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MINIREVIEW / MINISYNTHÈSE

Demystifying suberin
Mark A. Bernards

Abstract: Suberin is a term used to define a specific cell wall component that occurs, for example, in phellem (cork)
endodermal and exodermal cells and is characterized by the deposition of both poly(phenolic) and poly(aliphatic) do-
mains. Historically, the poly(phenolic) domain has been likened to lignin, and while there is an element of truth to this
comparison, recent evidence supports an alternative view in which the poly(phenolic) domain contains a significant
amount of nonlignin precursors (principally hydroxycinnamic acids and their derivatives) that are covalently linked to
each other in a manner analogous to the monolignols in lignin. Similarly, the conceptual model in which the
poly(aliphatic) domain of suberized tissues is represented as a random network of polyesterified, modified fatty acids
and alcohols has been replaced with one comprising a three-dimensional, glycerol-bridged network. Taken together, a
new model for suberin is emerging in which a hydroxycinnamic acid – monolignol poly(phenolic) domain, embedded
in the primary cell wall, is covalently linked to a glycerol-based poly(aliphatic) domain located between the primary
cell wall and the plasma membrane. The structural and biochemical evidence supporting this new suberin paradigm are
examined in this minireview, along with the presentation of a new structural model encompassing a current view of the
structure of suberin.

Key words: suberin, lignin, hydroxycinnamic acid, monolignol, poly(aliphatic) domain, poly(phenolic) domain, glycerol
polyester.

Résumé : Le terme subérine définit une composante pariétale cellulaire spécifique qu’on retrouve, par exemple, au ni-
veau du suber (écorce) et des cellules endodermales et exodermales; elle se caractérise par la déposition à la fois de
substances des domaines poly(phénolique) et poly(aliphatique), pour un tissus donné. Historiquement, le domaine
poly(phénolique) a été assimilé à la lignine et, bien qu’il y ait un élément de vérité dans cette comparaison, une preuve
récente supporte une interprétation alternative selon laquelle le domaine poly(phénolique) contient une quantité signifi-
cative de précurseurs sans affinité avec la lignine (principalement des acides hydrocinnamiques et leurs dérivés) qui
sont liés par covalence les uns aux autres de manière analogue aux monolignols de la lignine. De même, le modèle
conceptuel selon lequel le domaine poly(aliphatique) des tissus subérifiés est représenté comme un réseau aléatoire
d’acides gras modifiés et d’alcools polyestérifiés, a été remplacé par un modèle comportant un réseau tridimensionnel
lié par le glycérol. Pris dans son ensemble, un modèle nouveau est en émergence pour la subérine selon lequel un do-
maine poly(phénolique) basé sur l’acide hydrocinnamique – monolignol, enrobé dans la paroi cellulaire primaire, est lié
par covalence à un domaine poly(aliphatique) basé sur le glycérol et localisé entre la parois cellulaire primaire et la
plasmalemme. Dans cette minisynthèse, l’auteur examinent les preuves structurales et biochimiques supportant ce nou-
veau paradigme pour la subérine, et présente un nouveau modèle structural englobant une perception courante de ce
que pourrait être la structure de la subérine.

Mots clés : subérine, lignine, acide hydroxycinnamique, monolignol, domaine poly(aliphatique), domaine poly(phénolique),
polyester de glycérol.

[Traduit par la Rédaction] Bernards 240

Received 18 September 2001. Published on the NRC Research Press Web site at http://canjbot.nrc.ca on 15 March 2002.
Abbreviations: 4-CL, 4-coumaroyl-CoA ligase; CCR, hydroxycinnamnoyl-CoA oxidoreductase; CoA, coenzyme A; DFRC, derivatization
followed by reductive cleavage; DHAP, dihydroxyacetone-3-phosphate; HHT, hydroxycinnamoyl-CoA:ω-hydroxypalmitic acid
O-hydroxycinnamoyltransferase; NBO, alkaline nitrobenzene oxidation; NMR, nuclear magnetic resonance; SPAD, suberin poly(aliphatic)
domain; SPPD, suberin poly(phenolic) domain; TEM, transmission electron microscopy; THT, hydroxycinnamoyl-CoA:tyramine
hydroxycinnamoyltransferase; TMS, trimethylsilyl; VLC, very long chain; VLCFA, very long chain fatty acids.
M.A. Bernards. Department of Plant Sciences, University of Western Ontario, London, ON N6A 5B7, Canada
(e-mail: bernards@uwo.ca).

Can. J. Bot. 80: 227–240 (2002) DOI: 10.1139/B02-017 © 2002 NRC Canada

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228
Introduction
Suberin deposited in cells is characterized by the presence
of both a poly(aliphatic) domain, typically appearing as al-
ternating bands of light and dark staining material between
the cell wall and plasma membrane in TEM photomicro-
graphs, and a poly(phenolic) domain in the cell wall.
Suberized cells are found in underground plant parts (e.g.,
epidermis, endodermis, exodermis, root phellem, and tuber
phellem) as well as in bundle sheath cells and the phellem
(cork) of aboveground tissues of woody species that undergo
secondary thickening (Esau 1977). Historically, the aliphatic
domain has been described as a three-dimensional polyester
network of α,ω-dioic acids, ω-hydroxy acids, very long chain
fatty acids (VLCFAs), mid-chain-oxidized fatty acids
(Fig. 1) and esterified hydroxycinnamic acids (Fig. 2) (e.g.,
Kolattukudy 1980, 1984; Cottle and Kolattukudy 1982; Hol-
loway 1983; Graça and Pereira 1997). The phenolic domain,
on the other hand, has been likened to lignin (Kolattukudy
1980, 1984; Pereira 1988; Marques et al. 1994, 1999; Neto
et al. 1996; Conde et al. 1998), which is a three-dimensional
polymeric network derived from the monolignols (p-
coumaryl, coniferyl and sinapyl alcohols; Fig. 2). While this
view remains essentially correct, emerging evidence sup-
ports the notion that glycerol is a major component of the
aliphatic domain (Schmutz et al. 1993, 1994, 1996; Graça
and Pereira 1997, 1998, 1999, 2000a, 2000b; Moire et al.
1999) and that the phenolic precursors to the poly(phenolic)
domain are represented by a significant amount of
hydroxycinnamic acids and their derivatives (especially
ferulic acid and feruloyltyramine; Fig. 2) in addition to
monolignols (Bernards et al. 1995, 1999, 2001; Negrel et al.
1996; Schreiber 1996; Schreiber et al. 1999; Zeier and
Schreiber 1997, 1998; Zeier et al. 1999). Both lines of evi-
dence suggest that it is time to re-evaluate our concepts of
the biosynthesis and macromolecular structure of the cell
wall modifier we term “suberin”.
In this minireview, recent literature describing both the
aliphatic and phenolic domains of suberized tissues are high-
lighted in terms of their chemical composition, physiological
roles, and biosynthesis. With respect to the latter, a scheme
is presented to put the biosynthetic reactions of suberization
in a global metabolic perspective, as well as highlight some
of the obvious gaps in our knowledge. A tentative new struc-
tural model is presented in which recent chemical composi-
tion data and conceptual ideas are taken into account. Recent
reviews on the phenolic domain of suberized tissues,
(Bernards and Lewis 1998), suberin and cutin (Kolattukudy
2001), and lignin (Lewis et al. 1999) have been published,
and the reader is urged to consult these to gain a more com-
plete understanding of the current status of these major cell
wall modifications and the issues that define our understand-
ing of them.
The suberin enigma: What’s in a name?
Recently, Graça and Pereira (2000c) wrote a statement
that epitomizes the suberin enigma: “What is and what
should be called suberin remains an open question.” That is,
while it is recognized that suberized cells contain both
poly(aliphatic) and poly(phenolic) domains, even the most
cursory reading of the literature reveals apparent confusion
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over the use of the term suberin to describe the structural
and molecular entity that defines the walls of cells that are
“suberized”. For example, several groups have recently writ-
ten that cells from the cork layer of trees (i.e., the quintes-
sential suberized tissue) are composed of three main
structural components: suberin, lignin, and carbohydrates
(Marques et al. 1994, 1999; Neto et al. 1996; Conde et al.
1998). The implication is that the poly(phenolic) domain in
these tissues is a lignin and is therefore not a component of
suberin. This conclusion is further supported by the many
instances in the literature where the term suberin is loosely
used to describe only the aliphatic domain and its associated
“suberin phenolics” (i.e., esterified hydroxycinnamates)
(Holloway 1982; Marques et al. 1994, 1999; Graça and
Pereira 1997, 1998, 2000c; Conde et al. 1998; Zeier and
Schreiber 1997, 1998; Lopes et al. 2000a, 2000c). Indeed,
saponification, which readily cleaves the ester linkages of
the poly(aliphatic) domain, but not the more robust ether and
C–C linkages of the poly(phenolic) domain, is considered to
“desuberize” suberized tissues (e.g., Gil et al. 1997). Simi-
larly, lipid-soluble dyes (e.g., Sudan III–IV) that partition into
the lipid components of suberin, yet have no affinity for its
poly(phenolic) domain, are used to detect suberin
histochemically (e.g., Trockenbrodt 1994; Nunes et al. 1999).
If we accept the notion that suberized tissues contain both
poly(aliphatic) and poly(phenolic) domains, (and that the lat-
ter is not a lignin), then the term suberin must be used judi-
ciously and specifically in reference to a macromolecule
containing both. Consequently, different terms must be used
in reference to either domain when they are considered sepa-
rately from suberin as a whole. For example, the
poly(aliphatic) domain should be referred to as the suberin
poly(aliphatic) domain or SPAD, while recognizing that
there are nonpolymerized aliphatic (i.e., wax) components
associated with it. Similarly, the suberin poly(phenolic) do-
main can be abbreviated SPPD. These terms are used
throughout this minireview.
Chemical composition
Both the SPAD and the SPPD have their own unique chem-
ical composition and exist as distinct entities within suberized
cells (reviewed in Bernards and Lewis 1998). They do, how-
ever, appear to be covalently linked together (Stark and
Garbow 1992; Neto et al. 1996; Gil et al. 1997; Lopes et al.
2000a, 2000b), presumably at the inner surface of the cell
wall. While the structure of each domain is discussed in
greater detail in a later section, the following describes their
monomeric or precursor composition. It is important to keep
in mind that the majority of the chemical composition data
collected to date has come from the chromatographic analysis
of fragments isolated from suberized cell wall preparations af-
ter degradative procedures. Consequently, our ability to recon-
struct the original molecular domains is only as reliable as the
techniques used to degrade them.
The poly(aliphatic) domain (SPAD)
The SPAD of suberized tissues is assembled from a series
of specific, and in some cases unique, aliphatic components
(Fig. 1). Estimates based on the analysis of cork tissue from
Quercus suber suggest the SPPD accounts for up to 40–50%
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Bernards 229

Fig. 1. Aliphatic precursors of suberized tissues.

OH 1-Alkanols,
Saturated series, 16:0 to 32:0

- Alkanoic Acids,
COO
Saturated series, 16:0 to 28:0

- ω-Hydroxyalkanoic Acids,
HO COO
Saturated series, 16:0 to 24:0 and 18:1

- - α,ω-Alkandioic Acids,
OOC COO
Saturated series, 16:0 to 24:0 and 18:1

OH
- 9(10),ω-Dihydroxyalkanoic Acid,
HO COO Predominantly 18:0

OH
-
COO 9,10-Dihydroxyalkanoic Acid,
16:0, 18:0
OH

OH
-
HO COO 9,10,18-Trihydroxyalkanoic Acid,
Predominantly 18:0
OH
OH
- -
OOC COO 9,10-Dihydroxy-α,ω-Alkandioic Acids,
Predominantly 18:0
OH

O
- 9,10-Epoxy-ω-Hydroxyalkanoic Acid
HO COO Predominantly 18:0

O
- - 9,10-Epoxy-α,ω-Alkandioic Acids,
OOC COO Predominantly 18:0

O O Ferulates,
Homologous Series with C-16 to C-30
1-alkanols, including C-17, C-19 and C-21

OCH3 HO
OH
OH
Glycerol
OH

Fig. 2. Phenolic precursors of suberized tissues.

H
-
O O O N
CH 2OH

OH

R2 R1 R2 R1 R1
OH OH OH

R1=R2=H, p-Coumaric Acid R1=R2=H, p-Coumaryl Alcohol R1=H, p-Coumaroyltyramine

R1=OH, R2=H, Caffeic Acid R1=OCH3, R2=H, Coniferyl Alcohol R1=OCH3, Feruloyltyramine
R1=OCH3, R2=H, Ferulic Acid R1=R2=OCH3, Sinapyl Alcohol
R1=R2=OCH3, Sinapic Acid

of the dry weight of suberized cell walls (Marques et al.
1994; Graça and Pereira 1997). There are several compre-
hensive papers in which the aliphatic composition of
suberins from a wide variety of plant species are described
(e.g., Holloway 1983; Matzke and Riederer 1991; Graça and
Pereira 1997) and to which the reader is referred for a more
detailed analysis. Herein, a general overview is provided in
which the common components are described.
The main aliphatic structural components of the SPAD are
illustrated in Fig. 1, including 1-alkanols (trace amounts to
11% of monomers), ω-hydroxyalkanoic acids (13–61% of
monomers), α,ω-dioic acids (2–33% of monomers), mid-
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230
chain epoxide- and di- and tri-hydroxy-substituted
octadecanoates (trace amounts to 58% of monomers), and
glycerol (10–26% of monomers) (Holloway 1983; Graça and
Pereira 1997). In addition, hydroxycinnamic acids (approx.
1.5% of monomers) such as p-coumaric, caffeic, and espe-
cially ferulic acids (Graça and Pereira 1997; Fig. 1), as well
as ferulic acid esters of long-chain alcohols (Fig. 2), also
called ferulates (Adamovics et al. 1977; Chatterjee et al.
1977; Laver and Fang 1989; Baldé et al. 1991; Bernards and
Lewis 1992) have been identified in hydrolysates and ex-
tracts, respectively, from suberized tissues. According to
Matzke and Riederer (1991), the α,ω-dioic acids represent
aliphatics unique to suberized tissue, and can be used as di-
agnostic markers to differentiate between suberized and
cutinized tissues.
The principal analytical tools used to identify SPAD com-
ponents are (i) organic solvent extraction to isolate
unpolymerized wax components, (ii) hydrolysis (including
BF3–MeOH transesterification, reduction with LiAlH4, and
alkaline hydrolysis with NaOCH3 or NaOH) to isolate
esterified components, and (iii) subsequent gas chromatogra-
phy of their TMS-, methyl-, or acetyl-derivatives. The yield
and spectrum of aliphatics is dependent on hydrolysis condi-
tions, with complete removal only achieved by very harsh
treatment (Lopes et al. 2000a). With the use of these rela-
tively simple techniques, the composition of aliphatics and
waxes from suberized tissues is generally considered to be
completely known. However, as will be discussed below,
there is still much to learn about their biosynthesis and
macromolecular assembly in vivo. In considering the struc-
tures shown in Fig. 1, it is important to keep in mind that the
proportion of the individual aliphatic components differs
widely from species to species. For example, in potato
(Solanum tuberosum) suberin, α,ω-dioic acids (33% of
monomers) and ω-hydroxyalkanoic acids (32% of mono-
mers) predominate in the SPAD, while epoxides and mid-
chain hydroxylated fatty acids are present in only trace
amounts. By contrast, epoxides (31% of monomers) and ω-
hydroxyalkanoic acids (41% of monomers) predominate in
Q. suber (Holloway 1983).
The suberin poly(phenolic) domain (SPPD)
The chemical composition of the SPPD has been a matter
of controversy for some time (Bernards and Lewis 1998).
The first attempts to define the SPPD of suberized tissues re-
lied on alkaline nitrobenzene oxidation (NBO), a degradative
technique that results in cleavage of phenylpropanoid side
chains and yields benzaldehyde, benzoate, and acetophenone
derivatives with aromatic substitution patterns representative
of the phenylpropanoids in the intact tissue, but from which
the identity of the parent compound can be lost (Fig. 3). In
other words, the presence of p-hydroxybenzaldehyde,
vanillin, and syringin in NBO hydrolysates can be equally
indicative of hydroxycinnamic acids or monolignols (Bland
and Logan 1965) or indeed other phenolics such as tyramine
(Borg-Olivier and Monties 1993), as all yield similar substi-
tuted benzaldehydes upon hydrolysis. NBO was initially de-
veloped as an analytical tool to quantify lignins. When it
was then applied to suberized tissues, the presence of substi-
tuted benzaldehydes in the hydrolysate was taken as evi-
dence of lignin.
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With the advent of newer, more selective lignin degrada-
tion techniques, such as thioacidolysis, which cleaves only
8-O-4′ ethers of monolignols (Lapierre et al. 1986; Monties
1989; Fig. 3), and their application to suberized tissues
(Borg-Olivier and Monties 1989, 1993; Lapierre et al. 1996;
Schreiber 1996; Zeier and Schreiber 1997; Marques et al.
1999), a new picture of the SPPD began to emerge. For ex-
ample, thioacidolitic analysis of suberized potato tissues
yielded approximately 10% of the monomers released from
lignified tissues. Since thioacidolysis only yields between 20
and 40% of lignin monomers, this suggests that potato SPPD
contains much less “lignin” than lignified tissues. Compari-
sons between methylated and nonmethylated samples further
revealed that the monolignols in the SPPD are more highly
cross-linked than those in wood or wheat straw lignins
(Lapierre et al. 1996). Interestingly, careful analysis of the
products revealed evidence for feruloyltyramine, which is a
hydroxycinnamoyl amide (Negrel et al. 1996). Moreover, no
evidence for p-hydroxy-substituted phenolics was found
with thioacidolysis, despite the abundance of this mono-
substituted phenolic in NBO hydrolysates (Borg-Olivier and
Monties 1989). It is now known that a significant amount of
the p-hydroxybenzaldehyde units released via NBO were de-
rived from tyramine (Borg-Olivier and Monties 1993), pre-
sumably present as feruloyltyramine units in the SPPD
(Negrel et al. 1996). More recently, we have applied the rel-
atively new derivatization followed by a reductive cleavage
(DFRC) technique developed by Lu and Ralph (1997;
Fig. 3) to suberized tissues. The DFRC technique results in
cleavage of both 7-O-4′ and 8-O-4′ aryl ethers within pheno-
lic polymers (Fig. 3) with yields generally comparable to
those of thioacidolysis. However, in our hands, only 5–6%
of the amount of monolignols liberated from lignified tissues
could be recovered (Razem and Bernards 2002) from
suberized potato cell walls, again emphasizing the difference
between these two distinct phenolic polymers. NMR studies
(Bernards et al. 1995), in which the metabolic fate of spe-
cific carbon atoms in the phenylpropanoid side chain was
tracked using 1-[13C]-L-phe, 2-[13C]-L-phe, and 3-[13C]-L-
phe, demonstrated that hydroxycinnamic acids were major
components of the potato SPPD, and supported the conclu-
sion that they were covalently cross-linked via bonds other
than esters. Detailed phenolic analyses of suberized tissues
including potato (Razem and Bernards 2002), Clivia miniata
(Zeier and Schreiber 1997), maize (Zea mays) (Schreiber et
al. 1999; Zimmermann et al. 2000), and several other
monocots and dicots (Zeier and Schreiber 1998), have now
been conducted. In general there is evidence supporting the
hypothesis that suberized tissues contain a significant
amount of hydroxycinnamic acids and a relatively smaller
amount of monolignols than expected for a lignified tissue.
The results of these analyses can be summarized as follows:
as one moves from the stele to the epidermis of roots, there
is an increasing amount of hydroxycinnamic acid in the
poly(phenolic) components of the cell walls, ranging from
essentially 100% monolignol in the stele (as expected for
lignified walls of the vascular tissues) to a mixture of
monolignol and hydroxycinnamic acid in the epidermis. In
other words, the phenolic monomer profiles for lignified and
suberized tissues are different, and apparently tissue specific.
These differences may reflect the different physiological
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Bernards 231

Fig. 3. Schematic representation of the common lignin degradation techniques. (a) Alkaline nitrobenzene oxidation (NBO),
(b) thioacidolysis, and (c) derivitization followed by reductive cleavage (DFRC). The lignin macromolecule is depicted by a monomer
cross-linked to other monomers (labeled “R”). R1 and R2 denote the –OCH3 substitution sites for the common monolignols (See
Fig. 2). The basic reaction conditions for each technique are indicated, along with the principle reaction products. Note that for the
NBO reaction, both monolignol and hydroxycinnamic acid units are indicated, since polymers derived from either precursor yield the
same products. Figure adapted from Lu and Ralph (1997), and Zeier and Schreiber (1998).

(a)
(COOH)
CH 2OR
O Ary l O
Ary l O CHO COOH
NBO, NaOH
170°C, 4 h + +

R2 R1 R2 R1 R2 R1 R2 R1
O OH OH OH

(b)
CH 2OR EtS

O Ary l EtS
Ary l O Thioacidolysis SEt
EtSH, BF3–Et 2O
dioxane, 100°C, 4 h + Dimers and trimers
R2 R1 R2 R1
O OH
DFRC
1. AcBr
(c) 2. Zn
3. Ac 2O, pyridine
OAc

OAc

R2 R1 R2 R1
OAc OAc

roles played by lignified and suberized tissues, as well as
their distinct cellular origin (see below).
Physiological role(s)
The physiological roles of suberin are evident from its an-
atomical distribution and chemical composition. Suberized
cells are found in tissues of underground plant parts (e.g.,
epidermis, exodermis, root phellem, tuber phellem) as well
as in the Casparian band (endodermis and exodermis), and
in the bundle sheath cells and the phellem (cork) of above-
ground tissues of woody species that undergo secondary
thickening (Esau 1977). Interestingly, suberization is nor-
mally associated with cells that derive from a distinct cam-
bium (e.g., the phellogen in bark), or specific initials in the
root meristem (e.g., Malamy and Benfey 1997). For exam-
ple, the phellogen, or cork cambium, forms a continuous
tangential layer of rectangular, radially flattened cells out-
side of the phloem but usually inside the epidermis (Esau
1977). It is clearly distinct from the vascular cambium,
which gives rise to the cells of the secondary xylem and
phloem. The cells that form in files outward from the
phellogen ultimately become suberized and form the
phellem, or cork, layer. By contrast, the cells that form in-
side the phellogen (i.e., the phelloderm) are living,
unsuberized parenchyma cells. The root meristem initials
that ultimately give rise to suberized cells (e.g., epidermis,
exodermis, and endodermis) are different from those that
give rise to the lignified cells of the stele (e.g., Malamy and
Benfey 1997). Similarly, wounded plant tissue develops a
wound periderm (Borchert 1978; Rittinger et al. 1987;
Knobloch et al. 1989; Trockenbrodt 1994), and the files of
cells that form to the outside ultimately become suberized.
Thus, it can be concluded that the distinct chemical compo-
sition and physiological roles of suberized cell walls corre-
lates with their cellular origin.
The primary role of suberized cells appears to be one of
water retention. This conclusion is based on our knowledge
(i) that suberization involves the deposition of a significant
amount of waxes, the removal of which results in an in-
crease in water permeability (Soliday et al. 1979; Vogt et al.
1983; Gil et al. 2000), and (ii) that suberization occurs pri-
marily in epidermal tissues that do not form a cuticle (Esau
1977; Matzke and Riederer 1991). In addition, suberization
of the endodermis and exodermis provides an apoplastic bar-
rier (i.e., Casparian band) to solute transport through the cell
walls (e.g., Peterson et al. 1993, 1999).
It has long been hypothesized that suberized cells act as
preformed, as well as wound-induced, antimicrobial barriers
(Kolattukudy 1980, 1984, 1987), and this is supported by re-
cent evidence showing that Erwinia carotovora and
Fusarium sambucinum infection is greatly enhanced when
suberization is inhibited (Lulai and Corsini 1998). Interest-
ingly, invasion by E. carotovora is prevented by the SPPD

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232 Can. J. Bot. Vol. 80, 2002

Fig. 4. General biosynthetic scheme for the precursors of the suberin poly(aliphatic) and poly(phenolic) domains. The biosynthetic
reactions leading to the lipid and phenylpropanoid precursors of the SPAD (and its associated waxes) and the SPPD are depicted,
starting with starch and sucrose (i.e., the two main sources of biosynthetically available carbon in plants). The layout represents a
composite of knowledge derived from a number of different plant species (principally potato and maize). Details regarding some of
the key reactions are described in the text. No attempt is made to place any pathways or reactions into their appropriate subcellular
compartments although clearly these boundaries exist within the cell. The enzymes indicated are as follows (enzyme names fol-
lowed by an * are hypothetical): 1, starch degrading enzymes; 2, hexokinase; 3, glucose-6-phosphate dehydrogenase; 4,
phosphogluconate dehydrogenase; 5, ribulose-5-phosphate isomerase; 6, ribulose-5-phosphate epimerase; 7, transketolase; 8, sucrose
synthase; 9, UDP-glucose pyrophosphorylase; 10, glucose-6-phosphate isomerase; 11, phopshoglucose isomerase; 12, pyrophosphate
– fructose-6-phosphate 1-phosphorylase; 13, aldolase; 14, triose phosphate isomerase; 15, glyceraldehyde-3-phosphate
dehydrogenase; 16, phosphoglycerate kinase; 17, phosphoglyceromutase; 18, enolase; 19, pyruvate kinase; 20, pyruvate
dehydrogenase; 21, acetyl-CoA carboxylase; 21a, malonyl-CoA:ACP transacylase; 22, β-ketoacyl-ACP synthetase III; 23, β-ketoacyl-
ACP reductase; 24, β-hydroxyacyl-ACP dehydratase; 25, enoyl-ACP reductase; 26, β-ketoacyl-ACP synthetase I, II; 27, stearoyl-
ACP ∆9-desaturase; 28, β-ketoacyl-CoA synthase III; 29, β-ketoacyl-CoA reductase; 30, β-hydroxyacyl-CoA dehydratase; 31, enoyl-
CoA reductase; 32, fatty acyl-ω-hydroxylase; 33, ω-hydroxyacid dehydrogenase; 34, ω-oxoacid dehydrogenase; 35, fatty acyl-9-
hydroxylase*; 36, 9(ω)-hydroxy fatty acyl-10-hydroxylase*; 37, 9(10)-hydroxy fatty acyl-ω-hydroxylase*; 38, 9,10,ω-trihydroxyacid
dehydrogenase*; 39, 9,10-dihydroxy-ω-oxoacid dehydrogenase*; 40, ω-hydroxyacid-9,10-epoxide synthase*; 41, 9,10-epoxy-ω-
hydroxyacid dehydrogenase*; 42, 9,10-epoxy-ω-oxoacid dehydrogenase*; 43, reductases*; 44, glycerol-3-phosphate dehydrogenase;
45, hydroxycinnamoyl-CoA:1-alkanol hydroxycinnamoyl transferase; 46, DAHP synthase; 47, dehydroquinate synthase; 48, 3-
dehydroquinate dehydratase; 49, 3-dehydroshikimate reductase; 50, shikimate kinase; 51, EPSP synthase; 52, chorismate synthase;
53, chorismate mutase; 54, prephenate aminotransferase; 55, arogenate dehydratase; 56, arogenate dehydrogenase; 57, tyrosine
decarboxylase; 58, phenylalanine ammonia-lyase; 59, cinnamate-4-hydroxylase; 60, 4-coumaroyl-CoA ligase; 61, p-coumaric
acid 3-hydroxylase; 62, caffeic acid 3-O-methyltransferase; 63, ferulic acid 5-hydroxylase; 64, p-coumaroyl-CoA-3-
hydroxylase; 65, caffeoyl-CoA-3-O-methyltransferase; 66, hydroxycinnamoyl-CoA-5-hydroxylase*; 67, cinnamoyl-CoA
oxidoreductase; 68, hydroxycinnamaldehyde hydroxylase*; 69, coniferyl alcohol dehydrogenase; 70, hydroxycinnamoyl-CoA:tyramine
hydroxycinnamoyltransferase; 71, p-coumaroyltyramine-3-hydroxylase*; 72, caffeoyltyramine-O-methyltransferase*; 73,
hydroxycinnamoyl-CoA-7-hydroxylase*; 74, (7-hydroxy)-hydroxycinnamoyl-CoA reductase*; 75, thiolase*; 76, NAD(P)H-dependent
oxidase; 77, superoxide dismutase or spontaneous; 78, peroxidase; 84, glutamine synthase; 85, glutamine:2-oxoglutarate
aminotransferase. Reactions denoted by solid lines are known, while those denoted by broken lines are hypothetical or assumed.
Shaded boxes denote known precursors incorporated into the SPAD and SPPD. Adapted in part from Gang et al. (2001), and refer-
ences cited in the text.

while F. sambucinum infection is prevented by the SPAD. Aliphatics

Similarly, exodermal cells with suberin lamellae block the
ingress of Fusarium culmorum hyphae into roots of Zea
mays and Asparagus officinalis (Kamula et al. 1995).
Biosynthesis
The biosynthesis of the monomers of the SPAD and the
SPPD has not yet been elucidated in full, and only sketchy
details are available. Most of the biochemical studies that
have been conducted have used wound healing potato tubers
as a model system, while a few studies have also been car-
ried out using maize. A scheme for the overall biosynthesis
of the SPAD and SPPD precursors is depicted in Fig. 4, and
represents a composite of what is known and hypothesized.
At the most basic level, both aliphatic and phenolic compo-
nents are derived from the products of carbohydrate metabo-
lism, most notably pyruvate, phosphoenolpyruvate, and
erythrose-4-phosphate. A clear point of divergence occurs
where the fatty acid synthesis and shikimate pathways begin,
with the former ultimately giving rise to the 16:0 and 18:0
fatty acids that are the precursors to all of the aliphatics in
suberized tissues and the latter yielding phenylalanine, the
precursor for nearly all phenylpropanoids. As outlined be-
low, key reactions appear to include fatty acid oxidation and
elongation as well as those that govern the metabolic fate of
hydroxycinnamoyl-coenzyme A derivatives.
Close inspection of the monomer composition of the
SPAD (Fig. 1) reveals two basic classes of compounds:
shorter chain (principally C-18), highly oxidized fatty acids
and very long chain (e.g., C-24 to C-32) 1-alkanols, fatty ac-
ids, ω-hydroxy fatty acids and α,ω-dioic acids. Consequently,
oxidation (including that of ω- and mid-chain carbons) and
chain elongation are two features of particular note in the
biosynthesis of suberin aliphatics.
Oxidation
Early work published by Kolattukudy’s group in the late
1970s elucidated the steps in α,ω-dioic acid biosynthesis
(Agrawal and Kolattukudy 1977, 1978a, 1978b). Since then,
little attention has been paid to the unique aspects of
aliphatic monomer biosynthesis during suberization, except
for a series of papers (Pinot et al. 1992, 1993; Tijet et al.
1998) in which a Cyt P-450 from Vicia sativa, capable of the
ω-hydroxylation of both fatty acids and their 9,10-epoxide
and 9,10-hydroxylated derivatives are described. This work
was recently reviewed by Kolattukudy (2001). In summary,
Agrawal and Kolattukudy (1977, 1978a, 1978b) defined a
pair of NADP-dependent dehydrogenases that work in series
to first oxidize the ω-carbon of ω-hydroxyfatty acids to yield
the corresponding ω-oxo-fatty acid, and then the ω-oxo-
carbon to a carboxylic acid (Fig. 4). While the description of
these two dehydrogenases relied on the availability of C-16
and C-18 substrates, the presence of a wide range of α,ω-

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 NADPH
β-Ketoacyl-ACP

CO2 23
22
CO2 ACP
NADPH NADP+
21a NADP+
Bernards

ATP ADP NADP+ NADPH Acetyl-CoA Malonyl-CoA Malonyl-ACP

1 2 3 6-P-glucono- 21 23
Starch Glucose G6P γ-lactone β-Ketoacyl-ACP
CoASH Fatty Acid
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ATP ADP + Pi 26
10 H 2O spontaneous Biosynthesis
NADH CO2
UTP β-Hydroxyacyl-ACP
CO2 Acyl-ACP
G1P 6-P-gluconate

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20
UDP H2O PP 9 NADP+ 25 24
i 4

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8 NAD+ NAD(P)+
NADPH trans-∆2-Enoyl-ACP
Sucrose UDP-Glc CoASH
H 2O
Ru5P NAD(P)H O2 O2 NADP+
NADP+
+ 11 NADPH NADPH
Fructose 5 6 35 36
16:0
18:0 9-OH-FA 9,10-diOH-FA Lipid Modification
Pyruvate
ATP O2 O2
R5P Xu5P
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NAD+ NADPH NADPH

2 37 37
27
ATP NADP+
ADP 7 O2 NADP+ NADP+
19 NADPH
Pi NADH ATP ADP NADH 36
S7P NAD+ ADP 9,ω-diOH-FA 9,10,ω-triOH-FA 9,10-epoxy-ω-OH-FA
Carbohydrate 15 16 17 CO2
18 18:1
Metabolism F6P GAP GBP 3PG 2PG PEP 28
14 O2 NADP+ ? 40
H 2O β-Ketoacyl-CoA 20:0 NADPH
13 32 NADPH NADPH
NADPH 22:0
DHAP ω-OH-FA 18:1-ω-OH-FA 38 41
24:0
29
E4P Chain 26:0 NADP+ NADP+
13 NADPH
12 Elongation 28:0
NADP+ 33
F6P FBP 30:0
46
β-Hydroxyacyl-CoA 32:0
NADP+
Pi
30 ω-oxo-9,10-diOH-FA 9,10-epoxy- ω-oxo-FA
H 2O Pi NADH ω-oxo-FA
48 47
H 2O 43 O2 O2 O2
S3P SHK DHS DHQ DAHP NADPH
trans-∆2
- NADPH NADPH
50 49 Enoyl-CoA NAD+ 34 39 42
31
ADP ATP NADP+ NADPH
NADP+ NADP+ NADP+
NAD(P)H NAD(P)+
1o Alcohols α,ω-dioic acids 9,10-diOH- α,ω-dioic acids 9,10-epoxy- α,ω-dioic acids
51
Shikimic Acid (predominantly 18:0) (predominantly 18:0)
Pi NADH
Pathway
44
EPSP
52 NAD+ Poly(Aliphatic)
Pi Domain,
Chorismate VLCFA Waxes
53

Glycerol-P
Prephenate
Ferulates
Glu
85 54 45
CO2
2-OG CO2
56 57
CoASH
Arogenate Tyrosine Tyramine
Gln Glu
55 O2 SAM SAHC
NADP+
H 2O NADP+ NADPH NADPH
71 72 Feruloyl- 78?
84 CO2 p-Coumaroyl- Caffeoyl-
tyramine Poly(Phenolic)
tyramine tyramine
Phenylalanine Benzoates Domain
* 78?
CoASH
NH4+ CoASH AcetylCoA CoASH
58 70
70 75
CoASH † Hydroxycinnamoyl-
Ammonia 78 H 2O NADP+ NADPH
Cinnamate CoA’s
Recovery O2 NADH
74 77 76
NADPH H2O2 .- O2
73 O2
NAD+ H 2O
59
NADP+ O2 O2 Hydrogen Peroxide
NADPH NADP+ SAM SAHC NADPH SAM SAHC
NADP+ Generation
61 62
p-Coumaric Caffeic Ferulic 63 5-OH-Ferulic 62? Sinapic
Acid Acid Acid Acid Acid
ATP ATP ATP
ATP
Phenylpropanoid CoASH CoASH CoASH
CoASH
Metabolism 60 60 60 60
O2 ADP, Pi O2
ADP, Pi ADP, Pi ADP, Pi
SAM NADPH SAM
NADPH NADP+ SAHC NADP+ SAHC
64 65 66 65?
p-Coumaroyl- Caffeoyl- Feruloyl- 5-OH-Feruloyl- Sinapoyl-
CoA † CoA CoA † CoA CoA †
NADPH
67 67 67

NADP+ O2 NADP+ SAM SAHC O2 NADP+ SAM SAHC

NADPH NADPH
68 62 68 62
p-Coumaryl- Caffeyl Conifer- 5-OH-Conifer- Sinap-
Aldehyde Aldehyde aldehyde aldehyde Aldehyde
69 69 69
Monolignols

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234
dioic acids in suberized tissues (ranging from 18:0 to 32:0,
including 18:1; Fig. 1) suggests that either they have a rela-
tively broad substrate specificity or there is a family of re-
lated monooxygenases differing in their substrate specificity
vis-à-vis carbon-chain length or degree of oxidation.
In addition to α,ω-dioic acid biosynthesis, some mid-chain
oxidation of SPAD aliphatic monomers (principally 18:0)
also occurs (Fig. 4). Since the hydroxylated monomers are
predominantly 9-hydroxy, 9,10- and 9,ω-dihydroxy, and
9,10,ω-trihydroxyoctadecanoic acids, the logical sequence of
hydroxylation begins at carbon 9, followed by hydroxylation
at either carbon 10 or the ω-carbon. While all of these
hydroxylated monomers are found in suberized tissues
(Fig. 1), 9,10,ω-trihydroxy fatty acids can also undergo fur-
ther reaction to yield 9,10-epoxy-ω-hydroxy fatty acids.
Both the 9,10,ω-trihydroxy and 9,10-epoxy-ω-hydroxy fatty
acids can be further oxidized to yield the corresponding α,ω-
dioic acids. It is important to emphasize that, with the excep-
tion of underivatized α,ω-dioic acids, the metabolic se-
quences for fatty acid oxidation outlined in Fig. 4 are
speculative, and are based on the logical arrangement of the
aliphatic metabolites found in suberized tissues. A detailed
analysis of the enzymes and other potential intermediates in-
volved awaits future experimentation, although some have
been described in the context of cutin monomer biosynthesis
(reviewed in Kolattukudy 2001).
Chain elongation
As noted above, suberin aliphatic biosynthesis appears to
diverge at the level of the primary fatty acids, namely 16:0
and 18:0, with some undergoing chain elongation and others,
oxidation. Ultimately, some of the chain-elongated fatty ac-
ids also undergo oxidation reactions, but the site of oxida-
tion appears to be restricted to the ω-carbon and the
formation of VLC α,ω-dioic acids.
Chain elongation was initially demonstrated indirectly by
the incorporation of [14C]acetate into VLCFAs (Dean and
Kolattukudy 1977). More recently, chain elongation has
been shown to occur via a microsomal malonyl-CoA de-
pendent chain elongation pathway in maize (Schreiber et al.
2000). The elongation system preferred 18:0 and generated
22:0 and 24:0 products. The ketoacyl-CoA synthase catalyz-
ing the first step in the elongation of 18:0 via malonyl-CoA
has been cloned from maize (Schreiber et al. 2000) and is
currently under further investigation.
Other SPAD monomers
In addition to the aliphatic monomers described above,
glycerol, ferulates, and esterified hydroxycinnamic acids are
also found as part of the SPAD of suberized tissues. While
glycerol is a common metabolic intermediate, the scheme in
Fig. 4 depicts its origin from dihydroxyacetone phosphate
(DHAP) via glycerol-3-phosphate dehydrogenase. How this
monomer is incorporated into the suberin macromolecule as
an ester of fatty acids and hydroxycinnamates remains un-
known.
The biosynthesis of ferulates has been demonstrated in
wound-induced potato tubers (Negrel et al. 1993; Lotfy et
al. 1994, 1995), and is catalyzed in vitro by a feruloyl-
CoA:ω-hydroxyhexadecanoic acid feruloyltransferase
(based on the only available, readily soluble substrate ω-
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Can. J. Bot. Vol. 80, 2002
hydroxyhexadecanoic acid). It is presumed that the in vivo
substrates include a homologous series of long chain 1-
alcohols, matching those cleaved from ferulates isolated
from potato tubers (Bernards and Lewis 1992). The in-
volvement of feruloyl-CoA in the biosynthesis is consistent
with the strongly induced 4-CL activity in potato tubers af-
ter wounding (Bernards et al. 2000).
The incorporation of hydroxycinnamic acids into the
SPAD is depicted via the CoA esters (Fig. 4), but there is no
supportive evidence for this. No experiments have been con-
ducted to elucidate this aspect of suberin biosynthesis, al-
though it is abundantly clear that they are present as esters in
the mature macromolecule (Riley and Kolattukudy 1975;
Cottle and Kolattukudy 1982; Borg-Olivier and Monties
1993; Schreiber 1996; Zeier and Schreiber 1997; Bento et al.
1998; Schreiber et al. 1999; Graça and Pereira 2000c). It re-
mains to be seen whether there is, as of yet, an unidentified
intermediate involved in the process.
Aromatics
In the context of macromolecular phenolic polymers in
plants, the vast majority of research has focused on lignin
and the biosynthesis of monolignols, which are considered
their primary precursors. Recent advances in the
biosynthesis of monolignols help shed light on what may be
happening in actively suberizing cells, which incorporate ad-
ditional precursors along with the three monolignols.
Readers are referred to the excellent recent review by Dixon
et al. (2001), which summarizes the current status of our un-
derstanding of monolignol biosynthesis, and the implications
it has on how we envisage the flow of carbon through
phenylpropanoid metabolism.
Until recently, phenylpropanoid metabolism has been rep-
resented by a generalized metabolic grid in which all the
hydroxylation, methylation, CoA-ligation and reduction re-
actions leading to the three major monolignols from trans-
cinnamic acid via the hydroxycinnamates and their CoA de-
rivatives occurred in any of a number of reaction sequences
(e.g., Whetten and Sederoff 1995). There is growing evi-
dence, based partly on in vitro substrate specificity studies
of O-methyltransferases, and partly on the characterization
of transgenic plants with modified expression of monolignol
biosynthesis genes (see, for example, Guo et al. 2001; Ralph
et al. 2001), to support the notion that phenylpropane carbon
skeletons are channeled into syringyl alcohol biosynthesis as
early as p-coumaroyl-CoA via its reduction to p-
coumaraldehyde. Subsequent hydroxylation and methylation
reactions then occur with the aldehydes as substrates (Dixon
et al. 2001; Li et al. 1999, 2000; Osakabe et al. 1999). While
the formation of coniferyl alcohol may still occur via the re-
duction of feruloyl-CoA, the commitment step remains the
reduction of hydroxycinnamoyl-CoA esters via CCR. Thus
in lignifying tissues, there is an early commitment of carbon
into the biosynthesis of monolignols via CCR. In support of
this, it was recently shown that the activity of CCR induced
in lignifying Pinus taeda cells was considerably greater than
that in suberizing potato tubers (Bernards et al. 2000). This
aspect of monolignol biosynthesis is highlighted in Fig. 4 in
the distinct, divergent fates of hydroxycinnamoyl-CoA esters
and demonstrates how monolignol biosynthesis channels
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Bernards
away from the biosynthesis of the other phenolics, many of
which also become incorporated into the SPPD.
So what of the other phenolic precursors (i.e.,
hydroxycinnamates, hydroxycinnamate amides) found in the
SPPD? Is their biosynthesis also targeted or channeled? Un-
fortunately, less is known about this aspect of SPPD
biosynthesis. Nevertheless, there is sufficient evidence to be-
gin to piece together what might be happening. For example,
the hydroxylation and methylation of hydroxycinnamic acids
can (and does) occur at the hydroxycinnamoyl-CoA-
derivative level (reviewed in Dixon et al. 2001), and a puta-
tively suberin-specific O-methyltransferase has been cloned
from maize (Held et al. 1993). Thus the formation of
hydroxycinnamates may occur separately from that of
monolignols, and their metabolic fate channeled separately.
In this context, the activity of 4-CL is induced within 12–
24 h of wound-induced suberization (Bernards et al. 2000),
and there are presently at least three known (competing?)
fates for the CoA esters formed: (i) monolignol formation,
(ii) ferulate formation, and (iii) feruloyltyramine formation.
(A fourth fate would be the direct incorporation of
hydroxycinnamates into the SPPD via their CoA derivatives,
but this is discussed in the following section.) Regarding
monolignols, the activity of CCR is low in suberizing pota-
toes (Bernards et al. 2000), and likely represents a relatively
minor fate of hydroxycinnamoyl-CoA’s. By contrast, the in-
duction of HHT (Negrel et al. 1995) and THT (Negrel et al.
1993) (which catalyze the formation of ferulates and
hydroxycinnamoyltyramines, respectively) in potatoes dur-
ing suberization points to their potential role in channeling
phenolics into suberin and SPPD precursors. THT has been
purified (Negrel and Javelle 1997; Hohlfeld et al. 1995,
1996) and cloned (Schmidt et al. 1999) from potato. In
maize, wounding induces THT activity (Ishihira et al. 2000)
leading to the incorporation of hydroxycinnamoyltyramines
into the cell wall. HHT is widespread in plants (Lotfy et al.
1995) and has been purified from tobacco (Nicotiana
tabacum) (Lotfy et al. 1996). The ferulates that are their
metabolic products are common in suberized tissues from a
wide variety of woody and herbaceous plants (Adamovics et
al. 1977; Chatterjee et al. 1977; Laver and Fang 1989; Baldé
et al. 1991; Bernards and Lewis 1992).
Preliminary metabolite profiling data (Razem and
Bernards 2002) introduces several additional intriguing
possibilities into the biosynthesis of SPPD precursors dur-
ing suberization in potato tubers. For example, when the
macromolecular assembly of the SPPD of potato tubers is
inhibited (Bernards and Razem 2001; Razem and
Bernards 2002), one of the main phenolics that accumu-
lates is p-coumaroyltyramine, along with smaller amounts
of feruloyltyramine and no evidence of soluble
monolignols (Razem and Bernards 2002). This suggests
that p-coumaroyltyramine is a metabolic precursor to
feruloyltyramine, although the hydroxylation and
methylation of p-coumaroyltyramine has not been demon-
strated in vitro. It is of interest to note that for potato THT
in vitro, the KM for p-coumaroyl-CoA is lower than that
for feruloyl-CoA, even though the Vmax is also lower
(Hohlfeld et al. 1995). Arguably, the resulting Vmax/KM is
much higher for feruloyl-CoA as substrate, making it a
more efficient carbon skeleton donor, but if channeling is
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235
occurring, then feruloyl-CoA may not be available to THT
in vivo.
Macromolecular structure and assembly
At this point in time, virtually nothing is known about the
macromolecular assembly of the SPAD, and only sketchy,
preliminary details are known about that of the SPPD. De-
spite the paucity of details regarding their assembly, how-
ever, there has been a considerable amount of new published
information regarding the overall structure of both suberin
and the SPPD.
Macromolecular assembly of the SPAD
By analogy with the assembly of cutin (Kolattukudy
2001), it is assumed that the polyesters of the SPAD result
from the transfer of (derivatized) fatty acyl-CoAs to appro-
priate acceptors (i.e., glycerol, ω-, and mid-chain hydroxy
acids) within the matrix. Similarly, the phenolic acids (prin-
cipally p-coumaric and ferulic) are presumed to be incorpo-
rated via their CoA esters, or possibly after the formation of
other derivatives such as glycosides. However, to date no
such transferases or transformations have been described. In
any case, the apparent uniformity and regularity of the poly-
mer, suggested by the uniformity and regularity of suberin
lamellae, would argue that the assembly of suberin is a care-
fully orchestrated, enzyme-mediated process, rather than
random a one.
Macromolecular assembly of the poly(phenolic) domain
Recent progress suggests that the phenolic precursors of
the SPPD are polymerized with a requirement for H2O2
(Bernards and Razem 2001), adding convincing evidence
for the role of an anionic peroxidase that has been impli-
cated for a long time (Kolattukudy 1980, 1984; Espelie and
Kolattukudy 1985; Espelie et al. 1986). Recently it was
shown that polyphenol oxidase is not induced by wounding
in potato, thereby discounting a role for this class of
oxidase in the suberization process (Partington et al. 1999).
Thus, the macromolecular assembly of the SPPD begins
with the biosynthesis of appropriate precursors followed by
their transport to the cell wall (by an as yet unknown mech-
anism), and subsequent polymerization into a 3-
dimensional polymer by a wall-associated peroxidase.
While the precise identity of the peroxidase(s) involved re-
mains unproven, a specific anionic peroxidase has been im-
plicated (Espelie and Kolattukudy 1985; Espelie et al.
1986; Bernards et al. 1999). However, cationic peroxidases
also accumulate during wound-induced suberization
(Bernards et al. 1999) and have been proposed to be in-
volved in tomato (Lycopersicon esculentum) suberin
biosynthesis (Quiroga et al. 2000). The generation of
isoform-specific antibodies and their use in determining the
subcellular location of individual peroxidase isoforms is re-
quired to address this dilemma. Similarly, the source of
H2O2 required by the peroxidase(s) remains unproven, al-
though in potatoes it is likely to be an NADPH-dependent
oxidase (Bernards and Razem 2001; Razem and Bernards
2002), consistent with the role of this enzyme in the
Solanaceae (Doke 1985; Amacucci et al. 1999).
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236 Can. J. Bot. Vol. 80, 2002

Fig. 5. Tentative model for the structure of potato suberin. A portion of a suberized potato cell wall is pictured, including two lamellae
(SPAD) and a fraction of the SPPD. The poly(phenolic) domain is shown restricted to the primary cell wall and covalently attached to
carbohydrate units (C). The bold-faced phenolic components have been isolated using techniques such as thioacidolysis and DFRC, al-
though the linkages between them remain tenuous. The SPAD is represented by a linear, glycerol-based polyester, with esterified
“suberin phenolics”. Evidence has been presented for all of the components represented, although the stacked lipid “bilayer” arrange-
ment shown is hypothetical. In the model, the predominantly aliphatic zones would yield the light bands observed in TEM photomicro-
graphs, while the phenolic rich zones would yield the darker (electron rich) bands. The distance between two glycerol molecules
separated by a C-22 hydrocarbon would be approximately 2.5 nm (roughly equal to the thickness of suberin light bands), while the
combined thickness of a light and dark band would be between 3 and 4 nm. Ferulate esters of long chain fatty alcohols are shown in-
tercalated within the network. These molecules are thought to act as low molecular weight plasticizers within the SPAD. VLCFAs and
waxes are not represented in this structure, but they likely span successive lamellar bands or intercalate within the polyester network.
Overall, the model represents a thin, two-dimensional slice through what is envisioned to be a three-dimensional network. Accordingly,
attachments of the structure to other suberin components as well as the cell wall itself are shown with wavy lines. Connections are to
the following: C, carbohydrate; P, phenolic; S, suberin (phenolic or aliphatic).
OH
OCH 3

P
O O O
H 2C O O
P O S
O
O
O
O O O S
S HO
O
OCH 3 O
O O C O
O
CH 2OH O O O
O O
CH 2OH C OCH 3 S
HO HN OCH 3 O
O O O OH
C O
O C
O O O HO
OCH 3 O
O
(H3CO) OCH 3 OCH O O O S
3
O OCH 3 O O
OCH 3 HO
HOCH 2 O
( S) O O O
O C O O
C
O
O O HO
S O
CH 2OH HO O O S
- O
(COO ) O
O
O O O
HN OCH 3 H 3CO OCH 3 O
C O OH O O C
O O
O C
C O
O O
O OCH 3 HOH 2C O HO S
HOH 2C O O OCH 3 O
O O O
OCH 3
O OH ( S) O
S
P
O O S
(H 3CO) OCH 3 OCH 3 HO
O ( S)

C OH

Primary Cell wall Suberin lamellae

The structures of the SPAD and the SPPD tinct from the SPAD, and resides within the primary cell
In addition to the notion that the SPPD contains a signifi- wall. Thus, in conjunction with the chemical composition
cant amount of hydroxycinnamic acids, two major findings data described above, it is apparent that the SPPD resembles
have revolutionized the way in which we perceive the struc- lignin in both its subcellular location and macromolecular
tures of the SPAD and SPPD: (i) conclusive evidence that assembly, while differing in its precursor composition.
the two domains are spatially distinct (Rittinger et al. 1987; A role for glycerol in the SPAD has become increasingly
Knobloch et al. 1989; Lulai and Morgan 1992; Thomson et clear, and is thought to act as a bridge not only between
al. 1995), while remaining covalently linked (Stark and acyl units, but also between the SPPD and the SPAD
Garbow 1992; Neto et al. 1996; Gil et al. 1997; Lopes et al. (Schmutz et al. 1994; Graça and Pereira 2000b). In point of
2000a, 2000b), and (ii) the demonstration of specific acyl- fact, the presence of glycerol in suberized tissues has been
glyceryl and hydroxycinnamoyl-glyceryl esters in suberized known for some time (Schmutz et al. 1993, 1994) but only
tissues (Schmutz et al. 1993, 1994, 1996; Graça and Pereira recently has its structural role been hypothesized (Schmutz
1997, 1998, 1999, 2000a, 2000b; Moire et al. 1999). For ex- et al. 1996; Moire et al. 1999; Graça and Pereira 2000a,
ample, it is now known, through histochemical (Lulai and 2000b). The current thinking is that glycerol acts as a
Morgan 1992) and NMR studies (Stark and Garbow 1992; linker between successive layers of aliphatics, and provides
Lopes et al. 2000a, 2000b), that the SPPD is spatially dis- for a linear arrangement of acyl units within the three-

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Bernards
dimensional polymer. Indeed, the constant thickness of the
lamellae (i.e., 3–4 nm; Schmutz et al. 1996) is likely due to
this arrangement.
A new structural model
Taken together, the new compositional, biochemical, and
structural data derived from suberized tissues leads to the
need for a new model to help visualize the SPAD and the
SPPD, as well as how they combine to form suberin. Fig. 5
presents one conceptual view of what potato suberin might
look like, based on the evidence presented primarily for po-
tato tubers, but also for cotton (Gossypium hirsutum),
Q. suber and Pseudotsuga menziesii, as well as partial mod-
els recently proposed by Gil et al. (1997) and Lopes et al.
(2000a, 2000b). In Fig. 5, the SPPD is depicted as a
monolignol-hydroxycinnamoyl co-polymer, covalently
linked to primary cell wall carbohydrates. The nature of the
inter-unit linkages remains tenuous, and is based on a pre-
sumed similarity to lignin, as well as dimers isolated from
Q. suber cork after permanganate oxidation (Lopes et al.
1998). The fact that hydroxycinnamic acids are also cova-
lently linked through other than ester linkages, however, has
been demonstrated (Bernards et al. 1995; Negrel et al.
1996), as has their covalent attachment to carbohydrate moi-
eties (e.g., Stark and Garbow 1992). A covalent link between
the SPPD and SPAD, provided via glycerol, is depicted
(Graça and Pereira 1999), although direct links between acyl
groups and both the SPPD and carbohydrates of the primary
cell wall have also been predicted (Gil et al. 1997; Lopes et
al. 2000b).
The SPAD of suberized cells usually has the appearance
of alternating dark and lucent lamellar bands in TEM photo-
micrographs. Historically, this has been attributed to alter-
nating bands of electron dense and electron light
components within the structure. In the proposed model, this
is achieved via the “stacking” of successive layers of
aliphatics, with esterified phenolics (as well as intercalated
ferulates and waxes), in a manner analogous to bilayers. In-
deed, this hypothesis has been advanced earlier (Schmidt
and Schönherr 1982), albeit without the detail shown in
Fig. 5. Significantly, the SPAD is depicted as a linear poly-
ester, consistent with recent literature reports (e.g., Graça
and Pereira 2000a, 2000b).
Concluding remarks
Suberized tissues possess a specialized cell wall modifica-
tion that consists of poly(aliphatic) and poly(phenolic) do-
mains referred to herein as the SPAD and SPPD,
respectively. While these two domains are spatially distinct,
they coexist in suberized cells and are covalently linked to-
gether to form the macromolecule suberin. Their chemical
composition is unique and distinct from the related polymers
cutin and lignin. From biosynthetic and macromolecular as-
sembly points of view, the deposition of suberin, or indeed
either of its domains (i.e., SPAD and SPPD) remains only
partially characterized and is an area ripe for further re-
search. Structurally, however, significant advancements have
been made in recent years, and it is time to rethink how this
important cell wall modification might look in situ.
J:\cjb\cjb80\cjb-03\B02-017.vp
Monday, March 11, 2002 3:22:38 PM
237
While this minireview has attempted to both inform read-
ers about recent advances in suberin research as well as pro-
voke ideas about its identity, suberized tissues clearly remain
an enigma. And while the details about the chemical compo-
sition, biosynthesis, and macromolecular assembly and
structure of suberin are slowly coming to light, it is obvious
that a lot remains to be learned regarding the polymeric size,
types of inter-unit linkages between both phenolic and
aliphatic units, and perhaps more critically, the details re-
garding their macromolecular assembly.
Acknowledgements
The author gratefully acknowledges Dr. Lukas Schreiber,
University of Bonn, Bonn, Germany, and two anonymous re-
viewers for their careful reading of the manuscript and in-
sightful comments. The author’s work was supported by the
Natural Sciences and Engineering Research Council of Can-
ada.
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