PERIODICUM BIOLOGORUM UDC 57:61

VOL. 110, No 3, 257–262, 2008 CODEN PDBIAD

ISSN 0031-5362

Original scientific paper

Morphological and biochemical variations in St.

John’s wort, Hypericum perforatum L., growing over
altitudinal and UV-B radiation gradients

Abstract
MARIJA ROBLEK1
MATEJA GERM2 Background and Purpose: The climate of the Alpine region is marked
TADEJA TRO[T SEDEJ1 by a higher proportion of UV radiation which, combined with other stresses,
ALENKA GABER[^IK1 plays an important role in determining the differences between highland
1 and lowland plant populations. The study was aimed at identifying the
Dept. of Biology; Biotechnical faculty,
University of Ljubljana, Ve~na pot 111, 1000 plant habitus as well as selected morphological and biochemical properties
Ljubljana, Slovenia that enable optimised growth under given conditions, and at determining
the potential of plants to cope with UV-B radiation stress.
2
National Institute of Biology, Ve~na pot 111,
1000 Ljubljana, Slovenia Material and Method: St. John’s wort, Hypericum perforatum L.,
thrives in a continuum of habitats from lowlands to highlands. Chloro-
Correspondence: phylls a and b and UV absorbing compounds (AC) were determined. The
Mateja Germ height of the plants, internodes and shoots, and the angle of the shoots were
National Institute of Biology
Ve~na pot 111, 1000 Ljubljana, Slovenia
measured. Thicknesses of epidermis, palisade and spongy tissues were exam-
E-mail: [email protected] ined and evaluated by the computer program AnalySIS 3.0 (Soft Imaging
System, Münster, Germany). The number of seeds was counted and their
weight determined at the end of the growing season.
Abbreviations: Results and Conclusions: The content of total UV absorbing com-
DM – dry mass pounds (AC) in leaves increased with altitude. The average content of total
UV AC – UV absorbing compounds UV AC in plants growing at 1300 m a.s.l. was about 25% higher than at
Key words: altitude gradient, chlorophyll, 400 m a.s.l... The content of chlorophyll showed the opposite trend. At high
plant architecture, reproduction, UV altitudes plants were shorter, due to shorter and reduced numbers of inter-
absorbing compounds nodes. Reproductive success was much lower in plants growing at high alti-
tudes; they developed fewer flowers (on average 147 at 400 m a.s.l. and 50
at 1700 m a.s.l.) and seeds per plant than those growing in lowlands. The
present study indicates that St. John’s wort growing at high altitudes has de-
veloped an effective strategy to mitigate the effects of the severe alpine cli-
mate.

INTRODUCTION

T he climate of the Alpine region is very complex, due to interactions

between the mountains and the general circulation of the atmo-
sphere (1). The altitudinal gradient is associated with decreased air
temperature and pressure, increased precipitation, and changes in wind
exposure, soil fertility and duration of snow cover (2). Lower tempera-
tures and longer persistence of snow cover at higher altitudes result in a
Received January 4, 2006. shorter vegetation period. Marked differences are also observed in the
quality of the incident radiation. The intensity of UV-B radiation in-
Marija Roblek et al. Hypericum perforatum over altitudinal and UV-B radiation gradients

1
creases steeply at higher altitudes, even though the daily Jul

Relative amount of UV-B

total solar radiation is usually nearly the same along the 0.8 Avg
gradient (3). The increase in UV-B radiation (290-320
nm) has been reported to range from between 6 and 8%

radiation
0.6
(3) to 20% (4) per 1,000 m of elevation.
0.4
The higher proportion of UV radiation, combined
with other stresses in the mountains, plays an important 0.2
role in determining the differences between highland
and lowland plant populations (5). The environmental 0
400 800 1100 1300 1400 1700
constraints result in changes, not only in plant morphol-
Altitude m
ogy and anatomy, but also in their habitus, physiology
and productivity (2). Many adaptations found in moun- Figure 1. Relative amount of UV-B radiation in the locations over an
tain plants are similar to those shown to result solely elevation gradient in July and August.
from increased UV-B radiation. These include photo-
morphogenetic changes, disturbances to physiological
processes, modification of plant enzyme content, bio- (14.5160°E, 46.2741°N) at 800 m, Ambro` under
mass reduction, disturbances in pollen germination and Krvavec (14.5337°E, 46.2757°N) at 1100 m, half way be-
reduced reproductive success (6–9). An increased con- tween Ambo` and Alp Jezerca (14.5277°E, 46.2823°N) at
tent of UV-B screening compounds, in response to UV-B 1300 m, Alp Jezerca (14.5349°E, 46.2857°N) at 1400 m,
radiation, has been widely reported (9–14). and Krvavec (14.5358°E, 46.2846°N) at 1700 m. The av-
Sensitivity and adaptation to UV-B radiation are highly erage temperatures in 2001 at Brnik near Cerklje
species specific but also depend on the environment (10, (14.4882°E, 46.2461°N) at 384 m were 16.6 °C in June,
15–18). Species and populations originating from natu- 19.5 °C in July and 20.2 °C in August, and on Krvavec
rally high UV-B radiation locations, i.e. from high eleva- (14.5395°E, 46.3011°N) at 1740 m, 8.5°C in June, 11.8°C
tions or low latitudes, have been shown to be less sensi- in July and 13.6°C in August (27).
tive to UV-B radiation than those from low intensity The relative solar UV-B radiation at the localities
UV-B radiation locations (15, 19–23). Species adapt their studied (Figure 1) was estimated on clear days at noon
epidermal UV-B attenuation to match solar UV-B dose using a UV-B sensor with integrator (Delta T Device).
along latitudinal and altitudinal gradients, providing one
of the bases for tolerance to UV-B radiation in plants col- All analyses were made on first, fully developed leaves
onizing high altitude regions (24). Körner (25) claimed and on flowers.
that, due to the high level of adaptation of plants to solar
radiation, UV-B radiation does not present an important Biochemical analyses
constraint for growth and development of plants in al- Chlorophylls a and b (Chl a, b) were estimated after
pine regions. This hold true for present conditions, but Jeffrey & Humphrey (28). A weighed leaf was homogenised
given the presumed increase of ambient UV-B radiation in 8 cm3 of 90% (v/v) acetone and centrifuged (19 000×g,
in combination with global warming (26), the level of 3 min, 4 °C) in a top refrigerated ultracentrifuge (2K15,
changes might exceed the tolerance of high altitude plant Sigma, Osterode, Germany). Absorbance was measured
populations. In those regions snow-cover determines the with a UV/VIS Spectrometer System Lambda 12 (Per-
period over which plants intercept solar radiation. The kin-Elmer, Norwalk, CT, USA). The amount of Chl per
potential prolongation of the growth period due to global sample dry mass (DM) was calculated.
warming might also result in increased cumulative UV-B
doses received by plants. UV absorbing compounds (AC) were extracted from
freshly homogenised plant material with methanol : dis-
Hypericum perforatum (L.) (St. John’s wort) thrives in tilled water : HCl (37% (v/v)) = 79 : 20 : 1 (v/v/v). After
a continuum of habitats from lowlands to highlands. 20 minutes incubation the samples were centrifuged in a
The study was aimed at identifying the characteristics, top refrigerated centrifuge (1600xg, 10 oC, 10 min). The
i.e. habitus and selected morphological and biochemical supernatants of the samples were scanned in the range
properties, that enable optimised growth under given from 280 to 320 nm (UV-B) and from 320–400 nm
conditions, and at determining the potential of plants to (UV-A) at intervals of 1 nm. The absorbance values were
cope with UV-B radiation stress. integrated and expressed per dry mass (DM) of the sam-
ple, according to Mirecki & Teramura (29).
MATERIAL AND METHODS
Morphological analyses
Site description The height of the plants, internodes and shoots, and
Plants were collected for analysis from June to August the angle of the shoots were measured. Cross sections of
2001 from locations at different altitudes in the Slo- the leaves, thickness of epidermis, palisade and spongy
venian Alps. The chosen locations were P{ata near tissues were all prepared, using a microscope, and evalu-
Cerklje (14.5103°E, 46.2606°N) at 400 m, [krjan~evo ated by the computer program AnalySIS 3.0 (Soft Im-

258 Period biol, Vol 110, No 3, 2008.

Hypericum perforatum over altitudinal and UV-B radiation gradients Marija Roblek et al.

600
2500
400 m
400 m
500
Total UV-A AC rel. units

800 m 800 m
2000
1100 m 1100 m
1300 m 400
1500 1400 m 1300 m
1700 m 1400 m
300
1000
a a a b c 200
500
100
0
0
2500 260 280 300 320 340 360 380 400 420 440 460 480
Total UV-B AC rel. units

Month
2000
Figure 3. The absorbance spectra of methanol extracts of leaves of H.
1500
perforatum growing over an elevation gradient (n=4).
1000

500 a a a b b

0 RESULTS
June July August
Month
In July the production of substances filtering radia-
tion in the UV-B and UV-A ranges in leaves (Figure 2)
Figure 2. The amount of UV-A and UV-B absorbing compounds in
correlated with altitude (r2=0.62 and r2=0.72, respec-
leaves of H. perforatum growing over an elevation gradient. Letters
represent variance differences (one way ANOVA and Post Hoc tively). The average content of total UV AC in plants
Tukey’s test), (n=4). growing at 1300 m a.s.l. was about 25% higher than in
plants growing at 400 m a.s.l.. From Figure 2 it is evident
that the production of UV-B and UV-A AC was highly
aging System, Münster, Germany). The numbers of seeds dependent on seasonal changes. The timing of pheno-
were counted and their weight determined at the end of logical phases depends on the length of the vegetation
the growing season. period, which changes with elevation. Flowering started
in June at 400 and 800 m a.s.l., in July at 1100, 1300 and
1400 m a.s.l., but not until August at 1700 m a.s.l.. That is
Statistical analyses
why it was not possible to compare samples from all alti-
Measurements were made on 5 to 8 parallel samples. tudes at the same time. The spectra of methanol extracts
Variance (one way ANOVA and Post Hoc Tukey’s test, of leaves in July showed two peaks, at 290 nm and at 360
SPSS for Windows 11.0.0.) of all parameters was analysed nm (Figure 3). The increase of UV-B and UV-A AC with
to establish the differences between different locations. increased elevation was not found in the flowers (Table

TABLE 1
Chlorophyll a and b contents in leaves and the amounts of UV-A and UV-B absorbing compounds in flowers of H. perforatum
growing over an elevation gradient. Letters represent variance differences (one way ANOVA and Post Hoc Tukey’s test),
(n=4).

Altitude m
Month 400 800 1100 1300 1400 1700
June 3.70a 3.65a
Chl a July 4.94a 4.02b 2.83c 3.69b 3.16c
August 2.74a 2.77a 2.79a 2.76a 2.29b
June 2.43a 2.39a
Chl b July 3.28a 2.62b 1.78c 2.37b 2.03c
August 1.66a 1.65a 1.66a 1.65a 1.35b
June 1576a 1351a
UV-A AC July 1001a 1106a 968a 962a 1043a
August 372a 343a 420b 493c 451b
June 1174a 1062a
UV-B AC July 567a 715b 684a 752b 752b
August 309a 310a 345a 418b 352a

Period biol, Vol 110, No 3, 2008. 259

Marija Roblek et al. Hypericum perforatum over altitudinal and UV-B radiation gradients

125 40 50

100 40

No. of Internodes
30
Plant height cm

No. of branches
75 30
20
50 20

10
25 r2= -0.80 r2= -0.61 10 r2= -0.77

0 0 0
100 5 90

Internode length cm
Leaf thickness µg

o
Branch angle
60
3
50
2
30
r2= 0.13 1 2
r = -0.54 r2= 0.30

0 0 0
0 500 1000 1500 2000 0 500 1000 1500 2000 0 500 1000 1500 2000
Altitude m Altitude m Altitude m
Figure 4. Plant height, number and height of internodes, number of branches, branch angle and leaf thickness in H. perforatum growing over an ele-
vation gradient (n=8).

200 0.15 2000

Total seed mass mg
Seed dry mass mg

150 1500
No. of flowers

0.1

100 1000

0.05
50 500
2
r = -0.76 r2= -0.66 r2= -0.83
0 0 0
0 500 1000 1500 2000 0 500 1000 1500 2000 0 500 1000 1500 2000
Altitude m Altitude m Altitude m
Figure 5. Number of flowers per plant and seed dry mass in H. perforatum growing over an elevation gradient (n=8).

1), the correlation coefficients in July being 0.38 and 0.14, higher elevations. Analysis revealed a reduced number of
respectively. The synthesis of photosynthetic pigments reproductive organs and delayed time of reproduction.
Chl a and Chl b in July (per DM) was also dependent on The number of flowers and seeds per plant and the
elevation (r2=0.72 and r2=0.73, respectively) but there weight of each seed decreased with elevation (Figure 5).
were also marked differences during the vegetation pe- The average number of flowers per plant was 147 at 400
riod. The decrease in July was not the consequence of the m a.s.l. and 50 at 1700 m a.s.l.. There was a negative cor-
changes in the leaf thickness, since it did not differ be- relation between the measured UV-B dose and the num-
tween locations (Figure 4). ber of flowers and seeds (r2= –0.68 and –0.71, respec-
The analysis of H. perforatum growth was made at the tively) (Figure 5). The strong correlation between the
end of the growth period, at the time of fruiting, and number of flowers and the number of seeds (r2= 0.96)
showed the traits typical of alpine plants. A trend of lower showed that the length of the vegetative period for H.
growth with increased elevation was observed (Figure 4). perforatum was long enough to complete the reproduc-
The average height of plants was 89 cm at 400 m a.s.l., 38 tion cycle, even at the highest locations.
cm at 1400 m a.s.l. and 48 cm at 1700 m a.s.l.. This was
the consequence of both shorter and reduced numbers of
internodes. The height of plants was also slightly related DISCUSSION
to plant branching (r2= 0.54). High altitude plants de- St. John’s wort grown at different altitudes exhibited
veloped fewer branches (Figure 4) while the differences differences in plant architecture, biochemistry and repro-
in the branch angle were negligible. duction. These changes resulted from the combined ef-
Reproductive success was significantly affected by the fects of seasonal dynamics and the altitudinal gradients of
severe environment and enhanced UV-B radiation at environmental factors and, presumably, UV-B radiation.

260 Period biol, Vol 110, No 3, 2008.

Hypericum perforatum over altitudinal and UV-B radiation gradients Marija Roblek et al.

Important protection against UV-B in plants growing 5). The effects are in line with findings on UV-B treated
in high altitudes is provided by the production of UV AC, plants (43, 44, 9). The additional delay of reproduction
which protect vulnerable targets in the plant tissue (Fig- due to enhanced UV-B could have an effect on pollinators,
ure 2). These compounds are located in epidermal cells, which appear before or after the plants flower (45).
and constitute an effective, selective filter that absorbs The present study indicates that St. John’s wort grow-
UV radiation without interfering with photosynthetical- ing at high altitudes has developed an efficient strategy to
ly active radiation (30–32). Quantitative changes with el- mitigate the effects of the severe alpine climate. The pro-
evation of these substances in leaves of H. perforatum ap- duction of UV absorbing substances and other traits en-
pear to be important in determining sensitivity to UV-B. ables plants to cope with the high level of UV-B radia-
Jüngenliemk & Nahrstedt (33) report that St. John’s wort tion. However, the lower reproductive success at high
contains 22 different phenolic substances. The content is altitudes constitutes a risk for H. perforatum highland
also significant in flowers, being the highest in buds (34). populations if UV-B radiation dose were to increase or if
The amount of these substances produced in leaves obvi- some other events, such as air pollution and global warm-
ously depends on UV-B radiation level, but not in blos- ing, were to become more pronounced. Many character-
soms, as was seen from our results (Table 1). Fillela & istics of alpine species that enable their survival under
Penuelas (35) stated that the amount of UV-B AC was present condition might limit their responses to environ-
15% higher in the leaves of Quercus ilex thriving at 1200 mental changes, as well as to biological invasions, and
m a.s.l., than in those at 200 m a.s.l.. A positive correla- they are very likely to change their abundance and distri-
tion was also found between the amount of UV-B ab- bution range.
sorbing compounds and elevation for leaves of Fagus
sylvatica (36) and the previous year´s needles of Picea Acknowledgements: The authors are grateful to Prof.
abies (37). The content of photosynthetic pigments how- Roger Pain for critical reading of the manuscript. This re-
ever was higher in populations from lowlands (Table 1). search was supported by Ministry of Education, Science and
The decrease of chlorophyll content with elevation might Sport, Republic of Slovenia: the Plant Biology (P1-0212)
also be the consequence of increased UV-B radiation, and Communities, relations and communications in ecosys-
since this effect has been shown for many UV-B treated tems (P1-0255) programs. The financial support is grate-
plants (37–39). fully acknowledged.
Many effects of UV-B radiation concern morphoge-
netic changes (8). The plants grew less at higher eleva-
tions (Figure 4). Internodes were fewer and they were
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