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
3
.
1
. Leaf morphology and leaf dry weight No morphological changes or discolouration of
leaves independent of UV irradiation were ob- served. Leaf length was not affected by enhanced
UV-B not shown in detail. The leaf dry weight varied between 12.4 and 14.9 of fresh weight
and was not influenced by different UV-B irradiances.
3
.
2
. Light and electron microscopic obser6ations In comparison to controls Fig. 1a most of the
leaf tissues were intact even under high UV-B. The only remarkable deviation consisted of de-
formed or collapsed cells predominantly in the adaxial epidermal layer under enhanced UV-B. At
the ultrastructural level all organelles and mem- branes in such cells were affected. This pattern
was accompanied by a slight enlargement of the neighbouring intercellular space Fig. 1b. This
was confirmed by measurements revealing a sig- nificantly P 5 0.001 reduced thickness of epider-
mal cells under enhanced UV-B radiation Fig. 3. Because only the thin epidermal layer was af-
fected, the total leaf thickness was not signifi- cantly changed under high UV-B. By light
microscopic measurements, 33.9 9 12.5 com- pared to 11.7 9 17.2 under ambient UV-B of
epidermal cells were damaged significant differ- ence between variables with P 5 0.01. On this
occasion mostly adaxial cells were affected and showed deformed cell walls and reduced cell lu-
mina. The proportion of affected epidermal cells was especially increased in leaf samples below the
tip with an increase of 130 compared to the middle parts of leaves under high UV-B.
At the ultrastructural level Fig. 2a, no devia- tions have been observed in shape, size or struc-
ture of organelles in assimilation and bundle sheath cells. Chloroplasts and nuclei remained
intact even after enhanced UV-B radiation. The conducting tissues showed no alterations and
structure or amount of vacuolar contents ap- peared independent of UV-B exposure.
SEM observations revealed that the epicuticular wax layer was intact. Small regions with slight
modifications showing erosion in tubular waxes were found in all UV-B treatments and controls.
Wax structures in and around stomata were unaf- fected Fig. 2b.
3
.
3
. Carbohydrates Leaves were harvested during morning hours.
Therefore, carbohydrate content determined rep- resents mainly that remaining at the end of the
dark period. The values for starch, sucrose and
Fig. 1. Light microscopy of semi-thin transverse sections of Z. mays leaf blades. a Intact tissues grown under low UV-B; Bs, bundle sheath; E, epidermal cells; Ic, intercellular space; M, mesophyll cells; St, stomata. b UV-B treated leaf 70 enhanced in relation
to ambient radiation. Some epidermal cells are deformed or collapsed and below the intercellular space is slightly enlarged; mesophyll cells, leaf bundles and stomata are undisturbed; cE, collapsed epidermis; dE, deformed epidermis. Scale bars = 50 mm.
glucose are compared in Fig. 4a – c for leaves 23 and 4, 56, respectively. The content of fructose
was very low B 1 mg g
− 1
dry mass in all treatments. The sucrose and starch partitioning
varied more in dependence of the leaf exposure without showing a significant impact of UV-B
treatment Fig. 4b,c. Otherwise, the glucose con- tent was significantly lower P 5 0.01 in the
youngest leaves leaf 4 – 6 under high UV-B Fig. 4a. Generally the amount of starch was relatively
high. However, an increase in UV-B did not seem to cause starch accumulation if leaves grown un-
der ambient and under supplemental UV-B were compared Fig. 4c.
Fig. 2. a Transmission electron micrograph of leaf under supplemental UV-B. Normal structure of mesophyll asterisks and of bundle sheath chloroplasts with numerous starch grains. Other organelles are also intact. Scale bar = 1 mm. b Scanning electron
micrograph of adaxial leaf surface under enhanced UV-B. The epicuticular wax fine structure is predominantly unaffected, also around the stomatal opening. Scale bar = 20 mm.
3
.
4
. Pigment changes There was no significant impact of variable UV
levels simulated in this study on chlorophyll a or carotenoid contents. Under enhanced UV-B, a
slight decrease of chlorophyll b occurred. The ratio between chlorophyll a and b was increased
under enhanced UV-B but was decreased under ambient UV-B in comparison to low UV-B radia-
tion Table 2.
4. Discussion
