Suberin structure in potato periderm: Glycerol, long-chain monomers, and glyceryl and feruloyl dimers

Article abstract of DOI:10.1021/jf0006123

Suberin in extractive-free potato periderm amounts to 25% determined by NaOCH₃ methanolysis. Monomeric composition is characterized by glycerol (20% of monomers), long-chain α,ω-diacids, ω-hydroxyacids, alkanoic acids, and alkan-1-ols, with predominance of octadec-9-enodioic acid and 18-hydroxyoctadec-9-enoic acid (39 and 15% of long-chain monomers, respectively). Aromatic hydroxycinnamyl monomers were also present (<1%). Partial depolymerization of potato periderm suberin using a Ca(OH)₂-catalyzed methanolysis solubilized ~10% of suberin aliphatics. GC-MS analysis showed the presence of monomers, dimers, and trimers (87, 12, and 1% of identified compounds, respectively). A total of 26 dimers were identified by EIMS: monoacylglyceryl esters of α,ω-diacids, ω-hydroxyacids, and alkanoic acids (with predominance of the 1- and 2-isomers of the monoacylglyceryl ester of the octadec-9-enodioic acid), as well as feruloyl esters of ω-hydroxyacids and alkan-1-ols and a small quantity of a monoferuloylglycerol. Following a discussion of suberin macromolecular structure, it is proposed that in suberized cell walls, the polyaliphatic polymers have a three-dimensional development ensured by glycerol and exist independently from the associated polyaromatics.

Full text of DOI:10.1021/jf0006123

5476
J. Agric. Food Chem. 2000, 48, 5476−5483
Su ber in Str u ctu r e in P ota to P er id er m : Glycer ol, Lon g-Ch a in
Mon om er s, a n d Glycer yl a n d F er u loyl Dim er s
J ose´ Grac¸a* and Helena Pereira
Centro de Estudos Florestais, Instituto Superior de Agronomia, Universidade Te´cnica Lisboa,
1349-017 Lisboa, Portugal
Suberin in extractive-free potato periderm amounts to ∼25% determined by NaOCH3 methanolysis.
Monomeric composition is characterized by glycerol (20% of monomers), long-chain R,ω-diacids,
ω-hydroxyacids, alkanoic acids, and alkan-1-ols, with predominance of octadec-9-enodioic acid and
18-hydroxyoctadec-9-enoic acid (39 and 15% of long-chain monomers, respectively). Aromatic
hydroxycinnamyl monomers were also present (<1%). Partial depolymerization of potato periderm
suberin using a Ca(OH)2-catalyzed methanolysis solubilized ∼10% of suberin aliphatics. GC-MS
analysis showed the presence of monomers, dimers, and trimers (87, 12, and 1% of identified
compounds, respectively). A total of 26 dimers were identified by EIMS: monoacylglyceryl esters of
R,ω-diacids, ω-hydroxyacids, and alkanoic acids (with predominance of the 1- and 2-isomers of the
monoacylglyceryl ester of the octadec-9-enodioic acid), as well as feruloyl esters of ω-hydroxyacids
and alkan-1-ols and a small quantity of a monoferuloylglycerol. Following a discussion of suberin
macromolecular structure, it is proposed that in suberized cell walls, the polyaliphatic polymers
have a three-dimensional development ensured by glycerol and exist independently from the
associated polyaromatics.
Keyw or d s: Suberin; Solanum tuberosum; potato periderm; monoacylglyceryl esters; feruloyl esters
INTRODUCTION
ture of these polyaliphatic and polyaromatic materials
is not known, and it is not clear if they should be
considered as one or two different polymers.
Suberin is a major constituent of the cell walls of outer
plant tissues, namely, secondary growth periderms, as
well as interior tissues that function as isolation bar-
riers. The specificity of suberin location relates to its
function as a protective polymer involved in thermal
insulation, in water-loss prevention, and as a barrier
to pathogenic attack (Kolattukudy, 1977). Most of the
studies on the composition and structure of suberin and
associated materials have been made on the potato
natural or wound-induced periderm [e.g., Kolattukudy
and Agrawal (1974) and Stark and Garbow (1992)].
Other relevant works on suberin chemistry have been
made on an important forest product, commercial cork
[e.g., Marques et al. (1996) and Grac¸a and Pereira
(1997)], which is the outer bark of the cork oak (Quercus
suber L.).
What is and what should be called suberin remains
an open question. Suberin is known to include an
aliphatic polyester part, normally quantified as “suberin
content”, after depolymerization by reactions that break
ester bonds. The solubilized monomers after analysis
by GC-MS show mixtures of aliphatic long-chain R,ω-
diacids, ω-hydroxyacids, alkanoic acids, and n-alkanols
(Holloway, 1983; Kolattukudy and Espelie, 1989). Vary-
ing quantities of aromatic materials are solubilized
simultaneously with the above aliphatics and, except
for small quantities of monomeric phenylpropenoids, are
of unknown chemical nature (Riley and Kolattukudy,
1975; Borg-Olivier and Monties, 1993). Important quan-
tities of non-depolymerized polyaromatics remain in the
residual cell wall material. The macromolecular struc-
More recent results have brought a new insight into
the possible structure of suberin and its relations with
the other suberized cell wall components. Glycerol was
found to be a major monomer of suberins, and dimers
of glycerol esterified to all kinds of suberinic aliphatic
acids present as monomers were also identified, giving
body to the hypothesis that suberin is a glycerol-based
polyester (Grac¸a and Pereira, 1997, 1999). Dimers of
ω-hydroxyacids were also found as esters of ferulic acid,
showing one way to link aliphatics and aromatics (Grac¸a
and Pereira, 1998, 1999). Results from in situ analysis
by solid-state NMR showed that aliphatics and aromat-
ics occupied different areas in the suberized cells walls
(Gil et al., 1997; Bernards and Lewis, 1998), thus
allowing the existence of two independent polymers to
be considered.
The suberin dimers that have been obtained so far
were solubilized from bark corks, after a partial depo-
lymerization methanolysis with calcium oxide as cata-
lyst (Grac¸a and Pereira, 1997, 1999). Here we apply a
closely related procedure to obtain oligomers from potato
periderm suberin to get a better understanding of the
suberin polymer structure. The monomeric composition
of potato periderm suberin was also quantified, using a
technique that allows simultaneous analysis of glycerol
and the long-chain aliphatics (Grac¸a and Pereira, 2000).
MATERIALS AND METHODS
P ota to P er id er m . Potatoes (Solanum tuberosum) bought
in a local grocery were boiled and peeled. The potato peel strips
were thoroughly scraped of amilaceous tissue and oven-dried
at 50 °C (∼0.3% yield based on the initial wet weight). The
* Corresponding author (telephone 351.213653372; fax
351.213645000; e-mail jograca@isa.utl.pt).
10.1021/jf0006123 CCC: $19.00 © 2000 American Chemical Society
Published on Web 09/26/2000

Suberin Structure in Potato Periderm
J. Agric. Food Chem., Vol. 48, No. 11, 2000 5477
dried potato peel was ground in a coffee mill and sieved, and
the 40-60 mesh (0.25-0.42 mm) fraction was used for
analysis. Extractives were removed by Soxhlet extraction
successively with dichloromethane (8 h), ethanol (18 h), water
(24 h), and methanol (18 h). The extracts were, respectively,
3.3, 7.1, 21.5, and 0.8% (total ) 32.7%, based on the initial
dry weight). The extracted material was dried before metha-
nolysis reactions in a vacuum oven at 40 °C.
An alysis of Su ber in Mon om er s. NaOCH3-Catalyzed Meth-
anolysis. The dry, extractive-free (three samples, ∼500 mg
each) potato periderm was refluxed for 3 h in 50 mL of a 12
mmol L-1 solution of NaOCH3 in methanol, prepared by
dissolving metallic sodium in dry methanol (reagent grade,
kept over 3 Å molecular sieve). The mixture was filtered
through a 0.45 µm pore membrane filter, and the residue was
washed with 25 mL of methanol and 25 mL of chloroform.
Aliquots (2.5 mL) of the filtrate solution were taken for GC-
MS and GC-FID analysis as described below. The residue was
oven-dried and weighed. The methanolysis extract, corre-
sponding to the “suberin content”, determined by the mass loss,
averaged 24.6% (extractive-free basis).
Quantitative Analysis. 1,12-Dodecanediol (11.29 mg) and 12-
hydroxyoctadecanoic acid methyl ester (11.42 mg) were added
in the methanolysis mixture as internal standards for the
quantification of glycerol and long-chain monomers, respec-
tively. Details on the calibration and response factors used for
quantitative determinations of suberin monomers are de-
scribed in Grac¸a and Pereira (2000).
GC-MS and GC-FID Analyses. The aliquots taken from the
methanolysate filtrates were concentrated to dryness under a
flow of N2 in a warm bath, further dried in a vacuum oven at
40 °C, and derivatized with N,O-bis(trimethylsilyl)trifluoro-
acetamide (BSTFA) with 1% trimethylchlorosilane in pirydine
(1:1). After a 15 min staging in an oven at 60 °C, solutions
were analyzed by GC-EIMS (Masslab Trio 1000) and GC-FID
(HP 5890). Split injections were made in the same GC
conditions in both analyses: column, DB5-MS (J &W), 60 m,
0.25 mm internal diameter, 0.25 µm film thickness; injector,
300 °C; initial oven temperature, 100 °C (5 min), 10 °C/min
rate up to 240 °C, 2 °C/min rate up to 300 °C (15 min); FID
temperature, 300 °C. EIMS conditions: source, 200 °C, ioniza-
tion potential, 70 eV. Identification of monomer compounds
was made through their EIMS spectra, from published and
obtained spectra from model compounds, as described (Grac¸a
and Pereira, 1997).
Position of Double Bond in the Octadec-9-ene-1,18-dioic Acid.
Picolinyl and 2-alkenyl-4,4-dimethyloxazoline (DMOX) deriva-
tives were prepared from the isolated octadec-9-ene-1,18-dioic
acid following the method of Gunstone (1997). The picolinyl
derivative was synthesized from the acid chloride, prepared
by adding 0.5 mL of oxalyl chloride to ∼1 mg of the diacid
and letting it stand overnight. Excess reagent was evaporated
under N2, and 0.5 mL of a solution of 3-(hydroxymethyl)-
pyridine (HMP) in dichloromethane (40 mg/2 mL) was added
at 0 °C, left to warm to ambient temperature, and left for an
additional 2 h. Excess solvent was evaporated under N2 and
the mixture derivatized with BSTFA/pyridine for GC-MS
analysis. The DMOX derivative was prepared by adding 5 mg
of 2-amino-2-methylpropanol to ∼1 mg of the free diacid
and incubating the mixture for 5 h at 160 °C. After derivati-
zation with BSTFA/pyridine, the mixture was analyzed by GC-
MS.
An a lysis of Su ber in Dim er s. Ca(OH)2-Catalyzed Metha-
nolysis. Dry, extractive-free potato periderm (three samples,
2.5-3.3 g) was mixed with dry calcium hydroxide (2:1, w/w)
in powder form. Dry methanol (∼70 mL) was added, and the
mixture was refluxed at 72 °C (heating provided by an oil bath)
for 1 h, with stirring. The mixture was filtered hot (0.45 µm
pore membrane) under slight vacuum, and the residue was
washed with methanol and chloroform. The methanolysis
filtrate was concentrated under reduced vacuum, further
filtered through a 0.2 µm pore filter disk, and rediluted to a
known volume. Aliquots from these solutions were taken for
GC-MS analysis. Extracted material, quantified by the ex-
trapolation of the weight of the dried aliquots, averaged 2.3%
(extractive-free basis).
GC-MS Analysis. Aliquots taken from the methanolysate
solutions were dried and derivatized as described above. The
solutions prepared were analyzed by GC-EIMS, in split and
splitless mode, in two different chromatographic conditions:
(1) column, DB5-MS, 60 m, 0.25 mm internal diameter, 0.25
µm film thickness; injector, 325 °C; initial oven temperature,
100 °C (5 min), 10 °C/min rate up to 240 °C, 2 °C/min rate up
to 300 °C, 1 °C/min up to 340 °C (10 min); (2) column, HT5
(SGE), 50 m, 0.33 mm internal diameter, 0.10 µm film
thickness; injector, 325 °C; initial oven temperature, 100 °C
(5 min), 8 °C/min rate up to 240 °C, 3 °C/min rate up to 325
°C (30 min). EIMS conditions were as described above.
Ca(OH)2-Catalyzed Methanolysis of Model Compounds. Ca-
(OH)2-catalyzed methanolysis was applied to several sets of
model compounds, under the same conditions as described
above, in duplicate. The weight of Ca(OH)2 used in each case
was half the weight of the total of other organic compounds
present in the mixture: (1) 1-monostearoylglycerol (Sigma),
36 mg (0.1 mmol); (2) 2-monopalmitoylglycerol (Sigma), 26 mg
(0.08 mmol); (3) 1-monostearoylglycerol, 72 mg (0.2 mmol),
hexadecane-1,16-dioic acid (Tokyo Kasei), 29 mg (0.1 mmol),
and eicosane-1,20-dioic acid dimethyl ester (Tokyo Kasei), 37
mg (0.1 mmol); (4) glycerol (Merck), 45 mg (0.5 mmol),
hexadecane-1,16-dioic acid, 29 mg (0.1 mmol), and eicosane-
1,20-dioic acid dimethyl ester, 37 mg (0.1 mmol). After metha-
nolysis, the reaction mixtures were filtered and washed and
aliquots were taken, dried, derivatized, and GC-MS-analyzed
as described above. Quantitative data were determined from
the areas of compound peaks in the EIMS ion chromatograms
(TICs).
Isolation of Octadec-9-ene-1,18-dioic Acid. Octadec-9-ene-
1,18-dioic acid was obtained by preparative TLC from the
methanolysis products of potato periderm, followed by hy-
drolysis to obtain the free acid. Methanolysis with sodium
methoxide (3 g of Na in 500 mL of methanol) was carried out
in ∼15 g of extractive-free potato periderm. After filtering, the
methanolysate solution was acidified to pH 6, concentrated
close to dryness, and partitioned in chloroform/water. The
organic layer was washed two more times with water, con-
centrated, and rediluted with chloroform/methanol 7:3. This
solution was applied to 0.5 mm silica TLC plates, developed
with chloroform/ethyl acetate 8:2, and the band of the R,ω-
diacids dimethyl esters (Rf ∼0.7) was scraped off and extracted
(chloroform/methanol 2:1). Separation of the unsaturated
octadec-9-ene-1,18-dioic acid dimethyl esters from their satu-
rated counterparts was made by silver ion TLC (Nikolova-
Damyanova, 1992). TLC plates (0.5 mm silica gel) were
impregnated with silver by immersion in a solution of 20%
silver nitrate. The solution of the R,ω-diacid methyl esters was
applied to these plates, and after development with chloroform/
ethyl acetate 100:5, pure octadec-9-ene-1,18-dioic acid dimethyl
ester was recovered from a band at Rf 0.35 (a single peak in
the GC-MS analysis). The recovered octadec-9-ene-1,18-dioic
acid dimethyl ester was added to a 0.5 M solution of potassium
hydroxide in 95% ethanol and stirred at 80 °C during 1.30 h.
The mixture was acidified to pH 3.8 with 3 M HCl, evaporated,
and partitioned in chloroform/water. The organic phase, after
further washing, was dried over sodium sulfate. After solvent
removal, 18 mg of octadec-9-ene-1,18-dioic acid [checked by
GC-MS as its bis(trimethylsilyl)ester)] was obtained.
Synthesis of the 1-Monoferuloylglycerol. The synthesis was
based on the procedure of Neises and Steglich (1978). Ferulic
acid (Sigma), 20.99 mg (0.11 mmol), was dissolved in 250 µL
of dichloromethane plus 250 µL of dimethylformamide.
Under stirring, glycerol, 48.42 mg (0.53 mmol), and 50 µL of
a
solution of 4-(dimethylamino)pyridine (16.09 mg/mL of
dichloromethane) were added. After 5 min, 400 µL of a solution
of N,N′-dicyclohexylcarbodiimide (60.56 mg/mL of dichlo-
romethane) was slowly added. After 2 h of reaction, the
solution was filtered, an aliquot of 150 µL taken to dryness,
and TMS derivatized as described and analyzed by GC-
EIMS.

5478 J. Agric. Food Chem., Vol. 48, No. 11, 2000
Grac¸a and Pereira
Ta ble 1. Mon om er ic Com p osition of P ota to P er id er m
Su ber in , a fter Dep olym er iza tion by Na OCH3-Ca ta lyzed
Meth a n olysisa
Ta ble 2. Mon om er s a n d Oligom er s fr om P ota to P er id er m
Su ber in , a fter P a r tia l Dep olym er iza tion by
Ca (OH)2-Ca ta lyzed Meth a n olysis
mg/g
mg/ga
glycerol
220.3
m on om er s
glycerol
aromatic monomers
18-hydroxyoctadec-9-enoic acid
octadec-9-ene-1,18-dioic acid
other aliphatic monomers
431.7
2.9
97.5
96.0
98.4
coniferyl alcohol
ferulic acid
1.9
7.6
a lk a n -1-ols
octadecanol
docosanol
tricosanol
tetracosanol
hexacosanol
24.7
3.4
4.1
4.4
4.5
8.3
d im er s
monoacylglyceryl esters of alkanoic acids
1-monodocosanoylglycerol
2-monodocosanoylglycerol
1-monotetracosanoylglycerol
2-monotetracosanoylglycerol
1-monohexacosanoylglycerol
2-monohexacosanoylglycerol
1-monooctacosanoylglycerol
2-monooctacosanoylglycerol
0.9
0.2
1.9
0.8
2.5
0.9
2.1
0.5
0.2
a lk a n oic a cid s
hexadecanoic acid
docosanoic acid
tetracosanoic acid
hexacosanoic acid
octacosanoic acid
nonacosanoic acid
tricontanoic acid
84.1
2.0
5.4
12.4
13.7
38.5
4.3
1-monotricontanoylglycerol
monoacylglyceryl esters of R,ω-diacids
1-mono(octadecanedi-18-oic acid-1-oyl)glycerol
1-mono(octadec-9-enedi-18-oic acid-1-oyl)glycerol
2-mono(octadec-9-enedi-18-oic acid-1-oyl)glycerol
1-mono(tetracosanedi-24-oic acid-1-oyl)glycerol
2-mono(tetracosanedi-24-oic acid-1-oyl)glycerol
1-mono(hexacosanedi-26-oic acid-1-oyl)glycerol
monoacylglyceryl esters of ω-hydroxyacids
2-mono(18-hydroxyoctadec-9-enoyl)glycerol
1-mono(18-hydroxyoctadec-9-enoyl)glycerol
1-mono(22-hydroxydocosanoyl)glycerol
1-mono(24-hydroxytetracosanoyl)glycerol
1-mono(26-hydroxyhexacosanoyl)glycerol
monoacylglyceryl ester of ferulic acid
1-monoferuloylglycerol
7.8
0.4
61.0
13.9
0.6
0.2
0.2
r,ω-d ia cid s
318.7
6.5
300.8
0.9
5.0
5.5
hexadecanedioic acid
octadec-9-ene-1,18-dioic acid
9,10-dihydroxyoctadecanedioic acid
eicosanedioic acid
hexacosanedioic acid
1.5
4.0
0.2
0.4
0.3
ω-h yd r oxya cid s
148.6
13.1
120.8
0.3
6.1
3.8
16-hydroxyhexadecanoic acid
18-hydroxyoctadec-9-enoic acid
9,10,18-trihydroxyoctadecanoic acid
22-hydroxydocosanoic acid
24-hydroxytetracosanoic acid
26-hydroxyhexacosanoic acid
28-hydroxyoctacosanoic acid
0.7
2.2
2.3
feruloyl esters of alkan-1-ols
hexadecanyl ferulate
octadecanyl ferulate
docosanyl ferulate
0.6
0.3
0.1
unidentified
194.1
a
Compounds analyzed as methyl esters, trimethylsilyl ethers.
feruloyl esters of ω-hydroxyacids
16-O-feruloyloxyhexadecanoic acid
18-O-feruloyloxyoctadec-9-enoic acid
0.1
4.8
RESULTS AND DISCUSSION
tr im er s
1,1′-diglycerol-octadec-9-ene-1,18-dioate
1,2′-diglycerol-octadec-9-ene-1,18-dioate
2,2′-diglycerol-octadec-9-ene-1,18-dioate
4.9
2.0
<0.1
Su ber in Mon om er ic Com p osition . The NaOCH3-
catalyzed methanolysis solubilized ∼25% of the extrac-
tive-free potato periderm. This value is close to others
obtained for the quantification of the suberin content
in this material, using different depolymerization tech-
niques (Rodriguez-M. and Ribas-M., 1972; Kolattukudy
and Agrawal, 1974; Brieskorn and Binnemann, 1975).
Analysis of the methanolysis products (Table 1) showed
the presence of the long-chain alkan-1-ols, alkanoic
acids, ω-hydroxyacids, and R,ω-diacids, monomers also
previously identified (Brieskorn and Binnemann, 1974;
Kolattukudy and Agrawal, 1974; Holloway, 1983). In
addition, glycerol was found in large quantities, ac-
counting for ∼20% of all monomers. Less than 1% of
the mixture were hydroxycinnamyl aromatic monomers,
namely, ferulic acid and coniferyl alcohol. As quantified
by the calibrated internal standards, this mostly ali-
phatic monomer mixture accounted for ∼74% of the
mass loss after methanolysis.
The mixture of long-chain aliphatic acids is dominated
by two mono-unsaturated acids, the octadec-9-ene-1,-
18-dioic acid and the 18-hydroxyoctadec-9-enoic acid,
accounting for 39 and 15% of all monomers, respectively,
excluding glycerol. The R,ω-diacids and ω-hydroxyacids
are the main classes of acid compounds, accounting for
55 and 26% of the identified long-chain aliphatics,
respectively. Alkanoic acids are present in a fairly high
proportion compared to other suberins studied (Grac¸a
unidentified
167.5
a
Based in the integrated areas of the GC-MS reconstructed ion
chromatogram. Compounds analyzed as methyl esters, trimeth-
ylsilyl ethers.
and Pereira, 2000) and have very long chain lengths,
C28 being the more important homologue.
Earlier studies have shown that the position of the
double bond in the two main monomers of potato
suberin is midchain, at C-9 (Rodriguez-M. and Ribas-
M., 1972; Kolattukudy and Agrawal, 1974). Although
identification of monomers was made through their
EIMS, it is known that the mass spectra of fatty acid
methyl esters are little modified by changes in the
position of the double bond along the hydrocarbon chain.
The position of the double bond in the octadec-9-ene-
1,18-dioic acid, isolated from the potato suberin metha-
nolysis products, was checked after the synthesis of its
picolinyl and DMOX derivatives. The latter are nitrogen-
containing derivatives of the carboxyl group, which
under electron-impact conditions produce fragment ions
associated with each C-C cleavage along the chain,
without migration of the double bonds (when present),
thus allowing the determination of its position (Christie,
1997). Both derivatives confirmed the assignment of the

Suberin Structure in Potato Periderm
J. Agric. Food Chem., Vol. 48, No. 11, 2000 5479
F igu r e 1. EI mass spectrum of the 2-alkenyl-4,4-dimethyloxazoline (DMOX) bis-derivative of the octadec-9-ene-1,18-dioic acid,
isolated from the methanolysis products of the potato periderm suberin.
position of the double bond, and the mass spectrum of
the DMOX derivative is presented in Figure 1.
quantity of trimers (25 and 2%, respectively, of identi-
fied compounds excluding glycerol).
The geometric isomerism of the two main monene
acids was not studied here. The cis form was assigned
before to both acids (Rodriguez-M. and Ribas-M., 1972).
However, a poorly resolved division was observed here
within the peak of each of the acids, with the same mass
spectra. The proportion between the first eluted to the
later eluted part of the peaks was roughly 4:1 in the
octadec-9-ene-1,18-dioic acid and 2:1 in the 18-hydroxy-
octadec-9-enoic acid. As the cis form precedes the trans
form in the elution of unsaturated fatty acid methyl
esters in nonpolar columns, the possible presence of both
isomers should be considered here.
Su ber in P a r tia l Dep olym er iza tion . Partial depo-
lymerization of bark suberins has been achieved before
with a CaO-catalyzed methanolysis (Grac¸a and Pereira,
1997, 1999). This technique allowed the depolymeriza-
tion of between 5 and 10% of suberin aliphatics, of which
about one-third were dimers. These dimers were mostly
monoacylglyceryl esters of the long-chain aliphatic
suberinic acids, namely, of R,ω-diacids, ω-hydroxyacids,
and alkanoic acids. The acids appeared to be esterified
in both assignable positions of glycerol, 1- and 2-, the
first being predominant. The acids in the monoacyl-
glyceryl esters appeared roughly in the same propor-
tions as they appear as monomers after complete
depolymerization of the suberins.
A similar procedure was used here, with Ca(OH)2
instead of CaO as the catalyst of methanolysis reaction,
which provided a higher yield of suberin oligomers. The
Ca(OH)2-catalyzed methanolysis solublized ∼10% of the
“total” suberin, as measured by the NaOCH3-catalyzed
methanolysis extracted materials. The GC-MS analysis
of the mixture showed the presence of monomers,
dimers, and trimers (Table 2). Unidentified compounds
represented 17% of the mixture. Monomers accounted
for most of all of the identified compounds, ∼87%, of
which about half was glycerol with the remainder being
of the same type of aliphatic acids present after complete
depolymerization. Oligomers represented 13% of identi-
fied compounds, mostly composed of dimers and a small
Identification of the suberin dimers was made through
their EIMS as previously discussed (Grac¸a and Pereira,
1997, 1999). The mass spectra of two of the more
significant dimers, 1-mono(octadec-9-enedi-18-oic acid-
1-oyl)glycerol and 18-O-feruloyloxyoctadec-9-enoic acid,
including some of their fragmentation patterns are
presented in Figures 2 and 3. A total of 26 different
dimers were identified, including monoacylglyceryl es-
ters of the aliphatic acids, ferulates of ω-hydroxyacids
and alkan-1-ols, and a small quantity of a monoacylg-
lyceryl ester of ferulic acid (Table 2). The trimers were
diglycerol diesters of the octadec-9-ene-1,18-dioic acid,
the main acid monomer in the suberin of the potato
periderm. Discussion of these trimers, namely, evidence
regarding their identification, is presented elsewhere
(Grac¸a and Pereira, in press).
The Ca(OH)2-catalyzed methanolysis procedure was
applied to model compounds under the same conditions
as for potato periderm, to ascertain the extent of partial
hydrolysis of ester compounds and to check for the
eventual occurrence of resynthesis of glyceryl esters
from the depolymerized acids. The reaction conditions
were applied independently to 1-monostearoylglycerol
and 2-monohexadecanoylglycerol. In both cases only a
partial methanolysis of the monoacylglycerol esters was
observed, amounting to 65 and 57%, respectively, of
transesterification to the methyl esters of the corre-
sponding acids. However, interesterification was also
observed in the remaining nonmethanolyzed mono-
acylglycerol ester, a situation known to occur in other
reactions with acylglycerol esters (Sonnet, 1999). Migra-
tion of the acyl moiety in both directions between the
1- and 2-positions of glycerol occurred, with the move
from the 2- to the 1-position predominating. After the
Ca(OH)2 methanolysis, ∼10% of the 2-monostearoyl-
glycerol was obtained from the initial 1-isomer, and
∼95% of the 1-isomer was obtained from the initial
2-monohexadecanoylglycerol.
Glycerol and 1-monostearoylglycerol were also reacted
independently under the Ca(OH)2 methanolysis with

5480 J. Agric. Food Chem., Vol. 48, No. 11, 2000
Grac¸a and Pereira
F igu r e 2. EI mass spectrum of the 1-mono(octadec-9-enedi-18-oic acid-1-oyl)glycerol, methyl ester, bis(trimethylsilyl) ether, isolated
from the Ca(OH)2-catalyzed partial methanolysis products of the potato periderm suberin.
F igu r e 3. EI mass spectrum of the 18-O-feruloyloxyoctadec-9-enoic acid, methyl ester, trimethylsilyl ether, isolated from the
Ca(OH)2-catalyzed partial methanolysis products of the potato periderm suberin.
long-chain R,ω-diacids, as free acids and in the form of
dimethyl esters. The acyl part of the monoacylglycerol,
the free R,ω-diacid, and dimethylated R,ω-diacid were
all different in order to be able to check for changes in
positions. As expected under the conditions used, the
synthesis of glycerol esters was not observed, nor was
substitution of the acyl part of the monoacylglycerol by
the non-glycerol-linked R,ω-diacids. This shows that the
glycerol esters found after the partial methanolysis were
originally present in the suberin polymer.
Su ber in Dim er s. Almost all of the suberinic acids
of the potato periderm found as monomers were also
found as monoacylglyceryl esters in the solubilized
products of the Ca(OH)2-catalyzed methanolysis (Table
2). About two-thirds of all identified dimers were
monoacylglyceryl esters of R,ω-diacids, overwhelmingly
dominated by the 1- and 2-isomers of octadec-9-ene-1,-
18-dioic acid. Within each class of monoacylglycerols
(e.g., alkanoic acids, R,ω-diacids, and ω-hydroxyacids),
the proportion of each individual acid roughly followed
the same proportion as for monomers after complete
depolymerization of suberin. However, the ω-hydroxy-
acids were under-represented as monoacylglycerols,
even after accounting for the ω-hydroxyacids that ap-
peared as ferulate dimers (discussed below). We specu-
late that the position or chemical neighborhood of
ω-hydroxyacids in the suberin polymer makes their
ester linking to glycerol more susceptible to the metha-
nolysis reaction used.
The most abundant monoacylglycerols appeared as 1-
and 2-isomers, in a relative proportion of ∼3-5:1.
Because glycerol has two 1-positions (stereochemically
distinct only when differently substituted) and has only
one 2-position, the prevalence of 1-isomers is expected.

Suberin Structure in Potato Periderm
J. Agric. Food Chem., Vol. 48, No. 11, 2000 5481
F igu r e 4. EI mass spectrum of the 1-monoferuloylglycerol, tris(trimethylsilyl) ether, isolated from the Ca(OH)2-catalyzed partial
methanolysis products of the potato periderm suberin.
However, care has to be taken in the interpretation of
these results because reaction with model compounds
has shown that acyl migration between the 1- and
2-positions of glycerol (with preferential migration to
the 1-position) can occur under the conditions used.
1-monoacylglycerols and absent in the corresponding
2-isomers (Myher et al., 1974).
Su ber in Str u ctu r e. The results previously obtained
in bark corks and here in the potato peridem show that
suberin is a glycerol-structured polymer. Already a
significant number of suberin analyses have shown the
presence of glycerol (Carvalho, 1993; Schmutz at al.,
1993; Rosa and Pereira, 1994; Bento et al., 1998), and
its quantity has been positively related with the suber-
ization process (Moire at al., 1999). The role of glycerol
in the macromolecular assembly of the suberin polymer
seems now to be the interlinking between the long-chain
acid monomers. As one of the main monomer families
is the R,ω-diacids, their esterification to glycerol in both
ends can form the backbone for growth of the macro-
molecular polyester structure. The actual finding of such
diglycerol-R,ω-diacid diester trimers in the potato peri-
derm suberin (Grac¸a and Pereira, in press) gives sub-
stance to this hypothesis. The results of 13C solid-state
NMR studies in potato (Garbow et al., 1989; Stark and
Garbow, 1992) and cork suberin (Gil et al., 1997) do not
contradict the above hypothesis, showing carbons with
chemical vicinities compatible with primary and second-
ary ester group positions.
Earlier assumptions for the macromolecular develop-
ment of suberin involved the extensive cross-linking of
the long-chain aliphatics with aromatic phenylprope-
noids and the linear ω-hydroxyacid to ω-hydroxyacid
interesterification (Kolattukudy, 1977). This hypothesis
seems to be losing ground; direct evidence from solid
state 13C NMR shows that aliphatics and aromatics
occupy separate domains in suberized cell walls (Gil et
al., 1997; Bernards and Lewis, 1998). This supports
earlier histological and ultrastructural observations that
the suberin-containing cell walls have a multilayer
structure of two materials of alternate dark-clear
contrast (Sitte, 1962). Although glycerol has been found
here, and in other suberins (Grac¸a and Pereira, 1997,
1999), to be abundant enough to esterify all available
carboxylic groups, there is no evidence to exclude other
esters within the suberin polymer, such as the linear
The partial depolymerization of potato periderm
suberin also yielded feruloyl esters of ω-hydroxyacids
and alkan-1-ols (Table 2), as was the case for bark
suberins previously studied (Grac¸a and Pereira, 1998,
1999). The 18-hydroxyoctadec-9-enoic acid ester of feru-
lic acid was the most abundant ferulate (Figure 3). This
18-carbon acid is the most abundant ω-hydroxyacid and
the second most abundant suberin aliphatic acid mono-
mer. The peak of this ferulate in the GC-MS runs
appears to be divided, presumably due to the four
possible geometric isomers. Although the mass spectrum
remains basically the same, important variations are
observed in the abundance of some ions, namely, the
feruloyl ion (m/z 249 as the TMS ether) and the
molecular ion (m/z 560). The spectrum of the most
abundant subpeak is presented (Figure 3) and repre-
sents the trans-ferulic acid-cis-18-hydroxyoctadec-9-
enoic acid ester. Most of the ferulic acid found as
monomer is in the trans form, which is easy to assign
because the cis form exists in minor quantities and the
two isomers are well separated chromatographically. As
for the 18-hydroxyoctadec-9-enoic acid, the evidence
available indicates that it will be mostly in the cis form,
as discussed above.
A small quantity of another ester was found, with a
mass spectrum showing characteristics of both a feru-
late and a monoacylglycerol. After an open esterification
synthesis between ferulic acid and an excess of glycerol,
a GC-MS peak with the same mass spectrum was
obtained (Figure 4), and the compound was identified
as the 1-monoferuloylglycerol. Although the synthesis
conditions used could produce both the 1- and 2-isomers,
only one peak was obtained (45% yield based on the
initial ferulic acid) and was attributed to the 1-isomer
due to the presence of a significant ion at M - 103. We
know that this ion is present in the TMS derivatives of

5482 J. Agric. Food Chem., Vol. 48, No. 11, 2000
Grac¸a and Pereira
interesterication of ω-hydroxyacids. The ω-hydroxy-
acid-ω-hydroxyacid interester structure was proposed
to play a major role in cutins (Kolattukudy, 1977), and
a linear ester dimer of the 16-hydroxyhexadecanoic acid
has already been found in the tomato peel cutin, after
a partial hydrolysis reaction (Osman et al., 1995).
aliphatic polymer has a three-dimensional development
ensured by glycerol and exists independently from the
associated polyaromatics, to which they are peripherally
bonded. In this case, we propose that the name suberin
should be restricted to this glycerol-structured polyester.
ACKNOWLEDGMENT
Nevertheless, the linking points between the aliphatic
polyester part in suberins and the polyaromatics are
probably abundant. Solid state NMR has shown close
proximity of acyl chains and phenolics in wounded
potato tissue (Yan and Stark, 1998). Ferulic acid and
other related small phenolics are always found in the
suberin mixtures, as is also the case here in potato
periderm. Ferulic acid was also found here esterified to
the ω-hydroxyacids, as it has been before in two other
suberins (Grac¸a and Pereira, 1998, 1999). Because
ω-hydroxyacids are esterified through their acid end-
group to glycerol and through their primary hydroxyl
to ferulic acid, they may be the units in the suberin
glycerol-aliphatic polyester especially involved in link-
ing it to the surrounding polyaromatics. Such a trimeric
diester, glycerol-ω-hydroxyacid-hydroxycinnamic acid
(caffeic acid in this case) has been already identified in
the extractives of green cotton fiber (Schmutz et al.,
1994).
Thanks are due to Kevin Thurlow (Laboratory of the
Government Chemist, U.K.) for the naming of the
diglycerol trimers and to Isabel Baptista for technical
assistance.
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