Comparative FTIR and 13C CP/MAS NMR Spectroscopic Investigations
on Sporopollenin of Different Systematic Origins
Sabine W ilm esm eier3, Stefan Steuernagelb, and R o lf W ierm ann3
a Institut für Botanik der Westfälischen Wilhelms-Universität Münster,
Schloßgarten 3, D-48149 Münster, Bundesrepublik Deutschland
b Bruker Analytische Meßtechnik GmbH, Silberstreifen,
D-76287 Rheinstetten 4, Bundesrepublik Deutschland
Z. Naturforsch. 48 c, 697-701 (1993); received June 18,1993
Sporopollenin, Pollen, Microspores, Megaspores, FTIR Spectroscopy, 13C NMR Spectroscopy
Comparative phytochemical investigations were carried out on sporopollenins of the follow­
ing plant species: Bryophyta - Reboulia hemisphaerica and Polytrichum strictum; Pteridophyta
- Equisetum arvense, Selaginella selaginoides micro- and megaspores; Gymnospermae - Cycas
circinalis; Angiospermae - Typha angustifolia and Tulipa cv. “Apeldoorn”. Although the
range of the analyses included species from the Bryophyta to the Angiospermae, fundamen­
tal correspondences could be detected in the 13C NMR and FTIR spectra concerning the occur­
rence of aliphatics, aromatics, ether and carbonyl/carboxylic functions in varying degrees.
While the amount of aliphatics strongly dominated in the molecular structure of all other
sporopollenins investigated here, it was found in sporopollenin of Equisetum arvense in a much
lower extent. Despite the different morphology of Selaginella selaginoides micro- and mega­
spores, their sporopollenin spectra were nearly identical.
Introduction
Sporopollenin is an extrem ely resistan t biopoly­
m er, which is substantially involved in th e fo rm a­
tion of the ou ter spore and pollen wall. This b io ­
polym er is resistant against non-oxidative chem ical,
physical and biological degrad atio n to a high extent.
In th e past substantial results concerning th e struc­
tu re of sporopollenin have been o b tain ed [1], n ev er­
theless fundam ental aspects of th e definite polym er
structures and especially the biosynthetic pathw ays
leading to it are n o t yet clear in detail. T he results
of tracer experim ents [2 -5 ] revealed th at, in
com parison to all o th er precursors, such as
D,L-[2-14C]m evalonate, D -[l-14C]glucose, [U -14C]acetate, [2-14C]m alonic acid, L-[U-14C ]tyrosine,
[4-14C ]isopentenylpyrophosphate and [2-u C \-pcoum aric acid a high level of incorp o ratio n into the
sporopollenin fraction could be achieved using
L-[U-14C]phenylalanine as a tracer. D eg rad atio n of
the sporopollenin sam ples labelled via [ring-14C]Parts of the results were presented at the meeting of the
XII. International Congress on Sexual Plant Reproduc­
tion in Ohio, July 1992, at the Meeting of the German
Botanical Society in Berlin, September 1992, and at the
Joint Meeting of the German and Dutch Societies for
Cell Biology in Münster, March 1993.
Reprint requests to Prof. R. Wiermann.
Verlag der Zeitschrift für N aturforschung,
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phenylalanine by potash fusion resulted in the fo r­
m ation of p-hydroxybenzoic acid as the m ain label­
led d egradation product. Thus the integral incor­
p o ra tio n of the arom atic ring system into the poly­
m er could be clearly dem onstrated. T herefore it can
be assum ed th at the arom atic m etabolism is in ­
volved in sporopollenin biosynthesis.
O nly a low level of incorporation was achieved
using [U -14C ]-acetate and [2-14C]m alonic acid.
T hese results w ere unexpected because, according
to results of 13C N M R spectroscopic analyses o b ­
tain ed so far, sporopollenin mainly consists of longchain aliphatics w ith a substantial am ount of acid
an d /o r ester groups [6, 7]. T he oxygen content in
e th er, hydroxy, carboxylic acid, ester or ketonic
carbonyl groups m ay vary considerably.
O n th e basis of 13C N M R analyses, H em sley et al.
[8] observed that, despite far-reaching sim ilarities,
several differences occur in the spectra of fossil and
m o d ern sporopollenins. In contrast to recen t m a te­
rial they detected few er b ut bro ad er peaks in the
spectra of fossil m aterial. T hese findings w ere in­
te rp re te d as a hint of th e loss of functional groups
p articularly of those containing oxygen.
D u e to the higher com plexity of IR spectra, only
few applications of this m ethod for the structural
elucidation of sporopollenin can be found in the re ­
cent literatu re [9, 10]. T he IR spectroscopy, im ­
p roved by the F ourier transform technique with its
fast realization and high degree of resolution offers,
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S. W ilmesmeier et al. ■Com parative 13C CP/M AS N M R Spectroscopic Investigations on Sporopollenin
in contrast to old er studies [11, 12], various possi­
bilities for the characterization of sporopollenin
structures, especially in com parative studies of
sporopollenins from different origin and in com bi­
nation with th e results of 13C N M R spectroscopy.
T o o u r know ledge such phytochem ical investiga­
tions do not exist until now. T herefore, com para­
tive F T IR and 13C N M R spectroscopic studies w ere
carried out on a b road basis. It was the aim to o b ­
tain m ore know ledge about th e structure and
m olecular com position of sporopollenin and to
characterize the correspondences and differences
in th e com position of sporopollenin from various
system atic origin.
Materials and Methods
Sporopollenin was isolated from pollen or spores
of the following p lan t species: B ryophyta - Reboulia hemisphaerica and Polytrichum strictum; Pteridophyta - E quisetum arvense, Selaginella selaginoides micro- and m egaspores; G ym nosperm ae Cycas circinalis; A ngiosperm ae - Typha angustifolia and Tulipa cv. “A p eld o o rn ”. T o elim inate
soluble phenolic, lipoid o r terp en o id com ponents
being accum ulated in and/or on th e structures of the
exine/exospor, th e pollen and spore m aterial was
extracted exhaustively for several w eeks with dif­
feren t solvents. T he sporopollenin fraction was iso­
lated in a conventional way by tre a tm e n t with ph o s­
phoric acid (80% , 50 °C, 10 d) follow ed by a fu rth er
solvent extraction p rocedure of the residue. T he
obtain ed m aterial was lyophilized and stored in a
desiccator until exam ination.
F T IR analyses w ere carried out at th e Institut für
O rganische C hem ie der W estfälischen W ilhelmsU niversität M ünster on a N icolet 5 D X C spectrom ­
eter. F T IR spectra of the sam ples (K B r pellets)
w ere obtain ed betw een 4000 cm-1 and 400 cm-1.
13C CP/M A S N M R analyses w ere carried out with
a B ru k er M SL 200 or MSL 500 sp ectrom eter with
th e following set-ups: C-13 frequency: 50.3 or
125.7 MHz; sam ple rotation: 5 kH z round an axis
tilted 54.4° against the m ain m agnetic field axis;
contact tim e for cross polarization (CP): 1 ms;
aquisition period: 5 - 1 0 ms according to field in­
tensity; 90° pulse length: 4 ^is; spectral width:
5 0 - 6 0 kH z according to field intensity; repetitions:
800-16,000; pulse repetition time: 3 s. C hem ical
shifts are expressed in parts p er m illion (ppm ) and
are relative to external tetram ethylsilane.
Results and Discussion
T he 13C N M R and F T IR spectra of the analyzed
sporopollenin sam ples are shown in Figs. 1 and 2.
T h e 13C N M R spectra reveal th at long-chain aliphatics rep resen t a high am ount of the sporopolle­
nin structure, because all N M R spectra are dom i­
nated by the large signal No. 7 except for Cycas cir­
cinalis and E quisetum arvense. T he IR spectra con­
firm the occurrence of aliphatics by the bands C, H,
I and P (T able II). T he presence of arom atic com ­
p ounds is indicated by the N M R peaks 3, 4, 5
(T able I) and the IR signals B, G and O.
T he N M R signal 6 and IR bands L, M, N are char­
acteristic of e th er groups, w hereas the N M R bands
1 ,2 and the IR signals D , E, F are attrib u ted to car­
bonyl and carboxyl functions.
A com parative exam ination of the sporopollenin
spectra from B ryophyta, P teridophyta, G ym no­
sperm ae and A ngiosperm ae reveals a good accord-
BRYOPHYTA
Polytrichum commune
Reboulia hemisphaerica
PTERIDOPHYTA
Equisetum arvense
Selaginella setaginoides
megaspores = microspores
Fig. la . 13C CP/MAS NMR spectra of sporopollenin
isolated from different plant taxa; for interpretation
see Table I.
S. W ilmesmeier et al. ■Com parative 13C CP/MAS N M R Spectroscopic Investigations on Sporopollenin
699
GYMNOSPERMAE
Cycas circinalis
ANGIOSPERMAE
Typha angustifolia
Tulipa cv. 'Apeldoorn'
Fig. lb . 13C CP/MAS N M R spectra of sporopollenin
isolated from different plant taxa; for interpretation
see Table I.
ance in the type and position of th e d etected peaks.
O n the other hand th ere is a great diversity co n cern ­
ing the peak intensities; e.g. am ong the species
p resen ted here, Cycas exhibits th e highest relative
content of arom atic structures, w hereas sp o ro p o l­
lenin from Tulipa pollen is ch aracterized by its high
content of aliphatics.
Sporopollenin from spores of E quisetum arvense
seem s to be extraordinary d ifferent since the ali­
phatic signals in its spectra are strongly red u ced
(Fig. 2) or com pletely missing (Fig. 1). A s to th e
existence of this structural elem ent sporopollenin
of E quisetum arvense probably differs from o th e r
pteridophytic spores. A fu rth er peculiarity o f th e
E quisetum spores is fo r exam ple its sporoderm al
perm eability of w ater [13].
T he heterosporous Lycopsida Selaginella selaginoides was the o th er m em ber o f th e P terid o p h y ta
investigated. So far studies on th e walls of m icroand m egaspores m ainly concerned th e ir u ltrastru c­
tu re and fluorescence behaviour. O n the basis of
such results W illem se [14] show ed th a t sp o ro p o lle­
nin is not only a com ponent of th e m icrospore wall
b u t also of the wall surrounding th e fem ale gam etophyte of Cycas revoluta.
Fig. 2 a. F T IR spectra of sporopollenin isolated from
different plant taxa; for interpretation see Table II.
H ere, for the first tim e sporopollenin of m icroand m egaspores have b een com paratively analyzed
by F T IR and 13C N M R spectroscopy. B oth spec­
troscopic m ethods led to alm ost identical spectra
(for this reason only one of them is presented), re ­
vealing a high degree of consistence in the chem i­
cal com position of the micro- and m egaspore sp o ro ­
pollenin of this system. Considering the strongly
different m orphology of Selaginella selaginoides
m icro- and m egaspores these results are rem a rk ­
able. W hereas the sporoderm al sporopollenin of the
sm aller m icrospores is located in the para-exospore
and exospore, in the ten tim es larger m egaspores it
occurs in an bilayered exospore, whose volum inous
external layer consists of a spongy structure which
does n o t occur in any o th er recent pteridophytic
spore [15].
In th e obvious presence of aliphatic signals Selagi­
nella sporopollenin seriously differs from Equise-
700
S. W ilmesmeier et al. • Com parative 13C CP/M AS N M R Spectroscopic Investigations on Sporopollenin
Table I. 13C NMR assignments for the analyzed sporopollenins.
No. in
Fig. 1
Carbon type
Chemical shift
[ppm]
1
2
ketonic carbonyl
carboxylic acid
carboxylic ester
non-protonated
aromatic carbon
aromatic
alkene and/or aromatic
alcohol and ether
CH2 in polymethylenic chains
and CH3
200-210
170-180
160-170
3
4
5
6
7
Fig. 2 b. FTIR spectra of sporopollenin isolated from
different plant taxa; for interpretation see Table II.
turn sporopollenin, th e o th e r pteridophytic species
investigated.
In sum m ary it can b e concluded from the results
of F T IR and N M R spectroscopy th at the analyzed
sporopollenins show principle correspondences in
the occurrence of aliphatics, arom atics and ether,
carbonyl/carboxylic groups b ut also significant dif­
ferences. C onsequently, sporopollenin cannot be
considered as a uniform m acrom olecule; it rath er
rep resen ts a type of closely related biopolym ers
with different chem ical groups occurring in varying
am ounts.
Table II. Assignment of the FTIR signals marked in Fig. 2; v, stretching; 6, deformation.
Letter in
Fig. 2
Band origin, short comment
A
B
C
D
E
F
K
L
v (O -H )
v (C -H )
v (C -H )
v (O -H )
v(C = 0 )
v(C = 0 )
v(C = C)
v(C = C)
6 (C -H )
6 (C -H )
v (C -O )
6(OH)
v (C -O )
v (C -O )
M
v (C -O )
N
O
v (C -O )
6 (C -H )
P
8(CH2)
G
H
I
J
145
130
105
60-75
30
15
polymeric hydroxyls, broad band
aromatics
in methyl and methylene groups
in hydrogen bonds of carboxylic acids
carbonyl
in conjugated carbonyls
or in alkenes
aromatic skeletal vibrations
in methyl and methylene groups
in methyl groups
in arylalkyl ethers or
in alcohols
in ester groups
in methyl esters of aliphatic acids
or in ethers
in aliphatic ethers or
secondary alcohols
in ethers
in aromatics with
two neighbouring H
in aliphatic chains with n > 4
Wave number [cm-1] of the
absorption minimum
3420
3050-3030
2930/2860
3000-2500
1700-1680
1640
1610,1580,1517
1440
1380-1350
1270
1200
1165
1110
1010
830
720
S. W ilmesmeier et al. • Com parative 13C CP/MAS N M R Spectroscopic Investigations on Sporopollenin
Acknow ledgem ents
Financial support from the D eutsche Forschungs­
gem einschaft and th e F ond d er C hem ischen In d u ­
strie are gratefully acknow ledged. T he authors are
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in d ep ted to Prof. G erh ard E rk er and M artin Berlekam p for putting the F T IR spectrom eter at their
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