The Plant Journal (2010) 62, 277290

doi: 10.1111/j.1365-313X.2010.04144.x

A feruloyl transferase involved in the biosynthesis of suberin

and suberin-associated wax is required for maturation and
sealing properties of potato periderm
Olga Serra1, Carolin Hohn2,, Rochus Franke2, Salome Prat3, Marisa Molinas1, and Merce` Figueras1,,*
Laboratori del Suro, Departament de Biologia, Facultat de Cie`ncies, Universitat de Girona, Campus Montilivi s/n,
E-17071 Girona, Spain,
2
Institute of Cellular and Molecular Botany, University of Bonn, Kirschallee 1, D-53115 Bonn, Germany, and
3
Centro Nacional de Biotecnologa, Consejo Superior de Investigaciones Cientficas, Campus Universidad Autonoma de
Madrid, c/Darwin 3, E-28049 Madrid, Spain

Received 22 December 2009; accepted 6 January 2010; published online 25 February 2010.
*
For correspondence (fax +34 972 418150; e-mail merce.figueras@udg.edu).

These authors contributed equally to this work.

Present address: Institute of Molecular Physiology and Biotechnology of Plants, University of Bonn, Karlrobert-Kreiten-Strae 13, D-53115 Bonn, Germany.

SUMMARY
Suberin and waxes embedded in the suberin polymer are key compounds in the control of transpiration in the
tuber periderm of potato (Solanum tuberosum). Suberin is a cell-wall biopolymer with aliphatic and aromatic
domains. The aliphatic suberin consists of a fatty acid polyester with esterified ferulic acid, which is thought to
play an important role in cross-linking to the aromatic domain. In potato, ferulic acid esters are also the main
components of periderm wax. How these ferulate esters contribute to the periderm water barrier remains
unknown. Here we report on a potato gene encoding a fatty x-hydroxyacid/fatty alcohol hydroxycinnamoyl
transferase (FHT), and study its molecular and physiological relevance in the tuber periderm by means of a
reverse genetic approach. In FHT RNAi periderm, the suberin and its associated wax contained much smaller
amounts of ferulate esters, in agreement with the in vitro ability of the FHT enzyme to conjugate ferulic acid
with x-hydroxyacid and fatty alcohols. FHT down-regulation did not affect the typical suberin lamellar
ultrastructure but had significant effects on the anatomy, sealing properties and maturation of the periderm.
The tuber skin became thicker and russeted, water loss was greatly increased, and maturation was prevented.
FHT deficiency also induced accumulation of the hydroxycinnamic acid amides feruloyl and caffeoyl putrescine
in the periderm. We discuss these results in relation to the role attributed to ferulates in suberin molecular
architecture and periderm impermeability.
Keywords: ferulate esters, BAHD enzyme, suberin, wax, potato periderm, skin set.

INTRODUCTION
The plant periderm is an efficient external barrier in secondary stems, roots and tubers, in which it protects the fleshy parenchyma from infection and water loss for extended
time periods. Potato (Solanum tuberosum) is one of the
most important crops worldwide, and as such its tuber
periderm has been extensively studied (Lulai, 2007). The
periderm is composed of three layers: the phellem or cork
layer, the phellogen or mother layer, and the phelloderm
(Esau, 1965). In potato tuber, the phellogen differentiates by
periclinal divisions of epidermal/hypodermal cells in the
growing stolon tip, and remains active until tuber harvest
(Peterson and Barker, 1979). The dividing phellogen gives
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Journal compilation 2010 Blackwell Publishing Ltd

rise externally to 612 layers of suberized cells that form the

phellem or cork, which is almost impermeable to water and
gases. Internally, it gives rise to a few layers of parenchyma
cells, the phelloderm (Sabba and Lulai, 2002). While the
phellogen is active, its cell walls are thin and fragile, leading
to fracture and separation of the phellem (tuber skin) from
the underlying parenchyma (phelloderm). After the phellogen stops dividing, its cell walls become thicker, and the
phellem adheres to the phelloderm, and so the skin becomes
resistant to fracture, a process that is commonly known as
skin set (Lulai and Freeman, 2001). Skin set occurs after
harvest within a maturation period of 1421 days, during
277

278 Olga Serra et al.

which the phellem also acquires complete lipid coverage
and full water barrier properties (Schreiber et al., 2005;
Lendzian, 2006). Although the metabolic and hormonal
requirements for periderm formation and subsequent maturation are largely unknown, studies using potato discs
showed that abscisic acid (Lulai et al., 2008) but not ethylene
(Lulai and Suttle, 2004) induce suberization. Furthermore,
changes in polyamines have been related to tuberization
(Protacio and Flores, 1992; Mader, 1995) and to formation of
the wound periderm (Kim et al., 2008), although the physiological basis of polyamine activity in the periderm remains
obscure.
The water barrier properties of the periderm are commonly attributed to the hydrophobic nature of the lipids
deposited into phellem cell walls: suberin and suberinassociated wax (Franke et al., 2005). Suberin is a complex
biopolymer comprising polyaliphatic (aliphatic suberin) and
polyaromatic domains (Kolattukudy, 2001; Bernards, 2002;
Franke and Schreiber, 2007; Graca and Santos, 2007; Pollard
et al., 2008). Upon trans-esterification, the aliphatic suberin
releases x-hydroxyacids, a,x-diacids, fatty acids, primary
alcohols, glycerol and small amounts of hydroxycinnamic
acids, mainly ferulic acid (Graca and Pereira, 2000;
Schreiber et al., 2005). The polyaromatic domain is a
lignin-like polymer that mostly contains hydroxycinnamic
acids (Yan and Stark, 2000; Bernards and Razem, 2001).
Waxes embedded in suberin are mixtures of hydrophobic
compounds. In potato periderm, wax accounts for 4% of the
lipids, and mostly consists of linear very long chain
aliphatic compounds up to C32, including a large proportion of ferulate esters of primary alcohols (Bernards and
Lewis, 1992; Schreiber et al., 2005). The suberinwax complex is deposited between the primary cell wall and the
plasma membrane, and often appears as a layer of
alternating light and dark lamellae under electron microscopy (Schmidt and Schonherr, 1982). It has been proposed
that the translucent lamellae correspond to aliphatic constituents and the dense lamellae correspond to to aromatic
constituents (Schmutz et al., 1993, 1996; Graca and Santos,
2007). However, the molecular architecture of suberin
remains hypothetical (Pollard et al., 2008), but several
tentative models have been proposed (Bernards, 2002;
Graca and Santos, 2007). The models suggest an important
role for ferulate esters in linkage of the aliphatic suberin to
the polyaromatic domain (Graca and Santos, 2007) and to
polysaccharides (Iiyama et al., 1990; Pollard et al., 2008;
Graca, 2009). However, papers reporting on enzymes relevant to suberin biosynthesis are scarce, and crucial steps of
suberin biosynthesis remain controversial (Bernards, 2002;
Franke and Schreiber, 2007; Pollard et al., 2008). Reverse
genetic approaches in Arabidopsis and potato have demonstrated the involvement of a acyltransferase (GPAT),
CYP86A fatty acid x-hydroxylases and 3-ketoacyl-CoA
synthase (KCS) fatty acid elongases in suberin biosynthesis

(Beisson et al., 2007; Li et al., 2007; Hofer et al., 2008; Franke

et al., 2009; Serra et al., 2009a,b), ultrastructure (Serra et al.,
2009b) and sealing capacity (Serra et al., 2009a,b).
The BAHD family of plant acyl transferases (named based
on the first letter of each of the first four members biochemically characterized: BEAT, benzyl alcohol acetyltransferase;
AHCT, anthocyanin-O-hydroxycinnamoyltransferase; HCBT,
anthranilate-N-hydroxycinnamoyl/benzoyltransferase; DAT,
deacetylvindoline 4-O-acetyltransferase) are able to catalyze
aliphatic feruloylation (DAuria, 2006) using hydroxycinnamoyl CoA esters as acyl donors. The involvement of the
BAHD family in suberin biosynthesis was highlighted for the
first time using a transcriptomic approach of the phellem of
cork oak (Quercus suber) (Soler et al., 2007), which identified
a BAHD gene that was annotated as N-hydroxycinnamoyl/
benzoyltransferase-like (HCBT). Like other genes whose role
in suberin has been already demonstrated (GPAT5 and
CYP86A), HCBT transcripts showed highest accumulation
during the peak of cork seasonal growth (Soler et al., 2008).
In addition, a putative ortholog of this cork oak BAHD
acyltransferase gene was obtained from a subtracted potato
phellem library in our laboratory. Hence, this potato gene,
referred to below as FHT (fatty x-hydroxyacid/fatty alcohol
hydroxycinnamoyl transferase), is a strong candidate for
esterifying ferulic acid to suberin. At the same time, studies
with T-DNA mutants of the putative Arabidopsis ortholog
gene (At5g41040) proved involvement of the enzyme in
production of suberin ferulate esters (Gou et al., 2009;
Molina et al., 2009). The At5g41040 T-DNA mutants showed
low content of ferulic acid in suberin, increased water
permeability in the root and seed coat (Gou et al., 2009;
Molina et al., 2009), and an unaffected suberin ultrastructure
(Molina et al., 2009).
Here we report on the functional characterization of FHT
by a reverse genetic approach in potato, a model plant for
periderm and suberin studies. The effects of gene silencing
were analyzed in terms of the chemical composition, anatomy, ultrastructure and water permeability of the tuber
periderm. The chemical phenotype of FHT RNAi periderm is
consistent with a lack of feruloyl transferase activity on fatty
acid derivatives of suberin and suberin-associated wax, in
agreement with the in vitro activity of the purified FHT
protein. We discuss the role of ferulate esters in suberin and
periderm sealing properties. We also discuss changes
affecting the periderm maturation process and in the soluble
phenolics profile that occurred concomitantly with FHT
silencing.
RESULTS
An EST encoding a BAHD acyltransferase (FHT) was isolated
from a subtractive library for tuber skin of potato (S. tuberosum), constructed using tuber parenchyma as a control
tissue. The full-length sequence of this potato EST
(TC169622, http://compbio.dfci.harvard.edu) was obtained

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FHT function in potato periderm 279

from a potato periderm cDNA. A putative orthologous
function of FHT, At5g41040 (similarity 79%) and proteins
encoded by the previously mentioned cork ESTs (Soler
et al., 2007) (similarity 8892%) can be reasonably assumed,
as they share the highest homology in BLASTX analyses
(Altschul et al., 1990). Alignment of these proteins
(Figure S1) confirmed that FHT has the two conserved
motifs characteristic of BAHD acyltransferases: the HxxxD
motif involved in catalysis and the DFGWG motif located at
the C-terminal end (St Pierre and De Luca, 2000). WoLF
PSORT analysis (Horton et al., 2007) indicated that FHT is
most likely a cytosolic enzyme, as neither signal peptides for
targeting to organelles nor transmembrane domains were
recognized. The phylogenetic tree of BAHD enzymes
characterized to date (including FHT) is shown in Figure S2
(Appendix S2). The tree agrees with previously reported
trees by Stewart et al. (2005), DAuria (2006) and Yu et al.
(2009).

(a)

(b)

FHT down-regulation in potato plants

To confirm that FHT is involved in suberin biosynthesis, we
examined the expression pattern of this gene in various
potato tissues. Northern blot analysis showed high levels of
transcript in the tuber periderm and lesser amounts in the
root, where suberin is also deposited (Figure 1a). No transcript was detected in leaf, stem or tuber parenchyma.
Analysis of microarray data from The Institute for Genomic
Research (TIGR) Solanaceae Genomics Resource (http://
www.jcvi.org/potato/) confirmed this expression pattern,
with the level of transcript in periderm being seven times
higher than that in roots (113_Robin). Microarray data also
showed that FHT expression begins during stolonto-tuber transition in both in vitro cultured (049_Hannapel)
and soil-grown plants (087_Stupar).
The in planta function of FHT was investigated by stable
and constitutive down-regulation of the gene in potato. RNA
interference (RNAi) was performed using a non-conserved
fragment of 204 bp identified by BLASTN analysis against
the ESTs available in the TIGR potato database. The possibility of off-target silencing can be reasonably excluded as
the minimum number of identical consecutive nucleotides
for cross-silencing of unintended sequences is reported to
be 21 (Xu et al., 2006), and our RNAi fragment has at most
52% similarity and 14 identical consecutive nucleotides
compared with other ESTs in the TIGR database (Table S1).
Northern blot analysis of kanamycin-resistant plants confirmed FHT down-regulation in a number of lines, and those
showing a substantial decrease in the FHT transcript (FHT
RNAi lines 4, 11 and 37) were further selected for periderm
characterization (Figure 1b).
FHT RNAi lines developed normally, both in vitro and in
soil, and were identical to the untransformed controls with
regard to the aerial part of the plant, the roots and the
stolons. Tubers also formed in similar number, shape and

Figure 1. FHT transcript profile and FHT down-regulation in potato tuber skin
by Northern blot.
(a) Analysis of FHT transcript accumulation in stem, leaf, root, tuber
parenchyma (par.) and tuber periderm (per.).
(b) Accumulation of FHT transcripts in the tuber skin of nine independent
kanamycin-resistant lines and wild-type as a control.
Northern blots and ethidium bromide-stained RNA are shown in the upper
and lower panels, respectively, of (a) and (b).

size, but their skin displayed a clear phenotype caused by

loss of gene function. FHT RNAi tubers showed a darker
color at harvest, were less glossy and their skin was
appreciably rough and showed russetted scabby lesions
compared to the controls (Figure 2a,b). Russeting was found
to affect the whole tuber surface independently of tuber size,
suggesting an association with FHT loss of function. The
skin phenotype of the silenced tubers became more apparent during post-harvest storage, with transgenic tubers
showing a darker color and an increasingly russetted skin
upon maturation and storage. The tubers also lost weight
very fast and became progressively more and more wrinkled. Moreover, in contrast with wild-type tubers, which
completed the skin set process within the normal 1421 day
period, transgenic tubers were unable to complete skin
maturation and remained prone to excoriation through the
entire storage period (up to 60 days). In addition, peel
fragments from transgenic tubers were much smaller than
those from controls.
Effects of FHT deficiency on skin anatomy
The skin anatomy of the tubers was analyzed in mature soilgrown tubers of the selected silenced lines stored for
21 days. Under the stereoscopic microscope, the skin surface of the FHT RNAi tubers appeared duller and showed

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280 Olga Serra et al.

(a)

(b)

(c)

(d)

(e)

(f)

intense grid-like splitting and cracking, which is typical of

scabby lesions (Figure 2a,b). Under scanning electron
microscopy (SEM), the skin surface appeared less turgid
than in controls and the cell walls appeared wrinkly and
grainy due to collapse of the phellem cell layers (Figure 2cf).
As seen in cross-sections, the skin of transgenic tubers was
more than twice as thick as that of the wild-type, with more
phellem cell layers and a less regular cell organization
(Figure 2e,f). The crack lesions penetrated through the
phellem cell layers, and a second deeper phellem often
appeared where cracks were located, separated from the
upper phellem by one or two rows of parenchyma cells filled
with starch grains (Figure 2f, arrows). Directly exposed
parenchyma was never seen, and fungi or bacteria did not
associate with the cracks. To further rule out the possibility
that the scabby lesions of FHT-silenced tubers were not
caused by increased susceptibility to common scab infection
(Streptomyces spp.), we analyzed the skin phenotype in
tubers obtained from axenic cultured plants. The skin of the

Figure 2. Periderm anatomy for 21-day-stored

tubers grown in soil.
(a) Wild-type tuber.
(b) FHT RNAi tuber.
(c) SEM micrograph of the skin surface from a
wild-type tuber.
(d) SEM micrograph of the skin surface from an
FHT RNAi tuber.
(e) Cross-sectional SEM micrograph of wild-type
tuber periderm.
(f) Cross-sectional SEM micrograph of FHT RNAi
tuber periderm. The arrows indicate starch
grains between the periderms (P).

FHT RNAi in vitro cultured tubers showed a rougher

appearance and developed scabby lesions, and the controls
appeared glossy and smooth (Figure S3). This skin phenotype was corroborated by SEM analyses of periderm membranes obtained by cellulase and pectinase treatment (see
Experimental procedures). These membranes, which correspond to the enzymatically isolated phellem layer, were
examined on their inner and outer surfaces. The outer surfaces showed the same differences described in the skin of
FHT RNAi and wild-type tubers. No differences were
observed on the internal surface, which was rather uniform
and comprised undistorted cells with smooth cell walls
(Figure S4). Moreover, periderm membranes isolated from
FHT RNAi tubers were characterized by an increased
mass:surface ratio compared to controls (1.2 times heavier
at harvest and 3.6 times heavier after 21 days storage, on
average). They were also stiffer, broke more easily, and,
once dried, did not tend to roll up as wild-type membranes
do.

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FHT function in potato periderm 281

Effects of FHT deficiency on the water barrier capacity
As an indication of the effect of FHT down-regulation on
peridermal sealing properties, the percentage of tuber
weight loss was measured (Figure 3a). Tubers from wildtype and the three independent FHT down-regulated lines
(4, 11 and 37) were analyzed after storing them at room
temperature in the dark. After 11 days of storage, transgenic
tubers had lost approximately 10% more weight than wildtype controls (Figure 3a). Effects on water permeability were
also quantitatively determined using periderm membranes
enzymatically isolated from tubers stored for 21 days (Figure 3b). The water permeance per unit area was on average
15 times higher in FHT RNAi periderm compared to wildtype, whether membranes were from fresh tubers or those
stored for 21 days.

Effects of FHT down-regulation on aliphatic suberin and

wax composition
The effects of FHT down-regulation on the composition of
suberin and wax were analyzed by gas chromatography
using periderm membranes from tubers stored for 21 days.
The amount of suberin, which accounts for 96% of the
periderm lipids, remained practically unchanged in FHT
RNAi periderms (wild-type 422  71 lg cm)2; FHT RNAi
384  124 lg cm)2), but its composition was considerably
altered (Figure 4). Upon trans-esterification, suberin from
FHT RNAi periderms yielded much lower quantities of C18:1
x-hydroxyacid (76% reduction), ferulic acid (72% reduction)
and primary alcohols, except C20 primary alcohol, but the
relative amounts of fatty acids increased, with the exception

(a)

(a)

(b)

(b)

Figure 3. Water permeance of FHT RNAi lines.

(a) Evolution of tuber weight over the first 11 days of storage. Values are
means  SD for three independent FHT RNAi lines (n = 11, n = 3 and n = 9)
and wild-type (n = 12).
(b) Water permeance of enzymatically isolated periderm membranes
mounted in transpiration chambers. Values are means  SD for three FHT
RNAi independent lines (n = 3, n = 3 and n = 37 for freshly harvested tubers;
n = 4, n = 6 and n = 6 for tubers stored for 21 days) compared to the wild-type
(n = 11 for freshly harvested tubers; n = 14 for tubers stored for 21 days).

Figure 4. Relative amounts (lg) of aliphatic suberin monomers released after

methanolic trans-esterification of wax-free periderms from FHT RNAi and
wild-type tubers stored for 21 days.
(a) x-Hydroxyacid and a,x-diacid monomers.
(b) Aromatic, fatty acid and primary alcohol monomers.
Values are means  SD of three independent FHT RNAi lines (n = 2, n = 2 and
n = 1) and wild-type (n = 3) (t test for comparison against wild-type:
*P < 0.05; **P < 0.01).

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282 Olga Serra et al.

of C30 fatty acid (Figure 4). The amount of glycerol released
by trans-esterification and measured by a biochemical
method was not affected (wild-type 63  20 lg cm)2; FHT
RNAi 72  10 lg cm)2). With regard to wax, which accounts
for only 4% of the periderm lipids, the content was doubled
in FHT down-regulated lines (wild-type 14.8  1.9 lg cm)2;
FHT RNAi 33.0  3.3 lg cm)2), and there was also a
significant alteration in the lipid profile. As a whole, the
relative amount of ferulate esterified with primary alcohols
was reduced, while the relative amount of free fatty acids
and primary alcohols was increased, and that of alkanes
decreased (Figure 5). Similar results in terms of suberin and
wax composition were obtained from freshly harvested
tubers and those stored for 60 days (Figures S5 and S6).
FHT biochemical activity
To further demonstrate the enzymatic activity of FHT, the
coding sequence was inserted into a bacterial expression
vector to produce FHT fused to an N-terminal GST tag. The
recombinant protein, which was found to be soluble after
induction of expression at 18C, was purified by affinity
chromatography on glutathioneagarose beads, and the
native FHT protein was subsequently released by digestion

with thrombin. SDSPAGE analysis was used to test the

purity of the obtained protein, which appeared as a single
band with an apparent molecular mass of 55 kDa (Figure S7). The purified FHT was used to determine the ability
of the protein to transfer ferulic acid to the alcohol group of
an aliphatic compound. We used feruloyl CoA as acyl donor
and C16:0 x-hydroxyacid (x-hydroxypalmitic acid) and a
series of primary alcohols of various chain lengths (C7, C8,
C12, C14, C16, C18, C20) as acyl acceptors. The reaction
products were analyzed by HPLC, and new and unique peaks
appeared in the reactions containing C16:0 x-hydroxyacid
(Figure 6), 1-dodecanol (C12) and 1-tetradecanol (C14)
(Figure S8). The products had UV spectra similar to
that of ferulate. The reaction product containing C16:0
x-hydroxyacid was analyzed by LC-MS to confirm the nature
of the product. The mass of main molecular ion
([M ) H]) = 447 m/z and [M + H]+ = 449 m/z) indicates that
ferulic acid is conjugated with C16 x-hydroxypalmitic acid.
Consistently, the fragment ions obtained suggest the
presence of feruloyl residues, and are compatible with the

(a)

(a)

(b)
(b)

Figure 5. Relative amounts (lg) of periderm wax compounds released after

chloroform:methanol (1:1 v/v) treatment of FHT RNAi and wild-type tubers
stored for 21 days.
(a) Ferulic acid esters of primary alcohols, indicating the aliphatic carbon
chain length.
(b) Alkanes, free fatty acids and primary alcohols.
Values are the means  SD of three independent FHT RNAi lines (n = 2, n = 1
and n = 1) and wild-type (n = 3) (t test for comparison against wild-type:
*P < 0.05; **P < 0.01).

Figure 6. In vitro FHT activity.

(a) HPLC chromatograms monitored at 320 nm showing the reaction product
after incubating feruloyl CoA and C16:0 x-hydroxyacid (x-hydroxypalmitic
acid) with and without FHT. When FHT was added, a unique peak appeared at
a retention time of 16.4 min. The MS of this product showed diagnostic ions at
m/z 447 and 449 in negative and positive modes, respectively, corresponding
to x-feruloyloxypalmitic acid.
(b) Reaction scheme proposed for FHT, representing the transfer of feruloyl CoA to the alcoholic group of the fatty acid derivative x-hydroxypalmitic
acid.

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FHT function in potato periderm 283

fragmentation pattern of x-feruloyloxypalmitic acid (Figure S9).
Effect of FHT on suberin ultrastructure
To test the effects of FHT silencing on suberin ultrastructure,
samples of periderm from tubers stored for more than
1 month were examined by transmission electron microscopy (TEM). FHT deficiency produced no significant changes
in the fine architecture of the phellem cell walls in comparison to the wild-type (Figure 7). In FHT RNAi periderms, the
suberin wall was well developed and showed the typical
light and dark lamellation of the polymer. Moreover, no
consistent differences in either the thickness or the electron
density of primary and tertiary walls were observed. However, the vestiges of organellar structures inside the cells
were seen more frequently in transgenic periderms than in
wild-type controls. Analyses of periderms from in vitro
tubers confirmed the above observations.

analyzed by LC-MS at 320 nm (see Experimental

procedures). The LC profiles obtained are shown in Figure 8,
and the various compounds were identified based on their
fragmentation patterns (Appendix S1). Compared to wildtype, profiles of FHT down-regulated periderms showed
peaks corresponding to caffeoylputrescine (CafP) and
feruolylputrescine (FP), and also, in trace amounts, dimers
of feruloyltyramineferuloyltyramine (grossamide, GA) and
feruloyltyramineferuloyloctopamine (FT + FO). In both FHT
RNAi and wild-type periderms, chlorogenic acid (GCA),
feruloyltyramine (FT) and feruloyloctopamine (FO) were
present, but caffeic acid (Caf) was only detected in the wildtype. These results were observed in all three silenced lines
(4, 11 and 37).

(a)

FHT silencing induced changes in soluble phenolics

of periderm
To investigate whether down-regulation of FHT has
an impact on soluble hydroxycinnamates, phenolic
compounds extracted from the tuber skin were qualitatively

(b)

Figure 7. TEM micrograph of phellem cell wall from FHT-RNAi and wild-type
tubers, showing suberized secondary wall (SW) with the typical suberin
lamellae and the polysaccharide primary wall (PW) and tertiary wall (TW).
The lamellation of the suberin fine structure is similar in wild-type and FHT
RNAi tubers.

Figure 8. Qualitative HPLC analysis of soluble phenolics extracted from wildtype and FHT RNAi tuber periderm.
(a) Profiles at 320 nm of the methanolic extracts of skin. The alteration of the
soluble phenolic profile in FHT-deficient periderm occurs concomitantly with
the decrease in ferulate esters in suberin and the associated wax.
(b) Structures of soluble phenolics identified in the methanolic extracts of (a).

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284 Olga Serra et al.

confirmed here by in vitro activity assays, as for At5g41040
protein (Gou et al., 2009; Molina et al., 2009).

DISCUSSION
Here we report the role of a phellem BAHD acyltransferase,
FHT, using a loss-of-function approach. Phenotypic characterization of the silenced lines and FHT activity assays
showed an important function of this enzyme in the esterification of ferulic acid to fatty acid derivatives in both suberin
and periderm wax. Potatoes deficient in FHT exhibited
increased water loss, rough scabbed skin and impaired skin
maturation. Moreover, FHT deficiency also affected the
soluble phenolic composition of the periderm. The significance of these results is discussed in terms of suberin biosynthesis and ultrastructure and periderm sealing
properties. Our results provide relevant insights into the
function of ferulate esters in the periderm, and raise new
questions about factors involved in periderm anatomy and
maturation.
FHT is a fatty alcohol/fatty x-hydroxyacid hydroxycinnamoyl acyltransferase involved in suberin biosynthesis
Feruloyl transferase activity on x-hydroxyacids and/or primary alcohols had been demonstrated in protein extracts
from suberizing potato wound periderm (Lotfy et al., 1994),
in the roots of various angiosperms and gymnosperms
(Lotfy et al., 1995) and also in purified protein fractions
from tobacco cell cultures (Nicotiana tabacum; Lotfy et al.,
1996) and elicitor-treated French bean cells (Phaseolus
vulgaris; Bolwell et al., 1997). Very recently, Gou et al.
(2009) and Molina et al. (2009) identified and characterized
the candidate gene for this function in Arabidopsis,
At5g41040, referred to as hydroxycinnamoyl CoA:x-hydroxyacid O-hydroxycinnamoyl transferase (AtHHT) and
aliphatic suberin feruloyl transferase (ASFT) by the
respective authors. The proteins encoded by FHT and
At5g41040 show a putative orthologous relationship, and
share no close similarity with previously characterized
BAHD enzymes (Figure S2). In potato periderm, ferulate is
linked to x-hydroxyacid and primary alcohols in suberin
(Graca and Pereira, 2000), and to primary alcohols in the
suberin-associated wax (Schreiber et al., 2005). In both
lipid fractions, down-regulation of FHT produced a large
reduction of ferulic acid esters. In suberin, ferulic and
C18:1 x-hydroxyacid were equally reduced by approximately 70%, together with a reduction of primary alcohols
(except C20). Periderm wax showed a reduction of ferulate
esters of primary alcohols, together with an increase in
free primary alcohols. These results are consistent with a
lack of feruloyl transferase activity acting on C18:1
x-hydroxyacid and on primary alcohols. In agreement,
At5g41040 knockout mutants showed a striking decrease in
ferulic acid and changes in the suberin lipid profile (Gou
et al., 2009; Molina et al., 2009). The feruloyl transferase
activity of FHT on x-hydroxyacids and fatty alcohols was

FHT down-regulation does not alter suberin lamellation

Our observations confirm that the typical suberin lamellation is not altered by a deficiency in aliphatic ferulate esters,
as also shown in the root of At5g4140 mutants (Molina et al.,
2009). This fact calls into question the function commonly
attributed to ferulate esters in the suberin structure. It has
been proposed that the basis for suberin macromolecular
organization is the glycerola,x-diacidglycerol unit (Graca
and Santos, 2007), and that ferulate esters are key for linking
aliphatic suberin (translucent lamellae) to polyaromatics
(opaque lamellae) (Arrieta-Baez and Stark, 2006; Graca and
Santos, 2007). Accordingly, use of inhibitors of both fatty
acid elongation (Soliday et al., 1979; Schmutz et al., 1996)
and phenylpropanoid biosynthesis (Schmutz et al., 1993)
induce disorganization of the suberin lamellae. Even more
significant is the significant distortion of lamellae in
CYP86A33 down-regulated potato periderm, in which C18:1
a,x-diacid and glycerol amounts are greatly reduced (Serra
et al., 2009b). Conversely, FHT RNAi periderm, which
contains normal amounts of a,x-diacid and glycerol but significantly reduced amounts of ferulic acid and C18:1
x-hydroxyacid, maintains the typical lamellar structure.
Therefore, ferulic acid appears not to be required (or
required only in very minor amounts) to establish the
alternating structure of translucent and opaque layers.
FHT deficiency and the control of vapour water loss
The FHT RNAi periderm showed increased water permeability, as did the seed and root of Arabidopsis At5g41040
mutants (Gou et al., 2009). Periderm protection against
water loss is commonly attributed to suberin and its
associated wax, but how these lipids control transpiration
is still unclear (Lendzian, 2006). Wax plays a key role, as
shown by the increase in permeability (33100-fold) in waxfree potato periderm (Vogt et al., 1983; Schreiber et al.,
2005; Lendzian, 2006). Suberin also contributes, as
CYP86A33 RNAi periderm, with a reduced suberin load and
distorted suberin lamellae, shows a 3.5 times higher water
loss than wild-type (Serra et al., 2009b). Of note, FHT RNAi
periderm, with double the amount of wax, a normal
amount of suberin and typical suberin ultrastructure, had a
14-fold increase in water permeability. Although we cannot
rule out the possibility that qualitative changes in the
periderm aliphatic composition are responsible for the increase in transpiration, it is tempting to speculate a role for
ferulic acid in maintaining the water barrier. The decrease
in ferulic acid could lead to a weaker binding between
aliphatic and aromatic suberin domains and/or to cell-wall
polysaccharides, and therefore to a less cohesive and more
permeable periderm.

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FHT function in potato periderm 285

Effects of FHT deficiency on free and conjugated phenolic
compounds
In addition to providing protection against water loss, the
periderm also serves as an important barrier against pathogens, a function in which soluble phenolics play an important role (Dixon and Paiva, 1995). In periderm, feruloyl CoA is
central to the synthesis of suberin and wax compounds, and
also to that of soluble phenolics (Bernards, 2002). Thus, it can
be reasonably assumed that accumulation of feruloyl CoA as
a result of FHT deficiency may lead to a change in the soluble
phenolic content and profile. In potato periderm, soluble
phenolics mainly consist of chlorogenic and caffeic acids and
hydroxycinnamic acid amides (Razem and Bernards, 2002;
Helmja et al., 2007). Compared to wild-type, methanolic
extracts from FHT RNAi periderms contained new compounds, mainly feruloyl and caffeoyl putrescine, while other
compounds such as caffeic acid disappeared (Figure 8). It is
worth stressing that feruloyl and caffeoyl putrescine, which
are not normally observed in native periderms of the Desiree
cultivar, have been implicated in new formation of wound
periderm (Kim et al., 2008; Matsuda et al., 2005), and their
amount increased in Phytophtora infestans elicitor-treated
potato cells (Keller et al., 1996). Similarly, feruloyl amides
have been found to be associated with lesions of common
scab-infected potato (King and Calhoun, 2005).
FHT-deficient potatoes have russeted skin and impaired
periderm maturation
FHT down-regulation turned the smooth thin skin that is
characteristic of potatoes of variety Desiree to a rough
darker scabbed skin, typical of the Russet Burbank variety
(http://www.inspection.gc.ca/english/plaveg/potpom/var/
russetburbank/russetbe.shtml). Russeting is a natural tuber
growth trait typical of some varieties, whose genetic and
regulatory mechanisms are unknown. In Russet Burbank
potatoes, the periderm is thicker because of adhered cork
layers on the outer side (Okazawa and Okazawa, 1980; Sabba
and Lulai, 2002), and is more permeable and has the potential to lose more water vapor before maturing compared
to smooth-skinned varieties (Lulai and Orr, 1994). A russeted
skin is also associated to the common scab disease caused
by Streptomyces spp., which secretes a toxin (thaxtomin)
that stimulates the production of more cork layers (Spooner
and Hammerschmidt, 1992). Both Russet Burbank potatoes
and common scab lesions have a thicker and more brittle
skin, as occurs in FHT RNAi periderm. On the other hand, the
potato periderm acquires resistance to skinning through the
maturation process and the skin set is necessary for good
storage duration of the potatoes (Lulai and Orr, 1993). In the
mature periderm, the phellogen is meristematically inactive,
and acquisition of skinning resistance correlates with thickening and strengthening of the phellogen cell walls (Sabba
and Lulai, 2002). However, the biochemical and regulatory

processes involved in periderm maturation and skin set are

largely unknown (Lulai, 2007). It is noteworthy that FHT downregulation prevented skin set, as silenced tubers remained
prone to skinning even after two months of storage.
EXPERIMENTAL PROCEDURES
Plant material and growth conditions
Potato plants (S. tuberosum cv. Desiree) were propagated in vitro
and in soil as described previously (Serra et al., 2009b). For in vitro
propagation, stem cuttings were cultured in Murashige and Skoog
(MS) medium (Duchefa, http://www.duchefa.com) supplemented
with 2% w/v sucrose, and grown in growth cabinets under a light/
dark photoperiod cycle of 16/8 h at 22C and 67 lmol m)2 sec)1. In
vitro-grown plants were transferred to soil and grown for approximately 2 months in the greenhouse for tuber production. Tubers
were harvested from 8-week-old plants and stored at room temperature before analysis. For in vitro tuberization, stem cuttings of
two nodes were cultured in MS medium supplemented with 8% w/v
sucrose. Tuber initiation was induced under short-day conditions (8/
16 h light/dark) at 22C and 67 lmol m)2 sec)1 for 1 week, and then
potato cultures were kept under dark conditions to develop the
microtubers (Dobranszki, 2001). Three independent transgenic lines
were used throughout this research. Wild-type lines were grown in
parallel.

Full-length sequence of potato FHT and partial sequence

of cork FHT1
The full-length sequence of potato FHT (accession number
FJ825138) was obtained by TBLASTX analysis against the TIGR
potato database (http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/
Blast/index.cgi) using the FHT EST identified previously in the
subtracted library of potato tuber skin as the query. The tentative
consensus TC169622 was the best match. The FHT complete coding
region was PCR-amplified from tuber skin cDNA. First-strand cDNA
was synthesized using SuperScript III reverse transcriptase
(Invitrogen, http://www.invitrogen.com/) and an oligo(dT)16 primer.
PCR was performed using Advantage polymerase (Clontech,
http://www.clontech.com/) and the TC169622-specific primers
5-ATGGAGAGTGGTAAAAACAATGT-3 and 5-AGCTGCAATATG
GCTGTTTAG-3.
The partial sequence of cork oak FHT1 (accession number
CQ329869) was obtained from the two cork ESTs previously
identified (accession numbers EE743861 and EE745210) (Soler
et al., 2007). The upstream region of FHT1 was obtained using
cDNA from cork oak bark, the reverse primer complementary to EST
EE743861 (5-GGGTTGCGGGCCTTAAGTA-3) (Soler et al., 2008)
and 5 RACE (Invitrogen). The partial sequence of FHT1 was
obtained using primers complementary to this new upstream
sequence and to the 3 end of EST EE745210 (5-AGCCA
ACTTTGGTTCCTCCT-3 and 5-GACTTGGCCACGCATAATTT-3).

Phylogenetic analysis and sequence alignment

Amino acid sequence alignment of BAHD family members was
performed using the ClustalW program at the European Bioinformatics Institute (http://www.ebi.ac.uk/Tools/clustalw/). The alignment was edited using the BioEdit Sequence Alignment Editor
version 7.0.1. (Hall, 1999). Phylogenetic analyses were conducted
using MEGA version 4 (Tamura et al., 2007). The tree was constructed using a neighbor-joining algorithm, and bootstrap values
(%) for the nodes were obtained based on 1000 trials. Amino acid

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286 Olga Serra et al.

sequence alignment of cork oak FHT1, cork oak ESTS, EE743848 and
EE743849, Arabidopsis At5g41040 and potato FHT was performed
using the Multalin program (Corpet, 1988).

Plasmid construction
Silencing of FHT in potato plants was performed using a 204 bp
fragment that encompasses nucleotides 532736 from the first
nucleotide of the coding sequence. This fragment was PCR-amplified from the cSTS5P17 clone (supplied by Arizona Genomics
Institute, Tucson, accession number BG593863) using primers
5- AATTCTTGGGGTGAAACTGCT-3 and 5-TTCAAGCTTCTCAGGGTCAAA-3, which have attB1 and attB2 recombinant sequences,
respectively, at their 5 ends. The PCR product was cloned firstly into
the donor plasmid pDONR207 using BP clonase II (Invitrogen), and
secondly into the binary destination vector pBIN19RNAi using LR
clonase II (Invitrogen), as previously reported (Serra et al., 2009b).

Plant transformation for RNAi-mediated silencing

Potato leaves were infected with the Agrobacterium tumefaciens
strain GV2260 transformed with the RNAi recombinant plasmid
(Hofgen and Willmitzer, 1990) as previously described (Banerjee
et al., 2006). Kanamycin-resistant plants were regenerated and
grown until tuber development. Then they were analyzed for
accumulation of FHT mRNA in the tuber skin.

RNA isolation and Northern analysis

Total RNA was isolated from potato tissues using the guanidine
hydrochloride method (Logemann et al., 1987). The FHT probe was
obtained using the cSTS5P17 clone by the random primed method
(Roche, http://www.roche.com). Hybridization was carried as previously described (Amasino, 1986) using the conditions previously
reported (Serra et al., 2009b).

Isolation of periderm membranes

Removal of the unsuberized parenchymatic cells of periderm from
freshly harvested tubers and tubers stored for 21 or 60 days at room
temperature was performed using a mixture of 2% v/v cellulase and
2% v/v pectinase in 10)2 M citric buffer pH 3.0, as previously reported
(Schreiber et al., 2005; Serra et al., 2009b). Isolated periderms were
dried and stored at room temperature until used. Potato periderm
comprises three distinct tissues: the suberized tissue (phellem), the
cambial layer (phellogen) and the phelloderm. During this enzymatic
isolation, only suberized phellem tissue is obtained, and phellogen
and phelloderm are removed. However, we use the term periderm
instead of phellem, as a number of different authors have done
previously (Vogt et al., 1983; Stark et al., 1994; Schreiber et al., 2005).

values (P, m sec)1) were calculated from the slopes (F, g sec)1) of
the linear regression lines and fitted to the transpiration kinetics
using the following equation: P = F (A Dc))1, where A
(2.83 10)3 m2) corresponds to the exposed area and Dc
(106 g m)3) represents the driving force provided by the concentration of water in the chamber.
For assessment of tuber weight loss, freshly harvested tubers
grown in soil were weighed during 11 days of storage at intervals of
34 days. An analytical balance (ER-120A, A & D Instruments, Ltd.,
http://www.aandd.jp) and tubers from the three independent FHT
silenced lines were used.

Suberin and wax chemical analyses

Determination of the suberin and suberin-associated wax composition in potato tuber periderm was performed as previously
described by GC analyses (Schreiber et al., 2005; Serra et al.,
2009b). Three independent FHT down-regulated lines were used for
these studies. The total amount of periderm material used for each
analysis was 23 mg. Wax was extracted from isolated periderms at
room temperature for 18 h in a mixture of chloroform and methanol
(1:1 v/v). Chloroform/methanol extracts were used for wax analysis
without further purification. About 44-47% of the total wax amount
was identified. For depolymerization of aliphatic suberin, wax-free
periderms were trans-esterified by incubation at 70C for 18 h with
methanol/boron trifluoride (approximately 10% BF3 in methanol;
Fluka, http://www.sigmaaldrich.com) (Kolattukudy and Agrawal,
1974; Zeier and Schreiber, 1998). About 73-75% of the total suberin
amount was identified. All compounds were quantified and analyzed as trimethylsilyl (TMS) derivatives, obtained using N, O-bis
(trimethylsilyl)trifluoroacetamide (BSTFA, Macherey-Nagel, http://
www.mn-net.com). Compounds were quantified using an HP 5890
Series II GC-flame ionization detector (FID) (Hewlett-Packard,
http://www.hp.com) and analyzed using HPChemStation software
(Hewlett-Packard) by comparison with an internal standard (2 lg of
tetracosane for wax and 10 lg dotriacontane for suberin). Identification of the compounds was carried out using GC and a quadrupole mass selective detector (HP 5971A, Hewlett-Packard).
The glycerol content in suberin was quantified using a biochemical
method modified from Schmutz et al. (1996) and Moire et al. (1999)
and reported by Serra et al. (2009b). The isolated wax-free periderms
were trans-esterified (see above) to release the aliphatic suberin
monomers. The hydrolyzed cell-wall residues were rinsed once with
chloroform. The methanolysates were neutralized with saturated
sodium hydrogen carbonate, and the aqueous phase was used for
the determination of glycerol content. Free glycerol was quantified at
540 nm using Free Glycerol Reagent (Sigma, http://www.sigmaaldrich.com/) and a glycerol standard solution (triolein, Sigma). All three
independent FHT down-regulated lines were used for this study.

Measurement of peridermal permeance

Peridermal permeability was measured using transpiration chambers by a gravimetric method as previously described (Schonherr
and Lendzian, 1981; Vogt et al., 1983; Stark et al., 1994; Schreiber,
1996; Schreiber et al., 2005). Periderm membranes free of lenticels
were mounted on water-filled transpiration chambers, which were
turned upside down to ensure direct contact between water and
periderm. Chambers were kept in closed polyethylene boxes containing dry silica gel and stored at 25C, and the weight loss caused
by evaporation of water across the periderm to the silica gel was
determined at regular intervals using an analytical balance (Analytic
AC210S, Sartorius, http://www.sartorius-mechatronics.com). Transpiration kinetics were measured as described previously (Schreiber et al., 2005; Serra et al., 2009b), plotting the amount of water (g)
that had diffused across the periderm against time (sec). Permeance

FHT heterologous expression and enzyme activity assays

The cDNA of FHT was amplified using forward and reverse primers
with recognition sites for the restriction endonucleases BamHI and
NotI, respectively, at the 5 ends (5-GGATCCATGGAGAGTGGTAAAAACAATGT-3 and 5-GCGGCCGCGAGCTGCAATATGGCTGTTTAG-3) and a proofreading Taq polymerase (Advantage 2,
Clontech). The recombinant cDNA was subcloned into PCR4-TOPO
(Invitrogen), subsequently released by BamHI and NotI digestion,
and linked to pGEX4T-2 (GE Healthcare, http://www.gehealthcare.
com). This construct was transferred to Escherichia coli BL21 (DE3)
cells to express the fused protein. A 5 ml pre-culture was grown
overnight at 37C and used to inoculate 100 ml of fresh medium.
Bacteria were grown for 3 h at 37C, and expression of FHT was
induced overnight at 18C using 0.1 mM isopropyl-b-thiogalacto-

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FHT function in potato periderm 287

pyranoside (Hoffmann et al., 2003). After centrifugation for 10 min
at 2600 g, the bacteria were resuspended in 5 ml PBS containing
1 mM dithiothreitol. Cell lysis was performed by three passages
through a French press. The bacterial lysate was centrifuged at
16 000 g for 45 min, and the supernatant was incubated with glutathioneagarose beads at room temperature for 1 h. The beads
were washed three times with cold PBS, and FHT was released from
the GST tag by incubating the beads with thrombin for 1 h at room
temperature. The quality and amount of the protein were analyzed
using polyacrilamide gels stained with Coomassie brilliant blue
(Merck, http://www.merck-chemicals.com) and Nanodrop spectrophotometer D-1000 (Thermoscientific, http://www.nanodrop.com).
The purified protein was used to test the biochemical activity of FHT.
For the biochemical assay, the acyl acceptors were diluted with
acetone in a 20 mM stock solution. The enzyme reactions were
performed in a final volume of 100 ll containing 40 mM K-Pi buffer
pH 7, 1 mM dithiothreitol, 200 lM trans-feruloyl CoA (TransMIT
GmbH Flavonoid Research, http://www.transmit.de), 200 lM of acyl
acceptor (Sigma) and 0.21 lg of protein. Reactions were incubated
for 10 min at 30C, stopped with the same reaction volume of hot
acetonitrile (60C), heated at 96C for 1 min, and immediately
injected into HPLC/LC-MS or frozen at )20C. The protein remained
active after freezing and thawing in a buffer containing 50 mM Tris
pH 8, 150 mM NaCl, 2.5 mM CaCl2 and 0.1% 2-mercaptoethanol.

Extraction of soluble phenolic compounds of potato

periderm
Samples of approximately 1.1 cm2 of the tuber skin, obtained using
a cork borer, were manually and carefully detached from the
underlying parenchyma, weighed using a microbalance, frozen in
liquid nitrogen and quickly ground with a mortar and pestle.
Methanol (2 ml per 100 mg fresh weight) was added to the ground
tissue, which was re-extracted using 70% methanol (1 ml per
100 mg fresh weight). The sample was collected in a tube that was
centrifuged at 4000 g for 30 min. The supernatant was then clarified
by centrifugation at 16 000 g for 10 min, and immediately stored at
)20C or analyzed by LC-MS. Soil-grown tubers stored for 10 days
post-harvest were used in these analyses. Although the skin
separated cleanly from parenchyma in samples from FHT RNAi
tubers, most wild-type tubers had already completed the skin
set and removing the parenchyma was difficult and often
incomplete. Therefore, the obtained data must be considered only
qualitatively.

HPLC and LC-MS analysis

The compounds analyzed were resolved on a Novapak RP C18 column (4 lm of particle size, 4.6 250 mm inner diameter length;
Waters, http://www.waters.com) using a linear increasing gradient
of acetonitrile (solvent B) in water (solvent A), both with 0.05%
formic acid, at flow rate of 1 ml min)1 For soluble phenolic compounds, the gradient conditions were 9850% solvent A for 50 min,
returning to initial conditions of 98% solvent A for 5 min, and
re-equilibration of the column for 15 min. For characterization of the
reaction products of FHT activity, the elution conditions were 60%
solvent B for 5 min, a gradient of 60100% solvent B for 12 min,
100% solvent B for 5 min, from 100 to 40% solvent B over 6 min, and
re-equilibration of the column for 10 min (Lotfy et al., 1994).
LC analysis of FHT reaction products was performed on a Waters
analytical HPLC consisting of two 515 HPLC pumps, a 1996
photodiode array detector to record the UV absorption spectra,
and a 717 plus-autosampler equipped with an AF in-line degasser
(Waters). Compounds were identified based on their retention time
and their absorbance spectra.

LC-MS/MS analysis was used for the identification of periderm

soluble phenolic compounds and the reaction product of FHT
x-hydroxyferuloyloxypalmitic acid. LC analyses were performed
using an Agilent 1200 Series HPLC pump (Agilent, http://www.agilent.com) with detector set at 320 nm to monitor the hydroxycinna
moyl derivatives. The MS data were collected in negative or positive
mode using an Esquire 6000 ESI ion-trap LC-MS (Bruker Daltonics,
http://www.bdal.com) equipped with an electrospray ion source. The
sprayer needle was set to 4.1 kV, and the capillary was heated to
365C. Product peaks were identified by their retention time, their
expected mass, their MS fragmentation pattern (using an amplitude
of 3.5 V for soluble phenolics and 0.8 and 1.25 V for x-hydroxyferuloyloxypalmitic acid in positive and negative modes, respectively),
and relative to data reported in the literature.

Periderm microscopy
For SEM and TEM, periderm samples were prepared and observed as
described previously (Serra et al., 2009b). For SEM, small fragments
of tuber periderm were fixed under vacuum with 4% formaldehyde in
PBS (pH 7.5) at room temperature for at least 48 h. Fragments were
dehydrated with an increasing ethanol concentration series,
exchanged through amyl acetate, and critical point-dried. The
fragments were mounted on copper stubs and coated with gold.
Specimens were observed using a Zeiss DSM 960A SEM (http://
www.zeiss.com/). Digital images were collected and processed
using Quartz PCI version 5.10 (Quartz Imaging Corporation, http://
www.qrtz.com). For TEM, sections of periderm dissected into
1 1 mm2 sections were immediately placed in 100 mM sodium
cacodylate buffer (pH 7.0) containing 2.5% w/v glutaraldehyde and
2% w/v paraformaldehyde for 24 h. A vacuum was applied until
samples were submerged. Tissues were washed three times with
100 mM sodium cacodylate buffer (pH 7.0), and subsequently fixed
overnight in 100 mM sodium cacodylate buffer (pH 7.0) containing
1% w/v osmium tetroxide. Samples were then washed with 100 mM
sodium cacodylate buffer (pH 7.0), dehydrated in an acetone series
(30100% by 10% steps, 10 min each step), and infiltrated with
Spurrs epoxy resin (1:2, 1:1 and 2:1 v/v resin:acetone and pure resin
for 4 h, overnight, 3 and 5 h, respectively). Infiltrated tissues were
placed in moulds, and incubated at 60C for 2 days. Embedded
materials were thin-sectioned using an ultramicrotome RMC MT-XL
(RMC Products, http://www.rmcproducts.com). Sections were collected onto 200-mesh copper grids, stained with 2% w/v uranyl acetate for 15 min, and rinsed for 30 min, before being observed and
photographed using the Zeiss EM910 TEM at an accelerating voltage
of 60 kV. Images were obtained on Kodak electron microscope film
4489 (Kodak, http://www.kodak.com) and scanned using an HP
6100C (Hewlett-Packard).
All microscopic analyses were performed by the Microscopy
Service of the University of Girona, Spain.

ACKNOWLEDGEMENTS
The authors would like to thank Professor Lukas Schreiber
(Department of Ecophysiology, University of Bonn) and Dr Marcal
Soler (Laboratori del Suro, University of Girona) for their helpful
suggestions and positive feedback on the design of experiments, Dr
Manuel Ferrer (Instituto de Catalisis, Madrid, Spain) for kindly providing trans-feruloyl-CoA, Dr Llus Baneras and Dr Carles Borrego
(Departament de Biologia, Universitat de Girona, Spain) for kindly
lending the HPLC, Dr Concepcion Garca-Vallejo (Area de Industrias
Forestales del Center for International Forestry Research (CIFOR),
Instituto Nacional de Investigaciony Technologa Agraria y Alimentaria (INIA), Spain), and Dr Enriqueta Antico and Dr Anna
Roglans (Departament de Qumica, Universitat de Girona, Spain) for

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288 Olga Serra et al.

helpful advice on the identification of chemical compounds. We are
also very grateful to Ms Sara Gomez (Departament de Biologia,
Universitat de Girona, Spain) for her valuable assistance in the
laboratory work and taking care of the transgenic plants, Ms Anna
Costa (Serveis Te`cnics de Recerca, Universitat de Girona, Spain) for
her support and efficiency in the use of LC-MS, Mr Jordi Blavia and
Ms Carmen Carulla (Serveis Te`cnics de Recerca, Universitat de
Girona, Spain) for their highly skilled work with TEM and SEM, and
Mr Pau Boher (Laboratori del Suro, University of Girona) for his help
with purification of the protein. We also thank Dr Mike Pollard
(Department of Plant Biology, Michigan State University, East Lansing, USA) for helpful corrections and suggestions that improved
the manuscript. This work was financially supported by grants from
the Spanish Ministerio de Ciencia y Tecnologa and the Ministerio
de Educacion y Ciencia (AGL2003-00416, AGL200607342 and
AGL200913745, and Formacion de Personal Investigador (FPI)
grant and two mobility grants to O.S., and HA2007-0032). R.F.
gratefully acknowledges grants from the Deutsche Forschungsgemeinschaft and the Deutscher Akademischer Austausch Dienst.

SUPPORTING INFORMATION
Additional Supporting Information may be found in the online
version of this article:
Figure S1. Amino acid sequence alignment of FHT with its Arabidopsis putative ortholog (At5g41040) and the most homologous
partial ESTs identified from cork oak.
Figure S2. Phylogenetic tree constructed using the BAHD enzymes
whose function has been characterized, including FHT.
Figure S3. Periderm anatomy of in vitro-grown FHT RNAi and wildtype tubers.
Figure S4. SEM micrographs of isolated periderm membranes
(phellem) obtained by enzymatic treatment using cellulase and
pectinase.
Figure S5. Relative amounts (lg) of aliphatic suberin monomers
released after methanolic trans-esterification of wax-free periderms
from tubers of FHT RNAi and wild-type that were freshly harvested
or had been stored for 60 days.
Figure S6. Relative amounts (lg) of periderm wax compounds
released after chloroform:methanol (1:1 v/v) treatment of tubers of
FHT RNAi and wild-type that were freshly harvested or had been
stored for 60 days.
Figure S7. SDSPAGE gel stained with Coomassie brilliant blue for
purified FHT.
Figure S8. FHT activity on primary alcohols.
Figure S9. Fragmentation pattern by LC-MS/MS of x-feruloyloxypalmitic acid obtained in the FHT enzymatic assay using feruloyl CoA and C16:0 x-hydroxyacid as substrates.
Table S1. Potato ESTs most homologous to the FHT RNAi fragment
used for silencing.
Appendix S1. Soluble phenolic compounds identified by LC-MS/MS.
Appendix S2. Text file of the alignment used to generate the
phylogenetic tree in Figure S1.
Please note: As a service to our authors and readers, this journal
provides supporting information supplied by the authors. Such
materials are peer-reviewed and may be re-organized for online
delivery, but are not copy-edited or typeset. Technical support
issues arising from supporting information (other than missing
files) should be addressed to the authors.

REFERENCES
Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J. (1990) Basic
local alignment search tool. J. Mol. Biol. 215, 403410.
Amasino, R.M. (1986) Acceleration of nucleic-acid hybridization rate by
polyethylene-glycol. Anal. Biochem. 152, 304307.

Arrieta-Baez, D. and Stark, R.E. (2006) Using trifluoroacetic acid to augment

studies of potato suberin molecular structure. J. Agric. Food. Chem. 54,
96369641.
Banerjee, A.K., Prat, S. and Hannapel, D.J. (2006) Efficient production of
transgenic potato (S. tuberosum L. ssp. andigena) plants via Agrobacterium tumefaciens-mediated transformation. Plant Sci. 170, 732738.
Beisson, F., Li, Y., Bonaventure, G., Pollard, M. and Ohlrogge, J.B. (2007) The
acyltransferase GPAT5 is required for the synthesis of suberin in seed coat
and root of Arabidopsis. Plant Cell, 19, 351368.
Bernards, M.A. (2002) Demystifying suberin. Can. J. Bot. 80, 227240.
Bernards, M.A. and Lewis, N.G. (1992) Alkyl ferulates in wound healing potato
tubers. Phytochemistry, 31, 34093412.
Bernards, M.A. and Razem, F.A. (2001) The poly(phenolic) domain of
potato suberin: a non-lignin cell wall bio-polymer. Phytochemistry, 57,
11151122.
Bolwell, G.P., Gerrish, C. and Salaun, J.-P. (1997) Changes in enzymes
involved in suberisation in elicitor-treated french bean cells. Phytochemistry,
45, 13511357.
Corpet, F. (1988) Multiple sequence alignment with hierarchical clustering.
Nucleic Acids Res. 16, 1088110890.
DAuria, J.C. (2006) Acyltransferases in plants: a good time to be BAHD. Curr.
Opin. Plant Biol. 9, 331340.
Dixon, R.A. and Paiva, N.L. (1995) Stress-induced phenylpropanoid metabolism. Plant Cell, 7, 10851097.
Dobranszki, J. (2001) Effects of light on in vitro tuberization of the potato
cultivar Desiree and its relatives. Acta Biol. Hung. 52, 137147.
Esau, K. (1965) Periderm. In Plant Anatomy, 2nd edn. New York: John Wiley &
Sons, pp. 366381.
Franke, R. and Schreiber, L. (2007) Suberin a biopolyester forming
apoplastic plant interfaces. Curr. Opin. Plant Biol. 10, 252259.
Franke, R., Briesen, I., Wojciechowski, T., Faust, A., Yephremov, A., Nawrath, C.
and Schreiber, L. (2005) Apoplastic polyesters in Arabidopsis surface tissues
a typical suberin and a particular cutin. Phytochemistry, 66, 26432658.
Franke, R., Hofer, R., Briesen, I., Emsermann, M., Efremova, N., Yephremov, A.
and Schreiber, L. (2009) 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 J. 50, 8095.
Gou, J.Y., Yu, X.H. and Liu, C.J. (2009) A hydroxycinnamoyltransferase
responsible for synthesizing suberin aromatics in Arabidopsis. Proc. Natl
Acad. Sci. USA, 106, 1885518860.
Graca, J. (2009) Hydroxycinnamates in suberin formation. Phytochem. Rev.,
doi 10.1007/s11101-009-9138-4.
Graca, J. and Pereira, H. (2000) Suberin structure in potato periderm: glycerol,
long-chain monomers, and glyceryl and feruloyl dimers. J. Agric. Food.
Chem. 48, 54765483.
Graca, J. and Santos, S. (2007) Suberin: a biopolyester of plants skin. Macromol. Biosci. 7, 128135.
Hall, T.A. (1999) BioEdit: a user-friendly biological sequence alignment editor
and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41,
9598.
Helmja, K., Vaher, M., Gorbatsova, J. and Kaljurand, M. (2007) Characterization
of bioactive compounds contained in vegetables of the Solanaceae family
by capillary electrophoresis. Proc. Estonian Acad. Sci. Chem. 56, 172186.
Hofer, R., Briesen, I., Beck, M., Pinot, F., Schreiber, L. and Franke, R. (2008) The
Arabidopsis cytochrome P450 CYP86A1 encodes a fatty acid x-hydroxylase
involved in suberin monomer biosynthesis. J. Exp. Bot. 59, 23472360.
Hoffmann, L., Maury, S., Martz, F., Geoffroy, P. and Legrand, M. (2003) Purification, cloning, and properties of an acyltransferase controlling shikimate
and quinate ester intermediates in phenylpropanoid metabolism. J. Biol.
Chem. 278, 95103.
Hofgen, R. and Willmitzer, L. (1990) Biochemical and genetic-analysis of
different patatin isoforms expressed in various organs of potato (Solanum
tuberosum). Plant Sci. 66, 221230.
Horton, P., Park, K.-J., Obayashi, T., Fujita, N., Harada, H., Adams-Collier, C.J.
and Nakai, K. (2007) WoLF PSORT: protein localization predictor. Nucleic
Acids Res. 35, W585W587.
Iiyama, K., Lam, T.B.T. and Stone, B.A. (1990) Phenolic acid bridges between
polysaccharides and lignin in wheat internodes. Phytochemistry, 29, 733
737.
Keller, H., Hohlfeld, H., Wray, V., Hahlbrock, K., Scheel, D. and Strack, D.
(1996) Changes in the accumulation of soluble and cell wall-bound

2010 The Authors

Journal compilation 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 62, 277290

FHT function in potato periderm 289

phenolics in elicitor-treated cell suspension cultures and fungus-infected
leaves of Solanum tuberosum. Phytochemistry, 42, 389396.
Kim, J.H., Kim, H.S., Lee, Y.H., Kim, Y.S., Oh, H.W., Joung, H., Chae, S.K., Suh,
K.H. and Jeon, J.H. (2008) Polyamine biosynthesis regulated by StARD
expression plays an important role in potato wound periderm formation.
Plant Cell Physiol. 49, 16271632.
King, R.R. and Calhoun, L.A. (2005) Characterization of cross-linked
hydroxycinnamic acid amides isolated from potato common scab lesions.
Phytochemistry, 66, 24682473.
Kolattukudy, P.E. (2001) Polyesters in higher plants. Adv. Biochem. Eng.
Biotechnol. 71, 149.
Kolattukudy, P.E. and Agrawal, V.P. (1974) Structure and composition of
aliphatic constituents of potato-tuber skin (suberin). Lipids, 9, 682691.
Lendzian, K.J. (2006) Survival strategies of plants during secondary growth:
barrier properties of phellems and lenticels towards water, oxygen, and
carbon dioxide. J. Exp. Bot. 57, 25352546.
Li, Y., Beisson, F., Koo, A.J.K., Molina, I., Pollard, M. and Ohlrogge, J. (2007)
Identification of acyltransferases required for cutin biosynthesis and production of cutin with suberin-like monomers. Proc. Natl Acad. Sci. USA,
104, 1833918344.
Logemann, J., Schell, J. and Willmitzer, L. (1987) Improved method for the
isolation of RNA from plant tissues. Anal. Biochem. 163, 1620.
Lotfy, S., Negrel, J. and Javelle, F. (1994) Formation of x-feruloyloxypalmitic
acid by an enzyme from wound-healing potato tuber discs. Phytochemistry, 35, 14191424.
Lotfy, S., Javelle, F. and Negrel, J. (1995) Distribution of hydroxycinnamoylCoA:x-hydroxypalmitic acid O-hydroxycinnamoyltransferase in higher
plants. Phytochemistry, 40, 389391.
Lotfy, S., Javelle, F. and Negrel, J. (1996) Purification and characterization of
hydroxycinnamoyl-coenzyme A:x-hydroxypalmitic acid O-hydroxycinnamoyltransferase from tobacco (Nicotiana tabacum L.) cell-suspension
cultures. Planta, 199, 475480.
Lulai, E. (2007) The canon of potato science: 43. Skin-set and wound-healing/
suberization. Potato Res. 50, 387390.
Lulai, E.C. and Freeman, T.P. (2001) The importance of phellogen cells and
their structural characteristics in susceptibility and resistance to excoriation
in immature and mature potato tuber (Solanum tuberosum L.) periderm.
Ann. Bot. 88, 555561.
Lulai, E. and Orr, P. (1993) Determining the feasibility of measuring genotypic
differences in skin-set. Am. J. Potato Res. 70, 599609.
Lulai, E.C. and Orr, P.H. (1994) Techniques for detecting and measuring
developmental and maturational changes in tuber native periderm. Am.
Potato J. 71, 489505.
Lulai, E.C. and Suttle, J.C. (2004) The involvement of ethylene in woundinduced suberization of potato tuber (Solanum tuberosum L.): a critical
assessment. Postharvest Biol. Technol. 34, 105112.
Lulai, E.C., Suttle, J.C. and Pederson, S.M. (2008) Regulatory involvement of
abscisic acid in potato tuber wound-healing. J. Exp. Bot. 59, 11751186.
Mader, J.C. (1995) Polyamines in Solanum tuberosum in vitro free and
conjugated polyamines in hormone-induced tuberization. J. Plant Physiol.
146, 115120.
Matsuda, F., Morino, K., Ano, R., Kuzawa, M., Wakasa, K. and Miyagawa, H.
(2005) Metabolic flux analysis of the phenylpropanoid pathway in elicitortreated potato tuber tissue. Plant Cell Physiol. 46, 454466.
Moire, L., Schmutz, A., Buchala, A., Yan, B., Stara, R.E. and Roser, U. (1999)
Glycerol is a suberin monomer. New experimental evidence for an old
hypothesis. Plant Physiol. 119, 11371146.
Molina, I., Li-Beisson, Y., Beisson, F., Ohlrogge, J.B. and Pollard, M. (2009)
Identification of an Arabidopsis feruloyl-coenzyme A transferase required
for suberin synthesis. Plant Physiol. 151, 13171328.
Okazawa, Y. and Okazawa, N. (1980) On occurrence of defected potato tubers
with rough russeted skin. Jpn. J. Crop Sci. 49, 5865.
Peterson, R.L. and Barker, W.G. (1979) Early tuber development from explanted
stolon nodes of Solanum tuberosum var. Kennebec. Bot. Gaz. 140, 398406.
Pollard, M., Beisson, F., Li, Y. and Ohlrogge, J.B. (2008) Building lipid barriers:
biosynthesis of cutin and suberin. Trends Plant Sci. 13, 236246.
Protacio, C. and Flores, H. (1992) The role of polyamines in potato tuber
formation. In Vitro Cell. Dev. Biol. Plant, 28, 8186.
Razem, F.A. and Bernards, M.A. (2002) Hydrogen peroxide is required for
poly(phenolic) domain formation during wound-induced suberization.
J. Agric. Food. Chem. 50, 10091015.

Sabba, R.P. and Lulai, E.C. (2002) Histological analysis of the maturation of
native and wound periderm in potato (Solanum tuberosum L.) tuber. Ann.
Bot. 90, 110.
Schmidt, H.W. and Schonherr, J. (1982) Fine-structure of isolated and nonisolated potato-tuber periderm. Planta, 154, 7680.
Schmutz, A., Jenny, T., Amrhein, N. and Ryser, U. (1993) Caffeic acid and
glycerol are constituents of the suberin layers in green cotton fibres. Planta,
189, 453460.
Schmutz, A., Buchala, A.J. and Ryser, U. (1996) Changing the dimensions of
suberin lamellae of green cotton fibers with a specific inhibitor of the
endoplasmic reticulum-associated fatty acid elongases. Plant Physiol. 110,
403411.
Schonherr, J. and Lendzian, K. (1981) A simple and inexpensive method of
measuring water permeability of isolated plant cuticular membranes.
Z. Pflanzenphysiol. 102, 321327.
Schreiber, L. (1996) Chemical composition of Casparian strips isolated
from Clivia miniata Reg roots: evidence for lignin. Planta, 199, 596
601.
Schreiber, L., Franke, R. and Hartmann, K. (2005) Wax and suberin
development of native and wound periderm of potato (Solanum
tuberosum L.) and its relation to peridermal transpiration. Planta, 220,
520530.
Serra, O., Soler, M., Hohn, C., Franke, R., Schreiber, L., Prat, S., Molinas, M.
and Figueras, M. (2009a) Silencing of StKCS6 in potato periderm leads to
reduced chain lengths of suberin and wax compounds and increased
peridermal transpiration. J. Exp. Bot. 60, 697707.
Serra, O., Soler, M., Hohn, C., Sauveplane, V., Pinot, F., Franke, R., Schreiber,
L., Prat, S., Molinas, M. and Figueras, M. (2009b) CYP86A33-targeted gene
silencing in potato tuber alters suberin composition, distorts suberin
lamellae, and impairs the periderms water barrier function. Plant Physiol.
149, 10501060.
Soler, M., Serra, O., Molinas, M., Huguet, G., Fluch, S. and Figueras, M. (2007)
A genomic approach to suberin biosynthesis and cork differentiation. Plant
Physiol. 144, 419431.
Soler, M., Serra, O., Molinas, M., Garcia-Berthou, E., Caritat, A. and Figueras,
M. (2008) Seasonal variation in transcript abundance in cork tissue analyzed by real time RT-PCR. Tree Physiol. 28, 743751.
Soliday, C.L., Kolattukudy, P.E. and Davis, R.W. (1979) Chemical and ultrastructural evidence that waxes associated with the suberin polymer constitute the major diffusion barrier to water-vapor in potato-tuber (Solanum
tuberosum L.). Planta, 146, 607614.
Spooner, F. and Hammerschmidt, R. (1992) Lignification of potato tuber tissue
in response to pathogenic and non-pathogenic Streptomyces spp. Phytopathology, 82, 1166.
St Pierre, B. and De Luca, V. (2000) Evolution of acyltransferase genes: origin
and diversification of the BAHD superfamily of acyltransferases involved in
secondary metabolism. In Evolution of Metabolic Pathways (Romeo, J.T.,
ed). Amsterdam: Pergamon, pp. 285315.
Stark, R.E., Sohn, W., Pacchiano, R.A., Albashir, M. and Garbow, J.R. (1994)
Following suberization in potato wound periderm by histochemical and
solid-state 13C nuclear-magnetic-resonance methods. Plant Physiol. 104,
527533.
Stewart, C., Kang, B.-C., Liu, K., Mazourek, M., Moore, S.L., Yoo, E.Y., Kim,
B.-D., Paran, I. and Jahn, M.M. (2005) The Pun1 gene for pungency in
pepper encodes a putative acyltransferase. Plant J. 42, 675688.
Tamura, K., Dudley, J., Nei, M. and Kumar, S. (2007) MEGA4: molecular
evolutionary genetics analysis (MEGA) software version 4.0. Mol. Biol.
Evol. 24, 15961599.
Vogt, E., Schonherr, J. and Schmidt, H.W. (1983) Water permeability of
periderm membranes isolated enzymatically from potato tubers (Solanum
tuberosum L.). Planta, 158, 294301.
Xu, P., Zhang, Y., Kang, L., Roossinck, M.J. and Mysore, K.S. (2006) Computational estimation and experimental verification of off-target silencing
during posttranscriptional gene silencing in plants. Plant Physiol. 142, 429
440.
Yan, B. and Stark, R.E. (2000) Biosynthesis, molecular structure, and domain
architecture of potato suberin: a 13C NMR study using isotopically labeled
precursors. J. Agric. Food. Chem. 48, 32983304.
Yu, X.H., Gou, J.Y. and Liu, C.J. (2009) BAHD superfamily of acyl-CoA
dependent acyltransferases in Populus and Arabidopsis: bioinformatics
and gene expression. Plant Mol. Biol. 70, 421442.

2010 The Authors

Journal compilation 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 62, 277290

290 Olga Serra et al.

Zeier, J. and Schreiber, L. (1998) Comparative investigation of primary and
tertiary endodermal cell walls isolated from the roots of five monocotyle-

doneous species: chemical composition in relation to fine structure. Planta,

206, 349361.

Accession numbers for the sequence data: Sequence data from this article can be found in the EMBL/GenBank data libraries
under the following accession numbers. The full-length coding sequence of potato FHT (Ac. N. FJ825138) and the partial
sequence of cork oak FHT1 (Ac. N. CQ329869), which has been constructed from two ESTs previously identified (Ac. N. EE743861
and EE745210), have been isolated in this work. FHT1 and two other previously identified cork oak ESTs (Ac. N. EE743848 and
EE743849) and the Arabidopsis gene At5g41040 were described in this work as putative orthologous genes of potato FHT. The
microarray data analysis from TIGR Solanaceae Genomics Resource (http://www.jcvi.org/potato/) used in this work was from
the experiments 049_Hannapel, 087_Stupar and 113_Robin examining the expression of the clones corresponding to FHT:
STMIP96 (Ac. Ns.BQ51507879) and/or STMGR31 (Ac. Ns. BQ50760304). For construction of RNAi fragment and probe labeling
the potato clone used was cSTS5P17 (Ac. N. BG593863).

2010 The Authors

Journal compilation 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 62, 277290