uV radiation and temperature effects on functional traits in Helianthemum 
nummularium subsp. grandiflorum at the alpine and montane site  
in the Slovenian Alps
Učinki UV sevanja in temperature na funkcionalne značilnosti Helianthemum 
nummularium subsp. grandiflorum na alpinskem in montanskem rastišču  
v Julijskih Alpah
Tadeja Trošt Sedej*, Rok Damjanič
Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111,  
SI-1000 Ljubljana, Slovenia
*Correspondence: tadeja.trostsedej@bf.uni-lj.si
Abstract: Alpine plants have evolved strategies to survive harsh conditions, 
which include high UV and visible radiation, extreme temperatures, dryness and lack 
of nutrients. Survival strategies include biochemical, physiological and morphological 
responses, which are scarcely studied because of the time-demanding and complex 
experimental conditions. We researched functional traits in the alpine plant common 
rockrose Helianthemum nummularium subsp. grandiflorum growing under ambient 
UV-B and reduced UV-B radiation at different altitudes (1600 and 2000 m a.s.l.) of 
mount Vogel in the Slovenian Alps. Leaf anatomy, pigments and optical properties 
were investigated at the beginning and at the end of the growing season. Plants showed 
high constitutive UV-absorbing compounds content (UV-AC) throughout the season. 
Most leaf thickness parameters were not altered according to UV and altitude con-
ditions. Leaves did not transmit any UV spectrum, in agreement with high UV-AC. 
High photosynthetic spectrum transmittance at alpine altitudes was due to complex 
biochemical and anatomical responses to these conditions, rather than to UV radiation. 
Unchanged chlorophyll content of H� nummularium could be related to shrub life form, 
where leaves shade out high UV and PAR irradiance as well as contribute to lower 
leaf temperature. This study shows the complexity of alpine plant response, where 
specific characteristics of plant species should not be overlooked.
Keywords: Helianthemum nummularium, leaf optical properties, pigments, UV 
radiation
Izvleček: Alpinske in gorske rastline so razvile strategije za preživetje v skrajnih 
razmerah, ki vključujejo visoko jakost UV in vidnega sevanja, skrajne temperature, 
sušo in pomanjkanje hranil. Strategije preživetja alpinskih rastlin vključujejo bioke-
mične, fiziološke in morfološke odzive, ki jih raziskovalci zaradi zahtevne izvedbe 
redko proučujejo. V naši raziskavi smo proučevali funkcionalne značilnosti alpske 
rastline velecvetni popon Helianthemum nummularium subsp. grandiflorum, in sicer 
 ACTA BIOLOGICA SLOVENICA 
 LJUBLJANA 2021               Vol. 64, Št. 1: 41–55
42Acta Biologica Slovenica, 2021, 64 (1), 41–55
pod naravnim in zmanjšanim sevanjem UV-B na dveh različnih nadmorskih višinah 
(1600 in 2000 m n.m.) gore Zadnji Vogel v Julijskih Alpah.
Anatomijo listov, pigmente in optične lastnosti smo raziskovali na začetku in na 
koncu rastne sezone. Rastline so ob obeh merjenjih vsebovale veliko UV-absorbirajočih 
snovi (UV-AC). Večina parametrov, s katerimi smo ocenjevali debeline listnih tkiv, 
se ni odzivala na različne sevalne in temperaturne razmere. Listi niso prepuščali UV 
spektra sevanja, kar se je ujemalo z  veliko vsebnostjo UV-AC. Velika prepustnost 
fotosinteznega spektra sevanja na alpinski nadmorski višini je bila povezana z bioke-
mičnimi in anatomskimi značilnostmi rastline in ne z jakostjo UV sevanja. Nespremen-
jena vsebnost klorofila pri H� nummularium bi lahko bila povezana z grmičasto rastno 
obliko rastline, kjer zgornji listi zasenčijo spodnje liste ter tako prispevajo k manjšim 
prejetim odmerkom UV in fotosintezno aktivnega sevanja ter k nižji temperaturi listov. 
Študija kaže na kompleksen odziv alpinskih rastlin na UV sevanje in temperaturne 
razmere v gorah, pri čemer je pomembna tudi rastna oblika rastline.
Ključne besede: Helianthemum nummularium, optične značilnosti listov, 
pigmenti, UV sevanje
Introduction
Alpine plants come in a variety of life forms 
and have evolved various strategies to allow 
their survival under harsh mountain conditions 
(Körner 2003). Alpine plants adapt and acclimate 
to high ultraviolet (UV) and photosynthetically 
active radiation (PAR), low temperatures and 
frost, rapid temperature changes, soil dryness, 
lack of nutrients, low air humidity, strong winds 
and short growing season (Nicotra et al. 2010). 
Alpine environmental conditions can be stress-
ful for plant life, where topography, exposure, 
diseases and human activities can also contribute 
to the stressful environment (Turunen and Latola 
2005).
Ultraviolet-B radiation has been reported 
to cause changes in leaf morphology that lead 
to smaller and thicker leaves, with increased 
thickness of the epidermis or cuticle. These ap-
pear to be adaptive responses to stressful alpine 
environments (Jansen 2002; Robson et al. 2015). 
High UV-B radiation can negatively affect growth 
and productivity (Wargent et al. 2009; Gruber et 
al. 2010), carbon assimilation (Guidi et al. 2011; 
Hideg et al. 2013) and stomatal function (Nogues 
et al. 1999). Photosynthetic rates can be sustained 
under ambient doses of UV-B radiation through 
enhanced photoprotection, which depends on the 
induced synthesis of UV-B–absorbing compounds 
(UV-B–AC) and activation of the plant antioxidant 
defence system (Wargent et al. 2011; Kataria et 
al. 2014) or induction of photo-repair, which lead 
to the recovery of photochemical efficiency (Xu 
and Gao 2010). 
Damaging UV-B effects at high altitudes and 
low latitudes are an exception rather than norm 
(Searles et al. 2001). The responses of high-
altitude plants to UV-B radiation are often less 
pronounced compared to those of low-altitude 
plants, which suggests that alpine plants are ac-
climated and well-adapted to greater intensities of 
UV-B radiation. Indeed, these plants might have 
simultaneous co-tolerance to several stress factors, 
as acclimation or adaptation to harsh climates can 
also increase tolerance to UV-B radiation, and 
vice versa� Such interactions might aggravate or 
mitigate plant responses. Plant can increase their 
tolerance to UV-B, low temperatures or drought 
through increased acclimation to a second stressor 
(Chalker-Scott and Scott 2004; Trošt Sedej and 
Gaberščik 2008; Terfa et al. 2014).
The present study investigated adaptations 
and acclimatisation to increased UV-B radiation 
at two different alpine altitudes in the alpine plant 
common rockrose Helianthemum nummularium 
subsp. grandiflorum (Scop.) Schinz & Thell.). 
Common rockrose is widespread from upper 
montane (1200-1600 m a.s.l.) to alpine (1600-
2000 m a.s.l.) altitudes in Central and Southern 
European mountains. This indicates its successful 
adaptation and acclimation to the alpine environ-
43Trošt Sedej and Damjanič: UV and temperature effects on functional traits in H. nummularium
ment. Current research of Helianthemum has been 
limited to phylogenetics (Volkova et al. 2016). 
The objective of the present study was to 
investigate H� nummularium responses to differ-
ent UV radiation doses at different altitudes in its 
natural habitat. We aimed to study the interactive 
effects of diverse environmental conditions on the 
responses of H� nummularium in its natural habitat.
Materials and methods 
Plant species
Helianthemum grandiflorum subsp. gran-
diflorum is an alpine herbaceous perennial that 
sometimes has a woody base. It grows 10 to 30 
cm high, and flowers from June to September. H� 
nummularium is commonly distributed over warm, 
dry, rocky limestone meadows from montane to 
alpine altitudes throughout the Alps and other 
high mountains of Central Europe (Martinčič 
et al. 2010).
Experimental site
The experimental site was located at Mount 
Zadnji Vogel in the Julian Alps, Slovenia, and 
comprised a montane and an alpine altitude plot 
(1600 m a.s.l.: 46°19’04.4”N, 13°48’33.9”E; 2000 
m a.s.l.: 46°19’46.7”N, 13°48’07.3”E) (Fig. 1). 
Both plots were above the timberline, on south-
erly exposed slopes. Slope steepness was 70% 
to 100%. The vegetation consisted of meadows 
on calcareous ground that lacked grazing and 
tourism. Their mean annual precipitation from 
1981-2010 was 2600 to 3200 mm (Slovenian 
Environment Agency).
Two different UV exposures were provided to 
the plants using two types of filters: a Quinn XT 
UV filter (UV-), which absorbed UV-B and UV-
A; and a Quinn cast UVT filter (UV), which was 
transparent to UV-B and UV-A (Quinn-Plastics, 
UK). Five UV and five UV- filters (each 20 × 20 
cm) were positioned 15 cm above a group of three 
to six plants at each altitude. Similar soil water 
contents near and under the filters were maintained 
due to slope steepness, heavy rainfall and small 
size of the filters. 
Figure 1:  Experimental plot at Zadnji Vogel. A - at 1600 
m a.s.l. B - at 2000 m a.s.l. c - Helianthemum 
nummularium subsp. grandiflorum.
Slika 1:   Poskusna ploskev na Zadnjem Voglu. 
A - na 1600 m n.m. B - na 2000 m n.m. 
c - Helianthemum nummularium subsp� 
grandiflorum.
UV-B radiation was monitored over four 
days with a clear sky for all four different treat-
ments (two altitudes × two UV exposures), using 
a radiometer with a UV-B radiometric sensor 
(RM-22; Opsytec Dr. Gröbel, Germany). The data 
collected were used to determine the proportion 
of the UV-B radiation that reached the plants 
under the different UV filters. The standard daily 
UV-B doses (UV) and the reduced daily UV-B 
doses (UV-) were calculated over three months 
(July, August, September) and for the four treat-
44Acta Biologica Slovenica, 2021, 64 (1), 41–55
ments using a model (Björn and Murphy 1993) 
that included Caldwell’s generalised plant action 
spectra (Caldwell et al. 1986) for daily clear-sky 
measures (Tab. 1). 
5
10
15
20
25
Gr
ou
nd
 te
m
pe
ra
tu
re
 (˚
C)
Jul                                            Aug                                       Sep
1600m
2000m
Figure 2.  Mean daily ground temperatures at 1600 m and 2000 m a.s.l. at Zadnji Vogel, monitored once per hour 
in July, August and September, using temperature data loggers (HOBO TidbiTv2). 
Slika 2.   Srednje dnevne temperature tal na 1600 m in 2000 m n.m. na Zadnjem Voglu, merjene vsako uro julija, 
avgusta in septembra s pomočjo temperaturnih senzorjev (HOBO TidbiTv2).
For both research plots, temperatures were 
monitored 10 cm underground once per hour 
during the experimental period, using temperature 
data loggers (HOBO TidbiTv2; Onset Computer 
Corporation, USA) (Fig. 2). 
Measurements
Plants were analysed in July and September. 
The UV filters were placed above the plants at the 
end of June. The first sampling and measurements 
were conducted three weeks after the UV filters 
were placed. Measurements were performed on 10 
randomly selected fully developed upper leaves 
per treatment at two altitudes. All the analysed 
leaves developed under the filters.
table 1.  Total and biologically active UV-B doses calculated for daily clear-sky (UV) and reduced daily clear-sky 
 (UV-) UV-B according to month and altitude (Björn and Murphy 1993).
tabela 1.  Celokupni in biološko aktivni odmerki UV-B, izračunani za sončen dan (UV) in sončen dan pri zmanjšani 
  jakosti (UV-) UV-B glede na mesec in nadmorsko višino (Björn in Murphy 1993).
uV-B dose (kj m-2 day-1)
july  August September
Filter  uV-B 1600 m 2000 m 1600 m 2000 m 1600 m 2000 m
UV Total 55.91 57.58 47.75 49.27 34.52 35.73
Biologically 
active
7.13 7.34 5.86 6.05 3.9 4.04
UV- Total 16.77 17.28 14.32 14.78 10.36 10.38
Biologically 
active 
2.14 2.2 1.76 1.82 1.17 1.18
45Trošt Sedej and Damjanič: UV and temperature effects on functional traits in H. nummularium
Anatomical analysis
For the anatomical studies, 10 fully developed 
leaves were sampled from 10 plants per treatment 
at each altitude. Anatomical observations of the 
leaf cross-sections, which included palisade and 
spongy parenchyma thickness, and epidermis and 
cuticle thickness measurements, were performed 
under an optical binocular microscope (CX41; 
Olympus, Japan), with the measurements recorded 
and photographs taken with a digital camera 
(XC30; Olympus, Japan), using CellSens software 
(Olympus, Japan). The nail polish peel method 
was used to measure the stomatal features of the 
upper and lower leaf surfaces, under the same 
optical binocular microscope. 
Pigment analyses 
Photosynthetic pigments 
Fully developed leaves from 10 plants per 
treatment at each altitude were collected at noon 
and were kept on moist paper in a refrigerated box. 
Fresh mass (FM) of the leaf tissue (10 mg) was 
extracted in 80% (v/v) acetone in buffered distilled 
water (pH 7.8). Absorbances were measured us-
ing a UV/VIS spectrophotometer (Lambda 25; 
Perkin Elmer, USA). Chlorophyll a (Chl a) and 
chlorophyll b (Chl b) contents were calculated 
from the absorbances measured at 663.6 nm and 
646.6 nm (Porra et al. 1989). Pigment contents 
were expressed per leaf dry weight (mg g-1).
UV-B- and UV-A-absorbing compounds
The leaf tissue that was collected as described 
above (10 mg FM) from 10 fully developed leaves 
from 10 plants per treatment at each altitude were 
extracted in 5 mL of acidified MeOH (MeOH/
H2O/HCl (37%), 79:20:1, v/v) (Caldwell 1968). 
Absorbance was measured over the spectral ranges 
for UV-B (280-315 nm) and UV-A (316-400 nm) 
using a UV/VIS spectrometer (Lambda 25; Perkin 
Elmer, USA), and were calculated per dry weight 
(g-1) and integrated to estimate the total content 
of UV-B- (UV-B-AC) and UV-A-absorbing com-
pounds (UV-A-AC) (a.u. g-1).
Leaf optical properties
Fully developed leaves from 10 plants per 
treatment at each altitude were collected at noon 
and were kept on moist paper in a refrigerated 
box. The leaf transmittance and reflectance spectra 
were measured in the laboratory on fresh leaves, 
using the Jaz Modular Optical Sensing Suite 
(Ocean Optics, USA) with a measurement sphere 
(ISP-30-6-R; Ocean Optics, USA), applying UV/
VIS/near infrared light from a deuterium and 
halogen light source (DH-2000; Ocean Optics, 
USA). The leaf reflectance spectra were measured 
for the upper leaf surface. The spectrometer was 
calibrated to 100% reflectance using a white 
reference panel with >99% diffuse reflectance 
(Spectralon; Labsphere, North Sutton, NH, USA). 
The leaf transmittance spectra were measured for 
the lower leaf surface by illumination of the up-
per surface. The spectrometer was calibrated to 
100% transmittance with a light beam that passed 
directly into the interior of the integrating sphere. 
The transmittance and reflectance measurements 
were processed using SpectraSuite software (Ocean 
Optics, USA).
Data analysis
The plant responses and characteristic data 
were analysed using the SPSS Statistics 22.0 soft-
ware (IBM, USA). Statistical tests were performed 
on 10 samples. The normal distributions of the data 
were evaluated using Shapiro-Wilk tests. Homo-
geneity of variance was analysed using Levene’s 
tests. One-way ANOVA with multiple comparison 
tests and Tukey’s post-hoc tests or Kruskal-Wallis 
tests and Bonferroni post-hoc tests were used to 
compare differences between the four treatments 
at each experimental site. Factorial ANOVA was 
performed to investigate the effects of UV radia-
tion and temperature, and their interaction on the 
measured parameters. Redundancy analysis (RDA, 
CANOCO 5) was used to determine whether vari-
ations in the measured variables were related to 
the differences in temperature and UV radiation 
between experimental plots.
46Acta Biologica Slovenica, 2021, 64 (1), 41–55
Results
Leaf anatomical characteristics 
Most of the H� nummularium leaf thickness 
parameters were significantly higher at the montane 
altitude compared to the alpine altitude in July, 
which might be due to the delayed phenological 
events at the alpine altitude. The upper and lower 
cuticle thicknesses significantly increased under 
reduced UV radiation (UV-) at the montane and 
alpine altitudes in September, while spongy meso-
phyll thickness decreased. Total leaf thickness and 
specific leaf area did not change according to the 
different UV radiation and altitude conditions in 
September. The measured stomatal characteristics 
showed no changes at all (Tab. 2).
table 2:  Leaf anatomical characteristics for Helianthemum nummularium according to month and altitude for  
 near-ambient (UV) and reduced (UV-) UV radiation. 
tabela 2:  Anatomske značilnosti listov pri Helianthemum nummularium julija in septembra v montanskem in   
 alpinskem pasu pri naravnem (UV) in zmanjšanem (UV-) UV sevanju.
Measure Detail july                                 September
1600 m 2000 m        1600 m      2000 m Factorial ANOVA 
uV uV- uV uV- uV uV- t uV t*uV
Stomatal 
density 
(mm-2)
Upper
Lower
87.7 ±5.1a
157.8 ±5.3a
90.3 ±3.4a
154.5 ±3.9a
96.6 ±4.8a
164.6 ±12.5a
92.5 ±7.7a
161.1 ±9.3a
95.5 ±3.6a
160.2 ±3.9a
92.7 ±2.0a
152.1 ±3.5a
ns
ns
ns
ns
ns
ns
Stomatal 
length 
(µm)
Upper
Lower
32.9 ±0.5a
33.7 ±0.2a
32.3 ±0.5a
34.7 ±0.5a
34.7 ±0.4a
34.1 ±0.4a
34.8 ±0.8a
34.3 ±0.2a
33.9 ±0.3a
33.7 ±0.3a
34.2 ±0.5a
34.5 ±0.7a
ns
ns
ns
ns
ns
ns
Cuticle 
thickness 
(µm)
Upper
Lower
3.2 ±0.1a
2.2 ±0.2a
2.2 ±0.1b
1.5 ±0.1b
2.9 ±0.1a
2.5 ±0.2a
3.6 ±0.2b
2.9 ±0.2a
3.5 ±0.2b
3.0 ±0.2b
4.0 ±0.2c
3.1 ±0.2b
ns
ns
*
**
*
*
Epidermis 
thickness 
(µm)
Upper
Lower
22.5 ±0.9a
17.9 ±0.9a
21.7 ±1.0a
18.0 ±0.7a
22.8 ±0.8a
19.9 ±1.0a
25.2 ±0.8a
23.6 ±1.0a
24.3 ±0.6a
22.4 ±0.7a
22.6 ±0.7a
19.2 ±0.7a
ns
ns
ns
ns
ns
ns
Leaf 
thickness 
(µm)
- 302.8 ±6.3a 259.9 ±7.9b 320.6 ±12.5a 316.2 ±9.3a 309.2 ±3.9a 288.5 ±6.5a ns ns ns
Mesophyll 
thickness 
(µm)
Palisade
Spongy
133.0 ±3.7a
132.7 ±3.9a
112.4 ±3.9b
103.5 ±4.4b
140.7 ±7.7a
131.6 ±5.1a
140.2 ±5.3a
120.4 ±4.9a
127.8 ±2.0ab
128.0 ±3.0ab
124.6 ±4.0b
114.4 ±3.8b
**
ns
ns
*
*
ns
Specific 
leaf area 
(cm2 g-1)
- 0.2 ±0.0a 0.2 ±0.1a 0.1 ±0.0a 0.1 ±0.0a 0.1 ±0.0a 0.1 ±0.0a ns ns ns
Data are means ± standard error (n = 10 plants). Different letters indicate significant differences between the treat-
ments (p ≤0.05; one-way ANOVA); *, p ≤0.05; **, p ≤0.01; ns, p >0.05, as significant responses to environmental 
factors temperature (T) and UV radiation, and their interaction (Factorial ANOVA).
47Trošt Sedej and Damjanič: UV and temperature effects on functional traits in H. nummularium
Pigment contents
Pigment contents of H� nummularium showed 
the least changes for UV radiation and altitude 
for these three experimental plant species. The 
UV-B–AC contents in these leaves were lower at 
the montane than the alpine altitude in July and 
September (Tab. 3).
Leaf optical properties
Leaf reflectance for H� nummularium showed 
minor changes according to UV radiation and 
altitude, while leaf transmittance of the total 
photosynthetic spectrum (except for violet) was 
significantlly higher at the alpine than the montane 
altitude. Reduced UV radiation (UV-) increased 
the transmittance of the green and yellow spectra 
of all the three plant species. (Fig. 3, Tab. 4).
table 3:  Pigment contents of Helianthemum nummularium according to month and altitude for near-ambient 
 (UV) and reduced (UV-) UV radiation.
tabela 3:  Vsebnost barvil v listih pri Helianthemum nummularium glede na mesec in nadmorsko višino pri 
 naravnem (UV) in zmanjšanem (UV-) UV sevanju.
Measure                july                           September
1600 m 2000 m         1600 m          2000 m Factorial ANOVA
uV uV- uV uV- uV uV- t uV t*uV
Chlorophyll a 
(mg g -1 DW)
1.60 ±0.20a 1.93 ±0.16a 1.65 ±0.16a 1.61 ±0.15a 1.44 ±0.09a 1.40 ±0.13a ns ns ns
Chlorophyll b 
(mg g -1 DW)
0.83 ±0.18a 1.36 ±0.19a 1.44 ±0.42a 1.37 ±0.26a 1.06 ±0.16a 0.99 ±0.12a ns ns ns
UV-A–AC 
(a.u. g-1)
5.80 ±0.20a 6.14 ±0.70a 5.00 ±0.21a 4.46 ±0.30a 4.89 ±0.22a 4.65 ±0.43a ns ns ns
UV-B–AC 
(a.u. g-1)
5.08 ±0.40b 7.30 ±0.23a 4.80 ±0.28b 6.42 ±0.68ab 7.02 ±0.25a 6.37 ±0.54ab * ns ns
Data are means ±standard error (n = 10 plants). Different letters indicate significant differences between the treat-
ments (one-way ANOVA); *, p ≤0.05; ns, p >0.05, as significant responses to environmental factors temperature 
(T) and UV radiation, and their interaction (Factorial ANOVA).
48Acta Biologica Slovenica, 2021, 64 (1), 41–55
0
10
20
30
40
50
60
70
80
90
1000
10
20
30
40
50
60
70
80
90
100
280 380 480 580 680
Tr
an
sm
itt
an
ce
 [%
]
R
ef
le
ct
an
ce
 [%
]
1600 m  
R 1600 UV
R 1600 UV-
T 1600 UV
T 1600 UV-
0
10
20
30
40
50
60
70
80
90
1000
10
20
30
40
50
60
70
80
90
100
280 380 480 580 680
Tr
an
sm
itt
an
ce
[%
]
R
ef
le
ct
an
ce
 [%
]
Wavelength [nm]
       2000 m  
R 2000 UV
R 2000 UV-
T 2000 UV
T 2000 UV-
Figure 3:  Leaf optical properties in September, as leaf transmittance and reflectance of Helianthemum nummu-
larium at the indicated altitudes under near-ambient (UV) and reduced (UV-) UV radiation. Data are 
means for every 5-nm interval (n = 10 plants).
Slika 3:   Optične lastnosti listov v septembru pri Helianthemum nummularium na označenih nadmorskih višinah 
pod naravnim (UV) in zmanjšanim (UV) UV sevanjem. Podatki so aritmetične sredine 5-nm intervalov 
(n = 10 rastlin).
49Trošt Sedej and Damjanič: UV and temperature effects on functional traits in H. nummularium
In July, the diversity among the treated plants 
was low; the morphological variables and UV-
absorbing compounds content accounted for 66% 
of the total variation between the two altitudes and 
different UV-B radiation treatments according to 
RDA analyses. In September, the diversity between 
the treated plants was high; the morphological 
variables and UV-absorbing compounds content 
accounted for 19% of the total variation between 
the different UV-B radiation and altitudinal treat-
ments according to RDA analyses. Explanatory 
variables accounted for 19.9% of the total variation, 
where T explained 14.7% and UV-B 5.1%. (Fig. 4). 
table 4:  Significance of the effects of temperature and UV radiation on leaf reflectance and transmittance for   
 Helianthemum nummularium over the light spectral ranges in September. 
tabela 4:  Učinek temperature in UV sevanja na odbojnost in prepustnost listov Helianthemum nummularium v  
 različnih spektralnih območjih v septembru.
Light spectrum t uV t*uV
Reflectance
UV-B ns ns ns
UV-A ns ns ns
Violet ns ns ns
Blue ns ns ns
Green ns ns ns
Yellow ns ns ns
Red * ns ns
Near-infrared * ns ns
transmittance
UV-B ns ns ns
UV-A ns ns ns
Violet ns ns ns
Blue * ns ns
Green ** * ns
Yellow ** * ns
Red ** ns ns
Near-infrared * ns ns
*, p ≤0.05; **, p ≤0.01; ***, p ≤0.001; ns, p >0.05, as (light colour coded) significant effects of environmental 
factors temperature (T) and UV radiation, and their interaction (Factorial ANOVA).
50Acta Biologica Slovenica, 2021, 64 (1), 41–55
-0.8 0.6
-1
.0
1.
0
T
UV-B
StomUpp
CutUpp
CutLow
EpiUpp
Spongy UV-BAC
UV-AAC
SLA
UV-B
UV-A
Viol
Blue
Green
Yel
Ora
Red
NIR
Figure 4:  Redundancy analysis ordination diagram showing the strengths of the associations between the envi-
ronmental factors (T, UV-B) and functional traits of Helianthemum nummularium�
Slika 4:   Diagram redundančne ordinacijske analize prikazuje moč povezav med okoljskimi dejavniki (T, UV-B) 
in funkcionalnimi značilnostmi Helianthemum nummularium�
Discussion
Unaffected Chl a and Chl b contents of a shrub 
life form
Chl a and Chl b contents did not change in 
H� nummularium according to UV radiation and 
altitude throughout the season. Previous studies 
have reported that UV radiation can lead to reduc-
tions, no changes, and increases in chlorophyll 
contents (Smith et al. 2000; Bassman et al. 2003). 
It has been shown that UV radiation generates free 
radicals and leads to degradation of chlorophylls 
and carotenoids (Middleton and Teramura 1993), 
and also to increased biosynthesis of photosynthetic 
pigments under adequate intensities of PAR and 
UV-A (Verdaguera et al. 2017). Furthermore, 
heterogeneous responses can reflect differences 
in species and/or cultivars (Teramura 1983). 
Decreases in chlorophyll contents under strongly 
reduced solar UV-B radiation have been shown 
for Zea mays and Citrus aurantifolia (Barsig and 
Malz 2000; Ibanez et al. 2008). John et al. (2001) 
demonstrated that exposure to UV radiation can 
lead to the induction of genes associated with 
senescence, which can result in loss of plant chlo-
rophyll content. Trošt Sedej et al. (2020) showed 
a significant decrease in chlorophyll contents in 
an alpine plant Saxifraga hostii under the ambi-
ent UV radiation at alpine altitudes at the end of 
51Trošt Sedej and Damjanič: UV and temperature effects on functional traits in H. nummularium
the growing season, which might suggest earlier 
senescence. The different response of H� num-
mularium and S� hostii could be due of different 
life forms, where the leaves of S� hostii rosettes 
are more exposed to irradiance than the leaves of 
H� nummularium shrub growth form.
High levels of constitutive UV-absorbing 
compound contents
H� nummularium showed high UV-AC con-
tents throughout the season. In agreement with 
this, other studies have demonstrated that species 
from locations with naturally high UV radiation 
at either high altitude or low latitude are less 
sensitive to UV radiation than species from low 
UV radiation locations (Biswas and Jansen 2012). 
Flavonoids have key roles in plant UV protection 
due to their antioxidant and UV-screening prop-
erties (Smith et al. 2000). Flavonoid formation 
is not only induced by UV radiation, but is also 
affected by other environmental variables, such 
as photosynthetic irradiance, temperature and 
nutrient supply (Bassman et al. 2003). 
In H� nummularium, UV-AC contents were 
generally not affected by the different environmen-
tal factors, which demonstrated high constitutive 
UV-AC contents. Constitutive levels of UV-AC 
under increased UV radiation (flavonoids and other 
phenylpropanoid derivatives) have been correlated 
with plant tolerance to UV radiation (Qaderi et 
al. 2008). High constitutive and inducible UV-AC 
explain the good protection of the photosystem 
from UV radiation penetration of the plants at the 
alpine altitude. Constitutive and inducible UV 
radiation tolerances reflect phenotypic plasticity in 
response to the different UV environments; lowland 
Arabidopsis ecotypes show more inducible UV 
radiation tolerance, whereas highland Arabidopsis 
ecotypes show constitutive UV radiation tolerance 
(Jansen et al. 2010). These studies suggest that 
for alpine plants, constitutive defence is a more 
advantageous strategy than inducible defence, 
where UV stress is constantly high.
Most leaf thickness parameters showed no 
response to the UV and altitude conditions
Most of the H� nummularium leaf thickness 
parameters and all of the stomatal characteristics 
showed no changes according to the UV radiation 
and altitude in September. The exceptions were 
for spongy mesophyll thickness, upper and lower 
cuticle thickness, which increased and decreased, 
respectively, under the near-ambient UV radiation 
at the montane and alpine altitudes in September. 
UV radiation can induce several photomorphogenic 
responses, which include decreased leaf area 
and increased leaf thickness. Such modifications 
represent effective mechanisms that reduce the 
transmittance of UV radiation to the inner leaf 
tissue (Czégény et al. 2016). Increases in leaf 
thickness were attributed to increases in the number 
of spongy parenchyma cells in Brassica carinata 
and Medicago sativa (Bornman and Vogelmann 
1991). It has been reported that cuticle thickness 
of the upper needle surface of Abies balsamea 
decreases at high altitudes (DeLucia and Berlyn 
1984). These studies indicate that plants that are 
less adapted to UV radiation show more changes 
to their leaf parameters than those that are better 
adapted to UV radiation. This suggests that H� 
nummularium plants are well-adapted to UV 
radiation and high altitude.
Leaves did not transmit any UV spectrum 
The leaf reflectance of H� nummularium 
showed small changes according to UV radiation 
and altitude. Another study on S� hostii showed 
that alpine plants, but not montane plants, can be 
grouped according to their different UV radiation 
exposures in September, meaning that UV radiation 
had a distinct influence on leaf optical properties 
(Trošt Sedej et al. 2020).
The leaves of H� nummularium did not transmit 
any UV spectrum, which corresponded to their high 
constitutive and inducible UV-AC contents. Similar 
responses were shown for Arnica montana, where 
enhanced UV radiation was not the key factor that 
triggered changes in the flavonoid composition. 
The key factor was temperature, which decreased 
with altitude (Albert et al. 2009). A tropical alpine 
study indicated that high levels of UV radiation 
52Acta Biologica Slovenica, 2021, 64 (1), 41–55
screening are common to native and non-native 
plant species, and to different growth forms in an 
alpine environment. The plasticity of the epidermal 
UV radiation transmittance is a mechanism that 
is used by some, but not all, species to cope with 
varying solar UV radiation exposure. In the non-
native Verbascum thapsus, leaf transmittance of 
UV-A was shown to be variable along an alpine 
altitude gradient, and to be strongly correlated 
with UV-B radiation and altitude. However, in the 
native Vaccinium reticulatum, leaf transmittance 
of UV-A was consistently low and did not change 
with altitude (Barnes et al. 2017).
The leaf transmittance of the photosynthetic 
spectrum increased at the alpine altitude, while 
the transmittance of the green and yellow spectra 
increased under the reduced UV radiation in Sep-
tember. The leaf optical properties are dependent 
on the anatomical and biochemical characteristics 
of the leaf, such as the structure of the leaf, the 
concentration and type of flavonoids it contains, 
chlorophylls and other pigments, and the thickness 
of the epidermis, cuticle, waxes and hairs (Ziska 
et al. 1992), and they are correlated with plant 
life form (Day et al. 1992). The optical properties 
can be species-specific, but can also vary within a 
species, due to ontogenetic development of plants 
(Liew et al. 2008). The environmental conditions, 
to which a plant is exposed to, can alter leaf op-
tical properties, including for UV radiation and 
PAR, temperature, and water and soil properties 
(Ustin and Jacquemoud 2020). Therefore, the 
high photosynthetic spectrum transmittance of H� 
nummularium at the alpine altitude is more due to 
complex biochemical and anatomical responses 
to the alpine environmental conditions than to 
UV radiation itself.
conclusions 
H� nummularium showed high UV-AC con-
tents throughout the season. The UV-AC contents 
were generally not affected by the different environ-
mental factors, which indicated high constitutive 
UV-AC contents. Constitutive and inducible UV 
radiation tolerances reflect phenotypic plasticity in 
response to different UV environments. Most leaf 
thickness parameters showed no response to the dif-
ferent UV radiation and altitude conditions. Plants, 
less adapted to UV radiation, show more changes 
in leaf parameters than plants, well-adapted to UV 
radiation, which suggests that H� nummularium is 
well-adapted to UV radiation and high altitude. 
The leaf reflectance of H� nummularium showed 
small changes according to UV radiation and 
altitude. The leaves of H� nummularium did not 
transmit any UV spectrum, which corresponded 
to their high constitutive and inducible UV-AC 
contents. Leaf transmittance of the photosynthetic 
spectrum increased for the alpine altitude, while 
the transmittance of the green and yellow spect-
ra increased under the reduced UV radiation in 
September. The high photosynthetic spectrum 
transmittance of H� nummularium at the alpine 
altitude was due to complex biochemical and 
anatomical responses to the alpine environmental 
conditions, rather than to UV radiation. Unchanged 
chlorophyll content of H� nummularium could be 
related to shrub life form, where the leaves shade 
out the high UV and PAR irradiance as well as 
contribute to lower leaf temperature. The study 
shows complexity of alpine plant response, where 
specific characteristics of plant species should not 
be overlooked.
Povzetek
Alpinske in gorske rastline so razvile strategije 
za preživetje v skrajnih razmerah, ki vključujejo 
visoko jakost UV in vidnega sevanja, skrajne 
temperature, sušo in pomanjkanje hranil. Strategije 
preživetja alpinskih rastlin vključujejo biokemične, 
fiziološke in morfološke odzive, ki jih raziskovalci 
zaradi zahtevne izvedbe redko proučujejo. V naši 
raziskavi smo proučevali funkcionalne značilnosti 
alpske rastline velecvetni popon Helianthemum 
nummularium subsp. grandiflorum pod naravnim 
in zmanjšanim sevanjem UV-B na dveh različnih 
nadmorskih višinah (1600 in 2000 m n.m.) gore 
Zadnji Vogel v Julijskih Alpah. Anatomijo listov, 
pigmente in optične lastnosti smo raziskovali 
na začetku in na koncu rastne sezone. H� num-
mularium je ob obeh merjenjih vseboval veliko 
UV-absorbirajočih snovi (UV-AC) v listih. V 
splošnem različni okoljski dejavniki na vsebnost 
UV-AC niso vplivali, kar kaže na visoko vsebnost 
konstitutivnih UV-AC. Konstitutivne in inducibilne 
UV-AC odražajo fenotipsko plastičnost rastline v 
53Trošt Sedej and Damjanič: UV and temperature effects on functional traits in H. nummularium
odzivu na različna UV okolja. Večina parametrov, 
s katerimi smo ocenjevali debeline listnih tkiv, ni 
pokazala odziva na različno UV sevanje in tem-
peraturne razmere. UV sevanje pri manj prilago-
jenih rastlinah izzove večje spremembe anatomije 
listov kot pri rastlinah, prilagojenih na veliko UV 
sevanje, kar kaže, da je H� nummularium dobro 
prilagojena alpinska rastlina. Listi niso prepuščali 
UV sevanja, kar ustreza njihovi visoki konstitu-
tivni in inducibilni vsebnosti UV-AC. Prepustnost 
fotosinteznega spektra sevanja skozi liste se je 
povečala na alpinski nadmorski višini, medtem 
ko se je prepustnost zelenega in rumenega spektra 
povečala pod zmanjšanim UV sevanjem septembra. 
Velika prepustnost fotosinteznega spektra sevanja 
na alpinski nadmorski višini je bila povezana z 
biokemičnimi in anatomskimi značilnostmi rast-
line in ne z jakostjo UV sevanja. Nespremenjena 
vsebnost klorofila pri H� nummularium bi lahko 
bila povezana z grmičasto rastno obliko rastline, 
kjer zgornji listi zasenčijo spodnje liste ter tako 
prispevajo k manjšim prejetim odmerkom UV 
in fotosintezno aktivne svetlobe ter k nižji tem-
peraturi listov. Študija kaže na kompleksen odziv 
alpinskih rastlin na UV sevanje in temperaturne 
razmere v gorah, pri čemer je pomembna tudi 
rastna oblika rastline.
Acknowledgements 
We thank the Slovenian Research Agency for 
financing the research programme P1-0212. We are 
grateful to Dragan Abram for technical support.
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