Journal of Photochemistry and Photobiology B: Biology 62 (2001) 108–117

www.elsevier.com / locate / jphotobiol

UV-B absorbance and UV-B absorbing compounds ( para-coumaric acid)

in pollen and sporopollenin: the perspective to track historic UV-B levels
a, a b a a
Jelte Rozema *, Rob A. Broekman , Peter Blokker , Barbara B. Meijkamp , Nancy de Bakker ,
Jos van de Staaij, Adri van Beem a , Freek Ariese b , Saskia M. Kars c
a
Department of Systems Ecology,Vrije Universiteit, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands
b
Department of Analytical Chemistry and Applied Spectroscopy, Vrije Universiteit, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands
c
Department of Quaternary Geology and Geomorphology, Vrije Universiteit, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands
Received 21 December 2000; accepted 7 June 2001

Abstract

UV-B absorbance and UV-B absorbing compounds (UACs) of the pollen of Vicia faba, Betula pendula, Helleborus foetidus and Pinus
sylvestris were studied. Sequential extraction demonstrated considerable UV-B absorbance both in the soluble (acid methanol) and
insoluble sporopollenin (acetolysis resistant residue) fractions of UACs, while the wall-bound fraction of UACs was small. The UV-B
absorbance of the soluble and sporopollenin fraction of pollen of Vicia faba plants exposed to enhanced UV-B (10 kJ m 22 day 21
UV-B BE ) was higher than that of plants that received 0 kJ m 22 day 21 UV-B BE . Pyrolysis gas chromatography–mass spectrometry
(py-GC–MS) analysis of pollen demonstrated that p-coumaric acid and ferulic acid formed part of the sporopollenin fraction of the
pollen. The amount of these aromatic monomers in the sporopollenin of Vicia faba appeared to increase in response to enhanced UV-B
(10 kJ m 22 day 21 UV-B BE ). The detection limit of pyGC–MS was sufficiently low to quantify these phenolic acids in ten pollen grains of
Betula and Pinus.The experimental data presented provide evidence for the possibility that polyphenolic compounds in pollen of plants
are indicators of solar UV-B and may be applied as a new proxy for the reconstruction of historic variation in solar UV-B levels.  2001
Elsevier Science B.V. All rights reserved.

Keywords: Pollen; Polyphenolics; p-Coumaric acid; Ferulic acid; Vicia faba; Sporopollenin; UV-B absorbing compounds; Bioindicator; Proxy; UV-B
radiation

1. Introduction tion of phenylalanine into trans-cinnamic acid, which is a

Solar UV-B radiation may affect plant life directly or
indirectly. In addition to direct effects of enhanced solar
UV-B on photosynthesis and plant primary production
[1–4], UV-B effects relate to physiological and ecological
functions of various protective absorbing (poly)phenolics,
whose production is induced by UV-B. Flavonoids are
polyphenolic UV screens, products of the phenyl–pro-
panoid pathway [5–8,10–15]. UV-B stimulates synthesis
of phenylalanineammonia lyase (PAL) and chalcone synth-
ase (CHS) and other branch-point enzymes of the phenyl-
propanoid pathway [5,7–9]. PAL catalyzes the transforma-
*Corresponding author. Tel.: 131-20-444-7055; fax: 131-20-444-
7123.
E-mail address: jrozema@bio.vu.nl (J. Rozema).
central intermediate in the formation of complex phenolic
compounds such as flavonoids, condensed tannins and
lignin [7]. These UV-B-induced polyphenolics are not
easily degraded. A UV-B induced increase of lignin in
plant litter caused a reduced rate of litter decomposition
[16]. Sporopollenin is a lignin-like compound in plant
pollen and spores of algae and mosses. The chemical
nature of sporopollenin is incompletely known, but appears
to consist of an aliphatic chain with aromatic groups
[17–19]. The monomer para-coumaric acid, a phenolic
acid, has been demonstrated to form part of these aromatic
groups [19–21]. Sporopollenin is a major component of
pollen grains remaining in the fossil record.
In an earlier paper [22] we hypothesized that UV-B-
induced polyphenolics in pollen and spores of plants may
be used as indicators to track historic UV-B. Our line of
thinking is that enhanced solar UV-B promotes the ac-

1011-1344 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved.
PII: S1011-1344( 01 )00155-5
 J. Rozema et al. / Journal of Photochemistry and Photobiology B: Biology 62 (2001) 108 – 117
cumulation of phenolics in the pollen and spores of plants.
We assume that at least part of the UV-B-induced poly-
phenolics reside in the sporopollenin, and this has been
tested in the research presented here. Unlike soluble
UACs, UACs in the sporopollenin will remain in the fossil
record.
In the case of atmospheric CO 2 enrichment, historic
values of the atmospheric CO 2 content have been assessed
by analyzing air bubbles trapped in polar ice cores, or by
reconstruction based on stomatal densities of fossil leaves
and isotopic studies [23]. No such proxies are available to
reconstruct historic UV-B levels, yet there is need for a
proxy to track historic UV-B, since our knowledge of
UV-B levels before the occurrence of the ozone hole is
very limited. Antarctic stratospheric ozone measurements
started in 1957 (see [24]) and systematic recording of solar
UV-B with spectroradiameters in 1970 [25]. Over this
period, springtime stratospheric ozone measurements over
Antarctica have shown a sustained decrease since around
1980 [25]. It is very likely that anthropogenic CFCs have
led to current ozone depletion. Since no systematic in-
strumental measurements of stratospheric ozone and solar
UV were made before, the history of the stratospheric
ozone layer before the development of the ozone hole over
Antarctica is unknown. Levels of stratospheric ozone
reduction as high as 60% during the spring, have been
reported since 1985 for the Antarctic continent. Only
indirectly or theoretically past levels of solar UV-B are
considered in studies reconstructing the early atmosphere,
the evolution of the biosphere or the possibilities of
extraplanetary life [28,29]. Did stratospheric ozone deple-
tion and recovery occur in the 19th or 20th century or in
subrecent times? Is the current reduction of stratospheric
ozone part of a natural cycle?
With few exceptions [26,27], there have been no at-
tempts to directly or indirectly reconstruct past solar UV-B.
Markham et al. [26] found that there was an increase of
both UV-B absorbance and relative flavonoid levels in
leaves of Bryum argenteum with the decrease in Antarctic
stratospheric ozone levels from 1971 to 1980.
In this paper we aim to assess the UV-B absorbance of
both the soluble and sporopollenin fraction of pollen, the
inducibility of this by enhanced UV-B radiation and the
chemical nature of the UACs in the sporopollenin fraction.
Based on the experimental results we discuss the possi-
bility of using these parameters to reconstruct past UV-B
levels. With sequential extraction of UACs we aim at
quantifying soluble, wall-bound and sporopolleninous
UACs in pollen grains. We exposed the crop Vicia faba to
enhanced UV-B and collected the pollen produced for
chemical analysis of UACs. We also collected the pollen of
the abundantly flowering Helleborus foetidus in the botani-
cal garden of the Vrije Universiteit, Amsterdam and of
Betula pendula and Pinus sylvestris in the coastal dunes of
North Holland in the spring of 2000 for sequential
extraction of UACs and further chemical analysis of the
sporopollenin.
109
2. Material and methods
2.1. Cultivation of Vicia faba and UV-B treatment
Nine-day-old seedlings of Vicia faba cultivar Minica
were grown in a climatized greenhouse (temperature day
208C, night 158C; relative humidity day 72%, night 81%;
burning period PAR lamps (300 mmol PAR m 22 s 21 )
6.00–20.00 h) in 2.6-l pots filled with garden soil (Jon-
gkind, Aalsmeer, The Netherlands) enriched with 7.8 g
osmocote (controlled release fertilizer, Grace Sierra, Heer-
len, The Netherlands). The seedlings were exposed to two
radiation regimes throughout their growing and flowering
period (n56 plants per treatment): (a) 0 UV-B / PAR, (b)
1UV-B / PAR. The PAR level for the two radiation treat-
ments plants was 300 mmol m 22 s 22 PAR and was
supplied by Philips 400 W HPI / T lamps. Fluorescent tubes
above 1UV-B irradiated plants were wrapped in cellulose
acetate foil. A 3-h preburnt cellulose acetate foil was
replaced twice a week to avoid degradation effects. In the
greenhouse with fluorescent lamps UV-B radiation was
measured with a UV-X meter (San Gabriel, CA, USA)
calibrated against Optronics Model OL 752 readings. The
UV-B BE dose of 10.6 kJ m 22 day 21 simulating 30% ozone
depletion at clear sky on June 21 in Amsterdam was
obtained using the model of Green et al., 1980 (see
Rozema et al. [41]). The burning period of the fluorescent
lamps was from 12.00 to 16.00 h.
The UV-B BE dose simulating 30% ozone depletion was
calculated by multiplying UV-B spectra measured with an
Optronics spectroradiometer and weighting factors from
the Generalized Plant Action Spectrum (Caldwell, 1971;
see [41]) normalized at 300 nm. Non-burning fluorescent
tubes were placed above the PAR irradiated plants. Plants
were watered twice a week. After 24 days the Vicia plants
started to flower, which continued for about 6 weeks. The
corolla of Vicia faba flowers covers the anthers and stigma,
so that the anthers and pollen grains themselves are not
directly exposed to enhanced UV-B, assuming that the
corolla absorbs most of the incident UV-B [30]. Twice a
week thecas of Vicia flowers, that started to release pollen
grains were collected with a pair of tweezers and stored in
petri dishes at room temperature. During collection and
storage, pollen was not exposed to UV-B. Collection of
pollen was continued throughout the 6-week flowering
period.
The pollen grains collected over 6 weeks were combined
into a bulk sample for each plant. To obtain sufficient
acetolysis resistant pollen residue (sporopollenin), the
pollen of two plants was combined (three replicate sam-
ples) (Fig. 4).
2.2. Sequential extraction of UACs in pollen grains
For UV-B absorbance analysis, pollen grains of Vicia
faba were collected from the petri dishes with a pencil and
weighed with a Sartorius MP2 microbalance with a
110 J. Rozema et al. / Journal of Photochemistry and Photobiology B: Biology 62 (2001) 108 – 117
standard deviation of 0.001 mg (1 mg). The mass of pollen
weighed per sample ranged from 50 to 150 mg for Vicia
faba. Because limited amounts of pollen grains of Vicia
faba were available for sequential extraction, spring 2000
pollen was also collected from the abundantly flowering
Betula pendula and Helleborus foetidus. Ripe anthers from
ten flowering plants of Helleborus foetidus were collected
in the botanical garden of the Vrije Universiteit, March
6–20, 2000. Catkins of Betula pendula Roth were col-
lected April 3–17, 2000 from five trees in the North
Holland Dune reserve. Pollen grains of Pinus sylvestris
were collected May, 2000 from ten trees at the
Zwanewater nature reserve, a coastal dune area in Noord-
Holland.
The nomenclature follows [31]. The pollen was air dried
and stored in petri dishes wrapped in aluminium foil,
before |500 mg of Betula pendula and Helleborus foetidus
pollen grains per sample were used for sequential ex-
traction of UACs. Both in the catkins of Betula, the cones
of Pinus sylvestris, and the flowers of Helleborus, the
anthers and thecae may be exposed to solar radiation.
The pollen of each of the five Betula trees and ten
Helleborus plants was combined into bulk samples (five
and ten replicated samples).
2.2.1. Soluble, cytoplasmic fraction (acid methanol
extraction)
UV-B absorbing pigments were extracted with a
MeOH–H 2 O–HCl (79:20:1, v / v) solution. Samples (50–
150 mg pollen of Vicia faba, 500 (1000) mg of Betula
pendula and Helleborus foetidus) in 1 ml solution were
heated in a waterbath (908C) for 2 h. The absorbance,
integrated from 280 to 320 nm was measured using a
Shimadzu UV-1601PC UV–visible spectrophotometer
equipped with HYPER-UV32-BIT spectroscopy software spec-
trophotometer (see [22,55]). The solution was then cen-
trifuged at 3500 rpm (15 min) and the supernatant was
carefully removed, the pellet was air dried and used for the
NaOH extraction.
2.2.2. Wall bound fraction ( NaOH extraction)
A 1-ml volume of 2 M NaOH was added to the pellet in
a tube. The tubes were shaken on a tubeshaker, sonicated
(5 min), after 1 h at room temperature and occasional
intermittent shaking, centrifuged (15 min, 3500 rpm).
Concentrated HCl (220 ml) was added to the supernatant to
set the pH at 1.0 ([22,55]). The UV-B absorbance of the
supernatant was measured for the 280–320-nm bandwidth
as described in Section 2.2.1. The remaining pellet was air
dried and used for acetolysis extraction.
2.2.3. Sporopollenin fraction ( insoluble, acetolysis
resistant residue extraction)
A 1-ml volume of an acetolysis mixture [conc. (.95%)
H 2 SO 4 –acetic acid anhydride, 1:9, v / v] was added to the
pellet. The suspension was kept in a boiling water bath for
1 h and occasionally stirred. The acetolysis residue was
centrifuged at 3500 rpm (15 min) and washed twice in
acetic acid and three times in distilled water. The dark
brown residue (mainly consisting of sporopollenin) was
suspended in glycerol [32]. The absorbance of the suspen-
sion was measured from 280 to 320 nm and integrated as
described in Section 2.2.1.
2.3. Pyrolysis-gas chromatography–mass spectrometry
( py-GC–MS)
In this study a direct thermal desorption (DTD) interface
(ATAS, Veldhoven, The Netherlands) is used. This inter-
face makes it possible to use the liner of the GC injection
interface as a sample and / or reaction container. The
injection interface can be heated up to 6008C enabling
pyrolysis / chemolysis experiments. The heating rate of the
interface can be programmed from 1 to 168C / s. The DTD
interface is directly coupled to the CP-SIL 8CB fused-
silica capillary column (15 m30.32 mm I.D., 0.25 mm film
thickness) and is positioned directly on top of the gas
chromatograph (Varian, CP-3800). Mass spectra (0.6 scan
s 21 ) were recorded with an ion-trap mass spectrometer
(Varian, Saturn 2000) under electron impact conditions (70
eV).
For the chemical identification and quantification of
aromatic components ( p-coumaric acid and ferulic acid in
particular) air-dried pollen or the acetolysis resistant
residue was analyzed by the following techniques. The
acetolysis resistant residue related to 500 mg pollen of
Betula pendula (Fig. 4) and to 200 mg pollen of Vicia faba
(Fig. 5) was placed in a DTD liner insert. A 20-ml portion
of 0.25 M tetramethylammonium hydroxide (TMAH) in
MeOH (Acros Organics) was added to the insert and mixed
with the pollen. After drying of the pollen suspension
under vacuum, the samples were allowed to incubate
overnight under an inert N 2 atmosphere. For analysis, a
DTD liner insert containing an incubated sample was
placed in a DTD liner. The liner was next positioned into a
DTD interface at 508C and heated in the splitless mode at a
rate of 168C / s to 6008C (hold time 5 min). The products
released by the chemolysis / pyrolosis reactions were de-
sorbed onto a CP-SIL 8CB fused-silica capillary column
(15 m30.32 mm I.D., 0.25 mm film thickness). The GC
was programmed from 358C (hold time 3 min) to 1208C at
308C / min and subsequently to 3208C at 88C / min (hold
time 5 min). The presence of p-coumaric acid and ferulic
acid in the complex product mixtures was shown by mass
chromatograms of m /z 192 (or 1331161) and 222, respec-
tively; for more details see [52]. To attempt to assess the
detection limit of GC–MS analysis of phenolic acids in
pollen, ten pollen grains of Betula pendula and Pinus
silvestris, in an aqueous suspension, were transferred to a
40-ml liner insert inside a DTD liner. Subsequently 1 ml of
a 2 M MeOH–TMAH solution (Fluca) was injected into
the cold liner. After drying in vacuo, the DTD liner was
placed into the DTD-interface and heated from 40 to
3508C at 168C / s. The compounds released by the
 J. Rozema et al. / Journal of Photochemistry and Photobiology B: Biology 62 (2001) 108 – 117
Fig. 1. Relative UV-B absorbance in the soluble (cytoplasmic), wall-
bound and sporopollenin fraction of pollen of Betula pendula and of
Helleborus foetidus obtained after sequential extraction. The UV-B
absorbance was measured spectrophotometrically and integrated from 280
to 320 nm and expressed on a per milligram pollen basis. The percentage
contribution of each fraction of the total absorbance is given for each
species. Mean values of three replications with standard error of the mean
(S.E.M.). Within each species the three sequential fractions differed
significantly (P#0.05).
chemolysis reactions were transferred to the capillary
column, programmed from 358C (3 min hold time)
to1208C at 208C / min and further to 2608C at 88C / min (2
min hold time). The GC was coupled to an iontrap mass
spectrometer (40–650 m /z scan range) operated in the
electron impact mode. Results are shown in Fig. 3.
2.4. Scanning electron microscopy
The pollen grains were mounted using double-sided
adhesive tape and coated with gold for about 10 min.
Scanning electron microscopy analysis was performed with
a Jeol JSM scanning microscope with a sputter coating
Bio-Rad E5005 facility, according to methods described in
[33].
2.5. Statistics
Where possible (Fig. 1, Table 1)) the original data were
Table 1
Absorbance of acetolysis resistant residue (sporopollenin) obtained from
pollen grains of Vicia faba exposed to 0 and enhanced UV-B radiation
UV-B treatment UV-B absorbance S.E.M.
(kJ m 22 day 21 UV-B BE ) (280–320 nm)
0 14.1 2.3
10.6 27.6 3.1
The absorbance of the residue in glycerol suspension was scanned from
280 to 320 nm and integrated; mean values of three replications with
standard error of the mean. The effect of enhanced UV-B was significant
at P#0.05.
111
statistically analysed by one-way analysis of variance
using SYSTAT 5.1 [34]. Homogeneity of variance was tested
with the Bartlett test for homogeneity of group variances.
Significantly different means were detected with the Tukey
HSD multiple comparison.
3. Results
Sequential extraction of UACs in the Helleborus
foetidus pollen (Fig. 1) demonstrated that the absorbance
(280–320 nm) of the sporopollenin fraction contributes
57% of the total absorbance. The soluble, cytoplasmic
fraction (acid methanol extracted) and the wall-bound
fraction gave 37 and 6%, of the total absorbance, respec-
tively. For Betula pendula the absorbance of the acid
methanol (soluble) fraction (56%) was larger than that of
the sporopollenin fraction (41%). Fig. 2 shows the SEM
pictures of the pollen of Betula pendula and Helleborus
foetidus; the pollen of these two species differ in size and
morphology. The pollen of Betula pendula is triporate and
about 30 mm in diameter. The length of Helleborus
foetidus pollen is about 45 mm and the ornamentation is
distinctly reticulate and there are three colpe. It is unknown
to us whether these morphological and structural differ-
ences between pollen grain types relate to differences in
the the UV-B absorbance of the soluble and insoluble
pollen extracts of these two species.
Enhanced UV-B not only induced soluble UACs [22],
but also absorbance of the sporopollenin in Vicia faba was
enhanced (Table 1). We have been unable to identify and
quantify the soluble UACs in the pollen of Vicia faba
using the HPLC procedures described in Ref. [7].
On-line pyrolysis GC–MS analysis of ten pollen grains
of Betula pendula and Pinus sylvestris, in an aqueous
suspension and comprising the soluble, wall-bound and
part of the insoluble fraction of UACs of pollen, revealed
the occurrence of both p-coumaric acid and ferulic acid, in
addition to other pollen constituents. Fig. 3 shows the
normalized peaks of p-coumaric acid methyl ester and
ferulic acid methyl ester in the mass chromatograms of
m /z 192 and 222, respectively. The S / N ratio plotted
illustrates that the ten pollen grains used for these measure-
ments are not the lower limit for analysis. Smaller amounts
of pollen (one to three pollen grains) can possibly be
analyzed since a S / N of 3 is still acceptable for quantita-
tive purposes.
The py-GC–MS mass-chromatogram of Fig. 4 presents
a chemical analysis of the non-soluble and non-hydrolys-
able fraction of the Betula pendula pollen. The chromato-
gram demonstrates the presence of p-coumaric acid and
ferulic acid after the pyrolysis of a non-soluble (sporopol-
lenin) fraction of of Betula pendula.
This sporopollenin fraction refers to the residue after
grinding of Betula pendula pollen and ultrasonic extraction
(three times) with MeOH and DCM. Thereafter acid
112 J. Rozema et al. / Journal of Photochemistry and Photobiology B: Biology 62 (2001) 108 – 117
Fig. 2. Scanning electron micrograph of pollen of triporate Betula pendula (A) [49,50] and of Helleborus foetidus (B). The pollen of Helleborus foetidus is
3-zonecolpate and the ornamentation is distinctly reticulate [51].
hydrolyses took place using 2 M TFA and 12 M H 2 SO 4
and basic hydrolysis with KOH–MeOH (12 M1 4% H 2 O.
The py-GC–MS mass-chromatogram of pollen from
Vicia faba plants exposed to 0 and enhanced UV-B (Fig.
5), shows that in addition to aliphatic components (fatty
acids), the sporopollenin contains p-coumaric acid as well
as ferulic acid and that the content of these phenolic acids
is increased with enhanced UV-B. It is likely that these
aromatic monomers form part of polymers in the sporopol-
lenin.
Fig. 3. Partial mass chromatograms of TMAH pyrolysis of ten grains of
fresh pollen of Helleborus and Pinus, after py-GC–MS. The mass
chromatogram of m /z 192 reveals p-coumaric acid methyl ester and 222
ferulic acid methyl ester. The signal-to-noise ratio (S / N) provides a crude
indication of the detection limit for these compounds (per pollen) (S / N
should ideally be greater than 3).
4. Discussion
In this paper we report data on the absorbance of the
sporopollenin of higher plants and the occurrence of p-
coumaric acid and ferulic acid in the sporopollenin. These
results are placed in the perspective of reconstruction of
historic UV-B levels. First, we discuss the ecological and
physiological aspects of UACs in pollen and our ex-
perimental data. Then we consider the possibility of using
polyphenolics in sporopollenin to reconstruct the past UV-
B and stratospheric ozone climate.
Pollen and spores represent an important part of the life
cycles of higher and lower plants, respectively. Reproduc-
tion depends, amongst various factors, on the success of
pollination. Pollen walls consist of an inner layer, the
intine and an outer layer, the exine. The intine is a
cellulose–pectin layer, which is not preserved during
fossilization and is removed by acetolysis in modern pollen
preparation [35]. The exine (sporopollenin) is resistant to
various (bio)chemical and physical stresses, and is respon-
sible for the preservation of pollen grains during fossiliza-
tion [36,37].
Pollen grains are transported from the male stamen to
the female stigma. At the stigma the pollen tube may
develop through pores (Betula) or colpes (Helleborus, Fig.
2), in the pollen wall and penetrate the stigma tissue.
Pollen transport from the anthers to the stigma is by wind
or water, but also by insects. Protection of pollen is needed
against dehydration, herbivory, microbial attack, physical
and chemical breakdown, as well as against radiation
damage.
The pollen wall is effective in screening out more than
80% of the incident UV radiation [39]. In longer-lived
pollen grains, containing the haploid male gamete nuclei
[38], DNA is in a dehydrated state, which is particularly
 J. Rozema et al. / Journal of Photochemistry and Photobiology B: Biology 62 (2001) 108 – 117 113

Fig. 4. Partial mass chromatogram of a non-hydrolysable insoluble residue of Betula pendula pollen revealing the presence of p-coumaric acid. Plotting the
specific ion traces of m /z 133 and 161 allows the identification of p-coumaric acid in a complex mixture of compounds, which is more difficult when
plotting the whole mass range of m /z 40–650 since larger peaks can obscure the smaller ones. Although the ions 133 and 161 are specific fragments ions
of p-coumaric acid, which has a molecular mass of m /z 192, the unknown peak to the right of that of p-coumaric acid in the mass chromatogram contains
these fragments as well, but is a different type of compound. The non-hydrolysable insoluble residue was obtained from the Betula pendula pollen by a
sequential series of extraction, acid hydrolysis and saponification.

sensitive to UV-B irradiance [40]. Recently, DNA damage
has been demonstrated in plants growing in the Antarctic
ozone hole [53,54]. Dependent on the flower morphology
and structure, pollen grains developing and ripening on the
parental plant, may or may not be directly exposed to solar
radiation. The corolla of many flowers is opaque to UV-B
[30]. The corolla of Vicia faba plants may act as a UV
screen. In the open structured flowers of Deschampsia
antarctica and Colobanthus quitensis the anthers are likely
to be exposed to solar UV-B [22,41,42]. Day and Demchik
[43] measured a higher content of acid–methanol-extract-
able UV-B-absorbing compounds in pollen derived from
plants cultivated under elevated UV-B radiation. Similarly,
we reported [22] increased absorbance of soluble UACs in
Vicia faba with enhanced UV-B. In sequential extraction
we compared the UV-B absorbance of soluble, wall-bound
and insoluble fractions of the pollen of Betula pendula and
Helleborus foetidus. Although the absorbance of the
fractions varies between these species, the wall-bound
fraction appeared to be relatively small and that of the
soluble and sporopolenin fraction considerable. Compari-
son of the UV-B absorbances of the three sequentially
obtained extracts requires caution, since the absorbance of
extracted polyphenols is pH dependent and also because
the UV-B absorbance of the acetolysis resistant residue
pollen extract was measured in a glycerol suspension
which may show high turbidity.
The chemical structure of sporopollenin is still incom-
pletely understood. There is some agreement on aliphatic
compounds such as fatty acids and aromatic groups
forming main constituents of sporopollenin. The monomer
para-coumaric acid, a phenolic acid, forms part of these
aromatic groups [20]. Boom et al. [21] report para-
coumaric acid to be a main constituent of sporopollenin of
spores of Isoetes. Sporopollenin is insoluble in most
common acids and in most organic solvents [36]; sporopol-
lenin of some plant groups dissolves in 2-aminoethanol,
but this may alter the chemical nature of the sporopollenin.
A suspension of sporopollenin in glycerol allows measure-
ment of absorbance, while the chemistry of the sporopol-
lenin is unaffected. Sporopollenin of algal walls is the only
compound resistant to hot acetolysis [17–19,32,52]. There
is similar evidence for the sporopollenin from pollen of
higher plants to be acetolysis resistant [18,36,40]. Based
on this we considered the acetolysis resistant residue from
pollen to consist mainly of sporopollenin.
In this paper py-GC–MS of a chemolytically obtained
non-soluble pollen extract was used for the chemical
114 J. Rozema et al. / Journal of Photochemistry and Photobiology B: Biology 62 (2001) 108 – 117

Fig. 5. Mass chromatogram of non-hydrolysable insoluble residue of pollen of Vicia faba plants exposed to 0 or 10.6 kJ m 22 day 21 UV-B BE radiation,
indicating the presence of both p-coumaric acid and ferulic acid. The peak height of these compounds is higher in the enhanced UV-B treatment. Based on
|200 mg of Vicia pollen for each treatment.
 J. Rozema et al. / Journal of Photochemistry and Photobiology B: Biology 62 (2001) 108 – 117
characterization of sporopollenin, see Ref. [21]. As yet, it
is unknown to us whether this insoluble fraction of pollen
is chemically similar to the acetolysis resistant residue.
Anthers in the flowers of Vicia faba are covered by a
corolla, which screens out UV-B. Yet, the soluble UACs of
Vicia pollen are increased by UV-B. Plants grown in a high
UV-B environment apparently acclimate by providing their
pollen with protective UV-B absorbing polyphenolics in
the pollen wall. UV-B absorbing flavonoids in the pollen
are assumed to surround the generative nucleus [40]. The
outer pollen wall is probably deposited on the pollen cell
under the control of the parental genome [35,44]. This is
by a special tissue, the tapetum, surrounding the mi-
crospore mother cells. The tapetum may release sporopol-
lenin precursors and sporopollenin bodies onto the pollen
membrane [45]. This may imply that the content of UACs
of the pollen and moss spores is largely determined by the
solar UV-B dose, to which the parental plant is exposed
during the growing season and not by the UV-B dose
experienced by the pollen grains in the anthers or during
the transport to the stigma. This should be confirmed by
outdoor studies.
UACs similar to sporopollenin in pollen of higher plants
have been found in walls of spores of mosses and algae by
Xiong et al. [32]. Spores of many lower plants such as
mosses, ferns and algae contain compounds chemically
similar to sporopollenin of higher plants [18,19,32]. Such
spores or algal cell walls are often well preserved in the
fossil record [18]. Sporopolleninous walls in the alga
Chlamydomonas have been chemically characterized by
Blokker et al. [19]. The sporopollenin content in two algal
species during prolonged exposure to solar UV-B was
found to be enhanced [32]. In the present paper we found
the UV-B absorbance, as well as the amount of p-coumaric
acid and ferulic acid, to be increased with enhanced UV-B
in pollen of Vicia faba. This makes it likely that some
UV-B signal may be preserved in the fossil record.
It is likely that the UV-B absorbing properties of the
sporopolleninous wall help to protect the pollen proto-
plasm from UV-B radiation damage. Acid methanol ex-
tracted (soluble) UV-B absorbing pigments (including
soluble flavonoids), occurring in the pollen protoplasm,
may protect more particularly the developing pollen tube,
when this tube is not surrounded by a pollen wall.
In this paper we demonstrated that it is possible to
quantify p-coumaric acid and ferulic acid in ten pollen
grains of Betula pendula and Pinus sylvestris (Fig. 3). If
we assume that the mass spectrometric signal is linear, the
lower detection limit will be three pollen grains for p-
coumaric acid and one pollen grain for ferulic acid in case
of Helleborus and one pollen grain for both compounds in
Pinus sylvestris. This illustrates that it is very likely that
the detection limit of this py-GC–MS analysis is suffi-
ciently low to quantify these phenolic acids in individual
pollen grains and will allow the quantitative analysis of
115
UV-B induced coumaric acid and ferulic acid in fossil
pollen grains as well.
Flavonoids and other soluble phenolic compounds,
present in the cytoplasm of the pollen grains may be be
lost in the process of fossilization [35]. However, UACs in
the exine of pollen (e.g. p-coumaric acid [20] and possibly
wall-bound hydroxycinnamic acids such as ferulic acid
[46]) are more likely to be preserved in the fossil record.
Although flavonoids are relatively stable, breakdown or
loss of flavonoids or other UACs in the long run may help
to explain why two earlier attempts to track historic UV-B
have been only partially successful. There is evidence for
photochemical breakdown of flavonoids [7,27]. Also bac-
terial and fungal degradation of flavonoids has been
described [57]. Flavonoids in leaves of 35 samples of the
moss (Bryum argenteum) were analysed using herbarium
specimens collected over the period 1957–1989 from the
Ross Sea area of continental Antarctica [26]. With Antarc-
tic stratospheric ozone levels decreasing from 1971 to
1980, an increase of both UV-B absorbance and relative
flavonoid levels in Bryum argenteum could be detected.
This study indicates that flavonoids in historic plant
material may help to reconstruct past UV-B levels. The
content of two flavonoid aglycones (myricetin and quer-
citin) in the leaves of 44 herbarium specimens of the
(sub)arctic Cassiope tetragona, covering a period from
1820 to 1997, has been reported [27]. There was no
significant change of the flavonoid content over time.
However, ozone depletion in the Arctic is far less marked
that in the Antarctic [47] and is currently thought to be a
recent phenomenon.
It has been demonstrated [46] that sporopollenin and
aromatic groups therein are preserved after fossilization.
By oxidation and diagenesis an increase in aromatic
compounds of the pollen within the exines was found over
time. The content of aromatic groups may have been
increased because of their resistance to degradation. There
was no loss of aromatic compounds of pollen during
fossilization. While the aromaticity per total carbon area in
NMR studies may increase, the content of aromatic groups
per pollen grain will not. In this paper we indicated that
py-GC–MS is sufficiently sensitive to measure coumaric
acid and ferulic acid in individual pollen grains. Such a
UV-B signal preserved in fossil or subrecent pollen re-
cords, opens the perspective to track historic UV-B and
past stratospheric ozone levels. Here we suggest that when
historic and fossil plant material is available both flavo-
noids and pollen polyphenolics may help to track historic
UV-B levels.
Knowledge of the history of stratospheric ozone before
the Antarctic ozone hole is very limited. Stratospheric
ozone measurements in Arosa go back to the 1920s. These
data indicate stable stratospheric ozone levels until the
onset of stratospheric ozone breakdown in the 1970s [56].
Reconstruction of historic UV-B and ozone levels may
116 J. Rozema et al. / Journal of Photochemistry and Photobiology B: Biology 62 (2001) 108 – 117
indicate whether the levels of stable stratospheric ozone
were stable or fluctuating. Of course, the experimental data
presented here provide only some evidence and many
questions should be addressed in future research such as:
(i) what is the precise dose–response relationship between
UV-B radiation and UACs in sporopollenin; (ii) which
other factors affect UACs in pollen? (iii) what is the
chemical nature of sporopollenin and (iv) does the chemi-
cal nature of sporopollenin differ among various plant
groups?
Reconstructed UV-B levels of the past decades or
centuries may provide a reference point for current ozone
recovery scenarios [48], currently predicting a restored
stratospheric ozone layer around 2050. As yet (2000),
there are no signs of recovery and the reliability of such
predictions is limited.
Acknowledgements
The analysis of UACs in pollen and spores is partially
funded by the EU, DGXII Environment and Climate,
ENV4-CT97-0580) which is greatly acknowledged.
We are indebted to Dr. Sjoerd Bohncke, Department of
Quaternary Geology and Geomorphology, Faculty of Earth
Sciences, Vrije Universiteit, Amsterdam for palynological
advice. The authors are particularly indebted to Dr. Carlos
Ballare´ and Dr. Walter Helbling for inviting JR to the
SPARC 2000 UV-B impacts workshop, Mar del Plata,
November 6–11, 2000, where a keynote lecture ‘‘In search
of pre ozone hole UV-B levels’’ was presented by JR. The
authors acknowledge the critical and constructive com-
ments of two anonymous reviewers, which contributed to
the improvement of the paper.
References
[1] M.M. Caldwell, L.O. Bjorn, ¨ J.F. Bornman, S.D. Flint, G. Kulan-
daivelu, A.H. Teramura, M. Tevini, Effects of increased solar
ultraviolet radiation on terrestrial ecosystems, J. Photochem. Photo-
biol. B: Biol. 46 (1998) 40–52.
¨
[2] D.-P. Hader, The Effects of Ozone Depletion on Aquatic Eco-
systems, Academic Press, EIU. R.G. Landes, 1997, p. 275.
[3] J. Rozema, Stratospheric Ozone Depletion: The Effects of Enhanced
UV-B Radiation on Terrestrial Ecosystems, Backhuys, Leiden, 1999,
p. 355.
[4] J. Sullivan, J. Rozema, UV-B effects on terrestrial plant growth and
photosynthesis, in: J. Rozema (Ed.), Stratospheric Ozone Depletion;
the Effects of UV-B Radiation on Terrestrial Ecosystems, Backhuys,
Leiden, 1999, pp. 39–57.
[5] C.J. Beggs, E. Wellmann, Photocontrol of flavonoid biosynthesis, in:
R.E. Kendrick, G.H.M. Kronenberg (Eds.), Photomorphogenesis in
Plants, Kluwer, Dordrecht, 1995, pp. 733–751.
[6] K. Kubitzki, Phenylpropanoid metabolism in relation to land plant
origin and diversification, J. Plant Physiol. 131 (1987) 17–24.
[7] B. Meijkamp, R. Aerts, J.W.M. van de Staaij, M. Tosserams, W.H.O.
Ernst, J. Rozema, Effects of UV-B on secondary metabolites in
plants, in: J. Rozema (Ed.), Stratospheric Ozone Depletion; the
Effects of UV-B Radiation on Terrestrial Ecosystems, Backhuys,
Leiden, 1999, pp. 70–99.
[8] J. Rozema, A.H. Teramura, M.M. Caldwell, Atmospheric CO 2
enrichment and enhanced solar ultraviolet-B radiation: gene to
ecosystem responses, in: Y. Luo, H.A. Mooney (Eds.), Carbon
Dioxide and Environmental Stress, Academic Press, San Diego, CA,
1999.
[9] D.C. Gitz, L. Liu, J.W. McClure, Phenolic metabolism, growth and
UV-B tolerance in phenylalanine ammonia lyase-inhibiting red
cabbage seedlings, Phytochemistry 49 (1997) 377–386.
[10] T.A. Day, C.T. Ruhland, C.W. Grobe, F. Xiong, Growth and
reproduction of Antarctic vascular plants in response to warming
and UV radiation in the field, Oecologia 119 (1999) 24–35.
[11] P. Hutzler, R. Fischbach, W. Heller, T.P. Jungblut, S. Reuber, R.
¨
Schnmitz, M. Veit, G. Weissenbock, J.-P. Schnitzler, Tissue localisa-
tion of phenolic compounds in plants by confocal laser scanning
microscopy, J. Exp. Bot. 49 (1998) 953–965.
[12] G. Karabourniotis, G. Kofidis, C. Fasseas, V. Liakoura, I. Dros-
sopoulos, Polyphenol deposition in leaf hairs of Olea europaea
(Olaceae) and Quercus ilex (Fagaceae), Am. J. Bot. 85 (1998)
1007–1012.
[13] J.P. Schnitzler, T.P. Jungblut, W. Heller, P. Hutzler, U. Heinzmann,
E. Schmelzer, D. Ernst, C. Langebartels, H. Sandermann Jr., Tissue
localisation of UV-B screening pigments and chalcone synthase
mRNA in Scots pine (Pinus sylvestris L.) needles, New Phytol. 132
(1996) 247–258.
[14] Y. Manetas, Is enhanced UV-B radiation really damaging for plants?
Some case studies with European mediterranean plant species, in: J.
Rozema (Ed.), Stratospheric Ozone Depletion; the Effects of UV-B
Radiation on Terrestrial Ecosystems, Backhuys, Leiden, 1999, pp.
251–291.
[15] M. Stephanou, Y. Manetas, The effects of seasons, exposure,
enhanced UV-B and water stress on leaf epicuticular and internal
UV-B absorbing capacity of Cistus creticus: a Mediterranean field
study, J. Exp. Bot. 48 (1997) 1977–1985.
[16] J. Rozema, M. Tosserams, H.J.M. Nelissen, L. van Heerwaarden,
R.A. Broekman, N. Flierman, Stratospheric ozone reduction pro-
cesses: enhanced UV-B radiation affects chemical quality and
decomposition of leaves of the dune grassland species Calamagros-
tis epigeios, Plant Ecol. 128 (1997) 284–294.
[17] G. Shaw, The chemistry of sporopollenin, in: J. Brooks, P.R. Grant,
M.D. Muir, P. van Gijzel, G. Shaw (Eds.), Sporopollenin, Academic
Press, London, 1971, pp. 305–351.
[18] P.F. van Bergen, M.E. Collinson, D.E.G. Briggs, J.W. de Leeuw,
A.C. Scott, R.P. Evershed, P. Finch, Resistant biomacromolecules in
the fossil record, Acta Bot. Neerl. 44 (1995) 319–342.
[19] P. Blokker, S. Schouten, J.W. de Leeuw, J.S. Sinnighe Damste, ´ H.
van den Ende, Molecular structure of the resistant biopolymer in
zygospore cell walls of Chlamydomonas monoica, Planta 207
(1999) 539–543.
[20] K. Wehling, Ch. Niester, J.J. Boon, M.T.M. Willemse, R. Wiermann,
p-Coumaric acid — a monomer in the sporopollenin skeleton, Planta
179 (1989) 376–380.
[21] A. Boom, P. Blokker, J.S. Sinninghe Damste, ´ J.W. de Leeuw, J.J.
Boon, p-Coumaric acid-based sporopollenin of Isoetes killipi
(Lycopodiaceae) megaspores, submitted for publication.
[22] J. Rozema, A.J. Noordijk, R.A. Broekman, A. van Beem, B.M.
Meijkamp, N.V. de Bakker, J.W.M. van de Staaij, M. Stroetenga,
S.J.P. Bohncke, M. Konert, S. Kars, H. Peat, R.I.L. Smith, P.
Convey, Polyphenolic compounds in pollen and spores of Antarctic
plants as indicators of solar UV-B: a new proxy for the reconstruc-
tion of past solar UV-B?, Plant Ecol. 154 (2001) 7–23.
[23] R.F.B. Isarin, S.J.P. Bohncke, Mean July temperatures during the
Younger Dryas in Northern and Central Europe as inferred from
climate indicator plant species, Quater. Res. 51 (1999) 158–173.
[24] J.C. Farman, B.G. Gardiner, J.D. Shanklin, Large losses of total
ozone in Antarctica reveal seasonal CLO x / NO x interaction, Nature
315 (1985) 207–210.
 J. Rozema et al. / Journal of Photochemistry and Photobiology B: Biology 62 (2001) 108 – 117
[25] SORG, United Kingdom Stratospheric Ozone Review Group,
Stratospheric Ozone. Seventh report, Department of the Environ-
ment, Transport and the Regions, London, 1999.
[26] K.R. Markham, A. Franke, D.R. Given, P. Brownsey, Historical
Antarctic ozone level trends from herbarium specimen flavonoids,
Bull. Liaison Group Polyphenols 15 (1990) 230–235.
¨
[27] L.O. Bjorn, T. Callaghan, C. Gehrke, T. Gunnarson, B. Holmgren,
U. Johanson, S. Snogerup, M. Sonesson, O. Sterner, S.-G. Yu,
Effects on subarctic vegetation of enhanced UV-B radiation, in: P.J.
Lumsden (Ed.), Plants and UV-B, Responses to Environmental
Change, Cambridge University Press, Cambridge, 1997, pp. 233–
246.
[28] C.S. Cockell, Carbon biochemistry and the ultraviolet radiation
environments of F, G and K main sequence stars, Icarus 141 (1999)
399–407.
[29] F. Garcia-Pichel, Solar ultraviolet and the evolutionary history of
cyanobacteria, Origins of Life and Evolution of the Biosphere 28
(1998) 321–347.
[31] R. van der Meyden, Heukel’s Flora van Nederland, 22nd edition,
1996, p. 676.
[30] S.D. Flint, M.M. Caldwell, Influence on floral optical properties on
the ultraviolet radiation environment of pollen, Am. J. Bot. 70
(1983) 1416–1419.
[32] F. Xiong, J. Komenda, J. Kopeck, L. Nedbal, Strategies of ultra-
violet-B protection in microscopic algae, Physiol. Plant. 100 (1997)
378–388.
[33] S.K. Chapman, Working with a Scanning Electron Microscope,
Lodgemark Press, Kent, 1986, p. 113.
[34] R.R. Sokal, F.J. Rohlf, Biometry. The Principles and Practice of
Statistics in Biological Research, 3rd Edition, Freeman, San Fran-
cisco, 1995.
[35] A. Traverse, Spores and pollen morphology, in: A. Traverse (Ed.),
Paleopalynology, 1988, pp. 60–116.
[36] C. Jungfermann, F. Ahlers, M. Grote, S. Gubatz, S. Steuernagel, I.
Thom, G. Wetzels, R. Wiermann, Solution of sporopollenin and
reaggregation of a sporopollenin-like material: a new approach in
the sporopollenin research, J. Plant Physiol. 151 (1997) 513–519.
[37] A. Meuter-Gerhards, S. Riegert, R. Wiermann, Studies on sporopol-
lenin biosynthesis in Cucurbita maxima (DUCH). II. The in-
volvement of aliphatic metabolism, J. Plant Physiol. 154 (1999)
431–436.
[38] D.M. Paxson-Sowders, H.A. Owen, C.A. Makaroff, A comparative
ultrastructural analysis of exine pattern development in wild-type
Arabidopsis and a mutant defective in pattern formation, Proto-
plasma 198 (1997) 53–65.
[39] S.M. Demchik, T.A. Day, Effects of enhanced UV-B radiation on
pollen quantity, quality and seed yield in Brassica rapa (Bras-
sicaceae), Am. J. Bot. 83 (1996) 573–579.
[40] C.F. Musil, Differential effects of elevated ultraviolet-B radiation on
the photochemical and reproductive performances of dicotledonous
and monocotyledonous arid-environment ephemerals, Plant Cell
Environ. 18 (1995) 844–854.
117
[41] J. Rozema, R. Broekman, D. Lud, A. Huiskes, T. Moerdijk, N. de
Bakker, B. Meijkamp, A. van Beem, Consequences of depletion of
stratospheric ozone for terrestrial Antarctic ecosystems: the response
of Deschampsia antarctica to enhanced UV-B radiation in a
controlled environment, Plant Ecol. 154 (2001) 87–99.
[42] A. Holtom, S.W. Greene, The growth and reproduction of Antarctic
flowering plants, in: J.E. Smith (Ed.), A Discussion on the Terrestri-
al Antarctic Ecosystem, Trans. R. Soc. Biol. Sci., London 777
(1967) 323-337.
[43] T.A. Day, S.M. Demchik, Influence of enhanced UV-B radiation on
biomass allocation and pigment concentrations in leaves and re-
productive structures of greenhouse-grown Brassica rapa, Vegetatio
127 (1996) 109–116.
[44] C.M. Rogers, B.D. Harris, Pollen exine deposition: a clue to its
control, Am. J. Bot. 56 (1969) 1209–1211.
[45] D.D. Fernando, D.D. Cass, Plasmodial tapetum and pollen wall
development in Butomus umbellatus (Butomaceae), Am. J. Bot. 81
(1994) 1592–1600.
[46] A.R. Hemsley, A.C. Scott, P.J. Barrie, W.G. Chaloner, Studies of
fossil and modern spore wall biomacromolecules using 13 C solid
state NMR, Ann. Bot. 78 (1996) 83–94.
[47] J.A. Pyle, Global ozone depletion: observations and theory, in: P.J.
Lumsden (Ed.), Plants and UV-B, Responses to Environmental
Change, Cambridge University Press, Cambridge, 1997, pp. 3–11.
[48] S. Madronich, in: UNEP. Environmental Effects of Ozone Deple-
tion: Assessment, 1998, p. 192.
[49] K. Faegri, J. Iversen, Textbook of Pollen Analysis, Munksgaard,
Copenhagen, 1964, p. 295.
[50] P.D. Moore, J.A. Webb, An Illustrated Guide to Pollen Analysis,
Hodder and Stoughton, London, 1978.
[51] W. Punt, S. Blackmore (Eds.), The Northwest European Pollen
Flora, VI, Elsevier, Amsterdam, 1991, pp. 122–182.
[52] P. Blokker, 1999. Structural analysis of resistant polymers in extant
algae and ancient sediments, Doctorate Thesis, Utrecht University.
[53] C. Ballare,´ A.L. Scopel, C.A. Mazza, Effects of solar UV-B
radiation on terrestrial ecosystems: case studies from southern South
America, in: J. Rozema (Ed.), Stratospheric Ozone Depletion; the
Effects of UV-B Radiation on Terrestrial Ecosystems, Backhuys,
Leiden, 1999, pp. 293–311.
[54] M.C. Rousseaux, C.L. Ballare, ´ C.V. Giordano, A.L. Scopel, A.M.
Zima, M. Szwarcberg-Brachitta, P.S. Searles, M.M. Caldwell, S.B.
Diaz, Ozone depletion and UV-B radiation. Impact on plant DNA
damage in southern South America, Proc. Natl. Acad. Sci. USA 96
(1999) 15310–15315.
[55] C.T. Ruhland, T.A. Day, Effects of ultraviolet-B radiation on leaf
elongation production and phenyl propanoid concentration of De-
schampsia antarctica and Colobanthus quitensis in Antarctica,
Physiol. Plant. 109 (2000) 244–251.
[56] R.D. McPeters, S.M. Hollandsworth, Int. J. Environ. Studies 51
(1996) 165–182.
[57] J.B. Harborne, T.J. Mabry, H. Mabry (Eds.), The Flavonoids,
Chapman and Hall, London, 1975, p. 1204.