxy saturated
FA derivatives in root suberin and seed coat polyes-
ters. Surprisingly, downregulation of CYP86B1 did not
impair the water-barrier function of root suberin and
seed coat, showing that production of C16 and C18
x-hydroxy FA by other hydroxylases was sufficient
to maintain this major property of polyester-based
barriers.
Although CYP86A1 and CYP86B1 seem to be
responsible mostly for the synthesis of C16–C18 and
C22–C24 x-hydroxy FAs respectively, in plants
co-overexpression of these enzymes with the GPAT5
acyltransferase of suberin synthesis shows that their
specificity is probably in part overlapping. Indeed,
ectopic overexpression of CYP86A1 in Arabidopsis
resulted in the production of C22–C24 x-hydroxy FA
and diacids in stems cutin in addition to a major
increase in C16–C18 omega-oxidized monomers [27].
The reverse situation was observed for CYP86B1 [28].
By contrast to CYP86A1, which produced, in vitro,
the C16 and C18 monomers missing in the ralph
mutant, microsomal incubation of C22 and C24 FAs
with microsomes of yeast expressing CYP86B1 did not
produce any metabolite [26]. The same observation
was made with CYP86A33 involved in potato suberin
production [29]. One explanation would be that
CYP86B1 does not metabolize free FA, but rather
esterified FA (acyl-CoAs, glycerolipids, etc.). In this
respect, it is important to note that the demonstration

of x-hydroxylase-metabolizing esterified FA was
recently achieved with CYP86A22 from Petunia [30]
which x-hydroxylates saturated and unsaturated acyl-
CoA derivatives. In addition, it has also been shown
that CYP726A1 from Euphorbia lagascae acts on FAs
esterified to phosphatidylcholine to produce epoxy
fatty acids [31].
Intracellular localization studies revealed that both
proteins encoded by CYP86A1 and CYP86B1 localize
in the endoplasmic reticulum, in agreement with the
majority of plant FA hydroxylases [32]. It is notewor-
thy that the endoplasmic reticulum is also the major
location of C22 and C24 FA synthesis, which consists
of the elongation of FAs exported from chloroplasts.
FA in-chain hydroxylation
Using biochemical assays in Vicia faba, Soliday and
Kolattukudy [2,33] demonstrated three decades ago
the involvement of at least two distinct FA hydroxylas-
es in formation of the major cutin monomer
10,16-dihydroxypalmitic acid in broad bean. Genetic
and biochemical studies allowed Li-Beisson et al.
[34] to show that in Arabidopsis CYP77A6 hydroxyl-
ated on positions 8, 9 and 10, the 16-hydroxypalmitic
acid produced by CYP86A4. The sequential order
of the hydroxylation reactions was demonstrated by
both cutin monomer profiles in knockout mutants
for CYP77A6 and CYP86A4 and the fact that
recombinant CYP77A6 expressed in yeast was active
on 16-hydroxypalmitic acid, but not on palmitic acid
(Fig. 3). Null mutants in the gene coding for

CYP77A6 still had 66% of cutin load compared with
wild-type, but lacked the typical nanoridges present on
the surface of flowers. These specialized structures
have been suggested to help attract insect pollinators,
giving CYP77A6 an indirect role in reproduction. It is
possible that the introduction of in-chain hydroxyl
allows cross-linking of cutin, leading to reticulation
and strengthening of the polymeric envelope. The dis-
covery of an in-chain hydroxylase producing polyhy-
droxy FAs possibly important for the formation of
nanostructures increases the array of FA-modifying
enzymes with biotechnological interest that originate
from plant polyester metabolism [35].
Sporopollenin biosynthesis
Sporopollenin is a major polymeric component of
exine, the outer pollen wall, and represents a protective
F. Pinot and F. Beisson Cytochrome P450 metabolizing fatty acids in plants
FEBS Journal 278 (2011) 195–205 ª 2010 The Authors Journal compilation ª 2010 FEBS 199
envelope fundamental for pollen resistance [36]. Its
high resistance makes it difficult to study and its exact
structure remains to be elucidated. Preservation of
ancient pollen grains for millions of years illustrates its
stability and its protective properties. The participation
of FA in-chain and x-hydroxylases in sporopollenin
synthesis has recently been demonstrated with the
study of Arabidopsis mutants. Arabidopsis CYP703A2
[37] and CYP704B1 [38] knockout lines produced non-
maturated pollen grain lacking the normal exine layer.
Heterologous expression in yeast cells showed that
CYP703A2 and CYP704B1 are FA in-chain and

x-hydroxylases, respectively. The first preferentially
catalyses the hydroxylation of lauric acid (C12) at
position 7, whereas the second hydroxylates the x
position of C18 FAs. A second member of the
CYP704B subfamily has been described in rice [39].
One mutant line generated by treatment with
60
Co dis-
played complete male sterility. Exine was absent on
the pollen grain and analysis revealed a drastic loss of
cutin monomers in cyp704B2 anthers. The capacity of
CYP704B2 to x-hydroxylate C16 and C18 FA was
demonstrated after heterologous expression in the
yeast system.
Plant defence
Biochemical and genetics evidence indicates the involve-
ment of FA hydroxylases in plant defence. Parker and
Ko
¨
ller [40] showed that bean infection by Rhizocto-
nia solani was decreased when leaves were treated
with cutinases releasing x-hydroxy fatty acids. The
protection mechanism remains to be elucidated, but it
has been established that pathogen-challenged plants
perceive hydroxy FAs as key compounds in the induc-
tion of resistance [41,42]. These compounds also
induce elicitation of H
2
O
2

production [43]. It is note-
worthy that 9,10,18-trihydroxystearic and 18-hydroxy-
9,10-epoxystearic acids exhibit the strongest effect in
eliciting defence events. These FA derivatives are pro-
duced by CYP94A1 from V. sativa suggesting a poten-
tial role in plant defence for this enzyme. This
hypothesis receives support from experiments showing
that treatment of V. sativa seedlings with the stress
hormone methyl jasmonate enhanced CYP94A1 at the
transcriptional level [44]. Interestingly, clofibrate treat-
ment also enhanced CYP94A1 at the transcriptional
level [13] and increased the proliferation of peroxi-
somes [45] which have an important role in responses
to pathogens [46]. Induction of mammalian x-hydrox-
ylases by clofibrate and peroxisome proliferation occur
via activation of a peroxisome proliferator-activated
receptor. This peroxisome proliferator-activated recep-
tor can be activated by clofibrate [47] and by FA
derivatives such as prostaglandins. Clofibrate produces
similar effects in plants and animals [8,45]. Further-
more, there are evident structural analogies between
prostaglandins and jasmonates, which are both poly-
unsaturated FA derivatives involved in response to
stress. All these similarities strongly suggest that the
mechanisms of x-hydroxylase regulation by clofibrate
and FA derivatives are conserved between plants and
animals.
Study of the Arabidopsis att1 mutant confirmed the
implication of FA x-hydroxylases in plant defence
[24]. Pseudomonas syringea caused a more severe dis-

ease in att1 than in wild-type. This resulted from the
induction of type III genes necessary for parasitism, by
a still unknown process. In Arabidopsis, expression of
five members of the CYP86A subfamily involved in
FA x-hydroxylation was monitored by micro-array
and RT-PCR analysis [48]. They were found to be
expressed at different constitutive levels and their
expression varied with organs. They also responded
differently to chemicals and environmental stresses.
Sequence analysis of the promoters revealed cis-ele-
ments present in the promoters of other plant genes
that correlated with gene response.
Implication in plant defence events is not restricted
to FA x-hydroxylases. CYP709C1 is the first subtermi-
nal hydroxylase of long-chain FAs characterized in
plants [19]. This enzyme exclusively attacks x-1 and
x-2 carbons, and in the context of plant defence, it is
interesting to note that x-1 hydroxy derivatives of
FA have been described. Volicitin the x-1 hydroxy
HOOC
HOOC
HOOC
OH
OH
OH
A
B
C
CYP86A4
CYP77A6

Fig. 3. Synthesis of 10,16-dihydroxypalmitic acid from palmitic acid
in Arabidopsis thaliana. Putative pathway based on biochemistry
and mutant analysis [34]. In vivo endogenous substrates (free fatty
acids, acyl-CoAs and glycerolipids) are still unknown. (A) Palmitic
acid, (B) 16-hydroxypalmitic acid, (C) 10,16-dihydroxypalmitic acid.
Cytochrome P450 metabolizing fatty acids in plants F. Pinot and F. Beisson
200 FEBS Journal 278 (2011) 195–205 ª 2010 The Authors Journal compilation ª 2010 FEBS
linolenic acid coupled to glutamine [49] is responsible
for the majority of elicitor activity present in the oral
secretion of caterpillar species feeding on plants. It is
conceivable that products of reactions catalysed by
CYP709C1 have eliciting properties or are precursors
of molecules with eliciting properties. Studies of sub-
strate specificity showed that among the FAs tested,
CYP709C1 metabolized 9,10-epoxystearic with the
highest efficiency [19]. Hydrolysis of the resulting
17-hydroxy-9,10-epoxystearic acid by epoxide hydro-
lase would lead to the formation of a trihydroxy FA
with a chemical structure close to that of compounds
having antimicrobial properties [19]. The strong and
rapid induction of CYP709C1 by methyl jasmonate is
also in favour of its participation in plant defence. In
Arabidopsis, interplay of the epoxidase CYP77A4 with
other enzymes also accounts for the formation of
poly(hydroxy FA). CYP77A4 can produce vernolic
acid which is converted to diol by epoxide hydrolase
[21]. Hydroxylation of this diol by an x-1 hydroxylase
present in Arabidopsis [18] produces 12,13,17-trihydr-
oxyoctadeca-9-enoic acid (Fig. 4), which has been
shown to possess antifungal properties [50].

The importance of P450s metabolizing enzymes in
plants is illustrated by the atypical P450 family
CYP74. This family has been subjected to a tremen-
dous amount of work. For a detailed discussion the
reader in invited to refer to the specific review by
Stumpe and Feussner [51]. Briefly, contrary to the
majority of P450s, members of this family catalyse oxi-
dative reactions without O
2
and NADPH-cytochrome
P450 reductase. Three catalytic activities have been
assigned to these enzymes: allene oxide synthase,
hydroperoxide lyase and divinyl ether synthase
(Fig. 5). CYP74A1 with allene oxide synthase activity
from Arabidopsis was the first identified and character-
ized enzyme of the octanoid pathway leading to jas-
monates [7]. The so-called oxylipins (jasmonates,
aldehydes, divinyl ether, alcohols) generated by CYP74
members are signalling molecules as well as molecules
exhibiting antimicrobial and antifungal properties.
FA catabolism
No direct involvement of plant x-hydroxylases in FA
catabolism has been demonstrated. However, by anal-
ogy to knowledge concerning FA x-hydroxylases in
mammals and in microorganisms, it can be assumed
that plant x-hydroxylases could be major actors in this
process. This is particularly relevant for x-hydroxyl-
ases that are upregulated by a class of compounds (i.e.
clofibrate) [13,48] known to induce peroxisome prolif-
eration in mammals. In mammals, x-hydroxy deriva-

tives of FA produced by members of CYP4 family can
be further oxidized to dicarboxylic acids either by
dehydrogenases or by P450s able to perform the com-
plete oxidation of a methyl to a carboxyl group. In
both pathways, the resulting dicarboxylic acid can be
eliminated by b-oxidation in peroxisome. The key role
of x-hydroxylases in FA catabolism has also been
HOOC
HOOC
O
HOOC
OHHO
HOOC
OHHO
OH
A
B
C
D
CYP77A4
Epoxide hydrolase
In chain hydroxylase
Fig. 4. Formation of antifungic 12,13,17-trihydroxyoctadeca-9-enoic
acid in microsomes of Arabidopsis thaliana. Putative pathway lead-
ing to the formation of 12,13,17-trihydroxyoctadeca-9-enoic acid
based on distinct enzymes characterized in Arabidopsis [18,21].
(A) Linoleic acid, (B) vernolic acid, (C) 12,13-dihydroxyoctadeca-9-
enoic acid, (D) 12,13,17-trihydroxyoctadeca-9-enoic acid.
R R
1

R
1
HOO
R
R R
1
O
R
3
O
R
1
R
2
O
R
1
O
+
DES
AOS
HPL
LOX
Aldehyde
w-oxo fatty acid
Allene oxide
Divinyl ether
Fig. 5. Metabolism of polyunsaturated fatty acid by lipoxygenase
and CYP74. LOX: lipoxygenase; HPL: Hydroperoxide lyase; AOS:
Allene oxide synthase; DES: Divinyl ether synthase. Simplified from

Stumpe and Feussner [51].
F. Pinot and F. Beisson Cytochrome P450 metabolizing fatty acids in plants
FEBS Journal 278 (2011) 195–205 ª 2010 The Authors Journal compilation ª 2010 FEBS 201
established for yeast belonging to the genus Candida.
Members of the CYP52 family enable Candida maltosa
to grow on media containing aliphatic hydrocarbons
as a sole source of carbon and energy (reviewed in
[18]). In plants, besides enhancement of FA hydroxy-
lases at the transcriptional level, clofibrate [13,48] simi-
larly to what is observed in mammals, also induces the
proliferation of peroxisomes [45] in which FA b-oxida-
tion is of primary importance for energy production
[52]. CYP95A5 from tobacco [22] and CYP94C1 from
Arabidopsis [18] are both capable of producing dicar-
boxylic FAs either by a two-step oxidation of
x-hydroxy FA or by a three-step oxidation starting
from a FA (Fig. 1). It is postulated that dicarboxylic
FAs are degraded much faster through b-oxidation
than monocarboxylic FAs, and it has been proposed
that x-hydroxylases may function in deactivating FA-
derived lipid signals and in rapidly turning over free
FAs liberated by lipases during stress [52].
Reproduction
As mentioned above, maturation of pollen grains in
Arabidopsis and in rice depends in part on FA
x-hydroxylase activity [37,38]. Synthesis of the protec-
tive envelope in the rice anther also requires active FA
x-hydroxylase [39]. Downregulation of the enzymes
implicated in these processes leads to a male sterility
phenotype, giving a key role for FA x-hydroxylases in

reproduction. It has been known for a long time that
when present, stigma exudate is of primary importance
in pollination events. Di- and triglycerides of Nicoti-
ana tabacum are enriched in x-hydroxy FA which are
believed to be responsible for the recognition of stigma
by pollen [53]. Recently, transgenic Petunia expressing
CYP86A22-RNAi were produced and the lipid con-
tent of stigmas analysed [30]. Downregulation of
CYP86A22 was accompanied by a drastic decrease in
18-hydroxyoleic and 18-hydroxylinoleic acids, which
can even be lacking in some lines when they represent
96% of total stigma FA in wild-type. This was in
agreement with the enzymatic activity determined after
heterologous expression in insect cells, which showed
that CYP86A22 was able to x-hydroxylate C16 and
C18 fatty acids activated by CoA. Histochemical anal-
ysis located CYP86A22 exclusively in the stigma,
which is consistent with a specific role in flower devel-
opment and reproduction. In the context of reproduc-
tion, two characterized FA x-hydroxylases from
Petunia CYP92B1 [54] and Zea mays CYP78A1 [55],
and the partially characterized Petunia CYP703A1
[56], have also been shown to be preferentially
expressed in developing inflorescence.
Phylogeny and evolution
In plants, P450s have duplicated and diverged in order
to produce a tremendous array of compounds exhibit-
ing sometimes very similar structures. Fatty acid hy-
droxylases follow this rule. CYP86A1 and CYP86B1
represent a good example of this evolution: they both

hydroxylate the terminal methyl of acyl chains in the
suberin biosynthesis pathways and seem to differ only
by the chain length preference.
The CYP86 family is found in the genome of the
moss Physcomitrella patens in addition to the genomes
of Arabidopsis, rice and poplar [57]. It thus seems that
this family has played an important role in early plant
evolution. This is consistent with the demonstrated
role of CYP86As in the biosynthesis of the framework
matrix of the plant cuticle, a structure that is thought
to have played a great role in the adaptation of early
plants to life in a terrestrial desiccating environment.
CYP94 family members also duplicated during evolu-
tion of land plants while retaining their catalytic activ-
ity on FAs. The biological role of the CYP94 family is
still unknown, however.
The CYP703 family is also conserved in land plants
and typically each plant species contains only one
CYP703 [37]. CYP704B1 and CYP704B2 belong to a
conserved and ancient P450 family in moss and seed
plants. This suggests that reactions catalysed by
CYP703 and by members of the CYP704 subfamily
represent a key step in exine formation during plant
evolution.
The apparent absence of the CYP77 family in the
moss genome [58] might indicate that cuticles based on
cutin polyesters containing in-chain hydroxylated FAs
represent a more recent type of hydrophobic barriers.
The selective advantage conferred by cutins rich in
polyhydroxy FAs such as dihydroxypalmitates is

unknown, but it can be speculated that it is related to
the evolution of distinctive epidermal surface structures
such as flower nanoridges. Alternatively, it might be
that polyhydroxy FA-rich cutins have improved hydro-
phobic barrier properties.
Conclusion
Plants have developed a highly complex metabolic net-
work using the diversified catalytic properties of the
P450s [57]. Among these P450s, FA hydroxylases are
major actors involved in many aspects of plant biology.
When plants conquered dry land  400 million years
ago, one major problem they had to face was to resist
to dessication. They have developed cutin and suberin
two biopolymers constituted of FA and FA plus
Cytochrome P450 metabolizing fatty acids in plants F. Pinot and F. Beisson
202 FEBS Journal 278 (2011) 195–205 ª 2010 The Authors Journal compilation ª 2010 FEBS
phenolics, respectively. By introducing hydroxyl func-
tion in to monomers, FA hydroxylase will allow their
condensation and elongation of the polymers. Ensuring
reproductive success was also of primary importance
for land conquest. Cutin of the anther wall and sporo-
pollenin represent protective envelopes and the key role
of FA hydroxylases in their synthesis is now well estab-
lished. Plants are sessile organisms and rely on a battery
of chemicals for survival. Lipid metabolism is a major
player in the defence network of plants and it is tempt-
ing to speculate that hydroxyls, epoxides of FA as well
as dicarboxylic FA have properties similar to those
described for FA derivatives generated by members of
the CYP74 family implicated in plant defence [51]. Sim-

ilar to what is known for mammals and microorgan-
isms, FA x-hydroxylation in plants could be the starting
point for their catabolism leading to energy production
required for general development and plant defence.
More studies are needed to confirm and elucidate the
physiological meanings of P450 metabolizing FAs in
plants as well as in animals [59] and microorganisms
[60]. Concerning the plant kingdom, a major part of this
task should be achieved via the study of plant mutants.
Acknowledgements
FP was supported in part by a grant from KBBE 2009
program (grant ANR-09-KBBE-006-001). FB was sup-
ported in part by a grant from the 7th European
Community Framework Program (Marie Curie Inter-
national Reintegration Grant 224941).
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