Planta (1982)155:166-175
9 Springer-Verlag 1982
Composition and ultrastructure of the suberized cell wall
of isolated crystal idioblasts from Agave americana L. leaves*
Karl E. Espelie 1, Joachim Wattendorff 2, and P.E. Kolattukudy 1
1 Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164, USA
2 Institut ftir Botanische Biologic und Phytochemie der Universit~t Freiburg i.U., Albert-Gockel-Strasse 3,
CH-1700 Freiburg, Switzerland
Abstract. Styloid-calcium-oxalate-crystal-containing
idioblasts possess an interior cell-wall layer which has
a lamellar ultrastructure. Idioblasts were isolated by
centrifugation of an Agave americana leaf homoge-
nate through 2 M sucrose. The aliphatic monomers
of the polymeric material from an idioblast fraction
were primarily co-hydroxy acids (32%) and dicarbox-
ylic acids (35%), with CI8:1 dicarboxylic acid being
the most dominant monomer (25%). Nitrobenzene
oxidation of the idioblasts yielded syringaldehyde and
vanillin in a ratio of 0.46: 1. The major class of wax
associated with the idioblasts was free fatty acids
(34%). A major homologue of both the fatty acid
and fatty alcohol fractions of this wax was C22. The
hydrocarbon fraction of the wax had a broad chain-
length distribution with a large amount of even-num-
bered (47%) and shorter-chain homologues. The ul-
trastructure, the composition of the aliphatic and aro-
matic components of the polymeric material as well
as the composition of the wax show that the idioblast
cell wall is suberized. The wax and cutin polymer
of the epidermis of A. americana leaves were chemi-
cally characterized for comparative purposes.
Key words: Agave - Calcium oxalate - Cell wall
(idioblasts) 7 Crystal idioblast cell wall - Cutin - Su-
berin - Wax.
Introduction
Suberin, an insoluble polymer containing aliphatic
and aromatic domains, is deposited on the interior
side of the cell wall outside the plasma membrane.
This polymeric material is apparently interspersed
with layers of waxes giving a lamellar appearance
* Scientific paper No. 6115, Project 2001, College of Agriculture
Research Center, Washington State University, Pullman, WA
99164, USA
0032-0935/82/0155/0166/$02.00
Planta
to suberized walls. On the other hand, cutin, an insol-
uble polyester of aliphatic hydroxy and epoxy acids,
is deposited on a plant surface outside the pecto-
cellulosic epidermal cell wall and this polymer is also
embedded in waxes (for reviews, see Martin and Juni-
per 1970; Kolattukudy 1980 a; Wattendorff 1980). It
is not known whether all suberins thus anatomically
defined have chemical features by which they can
be clearly distinguished from all cutins. Isolation of
suberin-enriched preparations for chemical investiga-
tions involves removal of as much nonsuberinic mate-
rial as possible, and it is therefore not certain whether
all of the components thus obtained originated in
fact from suberin. Suberin-enriched preparations
from different anatomical regions of many plant spe-
cies must be investigated before general statements
about the chemical nature of suberin can be made.
However, the initial conclusions drawn from the re-
sults obtained with suberin preparations from potato
tubers (Kolattukudy and Agrawal 1974) and other
underground storage organs (Kolattukudy et al. 1975)
have remained valid for suberin preparations ob-
tained so far from other tissues (Dean and Kolat-
tukudy 1976; Espelie and Kolattukudy 1979a, b;
Espelie et al. 1979, I980b).
The density of calcium oxalate crystals in idio-
blasts, which are thought to have suberized walls,
provided a unique possibility for improved separation
of a suberin fraction. Calcium oxalate crystals in plant
cells are often encased in a sheath of wall material
(Wattendorff 1969, 1976a, 1980). In some cases this
sheath is continuous with an interior layer of the
cell wall which, because of its location and lamellated
structure, has been homologized to other suberinic
cell wall layers (Falk and el Hadidi 1961; Sitte 1962,
1975; Wattendorff 1974; Dean et al. 1977). The lucent
layers of this lamellar structure have been postulated
to be wax (Meyer 1938; Sitte 1957; Litvay and
Krahmer 1977) and selective inhibition of wax synthe-
K.E. Espelie et al. : Crystal idioblast suberin of Agave
sis p r e v e n t e d t h e d e v e l o p m e n t o f t h e s e l u c e n t l a y e r s
in w o u n d - h e a l e d p e r i d e r m a n d i n h i b i t e d t h e f o r m a -
t i o n o f a d i f f u s i o n b a r r i e r ( S o l i d a y e t al. 1979). I n
the present paper the chemical composition of the
l i p o p h i l i c i d i o b l a s t w a l l l a y e r is d e s c r i b e d . T h e c o m -
position of the aliphatic and aromatic components
of the polymer and of the associated wax shows that
t h e i d i o b l a s t cell w a l l is s u b e r i z e d a n d t h i s c h e m i c a l
c o m p o s i t i o n c o r r e s p o n d s r e m a r k a b l y to t h a t o f o t h e r
suberized tissues previously e x a m i n e d .
Materials and methods
Idioblast isolation. Fully grown leaves (60-75 cm long) from adult
plants of Agave americana L. were harvested during the mouths
of June to October, 1980 from the Botanical Garden, Freiburg
i.fJ., Switzerland. The leaf tip, the margins and their spines were
discarded. The cuticular membrane with some adjacent tissue was
peeIed off, air-dried at 30~ and stored at room temperature.
The remaining mesophyll tissue (seven batches of 200-450 g; total
1985 g) was cut into small pieces and homogenized in an Omni-
mixer (I. Sorvall Inc., Newtown, Conn., USA) with distilled water
(approx. 5 rain). The suspension was filtered through a glass-fiber
filter, the recovered solid was resuspended in water, mixed, and
filtered again for separation from the slime. The residue was sus-
pended in 1.5 1 of a solution containing 0.4% oxalic acid and
1.6% ammonium oxalate, and was incubated for 2 d at 30~ C.
The suspension was filtered and washed with water. The residue
was rehomogenized and filtered through a Bfichner funnel without
a filter to remove most of the fibers and vascular strands. Aliquots
(25 ml) of the filtrate thus obtained were layered onto 20 ml of
a 2 M solution of sucrose (density 1.26 g cm -3) and centrifuged
(2 min; 270 g). The crystal idioblasts with adhering tissue and some
pieces of vascular strands sedimented completely because of the
high density of calcium oxalate monohydrate (2.25 gcm 3) while
most of the other materials in the filtrate did not enter the sucrose.
The pellet was resuspended in water, filtered, washed with water,
and all pieces of vascular strands visible under a binocular micro-
scope (50 x ) were removed with forceps. The material remaining
above the sucrose solution was recovered, rehomogenized in water,
and centrifuged on sucrose as before. In some cases this treatment
was repeated a third time. The two or three yields of crystal idio-
blasts were collected on the same filter, dried at 30~ C, and stored
at room temperature. The amount of idioblasts in the seven differ-
ent isolations ranged from 0.51 to 1.61 mg g-1 fresh weight meso-
phyll tissue with a total yield of 2095.3 nag or 1.05 mg g - i meso-
phyll tissue. The percentage volume of pieces of vascular strands
remaining in the crystal idioblast fractions (crystals included) was
estimated to be l-5%. In these strands the cell walls of the xylem
vessels were lignified as indicated by the phloroglucinoI-hydrochlo-
ric acid reaction in the isolates as well as in situ in transverse
hand-sections of fresh leaves.
Electron microscopy. Part of the wet crystal idioblast fractions
(separately for the first and second yields) was placed without
fixation into acetone for 40 min or 3 h with several changes of
the solvent and then for 20 or 13 h in a 50% (v/v) mixture of
Durcupan ACM (Fluka, Buchs, Switzerland) and acetone at 30 ~ C
in a closed vessel. The embedding vessel was opened and the tem-
perature raised to 40~ for 3-5 d, then to 70-75~ C (2-3 d) for
complete polymerisation. Sections were stained with osmium te-
troxide, uranyl acetate, lead citrate, potassium permanganate or
167
with combinations of some of these. Other leaf tissue was fixed
in 0.07 M potassium phosphate-buffered (pH 6.8) 4% glutaralde-
hyde in situ for 22 h. Thin sections were stained using the sulphuric
acid - iodine/iodide - silver proteinate reaction (Wattendorff 1974,
section stain No. 5).
Wax extraction and analysis. The purified idioblasts were ground
to a fine powder in a Wig-L-Bug amalgamator (Crescent Dental
Manufacturing Co., Chicago, Ill., USA) and extracted with CHC13
for 24 h in a Soxhlet extractor. This solution was concentrated
to an oil which was subjected to thin-layer chromatography (TLC)
on Silica gel G (EM Laboratories, Elmsford, N.Y., USA) (1 mm
thick, 20 920 cm z) using hexane : diethyl ether :formic acid (40 : 10 : 1,
by vol.) as the solvent (system A). Individual classes of components
were eluted from the Silica gel with CHC13 : CH3OH (2 : 1, v/v).
A cutieular wax fraction was isolated by dipping the air-dried
epidermis mentioned above and also a mature leaf (approx.
200 cm 2, from a plant grown at Pullman) in CHC13 (500 ml) for
1 min at room temperature. The solvent was evaporated and the
resulting wax chromatographed as described above.
The free fatty acid and wax ester fractions were refluxed over-
night in 14% BF3 in CH3OH. The reaction mixtures were added
to H20 and the products, recovered by CHCt3 extraction, were
separated into methyl ester and fatty alcohol fractions by TLC
(solvent system A). The aldehyde fraction was reduced with NaBH4
in ethanol at room temperature for 4 h and the resulting alcohols
were purified by TLC (solvent system A).
The hydrocarbons and methyl esters were analyzed by com-
bined gas-liquid chromatography-mass spectrometry (GLC-MS)
with a gas chromatograph (Varian, Palo Alto, Cal., USA) attached
to a Perkin-Elmer-Hitachi RMU6D mass spectrometer (Perkin-
Elmer Co., Norwalk, Conn., USA) equipped with a Bieman separa-
tor interphase (Perkin-Elmer Co.) and a mass spectrometer readout
system (Columbia Scientific Industries, Austin, Tex., USA). The
samples were injected into a coiled glass column (180 cm length,
0.3 cm diameter) packed with 5% OV-1 on Gas-Chrom Q (Ana-
labs, North Haven, Conn., USA) at a temperature of 160~ C with
a programmed increase of 10~ C min-1. Peaks were identified by
their mass spectra, integrated by a Minigrator (Spectra-Physics,
Santa Clara, Cal., USA) and compared to authentic standards
for quantitation. Fatty alcohols were derivatized with N,O-bis(tri-
methylsilyl) acetamide (Sigma Chemical Co., St. Louis, Mo., USA)
at 90~ C before GLC-MS analysis.
Reductive depolymerization. To remove any traces of residual wax
the powdered idioblasts were extracted for an additional 24 h with
CHC13 and for 48 h with CH3OH. A portion of the dried residue
(0.5 g) was refluxed for 24 h in tetrahydrofuran with an excess
of LiA1D4 (Merck Chemical Co., St. Louis, Mo., USA) and the
CHC13-soluble material (0.014 g) recovered as before was subjected
to TLC with diethyl ether :hexane:methanol (8:2 : 1, by vol.) as
the solvent system to remove solvent impurities and highly polar
contaminants, all components with Rr 0.1-0.8 were eluted, deriva-
tized and analyzed by GLC-MS (Espelie et al. 1980b). The reduc-
rive depolymerization and GLC-MS analysis was repeated twice
with similar results and in one case separate classes of monomers
(alcohols, diols, triols and tetraols) were recovered from the thin-
layer chromatogram, o)-Hydroxypalmific acid (Aldrich Chemical
Co., Milwaukee, Wis., USA) was reduced with LiAIH4 and the
resulting diol was utilized as a standard.
The cuticular membrane was recovered from the air-dried
A. americana material from Freiburg after boiling for 1 h in a
solution containing ammonium oxalate (16 g 1-1) and oxalic acid
(4 g 1-1). The recovered cuticular membranes were treated with
a mixture of Aspergillus niger cellulase (5 g 1-1) and pectinase
(1 g 1-1) (Sigma Chemical Co.) at 30~ C for 16 h in 0.05 M acetate
buffer, pH 4.0, washed with water, air-dried, powdered (Wig-L-
168
Bug) and Soxhlet-extracted for 48 h each with CHC13 and then
CH3OH. Portions (0.1 g) of this leaf cutin were depolymerized
with LiAID 4 and analyzed as described above.
Nitrobenzene oxidation. A portion (0.695 g) of powdered and ex-
tracted idioblasts was added to 20 ml of 2 M NaOH and 1.0 ml
nitrobenzene and heated in a stainless steel bomb (1.9 cm internal
diameter, 8.0 cm high) (D and S Instruments, Pullman, Wash.,
USA) at 160~ C for 3 h. The reaction mixture was cooled, filtered,
and extracted with diethyl ether (4-50 ml). The aqueous fraction
was acidified with 6 M HC1 and extracted with diethyl ether
(4- 50 ml). These ether fractions were pooled, concentrated to an
oil (0.225 g) and subjected to TLC with ethyl ether:acetone:formic
acid (95:5:2, by vol.) as the solvent system. Components with
Rf 0.4 0.7 were eluted with CH3OH, concentrated to an oil
(0.020g), and treated with pyridine:acetic anhydride (2:1, v/v)
overnight at room temperature. This reaction mixture was added
to water and extracted with CHC13. The CHC13 layer was washed
with 0.1 M HCI, 0.1 M NaHCO3 and with water, concentrated and
subjected to TLC with CHC13:ethanol (9:1, v/v)~ as the solvent
system. The aromatic acetates, which had an Rf of 0.5, were eluted
and analyzed by GLC-MS. Acetate derivatives of vanillin and
p-hydroxybenzaldehyde (Sigma Chemical Co.) and syringaldehyde
(Aldrich Chemical Co.) were prepared and utilized as standards.
Results
Isolation and ultrastructure of idioblasts from leaves
of A. americana. A styloid 1 idioblast fraction with
little debris from mesophyll cells was isolated from
mature Agave leaves by centrifugation through 2 M
sucrose (Fig. 1). Although raphide idioblasts are also
present in Agave leaves (Wattendorff 1976b), they
were not observed in this styloid idioblast fraction
because they discharge their raphides during homo-
genization and thus do not sediment in 2 M sucrose.
The raphides are much smaller than the styloid crys-
tals and consequently sediment much less quickly.
Figure 2 clearly shows the lamellar structure typical
of the styloid idioblast cell wall (Wattendorff 1976 a).
The isolation process left many crystals within their
idioblasts (Figs. 1, 3). After the first centrifugation
of a leaf homogenate, the ratio in the pellet between
styloids enclosed in their idioblasts and those styloids
which showed no connection with a suberized cell
wall in a given section ranged between 2.3:1 and
0.95 : 1 (average from the counting of 893 crystal sec-
tions was 1.33 : 1). For crystals isolated after a second
centrifugation this ratio was between 0.89:1 and
0.17:1 (average from 742 sections was 0.28:1); subse-
quent centrifugation usually yielded much lower
quantity of material. Crystals that had no connection
with an idioblast wall in a given section might have
been attached to a wall in another plane. On the
other hand suberized material that was seen without
1 The styloid idioblast contains an elongated crystal with ridged
ends whereas the raphide idioblast contains a bundle of many
pointed, needle-shaped crystals per cell (Wattendorff 1976a;
Franceschi and Horner 1980)
K.E. Espelie et al. : Crystal idioblast suberin of Agave
a crystal (Fig. 4) was probably attached to a crystal
in another plane because it was found in the crystal
idioblast pellet.
The less-lamellated material of the suberized layer
of crystal idioblasts fixed in situ with glutaraldehyde
and osmium tetroxide (see "filling cork", in Fig. 2 b,
Wattendorff 1976a) was not observed in this study
in the material isolated by centrifugation and embed-
ded without prior fixation. Here the dark lamellae
of the "lamella cork" appeared after osmium or per-
manganate section staining, but often seemed to be
embedded in a clear, homogeneous material continu-
ous with the embedding resin (Fig. 5). Either the ace-
tone treatment must have dissolved the wax of the
filling cork, or the penetration of embedding material
into the layers of the filling cork was so perfect that
no limits could be perceived. After section staining
with permanganate there was often an irregular,
cloudy contrast where the filling cork is normally
observed (Fig. 2). It is possible that this cloudiness
was caused by a product of a reaction between the
filling cork and the permanganate. If this is the case,
it is likely that the wax in the filling cork was not
completely dissolved by the acetone and that the per-
manganate reacts more readily with its remnants than
does the osmium. The present results are consistent
with earlier opinions (Wattendorff 1969; Litvay and
Krahmer 1977) that the filling cork which also forms
the crystal sheaths might have a high wax content.
However, the chemical nature of the material called
filling cork which is less obviously lamellated in situ
than "lamella cork" has not been established.
The percentage volume of suberized wall layers
of the whole isolated cell wall was determined from
the corresponding surfaces in the sections. Estima-
tions of 43 electron micrographs of small magnifica-
tions (1,500-8,400 x ) resulted in an average value of
7.3% for a first yield or for mixed yields. Three of
these estimations were controlled by counting on a
point scanning pattern which gave values of the same
order of magnitude. In a separately isolated second
yield only 0.9% of the wall material was suberinic
(estimations from 16 micrographs, 1,500-7,100 x). In
this fraction many crystals had become enveloped
in pecto-cellulosic wall material of other cells during
the isolation process and had drawn this material
with them.
Thin sections made from A. americana leaf tissue
fixed in situ in glutaraldehyde gave a faintly positive
sulfuric acid-iodine/iodide-silver proteinate reaction
in the suberinic layer. Sometimes the silver granules
of the reaction were arranged in a lamellar pattern
(Fig. 6). This reaction seems to depend on the epoxide
content of the polymer (Holloway et al. 1981) and
the weak reaction seen may indicate that the suberin
Figs. 1, 2. Styloid idioblast fraction isolated from A. americana leaves, embedded without fixation. Fig. 1. General aspect of a section
through a fraction (isolated by the first centrifugation of homogenized mesophyll). Part of a vascular strand with two xylem vessels
(X) is sectioned, an extremely rare case. Arrows indicate some places where suberized layers are evident. The proportion of suberin
layers in the total wall material was estimated to be 4%. Section staining (s.s): 0.5% uranyl acetate followed by lead citrate, x 1,500.
Fig. 2. The lamellation in the lamella cork (L) has become visible following permanganate staining. The position of the filling cork
(F) is marked by a diffuse contrast where it is protected by the surrounding lamella cork but this contrast is weak in an open position
(upper left). The percentage of suberinic layers in relation to overall wall material is approx. 18%. s.s.: 2% K M n O 4 followed by
0.5% uranyl acetate. C, pecto-cellulosic cell wall. x 43,500
Figs. 3-5. Isolated styloid idioblast fraction from A. americana leaves (from initial or pooled centrifugations of homogenized mesophyll)
embedded without fixation. Fig. 3. Crystal idioblast with a crystal hole, especially rich in cell wall ramifications with suberinic layers,
the latter stained by KMnO4. The outgrowth from the right hand crystal angle is continuous with the suberinic outgrowth underneath
and only cut by the right edge of the figure, s.s.: 1% OsO4 followed by 2% KMnO,. x6,900. Fig. 4. Crystal idiohlast probably
cut through its tip which contains no crystal, s.s.: 0.5% uranyl acetate followed by 1% KMnO4. • Fig. 5. No clear limit is
visible between filling cork and embedding material in this outgrowth. Lamellation is visible in the lamella cork (L). s.s.: 2% OsO4
followed by 0.5% uranyl acetate and by lead citrate, x 38,200. Fig. 6. Styloid idioblast fixed in situ with 4% glutaraldehyde. The
H 2 S O 4 - I 2 / K I - A g P reaction results in a linear arrangement of silver granules in the suberinic layer (arrows). C, pecto-cellulosic cell
wall. • 35,500
K . E . Espelie et al. : C r y s t a l i d i o b l a s t s u b e r i n o f Agave 171

polymer of the idioblast contains a small amount T a b l e 1. C o m p o s i t i o n o f the a l i p h a t i c c o m p o n e n t s o f the lipid-

of epoxy acid. This, in fact, was shown to be the d e r i v e d p o l y m e r s f r o m the e p i d e r m i s a n d f r o m the i d i o b l a s t s o f
case by the chemical analysis described below. A. americana leaves. D - d e t e c t a b l e b u t less t h a n 0 . 1 %

Epidermis Idioblast
Reductive depolymerization of epidermal and idioblast
fractions from leaves of A. americana. The isolated Fatty alcohols
idioblasts of A. americana were depolymerized with C16 - 1.0
LiA1D4 in an effort to define chemically the polymeric CI8:I - 0.2
material responsible for the lamellar appearance seen C2o - 1.8
C22 - 0.7
in the cell wall of the idioblasts (Fig. 2). Since the
C24 - 0.3
cutin from the epidermis of the leaf was a possible
contaminant of the idioblast preparation, the cuticle Total - 4.0
from A. americana leaf was isolated and depolymer- Fatty acids
ized for comparison. The aliphatic monomers which C16 - 2.1
were released by LiA1D4 treatment and identified by CI8:1 0.6
GLC-MS are given in Table 1. The major classes of C20 - 3.3
aliphatic components of the polymer from the idio- C22 - 1.4
C24 -- 0.4
blasts were the co-hydroxy acids (32%) and the dicar-
boxylic acids (35%). Although co-hydroxyoctadecen- Total - 7.8
oic acid was the largest component (11%) of the ~o-hy-
o)-Hydroxy acids
droxy acid class, the C~6, C22 and C24 monomers C16 D 9.0
were comparable in amount (6-9%). However the C18:1 -- 10.8
C18:1 diacid was the dominant component in the di- C22 -- 6.7
carboxylic acid fraction comprising 72% of this group C24 - 5.5

of monomers. Total D 32.0

9,10-Dihydroxy C1 s dicarboxylic acid, which com-
prises 2% of the aliphatic portion of the idioblast Dicarboxylic acids
Ct~ - 6,3
polymer (Table 1), was identified on the basis of deu-
C18:I -- 25.1
terium incorporation. Reduction of this diacid with 9,10-Dihydroxy Cla - 2.0
LiA1D~ generated a C~8 tetraol distinguishable from Cz2 - 1.4
the tetraol generated from the reduction of 9,10,18-tri-
Total - 34.8
hydroxyoctadecanoic acid by the addition of two
more deuterium atoms during the reduction of the Polar acids
second carboxyl moiety. This difference was seen in D i h y d r o x y C~6 16.2 1.0
o J - H y d r o x y o x o C16 - 1.5
the c~-cleavage region of the mass spectrum where
9,10-Epoxy-18-hydroxy-C18 30.9 0.6
the dominant a-cleavage ions atm/e 305 (containing 9,10,18-Trihydroxy-C1 a 52.2 2.7
two deuterium atoms) and m/e 303 (with no deuteri-
um) appeared in the ratio of 2.7:1, indicating that Total 99.3 5.8
approx. 43% of the Cls tetraol had originated from Unknown 0.7 15.6
the 9,10-dihydroxy C1 s diacid.
Fatty alcohols and fatty acids comprised 4 and
8%, respectively, of the aliphatic portion of the idio-
blast polymer and in both cases, monomers with is a 10 (or 11), 18-dihydroxy Cls unsaturated acid
chain-lengths greater than Cas totalled 65-70% of with mid-chain positional isomerism.
the components in that class (Table 1). The degree and location of deuterium incorpora-
An additional monomer (11.2%) from the idio- tion into the C16 triol released from the idioblast
blast polymer could not be identified by its mass polymer, as shown by the c~-cleavage portion of the
spectrum. The two major a-cleavage fragments were mass spectrum of the trimethylsilylated derivative, in-
at m/e 317 and 331 which are possibly 10 and 11 dicated that 60% of this triol was derived from co-hy-
carbon fragments, respectively, both containing two droxyoxo Ct6 acid (as described in Espelie etal.
trimethylsilyloxy functions. The retention time of the 1980b). The remainder of the triol had originated
derivatized monomer in the gas chromatogram was from dihydroxyhexadecanoic acid. The mid-chain hy-
less than that of the derivatized C~8 triol which is droxyl group was subject to positional isomerism in
derived from 18-hydroxy-9,10-epoxyoctadecanoic both monomers with the 10-hydroxy isomer as the
acid and it is possible that this unidentified monomer dominant one (Table 2).
172 K . E . Espelie et al. : C r y s t a l i d i o b l a s t s u b e r i n o f Agave

T a b l e 2. P e r c e n t c o m p o s i t i o n o f p o s i t i o n a l i s o m e r s o f m i d - c h a i n (Table 1). These two monomers, which were seen in

o x i d i z e d o ) - h y d r o x y h e x a d e c a n o a t e f r o m the l i p i d - d e r i v e d p o l y m e r s the GLC-MS as the trimethylsilyl ethers of a C18
o f the e p i d e r m i s a n d i d i o b l a s t s f r o m A. americana leaves
tetraol and a Ct8 triol, respectively, were identified
Position
by their characteristic incorporation of deuterium
of mid-chain group upon LiA1D4 reduction as described previously (Wal-
ton and Kolattukudy 1972). Positional isomers of di-
C-7 C-8 C-9 C-10 hydroxy C16 acid were found and among them the
Epidermis
10,16-dihydroxy acid was the major one as in the
Dihydroxyhexadecanoate 3 27 14 56 idioblast polymer but in the cuticular membrane the
Idioblast
8,16-dihydroxy acid was more abundant than the
Dihydroxyhexadecanoate 0 8 27 65 9,16-dihydroxy acid whereas the reverse was true in
co-Hydroxyoxohexadecanoate 0 8 17 75 the idioblasts (Table 2).

Nitrobenzene oxidation of A. americana idioblasts. In

T a b l e 3. P h e n o l i c a l d e h y d e s r e l e a s e d b y a l k a l i n e n i t r o b e n z e n e oxi- an effort to examine the phenolic portion of the idio-
dation of idioblast preparation and vascular bundle fractious from
blast polymer, the isolated idioblasts were subjected
the l e a f o f A. americana. Values a r e given in g g m g - 1 d r y tissue
to alkaline nitrobenzene oxidation (Hartley 1971).
Idioblasts Vascular bundles Since lignified tissue was a possible contaminant of
the idioblast fraction (Fig. 1) vascular bundles with
p-Hydroxybenzaldehyde 0.02 0.36
fibers surrounding them were isolated from Agave
Vanillin 0.26 1.95
Syringaldehyde 0.12 10.91 leaves (from Pullman) and subjected to nitrobenzene
oxidation for comparison. The components, which
were ether-soluble after acidification of the reaction
In contrast to the composition of the idioblast mixtures, were examined as their acetates by gas li-
polymer, cutin from the leaf was composed of mainly quid chromatography-mass spectrometry (GLC-MS).
the typical C18 family of acids with trihydroxyoctade- Vanillin was the major (0.26 lag mg- 1 dry tissue) phe-
canoic acid (52%) and 9,10-epoxy- 18-hydroxyoctade- nolic component released from the idioblasts with
canoic acid (31%) as the two major components smaller amounts of syringaldehyde and p-hydroxy-

T a b l e 4. C o m p o s i t i o n o f t h e w a x a s s o c i a t e d w i t h the e p i d e r m i s a n d i d i o b l a s t s o f A. americana leaves. Values in p a r e n t h e s e s i n d i c a t e

the p e r c e n t a g e in e a c h class a

C15 C16 C17 C18 C19 C20 C21 C22 C23 C24 C25 C26 C27 C28 C29 C30 C31 C32 C33

Hydrocarbon
E p i d e r m i s (6.7) - 0.1 0.1 0.2 0.2 0.1 0.3 0.3 2.1 14.3 11.3 34.0 9.2 7.7 1.1 16.2 0.1 2.7
I d i o b l a s t (16.1) 0.1 0.2 1.7 3.5 3.6 6.1 7.0 10.9 9.9 i1.8 20.2 6.8 3.9 7.7 5.9 0.4 0.2 - -

W a x ester
E p i d e r m i s (16.3)
Acids 18.5 - 60.1 -- 4.9 - 16.3 -- 0.2 -- 0.1 . . . . . . .
Alcohols -- 0.2 -- 0.2 - 1.1 - 0.1 - 0.3 - 46.3 -- 34.2 - 5.4 -- 0.3 -
I d i o b l a s t (0)
Aldehyde
E p i d e r m i s (10.1) m m _ . . . . 0.1 2.1 16.0 2.8 70.5 2.8 3.6 0.9 0.4 -
I d i o b l a s t (0) _ m m

Fatty acid
E p i d e r m i s (t 9.7) 35.9 - 35.5 b - 3.5 0.6 3.9 0.6 3.6 1.1 4.0 0,1 6.3 0.1 0.3 - - -
I d i o b l a s t (33.9) 6.3 43.7 - 11.9 - 1.3 - 28.5 - 1.0 - 0.6 . . . . . . .

Fatty alcohols
E p i d e r m i s (45.2) 0.3 -- 0.2 - - - 0.1 -- 0.4 0.1 35.5 0.2 62.3 0.1 0.7 - 0.1 -

I d i o b l a s t (23.4) - 0.5 0.3 - 0 . 4 - 8 8 . 1 - 0 . 2 - - - 0.l . . . . .

" T h e w a x a s s o c i a t e d w i t h the e p i d e r m i s w a s e x t r a c t e d f r o m the c u t i c u l a r m e m b r a n e w h i c h h a d b e e n p h y s i c a l l y peeled o f f f r o m the

l e a f w i t h s o m e a d h e r i n g tissue (see M e t h o d s ) a n d t h e r e f o r e w o u l d i n c l u d e w a x e s w h i c h h a d been e m b e d d e d in cutin, a p o r t i o n
o f the w a x w h i c h h a d b e e n a d h e r i n g to the c u t i c u l a r s u r f a c e a n d v e r y small q u a n t i t i e s o f w a x w h i c h m i g h t h a v e b e e n p r e s e n t
in the a d h e r i n g tissue
b Includes unsaturated components
K.E. Espelieet al. : Crystalidioblast suberin of Agave
benzaldehyde. On the other hand, syringaldehyde was
the major phenolic component released by oxidation
of the vascular bundles (10.9 gg rag- 1) (see Table 3).
Composition of the wax associated with the cuticle and
the idioblasts of A. americana leaves. The wax was
recovered from both the epidermis (cuticle-associated
wax) and the idioblasts (suberin-associated wax), frac-
tionated into classes by thin-layer chromatography
(TLC) and analyzed by GLC-MS. The composition
of both waxes is given in Table 4. Fatty alcohols con-
stitute the major fraction (45%) of the epidermal wax
with fatty acids and wax esters comprising 20 and
16%, respectively. The major class of the idioblast
wax was free fatty acids (34%), and fatty alcohols
comprised only 23% of this wax. Although major
components of the free fatty acid fractions for both
the idioblast and epidermal waxes were C16 and Cls,
a unique feature of the idioblast wax was its high
content (29%) of C22. Long-chain (>C18) compo-
nents accounted for 24 and 31% of the free fatty
acids of the epidermal and idioblast fractions, respec-
tively. The major long-chain acid from the wax ester
fraction of the epidermal wax was C22 (16%). The
dominant fatty alcohol of the idioblast wax was C22
(88% of that class) whereas the major alcohols of
the epidermal wax were C26 (36%) and C28 (62%).
These latter two alcohols were also the dominant
members of the alcohols released from the wax ester
fraction of the epidermal wax. The idioblast wax had
no detectable wax ester fraction. The major compo-
nents in the aldehyde fraction of the epidermal wax
were C26 and C28, a class which was also not found
in the idioblast wax. The major hydrocarbons of the
epidermal wax were C2s (14%), C27 (34%) and C31
(16%). The largest component of the idioblast hydro-
carbon fraction was C25 (20%) with C22, C23 and
C24 each comprising approx. 10%.
Discussion
Ultrastructural and chemical analyses presented here
clearly indicate that the styloid calcium oxalate crystal
idioblasts from A. americana leaves are suberized. The
lamellar structure found in the idioblasts (Fig. 2) is
identical with the structures previously described in
situ in idioblasts (Wattendorff 1969, 1976a), and is
similar in appearance to other suberinic layers (Sitte
1962, 1975; Dean et al. 1977). Such lamellar structure
has also been seen and characterized chemically as
suberin in the natural periderm (Kolattukudy and
Agrawal 1974), wound periderm (Kolattukudy and
Dean 1974; Soliday et al. 1979) and "hollow heart"
regions of potato tubers (Dean et al. 1977) as well
173
as in the chalazal region of the inner seed coat of
Citrus paradisi seeds (Espelie et al. 1980b).
Based on the composition of suberin-enriched
preparations from a limited number of plants it was
concluded that the characteristic aliphatic compo-
nents of a suberin polymer are co-hydroxy acids and
dicarboxylic acids (see review by Kolattukudy 1975);
the present results are consistent with such a conclu-
sion. The dicarboxylic acids are biosynthetically de-
rived from the co-hydroxy acids and it was shown
that the o)-hydroxy acid dehydrogenase from wound-
healing potato tuber has a chain-length preference
for the C~s:l and shorter monomers (Agrawal and
Kolattukudy 1978). Such a chain-length specificity
appears to be reflected in the composition of the
idioblast suberin (Table 1). The presence of very long-
chain (> Cls) monomers, a characteristic of suberin
(Kolattukudy 1975, 1981), was also observed in the
idioblast polymer.
The composition of the polymer isolated from the
epidermis of the leaf is characteristic of a cutin with
the monomers being predominantly polar acids
(Table 1). In ultrastructural and chemical examina-
tions no cuticular contamination of the idioblast frac-
tion was detected. The chemical composition is in
general agreement with the results of Wattendorff
and Holloway (1980, 1982). The position of the mid-
chain hydroxyl group in the dihydroxy C16 and
co-hydroxyoxo Ci6 acids was fairly similar in both
the idioblast and epidermal polymers (Table 2) with
the C-10 isomers being the most prominent. In several
cases where cutin and suberin polymers from the same
plant have been studied the positional isomer com-
position of the mid-chain oxygenated C16 acids has
been very similar (Kolattukudy 1975; Espelie and
Kolattukudy 1979a, b; Espelie et al. 1980b). Such
a similarity may represent a species-related specificity
of the enzyme which hydroxylates co-hydroxy C 16 acid
(Soliday and Kolattukudy 1978).
The presence of both phenolic and aliphatic com-
ponents appears to be characteristic of suberin and
it has been estimated that 10~0% of the polymer
is composed of phenolics (Kolattukudy 1981). How-
ever, very little is known about the structure and
composition of the phenolic domain of suberin. Re-
cently nitrobenzene oxidation methods similar to
those used in studies on lignins (Chang and Allan
1971) have been used to examine the phenolic compo-
nents of suberin from potato periderm (Cottle and
Kolattukudy 1982). Such studies suggested that the
amount of p-hydroxybenzaldehyde and vanillin re-
leased by nitrobenzene oxidation can be used as a
measure of the phenolic components of suberin. Upon
nitrobenzene oxidation of the polymeric material of
the Agave idioblast small amounts of p-hydroxyben-
174
zaldehyde, vanillin and syringaldehyde were released.
These c o m p o n e n t s could arise f r o m the suberin of
the idioblasts or f r o m the lignin o f the vascular tissue
possibly contained in the idioblast preparations. Iso-
lated vascular bundles, u p o n oxidation with nitroben-
zene gave syringaldehyde as the m a j o r c o m p o n e n t
whereas vanillin was the m a j o r c o m p o n e n t released
f r o m the idioblasts. These results suggest that a r o m a t -
ic aldehydes released f r o m the idioblast preparation
did not originate f r o m the lignin o f vascular bundles
although syringaldehyde could have originated at
least in part f r o m such contamination. It is probable
that the p - h y d r o x y b e n z a l d e h y d e and vanillin origi-
nated f r o m the p h e n y l p r o p a n e moieties of the suberin
p o l y m e r of the idioblasts. A l t h o u g h the a m o u n t s of
such aldehydes released are small the observation that
both idioblasts and natural as well as w o u n d periderm
f r o m p o t a t o tubers give similar c o m p o n e n t s indicates
that the release of p - h y d r o x y b e n z a l d e h y d e and vanil-
lin by nitrobenzene oxidation can be used as a tool
in the chemical examination o f suberin.
The wax associated with the suberin o f the idio-
blasts had a composition distinct f r o m that o f the wax
associated with the cutin of the epidermis (Table 4).
Features of the idioblast wax composition were simi-
lar to those which had been previously shown to be
characteristic of suberin-associated waxes f r o m the
periderm of some u n d e r g r o u n d storage organs
(Espelie et al. 1980a). The p r o p o r t i o n of free fatty
acids (34%) was very high for a plant wax (Kolattuk-
udy 1980b) and C22 was a m a j o r homologue. The
chain lengths o f the m a j o r free alcohols were shorter
in the idioblast wax (C22) than in the epidermal wax
(C26 and Czs). The h y d r o c a r b o n s o f the idioblast
wax had a b r o a d e r chain-length distribution than
those of the cuticle in that a larger n u m b e r of hydro-
carbons were f o u n d as significant c o m p o n e n t s in the
former. Furthermore, the idioblast h y d r o c a r b o n s
showed a higher p r o p o r t i o n of even chain-length ho-
mologues (47 versus 24%) and a shorter total average
chain-length (23.8 versus 27.6) than the cuticular hy-
drocarbons. All of these have been previously f o u n d
to be characteristics of alkanes in suberin-associated
waxes (Espelie et al. 1980a).
Suberization of the idioblasts appears to be analo-
gous to previously characterized instances of suberiza-
tion in aerial parts of the plant (for example the endo-
dermal fraction in Sorghum, bundle sheaths in Zea
mays, and the chalazal region o f the inner seed coat
o f Citrus paradisi; Espelie and K o l a t t u k u d y 1979a,
b; Espelie et al. 1980b) where the p o l y m e r is laid
d o w n during the development o f the plant as a means
of providing a barrier to diffusion. Also, in the case
of the idioblast, suberization p r o b a b l y provides a bar-
rier to isolate the calcium oxalate crystals. Other inter-
K.E. Espelie et al. : Crystal idioblast suberin of Agave
nal lipid-derived layers which have been ultrastructur-
ally characterized m a y also be suberin polymers laid
d o w n as protective barriers by the plant.
The skillful technical assistance of Fr/iulein Yvette Isler was much
appreciated. We thank Dr. David Coahran for assistance with
the GC-MS system. Financial support from the Swiss National
Science Foundation is gratefully acknowledged. This work was
supported in part by National Science Foundation grant PCM
80-07908.
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Received 8 January; accepted 15 March 1982