Plant Science 193194 (2012) 2838

Contents lists available at SciVerse ScienceDirect

Plant Science
journal homepage: www.elsevier.com/locate/plantsci

Review

Plant fatty acyl reductases: Enzymes generating fatty alcohols for protective
layers with potential for industrial applications
Owen Rowland a, , Frdric Domergue b,c,
a
b
c

Department of Biology and Institute of Biochemistry, Carleton University, Ottawa, Ontario, K1S 5B6, Canada
Universit de Bordeaux, Laboratoire de Biogense Membranaire, UMR 5200, F-33000, Bordeaux, France
CNRS, Laboratoire de Biogense Membranaire, UMR 5200, F-33000, Bordeaux, France

a r t i c l e

i n f o

Article history:
Received 24 February 2012
Received in revised form 9 May 2012
Accepted 9 May 2012
Available online 16 May 2012
Keywords:
Fatty acyl reductase
Fatty alcohol
Wax ester
Alkyl hydroxycinnamate
Cuticle
Suberin
Sporopollenin

a b s t r a c t
Primary fatty alcohols are found throughout the biological world, either in free form or in a combined
state. They are common components of plant surface lipids (i.e. cutin, suberin, sporopollenin, and associated waxes) and their absence can signicantly perturb these essential barriers. Fatty alcohols and/or
derived compounds are also likely to have direct functions in plant biotic and abiotic interactions. An
evolutionarily related set of alcohol-forming fatty acyl reductases (FARs) is present in all kingdoms
of life. Plant microsomal and plastid-associated FAR enzymes have been characterized, acting on acylcoenzymeA (acyl-CoA) or acyl-acyl carrier protein (acyl-ACP) substrates, respectively. FARs have distinct
substrate specicities both with regard to chain length and chain saturation. Fatty alcohols and wax
esters, which are a combination of fatty alcohol and fatty acid, have a variety of commercial applications.
The expression of FARs with desired specicities in transgenic microbes or oilseed crops would provide a
novel means of obtaining these valuable compounds. In the present review, we report on recent progress
in characterizing plant FAR enzymes and in understanding the biological roles of primary fatty alcohols,
as well as describe the biotechnological production and industrial uses of fatty alcohols.
2012 Elsevier Ireland Ltd. All rights reserved.

Contents
1.
2.

3.

4.

5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Plant fatty acyl reductase (FAR) enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.
FAR protein structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.
Biochemical characterization of FAR enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.
Substrate specicities of plant FARs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Occurrence, synthesis and function of fatty alcohols in planta . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.
Fatty alcohols as energy storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.
Fatty alcohols in cuticular waxes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.
Fatty alcohols in aliphatic lipid polyesters: suberin and cutin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4.
Fatty alcohols in suberin-associated alkyl hydroxycinnamates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.5.
Fatty alcohols in pollen exine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Industrial applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.
Natural sources of fatty alcohols and wax esters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.
Biotechnological production of fatty alcohols in microbes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.
Biotechnological production of wax esters in oilseed crops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Abbreviations: FAR, fatty acyl reductase; CoA, CoenzymeA; ACP, acyl carrier protein.
Corresponding author. Tel.: +1 613 520 2600x4213; fax: +1 613 520 3539.
Corresponding author at: Universit de Bordeaux, Laboratoire de Biogense Membranaire, UMR 5200, F-33000, Bordeaux, France. Tel.: +33 0 5 57 57 15 83;
fax: +33 0 5 56 51 83 61.
E-mail addresses: Owen Rowland@carleton.ca (O. Rowland), frederic.domergue@u-bordeaux2.fr (F. Domergue).
0168-9452/$ see front matter 2012 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.plantsci.2012.05.002

29
29
29
30
31
32
32
32
33
33
34
34
35
35
36
36
36
36

O. Rowland, F. Domergue / Plant Science 193194 (2012) 2838

29

1. Introduction

2. Plant fatty acyl reductase (FAR) enzymes

Alcohol-forming fatty acyl reductases (FARs) produce fatty alcohols that have a single hydroxyl group at the terminal position
(Fig. 1). Plant primary fatty alcohols occur either in free form or
are linked by an ester-bond with a fatty acid (e.g. palmitic acid)
to give a wax ester or an aromatic compound (e.g. ferulic acid) to
give an alkyl hydroxycinnamate (Fig. 1). These various compounds
are often components of plant extracellular lipid barriers: cuticle
coating the aerial surfaces, suberin found in the cell walls of various internal and external tissue layers, and sporopollenin found
in the outer walls (exine) of pollen grains [14]. These barriers
consist of polymerized lipids and phenolics, along with associated
non-covalently linked waxes. These waxes are usually complex
mixtures of very-long-chain (C20C34) fatty acids and derivatives
including primary fatty alcohols and wax esters. Wax esters can also
serve as energy storage, such as in the case of jojoba (Simmondsia
chinensis) seed oil [5].
Pioneering work using cell-free homogenates from developing jojoba cotyledons demonstrated that the synthesis of fatty
alcohols from fatty acyl-CoAs is NADPH dependent [6]. This
led to the purication of the jojoba FAR and the subsequent
cloning of its corresponding cDNA [7]. Related proteins have
subsequently been cloned and characterized from other plants,
including Arabidopsis thaliana [4,811], rice [12] and wheat
[13], as well as insects [1417], mammals [18], birds [19], a
phytoagellate protist [20], a planktonic crustacean [21], and a
distantly related FAR from a prokaryote, Marinobactor aquaeolei
[22,23].
In this review, we focus on plant FARs and discuss the latest data obtained through reverse genetic studies, especially in
the model plant A. thaliana. The occurrence, synthesis and putative roles played by fatty alcohols in plants, as well as the
potential of FAR proteins for biotechnological applications are discussed.

Two general types of plant FARs have been described, distinguished according to their subcellular localization and acyl-linkage
substrate specicity. The rst type are microsomal enzymes acting
on acyl-CoA substrates, exemplied by the seed-expressed jojoba
FAR [7] and the cuticle-associated Arabidopsis ECERIFERUM4
(CER4)/FAR3 enzyme [8]. FARs of the second type have only recently
been characterized and are plastid-localized proteins that use acylACPs as substrates [4,11,12]. While the rst FAR type is responsible
for the synthesis of fatty alcohols in seed-storage wax esters, cuticle and suberin, the function of the second type remains elusive
although data point to at least a subset involved in sporopollenin
biosynthesis (see Section 3).
2.1. FAR protein structure
Active plant FAR enzymes are about 500 amino acids in length,
with the plastidial isoforms containing an N-terminal extension
containing a plastid (chloroplast) transit peptide [4,8,1012]. Plant
FARs share at least 25% identity (45% similarity) at the amino acid
level excluding the N-terminal extensions. According to the protein
structure prediction software Conserved Domains Database (CDD)
[24], they all share a NAD(P)H-binding Rossmann-fold domain
as well as a fatty acyl-CoA reductase (FAR C) domain (Fig. 2A).
FARs are thus predicted to be extended short-chain dehydrogenase/reductase proteins with an / folding pattern and a central
-sheet at the N-terminus (Rossmann fold) and a fatty acyl-CoA
reductase domain at the C-terminus. All plant FARs contain the
motif GXXGXX(G/A) at their N-terminus (Fig. 2A), which resembles
the canonical ADP binding domain and is probably involved in binding of NAD(P)H [25]. The FARs also contain the classic YXXXK active
site motif of the short-chain dehydrogenase/reductase superfamily and fall into the SDR117E family (Fig. 2A) [26,27]. The FAR C (or
fatty acyl-CoA reductase) domain is often annotated in databases

O
S

CoA or ACP

fatty acyl-CoA or -ACP

O

Fatty Acyl
Reductase
(FAR)

unreleased fatty aldehyde intermediate

O
S
HO

Alkyl HydroxyCinnamate
Synthase
Suberin/Cutin Fatty
(AHCS)
Alcohol Transferase

R (H, OH, or O-CH3)

O
O

CoA

hydroxycinnamate-CoA

HO

Wax Synthase (WS)

OH

FATTY ALCOHOLS
Cuticular waxes
Root waxes
O

WAX ESTERS
Cuticular waxes
Seed Storage (jojoba)

ALKYL HYDROXYCINNAMATES
Tuber periderm waxes
Root waxes

SURFACE LIPID POLYMERS

Suberin (e.g. root, seed coat,
wound-induced)
Cutin
Sporopollenin

Fig. 1. Biosynthesis of fatty alcohols and derivatives. Enzyme activities are shown in grey. Free fatty alcohols are generated by fatty acyl reductases (FARs) using fatty acyl-CoA
or fatty acyl-ACP as substrates (chain length ranges from C16C30 in plants). Fatty alcohols can then be linked with fatty acids or hydroxycinnamic acids to yield wax esters
and alkyl hydroxycinnamates, respectively. Fatty alcohols can also be incorporated into surface lipid polymers (suberin, cutin, and likely sporopollenin). The nal major
products are in uppercase and the locations where these compounds accumulate in plants are in italics.

30

O. Rowland, F. Domergue / Plant Science 193194 (2012) 2838

database ARAMEMNON [29]. The biggest group contains FAR1, 4,

5, 7 and 8, which are predicted to be ER-localized and to have two
putative transmembrane domains (around amino acids 310330
and 370390), albeit with low reliability. These same ve proteins contain a cysteine at position 5, which is recognized as a
putative palmitoylation site by CSS-Palm 2.0 [30]. Site directedmutagenesis together with subcellular localisation studies should
provide insights into the relevance of this palmitoylation site. The
second group contains FAR2/MS2 and FAR6, which are localized in
plastids [4,11,12]. Since FARs of this second grouping do not have
any putative transmembrane alpha helices, they are most probably soluble enzymes. Group 3 containing only FAR3/CER4 is similar
to group 1 FARs, but contains an additional predicted N-terminal
transmembrane helix (amino acids 2037). Although FAR3/CER4
is localized to the endoplasmic reticulum when heterologously
expressed in yeast [8], it is not known whether FAR3/CER4 has the
same subcellular localisation in planta. An ER localisation is nevertheless very likely given that the fatty acid elongase generating the
very-long-chain acyl-CoA substrates for FAR3/CER4 is ER-localized,
as is the wax synthase WSD1 that uses FAR3/CER4 products to generate wax esters [31]. It is notable that these three FAR groupings,
according to hydropathy and predicted subcellular localisation, are
retrieved as clades in phylogenetic trees (Fig. 2B) [8].
2.2. Biochemical characterization of FAR enzymes

Fig. 2. FAR structural domains and phylogenetic tree of plant FARs. (A) FAR
structural domains. The core FAR enzyme is 490500 amino acids and contains an NAD(P)H binding Rossmann Fold Domain at the N-terminus (highlighted
in light grey) and a FAR C domain (alternatively called male sterile domain) at
the C-terminus (highlighted in dark grey). The GXXGXX(G/A) sequence motif
for ADP/NAD(P)H binding is indicated as well as the predicted active site motif
YXXXK (X represents any amino acid). Some FARs have an N-terminal extension ranging in size from 50 to 120 amino acids and containing a plastid
targeting sequence. (B) Phylogenetic tree of plant FARs. Protein sequences were
aligned using ClustalW and a neighbor joining tree generated with Mega 5.05
software (http://www.megasoftware.net/). Branch lengths are proportional to the
amount of inferred evolutionary change. Bootstrap values were calculated from
1000 replicates. Euglena FAR is an outgroup and was used to root the tree. The
GenBank accession or genome identier numbers of the sequences are: Arabidopsis thaliana FAR1 (At5g22500), FAR2/MS2 (At3g11980), FAR3/CER4 (At4g33790),
FAR4 (At3g44540), FAR5 (At3g44550), FAR6 (At3g56700), FAR7 (At5g22420), FAR8
(At3g44560); Artemisia annua GFAR1 (ADK66305); Brachypodium distachyon FAR2
(XP 003562031); Brassica rapa MS2 (ABO14927); Euglena gracilis FAR (ADI60057);
Glycine max (Soybean) FAR1 (XP 003543435); Oryza sativa Japonica (Rice)
FAR1 (Os09g0567500), FAR2/DPW (Os03g0167600), FAR3 (Os04g0354600), FAR4
(Os08g0557800); Physcomitrella patens FAR1 (XP 001758118); Populus trichocarpa
(Poplar) FAR1 (XP 002323221), FAR2 (XP 002323348), FAR3 (XP 002305562); Simmondsia chinensis (Jojoba) FAR (AAD38039); Triticum aestivum (Wheat) TAA1a
(CAD30693).

as sterile or male sterile domain because of the early characterization of the rst cloned FAR gene encoding MALE STERILITY2
(MS2/FAR2) from A. thaliana [28]. This automated annotation is
outdated because Arabidopsis MS2/FAR2 protein and its rice homologue Defective Pollen Wall (DPW) are the only two FARs affecting
male fertility [12]. As Also, since both fatty acyl-CoA and fatty acylACP reductases have now been characterized, the general term fatty
acyl reductase for FAR is more appropriate.
An Arabidopsis multigenic family comprising eight FARs (FAR1FAR8) has been elucidated (Table 1) (Fig. 2B) [4,811]. They can
be classied into 3 distinct groups according to their predicted
subcellular locations and the presence or absence of predicted
transmembrane alpha helices using the plant membrane protein

The biochemical characterisation of FAR enzymes was achieved

in two successive phases. Studies in the 1970s using subcellular
fractions from organisms that produce wax esters, such as the
protist Euglena gracilis and jojoba, indicated that a single enzyme
produced primary alcohols from acyl-CoAs [6,32]. The cloning of
the rst FAR-coding genes in the past decade and their heterologous
expression in various systems (sometimes followed by purication
of the protein) enabled further biochemical studies, demonstrating that FAR enzymes have strong specicities for particular fatty
acyl chains, with regard to both acyl chain length and degree of
saturation.
It was demonstrated in 1970 that a cell free preparation of etiolated E. gracilis cells could reduce fatty acids to the corresponding
alcohols in the presence of ATP, CoA, and NADH [32]. Addition of
phenylhydrazine hydrochloride in the reaction mixture revealed
the presence of an aldehyde intermediate, and the same preparation could also reduce aldehydes to the corresponding alcohols.
Further experiments using dark-grown E. gracilis or pea (Pisum
sativum L.) leaves showed that this reduction activity was associated with microsomes [3335]. The synthesis of fatty alcohols from
acyl-CoAs in cell-free homogenates of developing jojoba cotyledons
was NADPH-specic and did not result in signicant quantities of
released aldehyde intermediate [6]. Altogether, these studies indicated that FAR enzymes can be found associated with microsomes,
are NAD(P)H dependant, and generate fatty alcohols via an unreleased aldehyde intermediate.
The purication of the FAR from jojoba embryos, the cloning of
its cDNA, and the rst biochemical characterization of this activity [7] allowed the identication of homologous FAR proteins from
both plants and animals, and opened the possibility of characterizing FAR enzymes in heterologous systems. The catalytic activity
of many putative FAR proteins has been demonstrated by heterologous expression in Escherichia coli, Saccharomyces cerevisiae, and/or
Nicotiana benthamiana leaves, followed by thin layer chromatography and/or gas chromatographymass spectrometry (GCMS)
analysis to demonstrate the appearance in the host of fatty alcohols upon expression of the transgene. The fatty alcohols were
produced from endogenous fatty acyl pools in these experiments.
The in vivo characterization of FAR activities resulted in fatty alcohol production without detection of the intermediate aldehyde,

O. Rowland, F. Domergue / Plant Science 193194 (2012) 2838

31

Table 1
Characterized Fatty Acyl Reductases (FARs) from plants.
Gene name

Species

AGI code or NCBI

accession

Protein size (a.a.)

Subcellular
localization

Primary substrate
specicity in planta

Functional association in
planta

Ref.

FAR1

Arabidopsis

At5g22500

491

ERc

C22:0

[9]

FAR2/MS2

Arabidopsis

At3g11980

616

Plastid

C16:0

FAR3/CER4

Arabidopsis

At4g33790

493

ERd

Root, seed coat and

wound-induced suberin,
cutin.
Pollen exine (likely
sporopollenin)
Cuticular waxes

Root, seed coat and

wound-induced suberin
cutin.
Root, seed coat and
wound-induced suberin,
cutin
Unknown
Likely a pseudogene
Possible pseudogene
Seed storage energy (wax
esters)
Anther cuticle, pollen exine
(likely sporopollenin)
Pollen wall

[9]

FAR4

Arabidopsis

At3g44540

493

ER

C24:0, C26:0,
C28:0, C30:0
C20:0

FAR5

Arabidopsis

At3g44550

496

ERc

C18:0

FAR6
FAR7
FAR8
ScFAR

Arabidopsis
Arabidopsis
Arabidopsis
Jojoba

At3g56700
At5g22420
At3g44560
AAD38039

548a
409b
496
493

Plastid
ERc
ERc
ERc

C16:0
None
Unknown
C20:1, C22:1

DPW

Rice

BAH00399

608

Plastid

C16:0

507

TAA1

a
b
c
d
e

Bread wheat

CAD67815

ER

C18:1, C20:1,
C22:1, C24:0,
C26:0e

[4,25]
[8]

[9]

[11]
[10]
[7]
[12]
[13]

Protein size is for long-form splicing variant, a short-form splicing variant has been identied but it encodes for an inactive 527 a.a. protein [10].
Amino acid number is from gene prediction [8], but the only cloned cDNA has a stop codon after 95 codons [10].
Predicted subcellular localization, needs experimental data.
Subcellular localization performed in yeast [8], needs in planta verication.
Chain length specicity of TAA1 deduced from expression in tobacco seeds, not native tissue [13].

suggesting that FAR proteins catalyze both reductions without signicant escape of the intermediate product. Comparison of various
host systems expressing the same FAR showed, however, that the
apparent substrate specicity of the FARs is strongly inuenced
by the host expression system used [9,10]. Although this approach
is useful for conrming FAR activity of a protein with unknown
function, the deduced substrate specicities in these heterologous
systems needs to be qualied (see Section 2.3).
Heterologous expression of tagged FARs in E. coli followed by
afnity purication has recently allowed for in vitro biochemical
characterization of FAR proteins. However, this methodology has
thus far been used successfully only for the soluble plastid FARs
[4,11,12]. The Arabidopsis MS2/FAR2 is specically active on C16:0ACP, with no signicant activity towards C16:0-CoA, and accepts
both NADPH and NADH as reductants [4]. Its rice homologue, the
DPW protein, was shown to exhibit more than 270-fold higher
specicity for C16:0-ACP than for C16:0-CoA substrates, but to be
strictly NADPH-dependent [12]. DPW and MS2/FAR2 activities are
most probably equivalent since rice DPW complements the Arabidopsis ms2 mutant [4]. Similarly, a second Arabidopsis plastid
FAR, FAR6, is specic for NADPH while accepting both C16:0-ACP
and C16:0-CoA as substrates in vitro [11]. The partially puried
FAR6 produced fatty aldehydes in addition to fatty alcohols, and
converted supplied fatty aldehydes to fatty alcohols [11]. Direct
evidence for the production of fatty alcohols via an aldehyde intermediate in vivo is still lacking, but these data strongly suggest that
this is at least the case in vitro.
2.3. Substrate specicities of plant FARs
FARs have distinct substrate specicities with regard to chainlength and acyl chain saturation (Table 1). Still, determining the
biologically relevant substrates(s) and specic physiological function for an individual FAR has been problematic. For example, the
apparent substrate specicity of the rst cloned FAR, the seed
expressed jojoba FAR, was highly dependent on the host expression
system used. C16:0 and C18:111 primary alcohols were produced

upon expression of jojoba FAR in E. coli, but C22:1 fatty alcohols

were detected when expressed in the seeds of high erucic acid
oilseed rape (Brassica napus) plants [7]. In addition, the wheat FAR
encoded by the TAA1a gene produces C14:0, C16:0 and C18:1 primary alcohols upon expression in E. coli, but C18:1, C20:1, C22:1,
C24:0 and C26:0 when expressed in mature transgenic tobacco
seeds [13].
When ve Arabidopsis FARs (FAR1, 2, 3, 6 and 8) and the jojoba
FAR (ScFAR) were expressed in E. coli, C14:0, C16:0 and C18:1 fatty
alcohols were the main products detected whatever FAR protein
was expressed, although in variable relative proportions (Fig. 3)
[10]. These products are clearly not in agreement with the fatty
alcohols synthesized by some of these FARs in planta. Analysis of the
wax esters present in jojoba seeds indicates that ScFAR mainly produces C20:1 and C22:1 primary fatty alcohols [5], while analysis of
the cuticular wax present on stems of Arabidopsis cer4/far3 mutant
plants indicates that CER4/FAR3 is responsible for the production
of C24:0 to C30:0 saturated fatty alcohols [8]. Also, analysis of the
aliphatic suberin polyester composition of Arabidopsis far1 mutant
plants revealed that FAR1 is primarily responsible for the synthesis
of C22:0 fatty alcohols in planta [9]. When FAR1 and CER4/FAR3 are
expressed in S. cerevisiae as a heterologous host, they produce fatty
alcohols very similar to those affected in the respective Arabidopsis
mutants, rather than the shorter chains implied through expression in E. coli (Fig. 3) [9]. Expression of CER4/FAR3 in yeast results
in production of C24:0 and C26:0 fatty alcohols [8], and furthermore when CER4/FAR3 is co-expressed in yeast with Arabidopsis
-ketoacyl synthase KCS5/CER60, which facilitates the elongation
of C26:0-CoA to C28:0-CoA [36], the presence of C28:0 fatty alcohols is also detected (F. Domergue, unpublished data). These same
chain lengths of fatty alcohols are affected in the cuticular wax of
cer4/far3 mutant plants [8]. Similarly, expression of FAR4 and 5
in yeast mostly generates C20:0-OH and C18:0-OH, respectively,
which is in agreement with the chain length of fatty alcohol most
affected in the aliphatic suberin polyester of Arabidopsis far4 and
far5 mutant plants [9]. Altogether these data suggest that substrate
availability in E. coli rather than FAR enzyme specicity is largely

O. Rowland, F. Domergue / Plant Science 193194 (2012) 2838

100

20:0OH

16:0
24:0

16:0OH

FAR6

26:0OH

FAR3

22:0OH

18:0OH

80

Expression in Bacteria

60

40

20

16:0OH

18:1OH

16:0OH

18:1OH

FAR1
0

14:0OH 18:1OH

20

16:0OH

40

60

80

14:0OH

24:0OH

18:0OH

Expression in Yeast

14:0

32

100

Fatty alcohol produced (% of total)

Fig. 3. Distribution of primary fatty alcohols produced by Arabidopsis FAR1, FAR3 and FAR6 expressed in yeast (left) versus bacteria (right). E. coli expression data were
taken from Doan et al. [10]. S. cereviseae data were taken from Domergue et al. [9] for FAR1/FAR3 and Doan et al. [11] for FAR6. Distributions were calculated as % of total
cell-associated fatty alcohols.

responsible for the prole of fatty alcohols produced. Using E. coli

as a heterologous expression system is therefore not appropriate
to characterize the substrate specicity of FAR proteins, with the
exception of the plastid-localized FARs FAR2/MS2 and FAR6 that
most probably produced shorter chain C16:0 fatty alcohols in planta
(Fig. 3; see Section 3.5). These altered substrate specicities in E. coli
may also be due, in part, to lack of post-translational modications
(e.g. glycosylation, fatty acylation) that can occur in eukaryotic systems. Finally, S. cerevisiae appears as a much more reliable system
to gain insight into the fatty alcohols that are produced by the FAR in
planta, but the yeast endogenous sub-cellular pool of available acyl
chains still governs to a certain extent the chemical composition of
primary alcohols produced (Fig. 3) [9].
Regardless of the heterologous host, these data indicate that
each FAR has unique substrate specicity in terms of both acyl chain
length and degree of saturation.
3. Occurrence, synthesis and function of fatty alcohols in
planta
Our present knowledge of the occurrence of fatty alcohols and
derivatives in plants is summarized in Fig. 1. Primary fatty alcohols,
in free and combined forms, are very often components of protective extracellular lipid mixtures, including (1) cuticular waxes on
aerial surfaces, (2) suberin-associated waxes of tuber periderms
and roots, and (3) the lipid polyesters, cutin and aliphatic suberin
[1,3,3739]. In an unusual and perhaps unique situation, the desert
plant jojoba has wax esters rather than triacylglycerols as its primary seed storage lipid [5]. Finally, the recent characterisation of
the ms2 and dpw mutants, both affected in pollen development
and fertility, strongly suggests that fatty alcohols are also part of
sporopollenin, the lipid polyester covering pollen grains [4,12]. The
following section focuses on the distribution of fatty alcohols in
planta as well as on their putative functions.
3.1. Fatty alcohols as energy storage
The bacterium Acinetobacter calcoaceticus and the algae-like
phytoagellate protist E. gracilis accumulate fatty alcohols in the
form of wax esters as energy storage. These examples are quite
exceptional, with triacylglycerols being the usual storage lipid for
energy supply in all kingdoms. Jojoba, a shrub native to the southwestern region of North America, is the only known plant that
accumulates wax esters in its seeds as the major energy reserve.
Jojoba oil accounts for 5060% of the seed dry weight and is composed of 98% wax esters, with C40 to C44 chain-length wax esters

being the major molecular species [5,40]. The fatty alcohol moiety is dominated by equal proportions of eicosenol (C20:1-OH)
and docosenol (C22:1-OH), together representing nearly 90% of the
total fatty alcohol. The fatty acid moiety is mostly C20:1 (70% of
the total fatty acid), and the remainder largely C22:1 (15%) and
C18:1 (10%) [5]. The jojoba wax esters play an equivalent role
as triacylglycerols found in oilseed plants (e.g. Brassica napus) in
that they provide the energy required by the seeds to achieve postgerminative growth before photosynthesis is established. Interest
in jojoba grew in the mid-1970s when the United States banned
the importation of spermaceti oil, which represented until then the
major source of wax esters for the lubricant and cosmetic industries
(see Section 4.1).
3.2. Fatty alcohols in cuticular waxes
Cuticular waxes coat the aerial organs of all terrestrial plants
[41]. Due to their strategic location at the interface between the
plant and its environment, they protect plants from various stresses
such as drought, UV light, and pathogen attack. Cuticular waxes
comprise two pools of waxes, those embedded within the cutin
polyester, named intracuticular waxes, and those deposited on the
outermost surface, named epicuticular waxes. Cuticular waxes are
typically a complex mixture of very-long-chain fatty acid derivatives (such as alkanes, aldehydes, ketones, primary alcohols, and
secondary alcohols), with free fatty alcohols and/or wax esters
being the predominant components in some species. Maize (Zea
mays) seedling leaf waxes contain up to 63% free C32:0 primary
alcohol (by weight), while cuticular waxes from mature leaves
contain up to 42% wax esters, with tetracosanol (C24:0-OH, 55%)
and hexacosanol (C26:0-OH, 27%) being the major fatty alcohol
moieties of these esters [42,43]. The leaf cuticular wax of barley
(Hordeum vulgare) contains about 75% free C26:0 primary alcohol [44], whereas Brazilian carnauba palm (Copernicia cerifera) wax
esters account for about 85% of leaf cuticular waxes [45].
CER4/FAR3 generates the vast majority of cuticular waxassociated primary alcohols of Arabidopsis (Table 1) [8]. Although
the cuticular waxes of Arabidopsis leaves and stems are dominated
by alkanes (along with alkane derivatives ketones and secondary
alcohols in stems), free primary alcohols and wax esters are also
present in signicant amounts. The primary alcohols are in a homologue series of even chain lengths C24:0-OH to C30:0-OH and the
predominant wax esters have C42, C44, and C46 chain lengths,
consisting of a C16:0 fatty acid moiety and C26:0, C28:0, or C30:0
fatty alcohol moieties [46]. As noted above, heterologous expression of the CER4/FAR3 protein in yeast generates C24:0-C28:0 fatty

O. Rowland, F. Domergue / Plant Science 193194 (2012) 2838

alcohols. Consistent with this prole, stem cuticular waxes of cer4

mutants are devoid of C24:0, C26:0 and C28:0 primary alcohols and
have signicantly reduced levels of the predominant C42, C44, and
C46 even-chain wax esters [8]. The noteworthy presence of residual
C30:0 primary alcohols in stem cuticular waxes of cer4 knock-out
alleles [8,47] suggests that another activity contributes to this fatty
alcohol. No other FAR in Arabidopsis has been found with activity
for acyl chains longer than C24, which suggests that there may be
non-FAR dependent routes to fatty alcohol production.
Although total wax load is not reduced in cer4 mutants due to
compensatory increases in products of the alkane-forming pathway, epicuticular wax crystal formation of stems is signicantly
affected [8,47]. cer4 mutants have a relatively smooth surface with
scattered plate-like protrusions compared to the stems of wild-type
plants that are covered with a dense array of wax crystals in the
shapes of tubes, short vertical rods, and irregular-shaped plates.
Approximately 75% of wild-type Arabidopsis stem cuticular wax
consists of alkane-pathway products and presumably they make
up much of the wax crystals observed. Nonetheless, the absence of
fatty alcohols dramatically disrupts wax crystal formation. In contrast, the absence of wax esters does not affect epicuticular wax
crystal formation in the wsd1 mutant [31]. This mutant is affected
in wax ester synthesis but not in fatty alcohol production, and has
unaltered epicuticular wax crystal formation. Detailed analyses of
epi- and intra-cuticular waxes compositions suggest that primary
alcohols preferentially accumulate in the intracuticular wax layer
[48]. We hypothesize that the presence of fatty alcohols within the
cutin matrix is necessary for the formation of epicuticular wax crystal structures at the plant surface. In the barley cer-j.59 mutant,
which is characterised by a large reduction of primary alcohols, the
leaf surfaces are nearly devoid of the plate-like wax crystals characteristic of wild-type plants [44]. More experiments are nevertheless
required to elucidate the specic roles of fatty alcohols, including
via wax crystal formation, in the various interactions of plants with
the biotic environment (e.g. experiments involving mutant plants
with altered levels of wax-associated fatty alcohols challenged with
microbial pathogens, insect pests, or herbivores).
Cuticular waxes also have an important role in pollenstigma
adhesion as well as pollen hydration [49,50], but it is unknown
whether the fatty alcohol constituents of these waxes have any
specic role there. An anther-specic FAR gene, TAA1, from bread
wheat (Triticum aestivum L.) is specically expressed at the time
of microspore wall thickening [13]. The amino acid sequence of
TAA1 does not have an N-terminal extension and is more related to
Arabidopsis CER4/FAR3 than to MS2/FAR2 (Fig. 2B). Since heterologous expression of TAA1 in tobacco seeds resulted in the production
of predominantly very-long-chain fatty alcohols (about 95% C20
and 40% C24), it is likely that TAA1 is involved in tryphine formation rather than sporopollenin formation (see Section 3.5 below).
Arabidopsis pollen grain tryphine lipids contain C24 to C30 fatty
alcohols [51]. However, cer4/far3 mutants are not affected in fertility [52], and thus the C24:0-C30:0 fatty alcohols produced by
CER4 do not appear to be required for pollen (tryphine)/stigma
recognition [53]. Promoter GUS-reporter gene analysis indicated
that CER4/FAR3 is expressed in the anther lament, but not in the
anther itself [8].
3.3. Fatty alcohols in aliphatic lipid polyesters: suberin and cutin
Suberin and cutin are apoplastic lipid polyesters that occur
adjacent to or within the cell walls of certain tissue layers such
as the root endodermis and peridermis (suberin), and aerial epidermal cells (cutin). Suberized cells contain both a poly(phenolic)
and a poly(aliphatic) component, with good evidence that these
two domains are covalently linked [54]. Cutin and aliphatic
suberin contain varying proportions of fatty alcohols depending

33

on the plant species [3]. Non-substituted fatty acids, hydroxy-fatty

acids (-hydroxy, mid-chain hydroxy, or both) and dicarboxylic
acids generally make up the bulk of aliphatic compounds found in
aliphatic suberin and cutin. Fatty alcohols account for about 15%
of the total (by weight) in the aliphatic suberin polymer of potato
tuber periderm and castor bean roots [38]. Free fatty alcohols are
also found in suberin-associated waxes (extracted by immersion
in non-polar solvent, such as chloroform, and thus not covalently
linked to the suberin polymer; see Section 3.5). Most of the fatty
alcohols in soybean roots are found in these solvent extracts rather
than as part of the aliphatic suberin polymer itself [55]. All of the
fatty alcohols are found in the soluble fraction of the soybean seed
cuticle and the Iris germanica root exodermis [56,57]. In contrast,
about equal proportions of fatty alcohols are found in the soluble
(i.e. as waxes) and insoluble (i.e. covalently linked with suberin)
fractions in wound healing potato tubers [58].
The suberin lipid polyester of Arabidopsis roots and seed coats
contains about 6.5% C18, C20 and C22 saturated fatty alcohols (by
weight), but these are only minor components in leaves [1,2]. The
Arabidopsis FAR1, FAR4 and FAR5 genes are specically expressed
in the root endodermis, while heterologous expression in yeast of
the corresponding proteins collectively results in the production
of the same fatty alcohols found in Arabidopsis aliphatic suberin
[9]. In addition, root and seed coat lipid polyesters of far1, far4 and
far5 Arabidopsis single mutant lines have reduced levels of C22:0,
C20:0 and C18:0 fatty alcohols, respectively (Table 1) [9]. These far1,
far4 and far5 mutant lines also have reduced levels of C22, C20 and
C18 saturated fatty alcohols, respectively, in leaf lipid polyester,
despite the very low levels of FAR1, FAR4 and FAR5 gene expression
in leaves [9]. Finally, wounding up-regulated the transcripts of all
three genes, resulting in about a doubling of the total fatty alcohols
level in the leaf lipid polyester [9], in agreement with suberin being
deposited in plant aerial tissues in response to wounding [38,57].
There is a simultaneous upregulation of two genes encoding ketoacyl synthases, KCS2/DAISY and KCS20, upon wounding stress
[59]. These synthases are involved in the production of the verylong-chain fatty acids specically found in suberin [59,60].
Although fatty alcohol compositions are systematically affected
in far1, far4 and far5 mutants, the other main lipid polyester
monomers (unsubstituted fatty acids, -hydroxyacids, and dicarboxylic acids) are not signicantly altered. Due to compensatory
effects, the total fatty alcohol levels in root aliphatic suberin, seed
coat and leaf cutin are also not signicantly affected by individual mutations of the Arabidopsis FAR1, FAR4 or FAR5 genes. Indeed,
only in far5 root aliphatic suberin, where C18:0-OH is the major
fatty alcohol, and in far1 seed coat, where C22:0-OH is the main
fatty alcohol, are the levels of total fatty alcohols slightly reduced
[9]. These subtle variations in fatty alcohol compositions in the single far mutants, but without major effects on the total fatty alcohols
content due to compensatory increases in other chain-length alcohols, may explain the absence of any obvious developmental or
physiological phenotypes in the single far1, far4 or far5 mutant
lines. Nonetheless, the ubiquitous presence of C18:0-C22:0 fatty
alcohols in aliphatic suberin together with the presence of three
genes with similar domains of expression coding for proteins with
overlapping substrate specicity suggests that fatty alcohols may
play a major role in suberin biosynthesis and/or function. The
characterisation of double and triple far1, far4 and far5 lines is
underway. This will certainly aid in the understanding of the roles
played by fatty alcohols in extracellular lipid polymers.
3.4. Fatty alcohols in suberin-associated alkyl hydroxycinnamates
Suberin-associated waxes are a collection of compounds similar to cuticular waxes that are not covalently linked to the suberin
polyester, but which play an important role in establishing the

34

O. Rowland, F. Domergue / Plant Science 193194 (2012) 2838

barrier properties of suberized cells [38]. Free fatty alcohols and

fatty alcohols esteried to a hydroxycinnamate compound such as
ferulic acid (alkyl hydroxycinnamates) were found in Douglas r
bark extracts [61], as well as in native and wound-induced suberin
periderm of subterranean plant storage organs [38,58,6264]. The
solvent extracted lipids (waxes) of native and wound potato periderms contain at least 50% alkyl hydroxycinnamates, mainly in the
form of C16 to C32 fatty alcohols esteried with ferulic acid [38].
Root wax is the term given to soluble lipids that are extracted
by a 1-min immersion of 7 week-old Arabidopsis roots in chloroform. They have a suberin-rich periderm in the outermost layer
at this time [65,66]. The most abundant class of compounds of
Arabidopsis root waxes are alkyl hydroxycinnamates (accounting for about 47%), with free C18C22 saturated fatty alcohols
additionally representing 10% of the total. These alkyl hydroxycinnamates are p-coumaric, caffeic and ferulic acids conjugated
with C18C22 saturated fatty alcohols [65,66]. Arabidopsis root
waxes also contain low levels of typical cuticular wax compounds
such as alkanes, as well as high levels of sterols and - and monoacylglycerols [6567]. It has not been established whether
root waxes are intimately associated with the suberin polyester.
Since the chain lengths of the fatty alcohol moieties of the alkyl
hydroxycinnamates match that of suberin polyester-bound fatty
alcohols (see above), it is highly likely that FAR1, FAR4, and FAR5
generate fatty alcohols for both fractions [9]. These three FAR genes
are specically expressed at sites of suberin deposition, and thus
the alkyl hydroxycinnamates are probably excreted by the same
cells, although they may not necessarily be accumulating in the
suberin layer.
The acyltransferase combining the fatty alcohols and the phenolic moiety generating root wax alkyl hydroxycinnamates has not
yet been elucidated in Arabidopsis, but it is likely a BAHD familymember acyltransferase related to the feruloyl-CoA transferase
(ASFT/RWP1) that is required for the incorporation of ferulate in
the suberin-polyester of roots and seeds of Arabidopsis [65,68].
The potato feruloyl transferase responsible for alkyl hydroxycinnamates synthesis has been identied and called FHT [64]. It
belongs to the BAHD-family and is most probably the orthologue of
ASFT/RWP1. Silencing of FHT not only affects alkyl ferulates, but also
the suberin polymer as the tuber peel of the corresponding RNAisilenced plants had increased water loss as well as an abnormal
suberin lamellar structure [64].
Alkyl hydroxycinnamates are natural antioxidants and thus may
affect lipid oxidation [69,70]. They also confer allelopathic properties [71], have anti-microbial activities [72], and are important for
the periderm function as a barrier to pathogens [73]. The inhibition
of various microbes in quantitative bioassays with alkyl hydroxycinnamates and testing for altered plantpathogen interactions
in mutants decient in root wax alkyl hydroxycinnamates will be
important future directions.
3.5. Fatty alcohols in pollen exine
Some sterile mutants affected in pollen exine development in
rice and Arabidopsis are due to mutations in genes coding for plastid FARs. This strongly suggests that fatty alcohols are necessary
for the formation of sporopollenin, the lipid polymer protecting
pollen grains [4,12,25,28]. Pollen grains are surrounded by an
extremely complex extracellular matrix made up of several layers
that have fundamental functions in pollen protection and conservation, pollen dispersal, as well as pollenpistil interactions. The
mature pollen cell wall is composed of a cellulosic and pectin-rich
intine layer deposited onto the plasma membrane, an intermediate exine layer principally made of the extremely resistant
sporopollenin polymer and an outermost tryphine layer surrounding the pollen grain with a waxy coat [74]. Sporopollenin resembles

suberin in that this polymer contains both an aliphatic component

[75] and a signicant aromatic component [76]. The exact composition of sporopollenin remains largely unknown because of its
resistance to depolymerization methods currently used with the
other extracellular lipid-based polymers cutin and suberin [77].
Reverse genetics in Arabidopsis and rice has identied many
co-regulated genes encoding enzymes most probably involved in
providing the acyl precursors required for sporopollenin/exine formation (reviewed in [78]). Arabidopsis MALE STERILITY2 (MS2)
was indeed the rst FAR sequence that became publicly available,
although no enzymatic function for the protein encoded by MS2
was proposed at that time [28]. MS2 corresponds to FAR2 in the
revised Arabidopsis FAR gene family nomenclature [9]. The deduced
MS2/FAR2 protein sequence was later found to be related to the
biochemically characterized jojoba FAR protein [7]. Together with
the absence of normal exine wall deposition on ms2 microspores,
this led to the hypothesis that MS2/FAR2 is responsible for the
production of fatty alcohols involved in sporopollenin biogenesis [25]. The functional characterization of the MS2/FAR2 protein
showed that it is localized to the plastid and highly specic for
acyl-ACPs and NADPH in in vitro assays (Table 1) [4]. Pollen grains
of both ms2 and cyp704B1 mutants have a dramatic zebra phenotype lacking a dened exine [75], indicating that fatty alcohols
and -hydroxyacids are both essential for the formation of the
sporopollenin polymer. This is particularly surprising because, in
contrast to the -hydroxyacids produced by CYP740B1, the fatty
alcohols produced by MS2/FAR2 only have one functional group
available for polymerization and therefore are a dead end products for polymerization.
The rice ortholog of MS2/FAR2 is Defective Pollen Wall (DPW)
[12]. MS2/FAR2 is localized in plastids and reduces C16:0-ACP to
the corresponding C16:0 primary fatty alcohol. DPW is specically expressed in the tapetal cells of rice anthers, as well as in
the microspores. A loss-of-function dpw mutant has a dramatically
altered pollen exine layer and abnormalities to the anther surface.
The dpw mutants have signicantly reduced levels of anther cutin
monomers, including fatty alcohols, but anther cuticular wax levels
are increased. Analysis of total soluble anther lipids revealed that
dpw has about a 40% reduction of both C16:0 and C18:0 primary
fatty alcohols and general increases in C12C24 free fatty acids.
These pleiotropic effects on anther lipid composition may in part be
explained by feedback regulation of gene expression, as a number
of genes involved in surface lipid biosynthesis have altered expression levels in the dpw mutant [12]. The fact that mutations in the
tapetal-expressed genes CYP704B2 and DPW cause defects in both
anther cutin and pollen exine strongly suggests that these two lipid
polymers have a common biosynthetic pathway [12,79].
No direct measurements of pollen exine composition have yet
been reported, and it is unclear how fatty alcohols may be assembled into this structure. Nevertheless, considering the extremely
resistant nature of sporopollenin and the presence of ether-bonds
in this extracellular lipid polymer according to NMR spectroscopy
analysis [80,81], it is tempting to speculate that increasing the
numbers of hydroxyl versus carboxyl functions would facilitate
the formation of ether bonds instead of ester bonds. This would
account for the extreme resistance of sporopollenin to classical
transmethylation-based depolymerization protocols.

4. Industrial applications
Long-chain primary alcohols are of industrial value, mostly
serving as surfactants in detergents and other cleaning products
[82]. Fatty alcohols are also used in cosmetics, agrochemical and
pharmaceutical formulations, as well as in food products, typically acting as thickening agents, emulsiers, or emollients [83].

O. Rowland, F. Domergue / Plant Science 193194 (2012) 2838

4.1. Natural sources of fatty alcohols and wax esters

Historically, the primary source of wax esters for industry was
the oil obtained from the spermaceti organ in the heads of
sperm whales (Physeter macrophalus). Spermaceti oil is primarily
1-hexadecanol (C16:0-OH) linked with palmitic acid (C16:0), and
is thought to be important for the whales natural sonar system
(echolocation) and possibly in regulating buoyancy during deep
dives [88]. The head of an adult male sperm whale contains up
to 2000 liters of wax ester, but overexploitation of this natural
resource led to signicant depletion of sperm whale populations.
The whales were saved from extinction with the ban on whale oil
use in 1972 followed by the species gaining full protection from
whaling in 1986. The botanical alternative to spermaceti oil is wax
esters found in jojoba seed oil (composition described in Section
3.1), which can be used for some of the same applications. However,
jojoba is not amenable to large scale cultivation and commercial
application of its use is generally limited to cosmetics (e.g. lotions
and shampoos) [89].
Another source of fatty alcohols and wax esters for present-day
applications is carnauba wax, which is one of the hardest natural
waxes with a melting point above 80 C. Leaves of the Brazilian palm
tree (Copernicia cerifera) secrete 12 pounds per tree/per year of
carnauba wax, which is a mixture of 8085% wax esters (aliphatic
and phenolic esters) and 1015% free fatty alcohols [45]. Carnauba
wax, also known as the queen of waxes, as it produces a glossy
nish, is commonly used in polishes (shoe, oor and furniture), car
waxes, as a coating agent for the paper industry, as a polisher in the
food industry (candies, gums and fruits), dental oss wax, and as a
shiny additive in many cosmetic formulas.
Candelilla (Euphorbia cerifera and E. antisyphilitica) and ouricouri (Syagros coronata) are alternative plant sources of jojoba- and
carnauba-type waxes, respectively, whereas the most important
animal resources for commercial production of waxes are beeswax
and sheep wool wax (lanolin) [90,91].
4.2. Biotechnological production of fatty alcohols in microbes
As described above in Section 2, the functional characterisation of FARs in bacteria and yeast indicated that these enzymes
have very distinct substrate specicities, particularly with regard
to chain length. Heterologous expression of FARs also revealed that
the fatty alcohols produced are found both in the transgenic cell

Fatty alcohol in the medium

(% of total)

C16:0-OH

C20:0-OH

C22:0-OH

C24:0-OH

C26:0-OH

80.0

60.0
40.0
20.0
0.0

Total FattyAlcohol produced

(g/unit OD)

C18:0-OH

FAR1

FAR3

FAR4

1.0

FAR5

5.43 0 .40

For example, Western Europe used more than 450 thousand tons
of fatty alcohols in 2006 to produce non-ionic polyglycol surfactants via the process of ethoxylation (reaction with ethylene oxide)
[84]. Fatty alcohols esteried with a fatty acid, i.e. wax esters, have
also been employed in a variety of industrial applications such as
waterproong leather, lamp oils, inks and polishes, dermatological
formulations, and cosmetics. The excellent lubrication properties
and stabilities of wax esters (e.g. resistance to oxidation) make
them of high intrinsic value in the lubricant industry, in particular
as high-performance factory machine lubricants and as automobile
transmission uids [85].
Straight chain fatty alcohols and wax esters can either be directly
extracted from natural sources or derived from reduction of vegetable oils (e.g. coconut or palm kernel oil) or animal oils (e.g.
tallow) [86]. Alternatively, they are prepared from petrochemical sources using the Ziegler process, involving oligomerization of
ethylene, or the Oxo process (hydroformylation), involving olen
chemistry [87]. None of these established processes involve FARs,
but with the recent cloning of numerous FAR coding sequences it is
now conceivable that novel biotechnological approaches for fatty
alcohol production can be developed in either transgenic microbes
or oilseeds (see below).

35

0.8
0.6
0.4
0.2
0.0
FAR1

FAR3

FAR4

FAR5

FattyAcid Reductase
Fig. 4. Amount and chain length distribution of primary fatty alcohols produced
by Arabidopsis FARs expressed in yeast. (A) Distribution of fatty alcohols found in
the culture medium (expressed as percent of total fatty alcohol produced). (B) Total
fatty alcohols produced by each FAR (expressed as g per unit OD600 ). Each value
corresponds to the mean SD from ve independent experiments. Expression in S.
cereviseae and analysis of cell-associated fatty alcohols were conducted as described
in Domergue et al. [9]. For analyzing lipids present in the culture medium, 5 ml of
medium was extracted once with chloroform/methanol (2:1, [v/v]) containing 50 g
heptadecanoic acid (C17:0) and 20 g pentadecanol (C15:0-OH) as internal standards, once with chloroform/methanol (2:1, [v/v]), and then once with chloroform.
Organic phases were combined, washed with 2.5 ml NaCl (2.5%, [w/v]) and dried
under a gentle stream of nitrogen. Derivatization and analyses were conducted as
already described for the yeast cell sediments (Domergue et al. [9]).

pellets and in the media used for bacterial or yeast growth [10,15].
When different FARs from Arabidopsis are expressed in yeast in
the absence of exogenously supplied fatty acids, large amounts
of fatty alcohols are found in the culture media of yeast expressing FAR1, FAR4 and FAR5, whereas no fatty alcohols are detected
in the culture medium of yeast expressing FAR3/CER4 (Fig. 4A).
These data suggest that yeast may tolerate or sequester a certain amount of fatty alcohols within the cell without signicantly
affecting growth, but that excess fatty alcohols are secreted. The
lack of C24:0 and C26:0 fatty alcohols in the culture medium of
FAR3/CER4-expressing yeast may be due to the inability of yeast
to secrete them or due to a lack of solubility of C24:0 and longer
chain lengths in the culture medium. Total fatty alcohol analysis
(cell pellet and medium) shows that FAR1 and FAR4 have rather
broad substrate specicities, whereas FAR3/CER4 and FAR5 are very
chain-length specic in yeast as they produce nearly exclusively
(>97%) a single fatty alcohol, C26:0-OH and C18:0-OH, respectively
(Fig. 4B). Although the mechanism of fatty alcohol secretion in
microbes remains to be investigated, it may be possible to exploit
this phenomenon for high-level production of fatty alcohols. The
engineering of E. coli for the production of fatty alcohols and wax
esters has indeed already been achieved [16,20,92,93]. Expression
of FAR enzymes in a oleaginous industrial yeast such as Yarrowia
lipolytica [94] or the use of a two-phase overlay system [95] may
allow for continuous production of high yields of fatty alcohols
extracted from the microbes or from the medium, respectively. In
addition, successful production of fatty alcohols in cyanobacteria
through the expression of plastid FARs [96] suggests that transgenic
microalgae could also be engineered in the future for the industrial
production of fatty alcohols.

36

O. Rowland, F. Domergue / Plant Science 193194 (2012) 2838

4.3. Biotechnological production of wax esters in oilseed crops

References

The excellent lubrication properties of wax esters as well as

their high resistance to hydrolysis or oxidation at high temperature makes them highly sought after for industrial applications.
They can be made synthetically, but it is a relatively expensive
process. In the 1990s, the biotechnology company Calgene (later
acquired by Monsanto) pursued the development of transgenic
oilseed crops that produce jojoba-like wax esters. They cloned and
characterized a FAR and a wax synthase from developing jojoba
embryos (see Section 2.2) [7,97]. Furthermore, they succeeded in
generating signicant amounts of wax esters in the seeds of transgenic Arabidopsis plants by co-expression of the jojoba FAR and
WS genes along with a fatty acid elongase (KCS) gene from Lunaria
annua [97]. The Lunaria fatty acid elongase was required to generate the very-long-chain acyl-CoA substrates (C20:1/C22:1/C24:1)
preferred by the jojoba FAR. The goal of producing high-value wax
esters in an industrial oilseed crop is currently being pursued by
an international group of plant lipid scientists primarily funded by
the European Union (http://icon.slu.se/ICON/). The target oilseed
plants are ones not generally used in food production (e.g. Crambe
abyssinica) to avoid contamination of edible oils with wax esters
intended for industrial applications. This collaborative project may
provide innovative oilseed crops producing tailor-made wax esters
that could replace fossil oil-derived lubricants.
The existence of diverse FARs in all life kingdoms provides a
variety of enzymes with different substrate specicities according
to both acyl chain length and degree of saturation. FARs having preference for short-chain monounsaturated or saturated fatty alcohols
have recently been characterized from an insect [15] and Euglena
[20]. These FARs therefore provide a platform for generating a
diverse array of high-value fatty alcohol and wax ester products.
Shorter chain and unsaturated wax esters have very low melting
points, which considerably increases the industrial applications of
these compounds. The genetic engineering of FAR enzymes will
be important for optimizing production through enhancement of
catalytic activity, modication of substrate specicity, and redirection of subcellular location. It may even be possible to engineer
FARs with specicities that do not exist in nature. This pursuit
needs to be guided by additional FAR enzyme structural information.

[1] R. Franke, I. Briesen, T. Wojciechowski, A. Faust, A. Yephremov, C. Nawrath,

L. Schreiber, Apoplastic polyesters in Arabidopsis surface tissues a typical
suberin and a particular cutin, Phytochemistry 66 (2005) 26432658.
[2] I. Molina, G. Bonaventure, J. Ohlrogge, M. Pollard, The lipid polyester composition of Arabidopsis thaliana and Brassica napus seeds, Phytochemistry 67 (2006)
25972610.
[3] M. Pollard, F. Beisson, Y. Li, J.B. Ohlrogge, Building lipid barriers: biosynthesis
of cutin and suberin, Trends in Plant Science 13 (2008) 236246.
[4] W. Chen, X.H. Yu, K. Zhang, J. Shi, S. De Oliveira, L. Schreiber, J. Shanklin, D. Zhang,
Male Sterile2 encodes a plastid-localized fatty acyl carrier protein reductase
required for pollen exine development in Arabidopsis, Plant Physiology 157
(2011) 842853.
[5] T.K. Miwa, Jojoba oil wax esters and derived fatty acids and alcohols: gas chromatographic analyses, Journal of the American Oil Chemists Society 48 (1971)
259264.
[6] M.R. Pollard, T. Mckeon, L.M. Gupta, P.K. Stumpf, Studies on biosynthesis of
waxes by developing jojoba seed-II the demonstration of wax biosynthesis
by cell-free homogenates, Lipids 14 (1979) 651662.
[7] J.G. Metz, M.R. Pollard, L. Anderson, T.R. Hayes, M.W. Lassner, Purication of a
jojoba embryo fatty acyl-coenzyme A reductase and expression of its cDNA in
high erucic acid rapeseed, Plant Physiology 122 (2000) 635644.
[8] O. Rowland, H. Zheng, S.R. Hepworth, P. Lam, R. Jetter, L. Kunst, CER4 encodes
an alcohol-forming fatty acyl-coenzyme A reductase involved in cuticular wax
production in Arabidopsis, Plant Physiology 142 (2006) 866877.
[9] F. Domergue, S.J. Vishwanath, J. Joubes, J. Ono, J.A. Lee, M. Bourdon, R. Alhattab,
C. Lowe, S. Pascal, R. Lessire, O. Rowland, Three Arabidopsis fatty acyl-coenzyme
A reductases, FAR1, FAR4, and FAR5, generate primary fatty alcohols associated
with suberin deposition, Plant Physiology 153 (2010) 15391554.
[10] T.T. Doan, A.S. Carlsson, M. Hamberg, L. Bulow, S. Stymne, P. Olsson, Functional
expression of ve Arabidopsis fatty acyl-CoA reductase genes in Escherichia coli,
Journal of Plant Physiology 166 (2009) 787796.
[11] T.T. Doan, F. Domergue, A.E. Fournier, S.J. Vishwanath, O. Rowland,
P. Moreau, C.C. Wood, A.S. Carlsson, M. Hamberg, P. Hofvander, Biochemical characterization of a chloroplast localized fatty acid reductase from Arabidopsis thaliana, Biochimica et Biophysica Acta (2011),
http://dx.doi.org/10.1016/j.bbalip.2011.10.019.
[12] J. Shi, H. Tan, X.H. Yu, Y. Liu, W. Liang, K. Ranathunge, R.B. Franke, L. Schreiber,
Y. Wang, G. Kai, J. Shanklin, H. Ma, D. Zhang, Defective pollen wall is required
for anther and microspore development in rice and encodes a fatty acyl carrier
protein reductase, Plant Cell 23 (2011) 22252246.
[13] A. Wang, Q. Xia, W. Xie, T. Dumonceaux, J. Zou, R. Datla, G. Selvaraj, Male
gametophyte development in bread wheat (Triticum aestivum L.): molecular,
cellular, and biochemical analyses of a sporophytic contribution to pollen wall
ontogeny, Plant Journal 30 (2002) 613623.
[14] K. Moto, T. Yoshiga, M. Yamamoto, S. Takahashi, K. Okano, T. Ando, T. Nakata,
S. Matsumoto, Pheromone gland-specic fatty-acyl reductase of the silkmoth,
Bombyx mori, Proceedings of the National Academy of Sciences of the United
States of America 100 (2003) 91569161.
[15] M.A. Lienard, A.K. Hagstrom, J.M. Lassance, C. Lofstedt, Evolution of multicomponent pheromone signals in small ermine moths involves a single fatty-acyl
reductase gene, Proceedings of the National Academy of Sciences of the United
States of America 107 (2010) 1095510960.
[16] P. Teerawanichpan, A.J. Robertson, X. Qiu, A fatty acyl-CoA reductase highly
expressed in the head of honey bee (Apis mellifera) involves biosynthesis of a
wide range of aliphatic fatty alcohols, Insect Biochemisry and Molecular Biology
40 (2010) 641649.
[17] J.M. Lassance, A.T. Groot, M.A. Lienard, B. Antony, C. Borgwardt, F. Andersson, E.
Hedenstrom, D.G. Heckel, C. Lofstedt, Allelic variation in a fatty-acyl reductase
gene causes divergence in moth sex pheromones, Nature 466 (2010) 486489.
[18] J.B. Cheng, D.W. Russell, Mammalian wax biosynthesis-I identication of two
fatty acyl-Coenzyme A reductases with different substrate specicities and
tissue distributions, Journal of Biological Chemistry 279 (2004) 3778937797.
[19] J. Hellenbrand, E.M. Biester, J. Gruber, M. Hamberg, M. Frentzen, Fatty acyl-CoA
reductases of birds, BMC Biochemistry 12 (2011) 64.
[20] P. Teerawanichpan, X. Qiu, Fatty acyl-CoA reductase and wax synthase from
Euglena gracilis in the biosynthesis of medium-chain wax esters, Lipids 45
(2010) 263273.
[21] P. Teerawanichpan, X. Qiu, Molecular and functional analysis of three fatty
acyl-CoA reductases with distinct substrate specicities in copepod Calanus
nmarchicus, Marine Biotechnology 14 (2011) 227236.
[22] P. Hofvander, T.T. Doan, M. Hamberg, A prokaryotic acyl-CoA reductase performing reduction of fatty acyl-CoA to fatty alcohol, FEBS Letters 585 (2011)
35383543.
[23] R.M. Willis, B.D. Wahlen, L.C. Seefeldt, B.M. Barney, Characterization of a fatty
acyl-CoA reductase from Marinobacter aquaeolei VT8: a bacterial enzyme catalyzing the reduction of fatty acyl-CoA to fatty alcohol, Biochemistry 50 (2011)
1055010558.
[24] A. Marchler-Bauer, S. Lu, J.B. Anderson, F. Chitsaz, M.K. Derbyshire, C. DeweeseScott, J.H. Fong, L.Y. Geer, R.C. Geer, N.R. Gonzales, M. Gwadz, D.I. Hurwitz,
J.D. Jackson, Z. Ke, C.J. Lanczycki, F. Lu, G.H. Marchler, M. Mullokandov,
M.V. Omelchenko, C.L. Robertson, J.S. Song, N. Thanki, R.A. Yamashita, D.
Zhang, N. Zhang, C. Zheng, S.H. Bryant, CDD: a Conserved Domain Database
for the functional annotation of proteins, Nucleic Acids Research 39 (2010)
225229.

5. Concluding remarks
Much remains to be learned about primary fatty alcohol
biosynthesis and functions. The priorities are: (1) elucidating the
three-dimensional structure of a FAR to unravel structure/function
properties, which should be most feasible with a soluble, plastid
FAR, (2) detailed mutagenesis and/or directed evolution of FARs to
identify residues important for activity and substrate specicity,
which will allow for development of FARs with desired properties for industrial applications; and (3) understand the specic
roles of fatty alcohols, wax esters, and alkyl hydroxycinnamates
associated with extracellular lipid barriers in plantenvironment
interactions.

Acknowledgements
The authors are part of the European Commission (EC) Framework 7 project entitled Industrial crops producing added value
oils for novel chemicals (ICON) and are supported by grants from
the EC, the Embassy of France in Canada (France-Canada Research
Fund), and the National Sciences and Engineering Research Council
of Canada.

O. Rowland, F. Domergue / Plant Science 193194 (2012) 2838

[25] M.G. Aarts, R. Hodge, K. Kalantidis, D. Florack, Z.A. Wilson, B.J. Mulligan, W.J.
Stiekema, R. Scott, A. Pereira, The Arabidopsis MALE STERILITY 2 protein shares
similarity with reductases in elongation/condensation complexes, Plant Journal 12 (1997) 615623.
[26] Y. Kallberg, U. Oppermann, B. Persson, Classication of the short-chain dehydrogenase/reductase superfamily using hidden Markov models, FEBS Journal
277 (2010) 23752386.
[27] K.L. Kavanagh, H. Jrnvall, B. Persson, U. Oppermann, Medium- and short-chain
dehydrogenase/reductase gene and protein families: the SDR superfamily:
functional and structural diversity within a family of metabolic and regulatory
enzymes, Cellular and Molecular Life Sciences 65 (2008) 38953906.
[28] M.G. Aarts, W.G. Dirkse, W.J. Stiekema, A. Pereira, Transposon tagging of a male
sterility gene in Arabidopsis, Nature 363 (1993) 715717.
[29] R. Schwacke, A. Schneider, E. Van Der Graaff, K. Fischer, E. Catoni, M. Desimone, W.B. Frommer, U.I. Flugge, R. Kunze, ARAMEMNON, a novel database
for Arabidopsis integral membrane proteins, Plant Physiology 131 (2003)
1626.
[30] J. Ren, L. Wen, X. Gao, C. Jin, Y. Xue, X. Yao, CSS-Palm 2.0: an updated software
for palmitoylation sites prediction, Protein Engineering and Design Selection
Journal 21 (2008) 639644.
[31] F. Li, X. Wu, P. Lam, D. Bird, H. Zheng, L. Samuels, R. Jetter, L. Kunst, Identication of the wax ester synthase/acyl-coenzyme A: diacylglycerol acyltransferase
WSD1 required for stem wax ester biosynthesis in Arabidopsis, Plant Physiology 148 (2008) 97107.
[32] P.E. Kolattukudy, Reduction of fatty acids to alcohols by cell-free preparations
of Euglena gracilis, Biochemistry 9 (1970) 10951102.
[33] A.A. Khan, P.E. Kolattukudy, A microsomal fatty acid synthetase coupled to acylCoA reductase in Euglena gracilis, Archives of Biochemistry and Biophysics 158
(1973) 411420.
[34] A.A. Khan, P.E. Kolattukudy, Solubilization of fatty acid synthetase, acyl-CoA
reductase, and fatty acyl-CoA alcohol transacylase from the microsomes of
Euglena gracilis, Archives of Biochemistry and Biophysicsl 170 (1975) 400408.
[35] J. Vioque, P.E. Kolattukudy, Resolution and purication of an aldehydegenerating and an alcohol-generating fatty acyl-CoA reductase from pea
leaves (Pisum sativum L.), Archives of Biochemistry and Biophysics 340 (1997)
6472.
[36] S. Trenkamp, W. Martin, K. Tietjen, Specic and differential inhibition of
very-long-chain fatty acid elongases from Arabidopsis thaliana by different herbicides, Proceedings of the National Academy of Sciences of the United States
of America 101 (2004) 1190311908.
[37] J. Zeier, K. Ruel, U. Ryser, L. Schreiber, Chemical analysis and immunolocalisation of lignin and suberin in endodermal and hypodermal/rhizodermal cell
walls of developing maize (Zea mays L.) primary roots, Planta 209 (1999) 112.
[38] L. Schreiber, R. Franke, K. Hartmann, Wax and suberin development of native
and wound periderm of potato (Solanum tuberosum L.) and its relation to peridermal transpiration, Planta 220 (2005) 520530.
[39] L. Schreiber, R. Franke, K.D. Hartmann, K. Ranathunge, E. Steudle, The chemical
composition of suberin in apoplastic barriers affects radial hydraulic conductivity differently in the roots of rice (Oryza sativa L. cv. IR64) and corn (Zea mays
L. cv. Helix), Journal of Experimental Botany 56 (2005) 14271436.
[40] V. Vrkoslav, M. Hakova, K. Peckova, K. Urbanova, J. Cvacka, Localization
of double bonds in wax esters by high-performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry utilizing the
fragmentation of acetonitrile-related adducts, Analytical Chemistry 83 (2011)
29782986.
[41] L. Samuels, L. Kunst, R. Jetter, Sealing plant surfaces: cuticular wax formation
by epidermal cells, Annual Review of Plant Biology 59 (2008) 683707.
[42] G. Bianchi, P. Avato, F. Salamini, Surface waxes from grain, leaves and husks of
maize (Zea Mays L.), Cereal Chemistry 61 (1984) 4547.
[43] A. Bianchi, G. Bianchi, P. Avato, F. Salamini, Biosynthetic pathways of epicuticular waxes of maize as assessed by mutation, light, plant age and inhibitor
studies, Maydica 30 (1985) 179198.
[44] P. Von Wettstein-Knowles, Gene mutation in barley inhibiting the production
and use of C26 chains in epicuticular wax formation, FEBS Letters 42 (1974)
187191.
[45] L.E. Vandenburg, E.A. Wilder, The structural constituents of Carnauba wax,
Journal of the American Oil Chemists Society 47 (1970) 514518.
[46] C. Lai, L. Kunst, R. Jetter, Composition of alkyl esters in the cuticular wax on
inorescence stems of Arabidopsis thaliana cer mutants, Plant Journal 50 (2007)
189196.
[47] M.A. Jenks, H.A. Tuttle, S.D. Eigenbrode, K.A. Feldmann, Leaf epicuticular waxes
of the eceriferum mutants in Arabidopsis, Plant Physiology 108 (1995) 369377.
[48] C. Buschhaus, R. Jetter, Composition differences between epicuticular and intracuticular wax substructures: how do plants seal their epidermal surfaces?
Journal of Experimental Botany 62 (2011) 841853.
[49] M. Wolters-Arts, W.M. Lush, C. Mariani, Lipids are required for directional
pollen-tube growth, Nature 392 (1998) 818821.
[50] G.M. Zinkl, B.I. Zwiebel, D.G. Grier, D. Preuss, Pollen-stigma adhesion in Arabidopsis: a species-specic interaction mediated by lipophilic molecules in the
pollen exine, Development 126 (1999) 54315440.
[51] D. Jessen, A. Olbrich, J. Knufer, A. Kruger, M. Hoppert, A. Polle, M. Fulda, Combined activity of LACS1 and LACS4 is required for proper pollen coat formation
in Arabidopsis, Plant Journal 68 (2011) 715726.
[52] M. Koornneef, C.J. Hanhart, F. Thiel, A genetic and phenotypic description of
eceriferum (cer) mutants in Arabidopsis thaliana, Journal of Heredity 80 (1989)
118122.

37

[53] M. Hulskamp, S.D. Kopczak, T.F. Horejsi, B.K. Kihl, R.E. Pruitt, Identication
of genes required for pollen-stigma recognition in Arabidopsis thaliana, Plant
Journal 8 (1995) 703714.
[54] M.A. Bernards, Demystifying suberin, Canadian Journal of Botany 80 (2002)
227240.
[55] R. Thomas, X. Fang, K. Ranathunge, T.R. Anderson, C.A. Peterson, M.A. Bernards,
Soybean root suberin: anatomical distribution, chemical composition, and relationship to partial resistance to Phytophthora sojae, Plant Physiology 144 (2007)
299311.
[56] S. Shao, C.J. Meyer, F. Ma, C.A. Peterson, M.A. Bernards, The outermost cuticle of
soybean seeds: chemical composition and function during imbibition, Journal
of Experimental Botany 58 (2007) 10711082.
[57] C.J. Meyer, C.A. Peterson, M.A. Bernards, A comparison of suberin monomers
from the multiseriate exodermis of Iris germanica during maturation under
differing growth conditions, Planta 233 (2011) 773786.
[58] W.L. Yang, M.A. Bernards, Wound-induced metabolism in potato (Solanum
tuberosum) tubers: biosynthesis of aliphatic domain monomers, Plant Signaling
and Behavior 1 (2006) 5966.
[59] R. Franke, R. Hofer, I. Briesen, M. Emsermann, N. Efremova, A. Yephremov, L.
Schreiber, The DAISY gene from Arabidopsis encodes a fatty acid elongase condensing enzyme involved in the biosynthesis of aliphatic suberin in roots and
the chalaza-micropyle region of seeds, Plant Journal 57 (2009) 8095.
[60] S.B. Lee, S.J. Jung, Y.S. Go, H.U. Kim, J.K. Kim, H.J. Cho, O.K. Park, M.C. Suh,
Two Arabidopsis 3-ketoacyl CoA synthase genes, KCS20 and KCS2/DAISY, are
functionally redundant in cuticular wax and root suberin biosynthesis, but
differentially controlled by osmotic stress, Plant Journal 60 (2009) 462475.
[61] M.L. Laver, H.H.L. Fang, Ferulic acid esters from bark of Pseudotsuga menziesii,
Journal of Agricultural and Food Chemistry 37 (1989) 114116.
[62] K.E. Espelie, N.Z. Sadek, P.E. Kolattukudy, Composition of suberin-associated
waxes from the subterranean storage organs of seven plants, Planta 148 (1980)
468476.
[63] M.A. Bernards, N.G. Lewis, Alkyl ferulates in wound healing potato tubers, Phytochemistry 31 (1992) 34093412.
[64] O. Serra, C. Hohn, R. Franke, S. Prat, M. Molinas, M. Figueras, A feruloyl transferase involved in the biosynthesis of suberin and suberin-associated wax is
required for maturation and sealing properties of potato periderm, Plant Journal 62 (2010) 277290.
[65] I. Molina, Y. Li-Beisson, F. Beisson, J.B. Ohlrogge, M. Pollard, Identication of an
Arabidopsis feruloyl-coenzyme A transferase required for suberin synthesis,
Plant Physiology 151 (2009) 13171328.
[66] Y. Li, F. Beisson, J. Ohlrogge, M. Pollard, Monoacylglycerols are components of
root waxes and can be produced in the aerial cuticle by ectopic expression of a
suberin-associated acyltransferase, Plant Physiology 144 (2007) 12671277.
[67] L. Schreiber, Transport barriers made of cutin, suberin and associated waxes,
Trends in Plant Science 15 (2010) 546553.
[68] J.Y. Gou, X.H. Yu, C.J. Liu, A hydroxycinnamoyltransferase responsible for
synthesizing suberin aromatics in Arabidopsis, Proceedings of the National
Academy of Sciences of the United States of America 106 (2009) 1885518860.
[69] H. Kikuzaki, M. Hisamoto, K. Hirose, K. Akiyama, H. Taniguchi, Antioxidant properties of ferulic acid and its related compounds, Journal of Agricultural and Food
Chemistry 50 (2002) 21612168.
[70] K. Yunoki, R. Musa, M. Kinoshita, H. Tazaki, Y. Oda, M. Ohnishi, Presence of
higher alcohols as ferulates in potato pulp and its radical-scavenging activity,
Bioscience, Biotechnology, and Biochemistry 68 (2004) 26192622.
[71] M.G. Nair, M.D. Epp, B.A. Burke, Ferulate esters of higher fatty alcohols and
allelopathy in Kalanche daigremontiana, Journal of Chemical Ecology 14 (1988)
589603.
[72] J.D. Baranowski, C.W. Nagel, Inhibition of Pseudomonas uorescens by hydroxycinnamic acids and their alkyl esters, Journal of Food Science 47 (1982)
15871589.
[73] R.L. Nicholson, R. Hammerschmidt, Phenolic compounds and their role in disease resistance, Annual Review of Phytopathology 30 (1992) 369389.
[74] S. Blackmore, A.H. Wortley, J.J. Skvarla, J.R. Rowley, Pollen wall development in
owering plants, New Phytology 174 (2007) 483498.
[75] A.A. Dobritsa, J. Shrestha, M. Morant, F. Pinot, M. Matsuno, R. Swanson, B.L.
Moller, D. Preuss, CYP704B1 is a long-chain fatty acid omega-hydroxylase
essential for sporopollenin synthesis in pollen of Arabidopsis, Plant Physiology
151 (2009) 574589.
[76] M. Matsuno, V. Compagnon, G.A. Schoch, M. Schmitt, D. Debayle, J.-E. Bassard,
B. Pollet, A. Hehn, D. Heintz, P. Ullmann, C. Lapierre, F. Bernier, J. Ehlting, D.
Werck-Reichhart, Evolution of a novel phenolic pathway for pollen development, Science 325 (2009) 16881692.
[77] K.H. Jung, M.J. Han, D.Y. Lee, Y.S. Lee, L. Schreiber, R. Franke, A. Faust, A. Yephremov, H. Saedler, Y.W. Kim, I. Hwang, G. An, Wax-decient anther1 is involved
in cuticle and wax production in rice anther walls and is required for pollen
development, Plant Cell 18 (2006) 30153032.
[78] T. Ariizumi, K. Toriyama, Genetic regulation of sporopollenin synthesis and
pollen exine development, Annual Review of Plant Biology 62 (2011) 437460.
[79] H. Li, F. Pinot, V. Sauveplane, D. Werck-Reichhart, P. Diehl, L. Schreiber, R.
Franke, P. Zhang, L. Chen, Y. Gao, W. Liang, D. Zhang, Cytochrome P450 family
member CYP704B2 catalyzes the -hydroxylation of fatty acids and is required
for anther cutin biosynthesis and pollen exine formation in rice, Plant Cell 22
(2010) 173190.
[80] F. Ahlers, J. Lambert, R. Wiermann, Acetylation and silylation of piperidine
solubilized sporopollenin from pollen of Typha angustifolia L, Zeitschrift fr
Naturforschung C 58 (2003) 807811.

38

O. Rowland, F. Domergue / Plant Science 193194 (2012) 2838

[81] M. Morant, K. Jorgensen, H. Schaller, F. Pinot, B.L. Moller, D. Werck-Reichhart,

S. Bak, CYP703 is an ancient cytochrome P450 in land plants catalyzing inchain hydroxylation of lauric acid to provide building blocks for sporopollenin
synthesis in pollen, Plant Cell 19 (2007) 14731487.
[82] C.A. Houston, Marketing and economics of fatty alcohols, Journal of the American Oil Chemists Society 61 (1984) 179184.
[83] S.M. Mudge, S.E. Belanger, A.M. Nielsen, Fatty Alcohols: Anthropogenic and
Natural Occurrence in the Environment, The Royal Society of Chemistry, Cambridge, UK, 2008.
[84] R.F. Modler, R. Gubler, A. Kishi. Surfactants, household detergents and their raw
materials, CEH Marketing Report, 2002.
[85] A. Carlsson, Production of Wax Esters in Crambe, CLC Press, Newbury, UK, 2006.
[86] U.R. Kruetzer, Manufacture of fatty alcohol based on natural fats and oils, Journal of the American Oil Chemists Society 61 (1984) 343348.
[87] H.J. Richter, J. Knaut, Proceedings second world conference of detergents, Journal of the American Oil Chemists Society 61 (1984) 160.
[88] M.R. Clarke, Function of the spermaceti organ of the sperm whale, Nature 228
(1970) 873874.
[89] H.H. Naqvi, I.P. Ting, Jojoba: Unique Liquid Wax Produced from the American
Desert, Timber Press, Portland, OR, 1990.
[90] A.P. Tulloch, Beeswax: structure of the esters and their component hydroxy
acids and diols, Chemistry and Physics of Lipids 6 (1971) 235.

[91] A.H. Warth, The Chemistry and Technology of Waxes, Reinhold Publishers, New
York, 1956.
[92] R. Kalscheuer, T. Stoveken, H. Luftmann, U. Malkus, R. Reichelt, A. Steinbuchel,
Neutral lipid biosynthesis in engineered Escherichia coli: jojoba oil-like wax
esters and fatty acid butyl esters, Applied and Environment Microbiology 72
(2006) 13731379.
[93] E.J. Steen, Y. Kang, G. Bokinsky, Z. Hu, A. Schirmer, A. Mcclure, S.B. Del Cardayre,
J.D. Keasling, Microbial production of fatty-acid-derived fuels and chemicals
from plant biomass, Nature 463 (2010) 559562.
[94] A. Beopoulos, J. Cescut, R. Haddouche, J.L. Uribelarrea, C. Molina-Jouve, J.M.
Nicaud, Yarrowia lipolytica as a model for bio-oil production, Progress in Lipid
Research 48 (2009) 375387.
[95] E.B. Goh, E.E. Baidoo, J.D. Keasling, H.R. Beller, Engineering of bacterial methyl
ketone synthesis for biofuels, Applied and Environment Microbiology 78 (2011)
7080.
[96] X. Tan, L. Yao, Q. Gao, W. Wang, F. Qi, X. Lu, Photosynthesis driven conversion of
carbon dioxide to fatty alcohols and hydrocarbons in cyanobacteria, Metabolic
Engineering 13 (2011) 169176.
[97] K.D. Lardizabal, J.G. Metz, T. Sakamoto, W.C. Hutton, M.R. Pollard, M.W. Lassner,
Purication of a jojoba embryo wax synthase, cloning of its cDNA, and production of high levels of wax in seeds of transgenic Arabidopsis, Plant Physiology
122 (2000) 645655.