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

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

Cytochrome P450 metabolizing fatty acids in plants:

characterization and physiological roles

Franck Pinot1and Fred Beisson2

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: franck.pinot@ibmp-cnrs.unistra.fr

(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.

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.

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 [1-14C]-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

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.

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].

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 silicogene 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

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 with60Co

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 H2O2 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.

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 O2 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 (jasjas-monates, 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

OH HO

HOOC

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-9-12,13-dihydroxyoctadeca-9-enoic acid.

R 1

HOO R

O

R 3

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].

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 producb-oxida-tion

[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 acprotec-tive 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

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)

References

1 Croteau R & Kolattukudy P (1974) Biosynthesis of

hydroxyfatty acid polymers Enzymatic synthesis of

cutin from monomer acids by cell-free preparations

from the epidermis of Vicia faba leaves Biochemistry

13, 3193–3202

2 Soliday C & Kolattukudy P (1978) Midchain

hydroxyl-ation of 16-hydroxypalmitic acid by the endoplasmic

reticulum fraction from germinating Vicia faba Arch

Biochem Biophys 188, 338–347

3 Kolattukudy P (1981) Structure, biosynthesis and

degra-dation of cutin and suberin Annu Rev Plant Physiol 32,

539–567

4 Benveniste I, Salau¨n J, Simon A, Reichhart D & Durst

F (1982) Cytochrome P450-dependent

omega-hydroxyl-ation of lauric acid by microsomes from pea seedlings

Plant Physiol 70, 122–126

5 Salau¨n J, Benveniste I, Reichhart D & Durst F (1981)

Induction and specificity of a (cytochrome

P450)-depen-dent laurate in-chain-hydroxylase from higher plant

microsomes Eur J Biochem 119, 651–655

6 Salau¨n J, Weissbart D, Durst F, Pflieger P & Mioskow-ski C (1989) Epoxidation of cis and trans delta-9-unsat-urated lauric acids by a cytochrome P450-dependent system from higher plant microsomes FEBS Lett 246, 120–125

7 Schuler M & Werck-Reichhart D (2003) Functional genomics of P450s Annu Rev Plant Biol 54, 629–667

8 Pinot F, Salau¨n J, Bosch H, Lesot A, Mioskowski C & Durst F (1992) Omega-hydroxylation of Z9-octadece-noic, Z9,10-epoxystearic and 9,10-dihydroxystearic acids

by microsomal cytochrome P450 systems from Vicia sativa Biochem Biophys Res Commun 184, 183–193

9 Pinot F, Bosch H, Alayrac C, Mioskowski C, Vendais

A, Durst F & Salau¨n J (1993) omega-Hydroxylation of oleic acid in Vicia sativa microsomes (Inhibition by sub-strate analogs and inactivation by terminal acetylenes) Plant Physiol 102, 1313–1318

10 Weissbart D, Salau¨n J, Durst F, Pflieger P & Mioskow-ski C (1992) Regioselectivity of a plant lauric acid omega hydroxylase Omega hydroxylation of cis and trans unsaturated lauric acid analogs and epoxygenation

of the terminal olefin by plant cytochrome P450 Biochim Biophys Acta 1124, 135–142

11 Kunze K, Mangold B, Wheeler C, Beilan H & Ortiz de Montellano P (1983) The cytochrome P-450 active site Regiospecificity of prosthetic heme alkylation by olefins and acetylenes J Biol Chem 258, 4202–4207

12 Cajacob C & Ortiz de Montellano P (1986) Mechanism-based in vivo inactivation of lauric acid hydroxylases Biochemistry 25, 4705–4711

13 Tijet N, Helvig C, Pinot F, Le Bouquin R, Lesot A, Durst F, Salau¨n J & Benveniste I (1998) Functional expression in yeast and characterization of a clofibrate-inducible plant cytochrome P-450 (CYP94A1)

involved in cutin monomers synthesis Biochem J 332, 583–589

14 Benveniste I, Tijet N, Adas F, Philipps G, Salau¨n J & Durst F (1998) CYP86A1 from Arabidopsis thaliana encodes a cytochrome P450-dependent fatty acid omega-hydroxylase Biochem Biophys Res Commun 243, 688–693

15 Kahn R, Le Bouquin R, Pinot F, Benveniste I & Durst

F (2001) A conservative amino acid substitution alters the regiospecificity of CYP94A2, a fatty acid hydroxy-lase from the plant Vicia sativa Arch Biochem Biophys

391, 180–187

16 Rupasinghe S, Duan H & Schuler M (2007) Molecular definitions of fatty acid hydroxylases in Arabidopsis thaliana Proteins 68, 279–293

17 Pinot F, Benveniste I, Salau¨n J, Loreau O, Noe¨l J, Schreiber L & Durst F (1999) Production in vitro by the cytochrome P450 CYP94A1 of major C18 cutin mono-mers and potential messengers in plant–pathogen interactions: enantioselectivity studies Biochem J 342, 27–32

18 Kandel S, Sauveplane V, Compagnon V, Franke R,

Millet Y, Schreiber L, Werck-Reichhart D & Pinot F

(2007) Characterization of a methyl jasmonate and

wounding-responsive cytochrome P450 of

Arabidopsis thalianacatalyzing dicarboxylic fatty acid

formation in vitro FEBS J 274, 5116–5127

19 Kandel S, Morant M, Benveniste I, Ble´e E,

Werck-Reichhart D & Pinot F (2005) Cloning, functional

expression, and characterization of CYP709C1, the first

sub-terminal hydroxylase of long chain fatty acid in

plants Induction by chemicals and methyl jasmonate

J Biol Chem 280, 35881–35889

20 Cabello-Hurtado F, Batard Y, Salau¨n J, Durst F, Pinot

F & Werck-Reichhart D (1998) Cloning, expression in

yeast, and functional characterization of CYP81B1, a

plant cytochrome P450 that catalyzes in-chain

hydroxyl-ation of fatty acids J Biol Chem 273, 7260–7267

21 Sauveplane V, Kandel S, Kastner P, Ehlting J,

Compa-gnon V, Werck-Reichhart D & Pinot F (2009)

Arabid-opsis thalianaCYP77A4 is the first cytochrome P450

able to catalyze the epoxidation of free fatty acids in

plants FEBS J 276, 719–735

22 Le Bouquin R, Skrabs M, Kahn R, Benveniste I,

Sala-u¨n J, Schreiber L, Durst F & Pinot F (2001) CYP94A5,

a new cytochrome P450 from Nicotiana tabacum is able

to catalyze the oxidation of fatty acids to the

omega-alcohol and to the corresponding diacid Eur J Biochem

268, 3083–3090

23 Wellesen K, Durst F, Pinot F, Benveniste I, Nettesheim

K, Wisman E, Steiner-Lange S, Saedler H &

Yephre-mov A (2001) Functional analysis of the LACERATA

gene of Arabidopsis provides evidence for different roles

of fatty acid omega-hydroxylation in development Proc

Natl Acad Sci USA 98, 9694–9699

24 Xiao F, Goodwin S, Xiao Y, Sun Z, Baker D, Tang X,

Jenks M & Zhou J (2004) Arabidopsis CYP86A2

represses Pseudomonas syringae type III genes and is

required for cuticle development EMBO J 23, 2903–

2913

25 Ho¨fer R, Briesen I, Beck M, Pinot F, Schreiber L &

Franke R (2008) The Arabidopsis cytochrome P450

CYP86A1 encodes a fatty acid omega-hydroxylase

involved in suberin monomer biosynthesis J Exp Bot

59, 2347–2360

26 Compagnon V, Diehl P, Benveniste I, Meyer D,

Schaller H, Schreiber L, Franke R & Pinot F (2009)

CYP86B1 is required for very long chain

omega-hy-droxyacid and alpha, omega-dicarboxylic acid synthesis

in root and seed suberin polyester Plant Physiol 150,

1831–1843

27 Li Y, Beisson F, Koo A, Molina I, Pollard M &

Ohlrogge J (2007) Identification of acyltransferases

required for cutin biosynthesis and production of cutin

with suberin-like monomers Proc Natl Acad Sci USA

104, 18339–18344

28 Molina I, Li-Beisson Y, Beisson F, Ohlrogge J & Pollard M (2009) Identification of an Arabidopsis feru-loyl-coenzyme A transferase required for suberin syn-thesis Plant Physiol 151, 1317–1328

29 Serra O, Soler M, Hohn C, Sauveplane V, Pinot F, Franke R, Schreiber L, Prat S, Molinas M & Figueras

M (2009) CYP86A33-targeted gene silencing in potato tuber alters suberin composition, distorts suberin lamel-lae, and impairs the periderm’s water barrier function Plant Physiol 149, 1050–1060

30 Han J, Clement J, Li J, King A, Ng S & Jaworski J (2010) The cytochrome P450 CYP86A22 is a fatty acyl-CoA omega-hydroxylase essential for Estolide synthesis

in the stigma of Petunia hybrida J Biol Chem 285, 3986–3996

31 Cahoon E, Ripp K, Hall S & McGonigle B (2002) Transgenic production of epoxy fatty acids by expres-sion of a cytochrome P450 enzyme from Euphorbia lagascaeseed Plant Physiol 128, 615–624

32 Kandel S, Sauveplane V, Olry A, Diss L, Benveniste I

& Pinot F (2006) Cytochrome P450-dependent fatty acids hydroxylases in plants Phytochem Rev 5, 359– 372

33 Soliday C & Kolattukudy P (1977) Biosynthesis of cutin omega-hydroxylation of fatty acids by a microsomal preparation from germinating Vicia faba Plant Physiol

59, 1116–1121

34 Li-Beisson Y, Pollard M, Sauveplane V, Pinot F, Ohl-rogge J & Beisson F (2009) Nanoridges that character-ize the surface morphology of flowers require the synthesis of cutin polyester Proc Natl Acad Sci USA

106, 22008–22013

35 Li Y & Beisson F (2009) The biosynthesis of cutin and suberin as an alternative source of enzymes for the pro-duction of bio-based chemicals and materials Biochimie

91, 685–691

36 Blackmore S, Wortley A, Skvarla J & Rowley J (2007) Pollen wall development in flowering plants New Phytol

174, 483–498

37 Morant M, Jørgensen K, Schaller H, Pinot F, Møller B, Werck-Reichhart D & Bak S (2007) CYP703

is an ancient cytochrome P450 in land plants catalyzing in-chain hydroxylation of lauric acid to provide building blocks for sporopollenin synthesis in pollen Plant Cell 19, 1473–1487

38 Dobritsa A, Shrestha J, Morant M, Pinot F, Matsuno

M, Swanson R, Møller B & Preuss D (2009) CYP704B1

is a long-chain fatty acid omega-hydroxylase essential for sporopollenin synthesis in pollen of Arabidopsis Plant Physiol 151, 574–589

39 Li H, Pinot F, Sauveplane V, Werck-Reichhart D, Diehl P, Schreiber L, Franke R, Zhang P, Chen L, Gao Y et al (2010) Cytochrome P450 family member CYP704B2 catalyzes the omega-hydroxylation of fatty acids and is required for anther cutin biosynthesis