plant tissue Tevini, 1993. In some studies, it was shown that the ultrastructure of photosynthetic
plant organs may be changed by UV-B radiation Santos et al., 1993; Lu¨tz et al., 1997; Barsig et al.,
1998. However, the information on ultrastruc- tural changes by increasing UV-B is limited. Even
though Zea mays is one of the most important cereal crops, knowledge of its resistance to en-
hanced UV radiation is limited. Previous investi- gations have shown growth reductions or leaf
damage in different maize cultivars Santos et al., 1993; Mark and Tevini, 1996.
The sun simulator at the GSF Research Center near Munich, Germany, provides realistic white
light conditions which is a prerequisite for ecolog- ically relevant UV experiments Meindl and Lu¨tz,
1996. For this study a realistic scenario of UV-B enhancement under intensive light conditions was
simulated. Unrealistic high UV-B doses as used in previous investigations were avoided. The aim of
this study is to describe UV-B effects on cell fine structure and on carbohydrate and pigment pools
simultaneously. We investigated the impact of different UV-B irradiances on epicuticular wax
structure, on the epidermis and the underlying cell layers. The structural stability of leaf components
under enhanced UV-B is a prerequisite for their functional vitality. Therefore, carbohydrate and
pigment content may be a possible marker. The initial hypothesis of this study was that the sugar
maize cultivar Z. mays cv. saccharata Koern ‘Tasty Sweet’ is a UV-sensitive plant species at
the level of leaf vitality.
2. Material and methods
2
.
1
. Plant material and growth conditions Seeds of sugar maize Z. mays L. cv. saccharata
Koern ‘Tasty Sweet’ supplied by Agrisaaten were planted in 20-cm pots containing a loamquartz
sand mixture and grown under Central European mid summer conditions measured at Neuherberg
48.2° N, 11.5° E, altitude 500 m in July. After emergence of the sixth leaf, plants were exposed in
the GSF sun simulator. The GSF sun simulator provides light conditions of ambient Central Eu-
ropean mid summer conditions. A combination of different lamps simulates the visible light, com-
pared to spectroradiometric field data obtained on a cloudless summer day at 50° N with approx. 60°
maximum solar elevation Do¨hring et al., 1996; Thiel et al., 1996. By the lighting technology in
the GSF Phytotron the self-shading of the leaves was minimized, thus possible interactions with the
response of maize plants to UV-B can be neglected.
The climate used in the simulators was a weekly repeated radiation, temperature and humidity
regime with temperature variation of 16 – 28°C with a range of daily maxima between 22 and
28°C. The relative humidity was kept at 90 at night and continuously reduced to a minimum of
60 in the afternoon. As a UV-B free control UV-B B 0.1 W m
− 2
, a WG 360 filter Schott was used. All irradiation data were obtained from
spectroradiometry using a double monochroma- tor system Bentham DM 300 with a cosine
corrected input optics Thiel et al., 1996.
2
.
2
. UV-B simulation UV-B radiation is supplemented by 96 UV-B
lamps Philips TL 12, 40 W. Ecologically irrele- vant wavelengths B 280 nm were excluded by
soda-lime glass filters. Different combinations of selected UV colour glass filters with specified UV-
B transmittance allowed a variation of cut-off wavelengths to simulate various UV-B scenarios
Table 1. Details of the UV-B filtering technique are described by Do¨hring et al. 1996. For this
study the sugar maize plants were exposed to four different UV-B radiation scenarios with replicates
simulating between 1 and 170 of the natural UV-B radiation measured at clear sky conditions
in Neuherberg, Germany 48.2° N, 11.5° E, alti- tude 500 m, July 30, 1992. The simulation exper-
iment with the ‘306’ and ‘293’ nm cut-off are controls without or with 45 reduced UV-B in
relation to ambient conditions. The treatments with ‘288’ and ‘281’ nm cut-off represent ambient,
respectively supplemental UV-B radiation 70 enhanced UV-B with a simulated 40 – 50 ozone
depletion under clear sky conditions, personal information, H.K. Seidlitz, GSF. The experiment
has been conducted in two sun simulator cham- bers for 16 days.
2
.
3
. Macroscopic obser6ations Immediately after the end of their exposure in
the sun simulator, leaf samples from the treat- ments were observed for growth characteristics
and vitality under a binocular microscope 25 × .
2
.
4
. Light and electron microscopy For transmission and scanning electron mi-
croscopy, segments 5 mm below the tip and of the middle part of the fourth leaf of each treated
plant were fixed in 2.5 glutaraldehyde and postfixed in 2 osmium tetroxide in 0.1 M phos-
phate buffer, pH 7.1. Dehydration was accom- plished in a graded acetone series. The samples
were infiltrated and embedded in Epon Spurr. Semi-thin sections 1-mm cross sections were cut
with a Reichert OM U3 ultratome and stained with
azur-II-methylene-blue for
preliminary screening with a light microscope. For total leaf
and epidermal cell thickness they were measured with a light microscope connected to the imaging
analysis system SYS
®
. Thin sections were cut with a diamond knife, stained with Reynold’s lead
citrate and examined with a Siemens Elmi 1 A TEM at 80 kV. For scanning electron microscopy,
leaf segments were fixed in glutaraldehyde, dehy- drated in acetone, critical-point dried, sputtered
with gold and examined in a Hitachi-S-4000 REM at 20 kV.
2
.
5
. Determination of dry weight Dry weight of all leaves was determined by
freeze-drying of the material.
2
.
6
. Determination of carbohydrate metabolites Leaf samples were separated according to their
phyllotaxis into three groups a: leaf 2 – 3; b: leaf 4; c: leaf 5 – 6, were immediately quick-frozen in
liquid nitrogen, freeze-dried for 24 h Christ Al- pha I-5, homogenized by grinding in a ball mill
Retsch and stored at − 25°C.
2
.
6
.
1
. Starch Five hundred milligrams lyophilized powder
was extracted at room temperature with 80 vv ethanol. Following centrifugation 10 000 × g, 10
min, the supernatant containing the low-molecu- lar, soluble carbohydrates was discarded. Follow-
ing two further treatments with 80 ethanol, the pellet was hydrolyzed by heating in 25 – 30 ml
bidistilled water 95°C, 1 h and cooled. The pH was adjusted to 4.68 – 4.74 by addition of sodium
acetate. Amyloglucosidase Aspergillus niger L., EC 3.2.1.3 was added incubation for 1 h, 58°C,
water-bath, followed by boiling 95°C, addition of charcoal 75 mg and centrifugation 10 000 ×
g, 10 min, twice. After filtration, an aliquot of the supernatant was used for enzymatic determination
with equipment by Boehringer according to Beut- ler 1985a.
Table 1 Integrated data of the irradation regimes used in the UV-B simulation
Spectrum no. 1
2 3
4 Sanalux
Filter 1 Sanalux
Sanalux Sanalux
Filter 2 Float glass
Pyran Sanalux
Plexiglas 4.73
2.15 UV-B 280–320 nm W m
− 2
1.27 0.03
35.4 33.2
UV-A 320–400 nm W m
− 2
30.0 28.2
Max. 1800 PAR 400–700 nm mol m
− 2
s
− 1
103 105
102 100
Max. illuminance IL kLux 281
288 293
306 Cut-off wavelength nm
2
.
6
.
2
. Sucrose, glucose and fructose One hundred milligrams of maize-powder was
extracted in 65 aqueous ethanol vv at 68°C for 1 h after pH-adjustment to ] 6 to avoid
hydrolysis of sucrose. After centrifugation as above the starch-containing sediment was re-
moved and charcoal was added to reduce the blank reading. Following centrifugation, aliquots
were assayed according to Beutler 1985b and Boehringer 1989. All extraction procedures de-
scribed here were optimized in order to obtain a recovery of external standards of more than 90.
2
.
7
. Pigment analysis Pigments were analysed in the fourth leaves.
They were lyophilized as described above. Chloro- phylls and carotenoids were extracted in 80
acetone. The extract was centrifuged twice at 5300 × g for 10 min and analysed spectrophoto-
metrically Shimadzu UV-160A at 646 and 663 nm
for chlorophylls
and at
470 nm
for carotenoids. Chlorophylls and carotenoids were
calculated per unit dry weight according to Licht- enthaler and Wellburn 1983.
2
.
8
. Statistical analyses Significance of differences between treatments
was determined by either Mann – Whitney U-test for nonparametric data or by one-way and multi-
ple ANOVA followed by LSD or Scheffe´ means tests for parametric data.
3. Results
