Environmental and Experimental Botany 109 (2015) 264–275
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Environmental and Experimental Botany
journal homepage: www.elsevier.com/locate/envexpbot
Effects of enhanced UV radiation and water availability on
performance, biomass production and photoprotective mechanisms of
Laurus nobilis seedlings
Meritxell Bernal a , Dolors Verdaguer a, *, Jordi Badosa b , Anunciación Abadía c ,
Joan Llusià d,e, Josep Peñuelas d,e , Encarnación Núñez-Olivera f , Laura Llorens a
a
Environmental Sciences Department, Faculty of Sciences, University of Girona, C/ Ma Aurèlia Campmany 69, 17071 Girona, Spain
Laboratoire de Météorologie Dynamique, Ecole Polytechnique, 91128 Palaiseau, France
c
CSIC, Department of Plant Nutrition, Aula Dei Experimental Station, Apdo. 13034, 50080 Zaragoza, Spain
d
CSIC, Global Ecology Unit CREAF-CEAB-UAB, Cerdanyola del Vallès 08193 Catalonia, Spain
e
CREAF, Cerdanyola del Vallès 08193 Catalonia, Spain
f
Complejo Científico-Tecnológico, Universidad de La Rioja, Av.Madre de Dios 51, 26006 Logroño, Spain
b
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 3 February 2014
Received in revised form 29 May 2014
Accepted 16 June 2014
Available online 2 July 2014
Climate models predict an increase in ultraviolet (UV) radiation and a reduction in precipitation in the
Mediterranean region in the coming decades. High levels of UV radiation and water shortage can both
cause photo-oxidative stress in plants. The aim of this study was to investigate the effects of enhanced UV
radiation and its interaction with low water availability on seedling performance, biomass production,
and photoprotective mechanisms of the sclerophyllous evergreen species Laurus nobilis L. (laurel). To
achieve this goal, one-year-old seedlings of L. nobilis were grown outdoors under three UV conditions
(ambient UV, enhanced UV-A, and enhanced UV-A + UV-B) and under two watering regimes (watered to
field capacity and reduced water supply). The results show that plants produced more biomass when
exposed to above ambient levels of UV-A or UV-A + UV-B radiation, especially under low water
availability. This was probably related to a UV-induced increase in leaf relative water content and in leaf
water use efficiency under water shortage. Even though our results suggest that UV-A supplementation
may play an important role in the stimulation of biomass production, plants grown under enhanced UV-A
plots showed higher levels of energy dissipation as heat (measured as NPQ) and a higher de-epoxidation
state of the violaxanthin cycle. This suggests a greater excess of light energy under UV-A
supplementation, in accordance with the observed reduction in the foliar content of light-absorbing
pigments in these plants. Strikingly, the addition of UV-B radiation mitigated these effects. In conclusion,
UV enhancement might benefit water status and growth of L. nobilis seedlings, especially under low
water availability. The results also indicate the activation of different plant response mechanisms to UV-A
and UV-B radiation, which would interact to produce the overall plant response.
ã 2014 Elsevier B.V. All rights reserved.
Keywords:
UV radiation
Drought
Phenolic compounds
Xanthophyll cycle
Photosynthetic pigments
Mediterranean species
1. Introduction
The levels of UV-B radiation reaching the Earth's surface have
increased in recent decades due to the depletion of the ozone layer,
which is not expected to recover until 2025–2040 at mid-latitudes
(World Meteorological Organization, WMO/UNEP, 2010).
* Corresponding author. Tel.: +34 972418174; fax: +34 972418150.
E-mail addresses: [email protected] (M. Bernal),
[email protected] (D. Verdaguer), [email protected]
(J. Badosa), [email protected] (A. Abadía), [email protected] (J. Llusià),
[email protected] (J. Peñuelas), [email protected]
(E. Núñez-Olivera), [email protected] (L. Llorens).
http://dx.doi.org/10.1016/j.envexpbot.2014.06.016
0098-8472/ ã 2014 Elsevier B.V. All rights reserved.
Furthermore, in the Mediterranean Basin, decreases in the
mean cloudiness (Giorgi et al., 2004) will likely expose plants in
terrestrial ecosystems to higher fluxes of UV-B and UV-A radiation
in the near future. In addition, Mediterranean plants are expected
to experience longer dry periods in the forthcoming decades
(Intergovernmental Panel of Climate Change, IPCC, 2012).
Considered individually, increases in UV-B radiation and
drought have often been related to reductions in plant growth
(Chaves et al., 2003; Llorens et al., 2004; Caldwell et al., 2007;
Ballaré et al., 2011). However, the interactive effects of a
combination of these two factors on plant growth are still largely
unknown, and most of the available information is on crops or high
latitude species (see Caldwell et al., 2003, 2007 and Ballaré et al.,
M. Bernal et al. / Environmental and Experimental Botany 109 (2015) 264–275
2011 for reviews). Several studies have reported that plants
growing under enhanced UV-B radiation are more tolerant to
water stress, which has been related to an increase in leaf water
potential (Hofmann et al., 2003) or in leaf relative water content
(Poulson et al., 2006) in response to increased levels of UV-B.
However, little is known about the mechanisms that would explain
an improved plant water status under a combination of enhanced
UV-B radiation and drought. In Arabidopsis, a higher leaf water
content in response to such a combination was attributed to the
production of osmolytes and stress-related proteins such as
dehydrins (Schmidt et al., 2000), an increase in proline content,
and a decrease in stomatal conductance (Poulson et al., 2006). A
reduced leaf area production upon exposure to enhanced UV
radiation and drought has also been described in wheat (Feng et al.,
2007) and in peas, in this latter case together with a decrease in
stomatal conductance (Nogués et al., 1998), which would reduce
plant water loss. Unfortunately, very few data are available on
Mediterranean species, and even less on woody evergreen
sclerophyllous species (Paoletti, 2005). Nevertheless, it is surprising that in most of the Mediterranean species studied, such as
Nerium oleander (Drilias et al., 1997), Olea europaea, Rosmarinus
officinalis and Lavanda stoechas (Nogués and Baker, 2000) or
Ceratonia siliqua (Kyparissis et al., 2001), no interactive effects
between UV-B radiation and drought on plant growth have been
reported. Only in some pine species, a beneficial effect of
supplemental UV-B radiation under water stress was found. In
this case, it was attributed to an increase in cuticle thickness, which
would restrict cuticular transpiration thereby improving leaf water
content (Manetas et al., 1997). Taking into account the climate
model predictions for the Mediterranean Basin, it is clear that more
studies are needed to understand how enhanced UV radiation can
modulate plant responses to drier conditions.
Among the most common plant mechanisms described to avoid
or counteract the damaging effects of UV-B radiation are those
aimed at preventing UV-B penetration into the leaf, either by
developing thicker, hairier, or waxier leaves (Jansen, 2002;
Karabourniotis et al., 1993), or by increasing their content in
UV-B-absorbing compounds, mainly phenols such as hydroxycinnamic acids and flavonoids (Jansen, 2002; Julkunen-Tiitto et al.,
2005). Recent studies suggest that hydroxycinnamic acids are
predominantly involved in UV-B screening, whereas flavonoids,
especially dihydroxy B-ring substituted flavonoids, are mainly
involved in counteracting the generation of reactive oxygen species
(ROS) (Agati et al., 2012; Brunetti et al., 2013). Increases in phenols
have also been related to other environmental stresses, such as
drought (see Selmar and Kleinwächter, 2013 for a recent review).
Indeed, in most of the species analysed till now – mainly crops – a
synergistic effect between UV-B radiation and drought on the
accumulation of foliar flavonoids and phenols has been found
(e.g., Balakumar et al., 1993; Feng et al., 2007; Hofmann et al., 2003;
Nogués et al., 1998). However, other studies have reported
contrasting results. For instance, Alexieva et al. (2001) and
Sangtarash et al. (2009) found no interactive effect between these
two factors on total foliar phenol content, whereas Turtola et al.
(2005) reported a lower UV-B-induced accumulation of phenols
when willow seedlings were simultaneously stressed by drought.
For Mediterranean sclerophyllous species, the few studies
available have reported no interactive effect between UV-B
radiation and drought on total leaf phenol content (Kyparissis
et al., 2001; Nogués and Baker, 2000). However, these studies did
not give information on possible changes in specific phenols.
Under conditions of high irradiance or water shortage (which
can limit CO2 fixation), an excess of absorbed light by plants can
cause photo-oxidative damage (Foyer et al., 1994). To counteract
these stressful conditions, Mediterranean plants have developed
different photoprotective mechanisms, some of them involving
265
reductions in the foliar pool of chlorophylls (Munné-Bosch and
Alegre, 2000), or changes in carotenoids. In terrestrial plants, two
carotenoid cycles have been associated with the thermal dissipation of excess light: the ubiquitous violaxanthin cycle (V-cycle),
also known as the xanthophyll cycle (Demmig-Adams et al., 1996),
and the lutein-epoxide cycle (Lx-cycle), which seems to be specific
to certain species (García-Plazaola et al., 2002; Llorens et al., 2002).
In the V-cycle, zeaxanthin (Z), the most effective xanthophyll in the
dissipation of excess energy as heat (Demmig-Adams et al., 1996;
García-Plazaola et al., 2007), is formed by de-epoxidation of
violaxanthin (V) via the intermediate antheraxanthin (A). The deepoxidation state of the V-cycle has been reported to increase
under enhanced UV-B radiation in leaves of Fagus sylvatica (Šprtová
et al., 2003; Láposi et al., 2009) or to decrease under UV exclusion
in leaves of Vitis vinifera (Núñez-Olivera et al., 2006) and in pine
needles (Martz et al., 2007). Other studies have failed to find any
effect of ambient UV-B radiation on the V-cycle de-epoxidation of
some species, such as pea or spruce, compared to plant growth
without UV-B (Bolink et al., 2001; Kirchgebner et al., 2003). The deepoxidation of lutein epoxide to lutein in the Lx-cycle has also been
associated with the thermal dissipation of energy in some species
(Llorens et al., 2002; García-Plazaola et al., 2007), but, to our
knowledge, it has never been investigated in relation to changes in
UV-B radiation. Neither is it known whether UV-B modulates
water availability effects on the de-epoxidation state of the V- and
Lx-cycles in sclerophyllous species.
Therefore, the aim of this study was to examine whether above
ambient UV radiation levels (specifically, a 20–25% increase in
erythemally weighted UV doses) would affect growth, foliar gas
exchange, and water relations of Laurus nobilis L. (laurel) seedlings,
and whether these effects would improve the tolerance of this
species to water limitation. We also investigated the effects of
these two abiotic factors on the foliar content of specific phenolic
compounds and chloroplastic pigments of this species. L. nobilis
was selected because it is a native Mediterranean evergreen tree of
great ecological and economical interest, which has been widely
studied due to its pharmaceutical and alimentary properties.
2. Materials and methods
2.1. Plant material and experimental design
One-year-old seedlings of Laurus nobilis L. with a root ball were
planted in 2 L pots (5 cm wide 20 cm deep) with 530 g of a
mixture containing composted pine bark and Sphagnum peat
(1:1 by volume). The growth medium was fertilised with Osmocote
(4 kg m3), basal dressing (1 kg m3), and dolomite (4 kg m3) to
avoid nutritional deficiencies during the experiment. Seedlings
were grown under controlled irrigation and supplemented with
UV radiation in an outdoor setting to obtain more realistic and
balanced ratios of UV radiation/PAR (photosynthetically active
radiation).
Seedlings were distributed in nine plots built with 1.3 m 1.2 m
metallic frames equipped with four fluorescent lamps mounted
overhead. These nine plots were organised in three blocks, with
each block having one plot with the following UV radiation
conditions: ambient UV radiation, enhanced UV-A radiation, and
enhanced UV-A + UV-B radiation. Within each plot, two irrigation
regimes were applied (see below). Each combination of UV
radiation and irrigation was, thus, replicated three times in a
randomised complete block design.
The experiment was conducted in an experimental field (Can
Vilallonga) in the vicinity of Cassà de la Selva (Girona,
northeastern Iberian Peninsula, 41530 N, 2 520 E) from May 29,
2009 to January 21, 2010. Before the UV treatment began, plants
were allowed to acclimate to the environmental UV and PAR
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M. Bernal et al. / Environmental and Experimental Botany 109 (2015) 264–275
conditions of the site for 2 weeks. On May 25, immediately prior
to starting the experiment, we measured the length and basal
diameter (at 2 cm above the cotyledonary node) of the main stem
of four plants from each UV radiation and irrigation treatment.
Statistical analyses indicated that initial differences in these
parameters among plants in the different UV radiation and
irrigation treatments were not significant (data not shown). All
samples were obtained around noon (2 h) under clear-sky days
from randomly chosen seedlings, and, unless otherwise noted,
they were collected in September 2009.
2.1.1. UV-radiation treatment
The three UV-radiation conditions (Table 1) were:
Enhanced UV-A UV-B: Solar UV radiation (UV-A UV-B) was
supplemented with four 40 W fluorescent lamps of 1.2 m in
length (TL 40 W/12 RS, with a peak at 313 nm; Phillips, Spain)
mounted in metal frames (1.3 1.2 m). Ultraviolet radiation was
applied daily for 2.5–3.5 h (depending on the month) centered at
solar noon. The fluorescent lamps were wrapped with cellulose
diacetate foil (Ultraphan URT, 0.1 mm, Digefra GmbH, Munich,
Germany) to remove wavelengths <280 nm (UV-C radiation).
Cellulose diacetate films were pre-burned for 3 h before being
applied to the lamps and were replaced after 36 h of use to avoid
the effects of plastic photodegradation.
Enhanced UV-A: These plots served as a control for the effect of
UVA in the UV-A + UV-B plots. Ultraviolet radiation was supplied
as described in the UV-A + UV-B plots, but the lamps were
wrapped with polyester instead of cellulose diacetate films
(Mylar D, 0.13 mm; PSG Group, England) to block both UV-B and
UV-C radiation (transmittance >315 nm).
Ambient UV: Seedlings in these plots received only solar UV
radiation. Instead of fluorescent holders, plots were equipped
with wood strips to ensure that plants were exposed to the same
conditions of shading than plants in the other two radiation
conditions.
Within each plot, plants were rotated every week to minimise
microenvironmental and border effects. To prevent UV radiation
contamination among plots, two 120 cm 30 cm clear sheets of
polycarbonate (no transmission below 400 nm) were fastened
along the two sides of the metal frames parallel to the fluorescent
lamps. Erythemally weighted UV doses (UVE, McKinlay and Diffey,
1987 McKenzie et al., 2004) were measured using an UVS-E-T
radiometer (Kipp and Zonen, The Netherlands) at the experimental
field within enhanced UV-A + UV-B plots. Measurements were
always taken at the top of plant canopies. The UVS-E-T radiometer
used has a peak response around 300 nm and the response
decreases by three orders of magnitude at 330 nm and an extra
order of magnitude at 400 nm. UVE doses within ambient plots
were estimated subtracting fluorescent's contribution to UVE
values obtained in UV-A + UV-B plots. UVE doses within enhanced
UV-A plots were calculated by adding the UV-A contribution,
which was estimated from field spectroradiometric measurements, to ambient UVE doses. On average, UV supplementation in
enhanced UV-A + UV-B plots increased UVE doses a 23% above
ambient levels, although increases ranged from 19% to 38%
depending on the month (Table 1). Despite UVE is the irradiance
weighted according to the action spectrum for UV-induced
sunburn in human skin (CIE, 1987 International Committee of
Lightening), UVE measures on plant growth studies have been
widely used giving reliable estimates of UV doses (Kotilainen et al.,
2011). In enhanced UV-A plots, UVE calculated values were similar
to that of ambient UV plots (Table 1) due to the very weak spectral
response of the UVS-E-T sensor in the UV-A range, as commented
above. To illustrate this, while irradiance in the UV-B range
contributes 83% to UVE, UV-As contribution is only 17% (although
UV-A represents 94% and UV-B 6% of the global radiation that
reaches the ground). In addition, UV-A doses emitted by the
fluorescents lamps were small (less than 2% of the ambient UV-A
under clear-sky conditions, according to our field measurements)
in comparison to ambient UV-A radiation, as it has been previously
reported (e.g., Aphalo et al., 2013). Photosynthetically active
radiation (PAR) was measured with a LI-190SA Quantum Sensor
(Li-Cor, USA) in the same plots and at the same height than UVE
doses (Table 1).
2.1.2. Irrigation treatment
Plants received water from rainfall and from a controlled dripirrigation system, which was programmed according to the
established irrigation conditions and monthly rainfall (Table 1).
In June 11, two irrigation regimes were applied: half of the
seedlings of each plot (chosen randomly) were irrigated to field
capacity (well-watered plants, WW), which involved adding from
0.2 to 1.34 L of water per day depending on the precipitation, while
the other half (low-watered plants, LW) received approximately
40% and 25% less water from June to September and from October
to December, respectively, than WW plants.
2.2. Biomass production and foliar morphological traits
Above- and below-ground biomass were determined for four
seedlings from each plot and irrigation treatment (three were
harvested in September and one in January). The leaves, stems, and
roots were separated and oven-dried at 70 C for 72 h to assess
their dry mass (DM).
One fully expanded and light-exposed leaf from four
individual plants per plot and irrigation condition was sampled
to determine leaf thickness and leaf mass per area (LMA,
mg cm2). Foliar thickness was measured with a portable
micrometer (mod. 4000DIG, Baxlo, Spain). The leaves were then
scanned (Epson perfection 1250, USA), and leaf areas determined
using an image-analysis programme (ImageTool, University of
Texas Health Science Center, USA). Subsequently, the DM of leaves
Table 1
Total monthly rainfall in Cassà de la Selva, monthly average UVE doses (kJ m2 day1) in ambient UV, enhanced UV-A + UV-B and enhanced UV-A plots, and monthly average
photosynthetically active radiation (PAR) doses (kJ m2 day1) S.E.
Month
Rainfall
(L m2)
UVE (kJ m2 day1) ambient UV plots
UVE (kJ m2 day1)
enhanced UV-A + UV-B plots
UVE (kJ m2 day1)
enhanced UV-A plots
PAR
(kJ m2 day1)
June
July
August
September
October
November
December
January
11
4.8
14.6
43.2
73.7
21.5
18.4
71.1
4.191 0.156
3.875 0.230
2.759 0.081
2.086 0.081
1.724 0.082
0.934 0.057
0.398 0.057
0.494 0.046
4.976 0.175
4.658 0.249
3.510 0.094
2.603 0.103
2.261 0.104
1.263 0.074
0.525 0.072
0.601 0.048
4.199 0.156
3.884 0.230
2.767 0.081
2.092 0.081
1.730 0.082
0.937 0.057
0.399 0.057
0.495 0.046
9874 427
7910 644
6828 369
5041 253
4524 268
2364 198
458 173
1427 235
M. Bernal et al. / Environmental and Experimental Botany 109 (2015) 264–275
was assessed and the LMA calculated as the quotient between
foliar DM and foliar area for each leaf.
2.3. Relative water content
The relative water content (RWC, %) was measured in the
same leaves used to analyse morphological traits. After collection, leaves were weighed to obtain leaf fresh mass (FM) and then
they were stored in darkness with distilled water for 48 h to
determine leaf turgid mass (TM). The DM was subsequently
measured (as described above), and the RWC was calculated as:
(FM-DM/TM-DM) 100.
2.4. Gas-exchange measurements
Foliar gas exchange was measured in one plant per plot and
irrigation condition, using one young, fully expanded lightexposed leaf located at the top of the canopy. Measurements
were taken using a gas-exchange system (CI-340 Hand-Held
Photosynthesis System, CID, Inc. Camas, WA 98607, USA) as
described in Llusià et al. (2012). With this system, L. nobilis net
photosynthesis (A, mmol CO2 m2 s1) and stomatal conductance
(gs, mmol H2O m2 s1) were determined under environmental
conditions of CO2 concentration, humidity and temperature, but
fixing PAR to 1000 mmol m2 s1 to saturate the photosystems and
to avoid possible light differences among measurements.
267
ethyl acetate 7/0.96/0.04/8 (v/v/v/v)] was pumped for 4.6 min.
Triethylamine was added to each phase to obtain a final
concentration of 0.7% in the mixture. The injection volume was
20 mL, and the analysis time for each sample was 15 min, including
the equilibration time, which consisted of flushing phase A for
5 min at the beginning. Results were expressed in mg of pigment
per g of foliar DM using LMA conversion.
2.7. Total phenols
One fully expanded light-exposed leaf from four plants per plot
and irrigation condition was sampled. From each leaf, 10 mg of
dried material was ground to a powder and mixed with 2.5 mL of
50% methanol. The extract was then shaken for 1 h and
subsequently centrifuged for 5 min at 800 g. Total phenolic content
was determined following the method described in Rozema et al.
(2006). A fraction of the extract (50 mL) was mixed with 3.5 mL of
distilled water and 250 mL of Folin–Ciocalteu reagent (Merck,
Germany). After 8 min, 750 mL of Na2CO3 (20%) was added.
Absorbance was measured 2 h later at 760 nm with a spectrophotometer (U-2000, Hitachi, USA). The total phenolic content of
leaves was calculated from the standard curve for gallic acid,
prepared from 50 mL of a standard solution of gallic acid (40, 80,
150, 200, 400, 600, 800, and 1000 mg L1) and expressed as mg of
gallic acid equivalents per g of DM.
2.8. UV-absorbing compounds (UACs)
2.5. Chlorophyll fluorescence
At predawn and midday, the components of foliar chlorophyll
fluorescence were quantified by means of a PAM-2100 portable
modulated fluorometer (Heinz Walz GmbH, Effeltrich, Germany)
for four seedlings per plot and irrigation condition, using one
exposed and fully developed leaf per seedling. Minimum (Fo) and
maximum (Fm) dark-adapted fluorescence was determined and
used to calculate the potential photochemical efficiency of
photosystem II as Fv/Fm, where Fv is variable fluorescence and
Fv = Fm Fo. The actual photochemical efficiency of photosystem II
in the light-adapted state was calculated as DF/Fm0 = (Fm0 F)/Fm0 ,
where F is the steady-state fluorescence yield under the given
environmental conditions and Fm0 is the maximum level of
fluorescence obtained during a saturating flash of light. The
apparent electron transport rate (ETR) was then calculated as
ETR = DF/Fm0 PAR 0.84 0.5, where PAR was the incident
photosynthetically active radiation (expressed in mmol m2 s1),
0.84 was the assumed coefficient of absorption of the leaves, and
0.5 was the assumed distribution of absorbed energy between the
two photosystems (Galmés et al., 2007). The non-photochemical
quenching coefficient (NPQ) was determined as (FmFm0 )/Fm0 .
2.6. Photosynthetic pigments
At predawn and midday at least three foliar discs of 0.64 cm2
per plot and irrigation condition were sampled, always from fully
expanded light-exposed leaves from different plants. Leaf discs
were immediately frozen in liquid nitrogen, and stored at 80 C
until analysis. Photosynthetic pigments were extracted with 1 mL
of acetone in the presence of liquid nitrogen and ascorbate by
grinding the foliar discs in a mortar. Four mL of acetone were then
added, and the mixture was stored at 20 C, as previously
described (Abadía and Abadía, 1993). Pigment extracts were
thawed on ice, filtered through a 0.45-mm filter, and analyzed by
high-performance liquid chromatography (HPLC) following the
method described by Larbi et al. (2004). Two phases were pumped
instead of three: phase A [acetonitrile:methanol 7/1 (v/v)] was
pumped for 3.4 min, and phase B [acetronitrile:methanol:water:
Methanol-soluble and methanol-insoluble (alkali-extractable
cell wall-bound) UV-absorbing compounds were analyzed for four
plants per plot and irrigation condition. Three foliar discs of
0.64 cm2 were collected from fully expanded and light-exposed
leaves of each plant, frozen in liquid nitrogen, and stored at 80 C
until analysis. Total foliar content of UACs and foliar concentrations
of several phenolic compounds were measured by spectrophotometry (Perkin-Elmer, Wilton, CT) and HPLC (Agilent
HP1100 HPLC system, Agilent Technologies, Palo Alto, CA),
respectively. The extraction and analytical methods were as
reported in Fabón et al. (2010). Briefly, after grinding the plant
material in a tissue lyser (Qiagen, Hilden, Germany), 5 mL of
methanol:water:7 M HCl (70:29:1) was added and the mixture
stored for at least 20 h at 4 C in the dark. The extract was
centrifuged at 6000 g for 15 min at 10 C, and the supernatant was
removed and used for the determination of methanol-soluble UACs
(mainly located in the vacuoles). The pellet was stored at 80 C
and later used for the determination of methanol-insoluble UACs
(mainly located in the cell wall).
For the determination of methanol-soluble phenols by HPLC,
250 mL of the supernatant were filtered (0.22 mm) and pumped
into the HPLC. The remainder of the supernatant was used to
determine total methanol-soluble UACs spectrophotometrically in
arbitrary units, as the area under the absorbance curve in the
intervals 280–315 nm and 280–400 nm (UAC280–315 and
UAC280–400) per unit of DM. For the methanol-insoluble UACs,
the pellet remaining from the methanol extraction was hydrolysed
with 2 mL of 1 M NaOH, and the mixture was heated at 80 C for 3 h.
One mL of 5.6 N HCl was added, and the UACs were extracted three
times with 2 mL of ethyl acetate. The supernatants from each
extraction were collected and evaporated. The residue was
dissolved in methanol, and the amount of phenols specific to
the cell wall were determined by HPLC, while the total content of
UACs was determined spectrophotometrically in the same units as
above. Specific phenolic compounds were identified taking into
account their absorption spectra and retention times, and
quantification was made by calibration curves using appropriate
commercial external standards.
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M. Bernal et al. / Environmental and Experimental Botany 109 (2015) 264–275
2.9. Statistical analyses
3.2. Leaf physiological parameters
The main effects of UV radiation treatment, irrigation
treatment, block, and their interactions, on all variables, except
those obtained from foliar gas-exchange measurements, were
assessed by three-way analysis of variance (ANOVA). When the
overall effect of UV treatment and/or the interaction between UV
and irrigation treatments were/was significant, we tested the
effects of our UV treatment within each watering regime using
two-way ANOVAs, with block and UV treatment as fixed factors.
Pairwise comparisons between UV conditions were analyzed using
Duncan's test. For the foliar gas-exchange data, the effects of the
UV and irrigation treatments were analyzed using two-way
ANOVAs, since only one plant per plot and irrigation treatment
was sampled. Kolmogorov–Smirnov and Levene's tests were used
to test normality and homoscedasticity, respectively, and data was
log-transformed when necessary. When normally distributed data
did not meet the assumption of homoscedasticity, the GamesHowell post-hoc test was applied. A significance level of
p 0.05 was used for all statistical tests.
A significant interaction between the effects of UV and
irrigation treatments was found for leaf RWC (Table 2). For
control plants, well-watered seedlings had a 3.4% higher foliar
RWC than did low-watered seedlings (F 1,23 = 9.01, p < 0.01)
(Fig. 2). Besides, while the UV treatment did not affect the foliar
RWC of well-watered plants, low-watered seedlings had 2.2% and
2.6% higher foliar RWCs under enhanced UV-A and UV-A + UV-B
radiation, respectively, than control seedlings, although the
difference was only significant in the UV-A + UV-B-supplemented
plants (Fig. 2).
The effect of the irrigation treatment on leaf gas exchange
parameters differed among UV-conditions, which would explain
the significant interaction found between treatments (Table 2).
Basically, low-irrigated plants supplemented with UV-A radiation
had higher photosynthetic rates (A) and stomatal conductance (gs)
than those grown under optimal irrigation (Fig. 3). In contrast, no
significant differences were observed in leaf gas exchange
parameters between low- and well-watered plants supplemented
with UV-A + UV-B radiation (Fig. 3). Overall, A and water-use
efficiency (WUE or A/gs) were significantly higher in low- than in
well-watered plants (Table 2), which was basically due to the
higher values found in plants supplemented with UV-A and
UV-A + UV-B radiation (100% and 56%, respectively, in the case of
WUE, despite differences were not significant; Fig. 3).
Parameters derived from foliar chlorophyll fluorescence measurements (ETR, Fv/Fm, and NPQ) were globally unaffected by the
treatments, either at predawn (data not shown) or at midday
(Table 2). Nevertheless, low-watered plants exposed to enhanced
UV-A radiation fluxes had significantly higher NPQ values at
midday than plants grown under ambient UV levels or under
UV-A + UV-B supplementation (Fig. 4).
3. Results
3.1. Seedling biomass and leaf morphological traits
Exposure to enhanced levels of UV-A and UV-A + UV-B radiation
increased the production of biomass in L. nobilis seedlings. Seedlings
grown under these conditions had, respectively, 36% and 41%
greater stem biomass and 26% and 30% greater root biomass than
seedlings grown under ambient levels of UV radiation (Table 2).
However, when the analyses were conducted within each
irrigation condition, differences were only significant for lowwatered plants. Indeed, low-watered plants exposed to enhanced
UV-A and UV-A + UV-B radiation had greater foliar, stem and root
biomass than control plants (F2,36 = 3.90, p = 0.03; F 2,36 = 5.56,
p = 0.01, and F 2,36 = 3.55, p = 0.04, respectively) (Fig.1), although they
did not show changes in the root-to-shoot ratio (data not shown).
Leaves from UV-A + UV-B-supplemented plants were about
11% thicker than those of control plants, but neither foliar area nor
LMA changed significantly in response to our UV treatment
(Table 2). Overall, plant biomass and leaf morphological traits did
not differ significantly between the two irrigation conditions
(Table 2).
3.3. Leaf photosynthetic pigments
Results consistently indicated that, at predawn and midday,
L. nobilis seedlings growing in enhanced UV-A plots had the
lowest foliar content of most of the photosynthetic pigments
related to the absorption of light: chlorophyll a + b, neoxanthin,
b-carotene and lutein (although differences in this last pigment
were not significant) (Table 3). At predawn, however, UV
radiation effects on the foliar content of chlorophylls and
Table 2
Overall mean S.E. for plant biomass and leaf morphological and physiological parameters of Laurus nobilis seedlings grown under three different UV radiation conditions
(ambient UV, enhanced UV-A, and enhanced UV-A + UV-B) and two irrigation levels (WW, well-watered and LW, low-watered). Different letters and values in boldface
indicate statistical differences (p 0.05) in pairwise comparisons. N = 72 for all variables except for A, gs and WUE (N = 18). A, photosynthetic rate; ETR, apparent electron
transport rate; gs, stomatal conductance; LMA, leaf mass per area; ns, not significant; NPQ, non-photochemical quenching; WUE, water-use efficiency.
UV radiation treatment
Leaf thickness (mm)
Leaf area (cm2)
LMA (mg cm2)
Total biomass (g)
Stem biomass (g)
Root biomass (g)
Leaf biomass (g)
RWC (%)
A (mmol CO2 m2 s1)
gs (mmol H2O m2 s1)
WUE (mmol CO2 mmol1 H2O)
ETR midday
Fv/Fm midday
NPQ midday
UV Irrigation
Irrigation treatment
Ambient
UV
Enhanced
UV-A
Enhanced
UV-A + UV-B
p- value
WW
LW
p-value
p-value
0.40 0.009 a
11.08 0.65
12.50 0.21
11.46 0.64 a
2.44 0.17 a
6.08 0.31 a
2.94 0.21
89.93 0.62
6.11 1.07
59.69 6.34
0.10 0.02
68.30 9.22
0.66 0.01
1.72 0.19
0.42 0.012 ab
10.14 0.56
13.12 0.27
14.41 1.15 b
3.32 0.03 b
7.67 0.59 b
3.42 0.28
89.73 0.84
6.76 1.87
61.06 7.03
0.10 0.02
69.13 7.88
0.66 0.01
1.82 0.21
0.45 0.015 b
11.15 0.69
13.15 0.41
14.94 1.14 b
3.45 0.31 b
7.92 0.61 b
3.56 0.28
90.79 0.52
5.99 0.92
53.06 4.13
0.11 0.01
71.06 9.33
0.66 0.01
1.52 0.04
0.04
ns
ns
0.03
0.01
0.02
ns
ns
ns
ns
ns
ns
ns
ns
0.42 0.010
10.73 0.62
12.91 0.30
14.28 0.94
3.30 0.25
7.49 0.49
3.49 0.24
90.14 0.62
4.49 0.74
56.73 4.64
0.09 0.01
75.84 7.17
0.66 0.01
1.78 0.18
0.43 0.011
10.84 0.40
12.93 0.20
12.93 0.73
2.84 0.20
6.96 0.38
3.12 0.18
90.14 0.48
7.63 1.15
59.14 5.06
0.13 0.01
63.16 7.00
0.65 0.01
1.59 0.06
ns
ns
ns
ns
ns
ns
ns
ns
0.05
ns
0.03
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
0.02
0.05
0.05
ns
ns
ns
ns
M. Bernal et al. / Environmental and Experimental Botany 109 (2015) 264–275
a)
a)
b
ab
A
Ambient UV
Enhanced UV-A
Enhanced UV-A+UV-B
10
8
2
A A
*
12
Net photosynthetic rate (A)
(μmol CO m-2s-1)
Leaf biomass (g)
5
4
269
a
3
2
6
4
2
1
Stem biomass (g)
4
A
b
b
A
A
3
60
a
2
40
20
a*
A
c)
1
WW
0,16
LW
X Data
10
ab
b
A A
8
A
a
6
WUE (A/gs )
(μ mol CO2 mmol H2O)
c)
Root biomass (g)
*
80
2
Ambient UV
Enhanced UV-A
Enhanced UV-A+UV-B
5
Stomatal conductance (gs)
(mmol H O m-2s-1)
b)
b)
0,12
0,08
0,04
4
2
WW
LW
Irrigation treatment
LW
Irrigation treatment
Fig. 1. Leaf, stem, and root biomass for well- (WW) and low-watered (LW) seedlings
of Laurus nobilis grown under three UV conditions. Values are means S.E. (N = 12).
Different letters indicate statistically significant differences (p 0.05) among UV
conditions within each irrigation level (upper and lower case letters for WW and LW
plants, respectively).
RWC (%)
100
Ambient UV
Enhanced UV-A
Enhanced UV-A+UV-B
A
90
A
A
a*
ab b
80
70
WW
Fig. 3. (a) Net photosynthetic rate (A), (b) stomatal conductance (gs), and (c) wateruse efficiency (WUE) of Laurus nobilis seedlings exposed to different UV and
irrigation conditions. Values are means S.E. (N = 3). The asterisks indicate
statistically significant differences between well-watered (WW) and low-watered
(LW) plants grown under the same UV conditions (p 0.05). Within each irrigation
level, plants under different UV conditions did not show significant differences in
the studied parameters.
Non-photochemical quenching
(NPQ)
WW
2,0
Ambient UV
Enhanced UV-A
Enhanced UV-A+UV-B
1,8
b
A
1,6
A
A
a
a
1,4
1,2
LW
Irrigation treatment
WW
LW
Irrigation treatment
Fig. 2. Foliar RWC for well- (WW) and low-watered (LW) seedlings of Laurus nobilis
grown under three UV radiation conditions. Values are means S.E. (N = 12).
Different letters indicate statistically significant differences (p 0.05) among UV
conditions within each irrigation level (upper and lower case letters for WW and LW
plants, respectively). The asterisk indicates a significant difference between WW
and LW plants exposed to ambient UV conditions.
Fig. 4. Non-photochemical quenching (NPQ) for well- (WW) and low-watered (LW)
seedlings of Laurus nobilis under three UV conditions at midday. Values are
means S.E. (N = 12). Different letters indicate statistically significant differences
(p 0.05) among UV conditions within each irrigation level (upper and lower case
letters for WW and LW plants, respectively).
270
M. Bernal et al. / Environmental and Experimental Botany 109 (2015) 264–275
Table 3
Overall means S.E. at predawn (A) and midday (B) for the foliar content of photosynthetic pigments of Laurus nobilis seedlings grown under three different UV conditions
(ambient UV, enhanced UV-A, and enhanced UV-A + UV-B) and two irrigation levels (WW, well-watered and LW, low-watered). The effects (p-values) of the UV and irrigation
treatments and the interactions between both factors were tested by three-way ANOVA, with a total of 18 degrees of freedom for the UV treatment and 27 for the irrigation
treatment. Different letters and values in boldface indicate statistical differences (p 0.05) in pairwise comparisons. AZ, Anteraxanthin plus Zeaxanthin; VAZ, Violaxanthin
plus Anteraxanthin plus Zeaxanthin; ns, not significant.
UV treatment
(A) Predawn
–
Chlorophyll a + b (mg g DM1)
Chlorophyll a/b
Neoxanthin (mg g DM 1)
b-carotene (mg g DM 1)
a-carotene (mg g DM 1)
Violaxanthin (mg g DM 1)
Anteraxanthin (mg g DM 1)
Zeaxanthin (mg g DM 1)
VAZ pool
AZ/VAZ
Lutein (mg g DM 1)
Lutein-5,6-epoxide (mg g DM 1)
Lutein + Lutein-5,6-epoxide (mg g DM
(B) Midday
–
Chlorophyll a + b (mg g DM 1)
Chlorophyll a/b
Neoxanthin (mg g DM 1)
b-carotene (mg g DM 1)
a-carotene (mg g DM 1)
Violaxanthin (mg g DM 1)
Anteraxanthin (mg g DM 1)
Zeaxanthin (mg g DM 1)
VAZ pool
AZ/VAZ
Lutein (mg g DM 1)
Lutein-5,6-epoxide (mg g DM 1)
Lutein + Lutein-5,6-epoxide (mg g DM
1
1
UV irrigation
Irrigation treatment
Ambient
UV
Enhanced
UV-A
Enhanced
UV-A + UV-B
p-value
WW
LW
p-value
p-value
)
4.32 0.24 a
3.89 0.06
0.12 0.005 a
0.28 0.01 a
0.01 0.003
0.26 0.01
0.02 0.002
0.005 0.001 a
0.28 0.01
0.09 0.01
0.39 0.01
0.04 0.004 a
0.43 0.02 a
3.33 0.22 b
4.11 0.10
0.10 0.01 b
0.23 0.01 b
0.01 0.003
0.23 0.01
0.02 0.002
0.008 0.001 b
0.26 0.01
0.12 0.01
0.34 0.01
0.03 0.004 b
0.37 0.02 b
4.52 0.33 a
3.88 0.07
0.13 0.01 a
0.29 0.01 a
0.01 0.002
0.25 0.01
0.02 0.002
0.006 0.001 ab
0.28 0.01
0.10 0.01
0.39 0.02
0.04 0.003 a
0.43 0.02 a
0.01
ns
<0.01
<0.01
ns
ns
ns
0.05
ns
ns
ns
0.02
0.03
4.04 0.20
3.89 0.06
0.12 0.01
0.26 0.01
0.01 0.002
0.24 0.01
0.02 0.002
0.007 0.001
0.27 0.01
0.11 0.01
0.37 0.01
0.03 0.003
0.40 0.01
4.07 0.30
4.02 0.07
0.12 0.01
0.28 0.01
0.01 0.002
0.25 0.01
0.02 0.001
0.007 0.001
0.28 0.01
0.10 0.01
0.38 0.02
0.04 0.004
0.42 0.02
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
0.02
ns
ns
0.01
0.02
ns
ns
ns
ns
ns
ns
<0.01
0.03
)
4.16 0.16 a
3.63 0.06
0.11 0.01 ab
0.27 0.01 a
0.01 0.003
0.19 0.01 a
0.04 0.003
0.03 0.01 ab
0.26 0.01
0.26 0.03ab
0.39 0.01
0.04 0.005ab
0.43 0.01 a
3.50 0.19 b
3.68 0.07
0.09 0.01 b
0.22 0.01 b
0.01 0.002
0.15 0.01 b
0.04 0.004
0.05 0.01 a
0.24 0.01
0.35 0.06 a
0.34 0.02
0.03 0.004 a
0.37 0.02 b
4.42 0.24 a
3.77 0.09
0.12 0.01 a
0.29 0.01 a
0.02 0.003
0.22 0.01 a
0.03 0.003
0.02 0.003 b
0.27 0.01
0.19 0.02 b
0.39 0.02
0.05 0.004 b
0.43 0.02 ab
0.01
ns
0.03
<0.01
ns
<0.01
ns
0.03
ns
0.02
ns
0.01
0.02
4.05 0.20
3.68 0.06
0.11 0.01
0.26 0.01
0.01 0.002
0.18 0.01
0.03 0.003
0.03 0.01
0.25 0.01
0.28 0.04
0.37 0.02
0.04 0.04
0.41 0.02
4.03 0.15
3.71 0.06
0.11 0.01
0.26 0.01
0.02 0.003
0.19 0.01
0.03 0.003
0.03 0.01
0.26 0.01
0.25 0.03
0.37 0.01
0.04 0.004
0.41 0.01
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
b- and a-carotene were dependent on the amount of water
supplied to the plants (Table 3), since differences were only
significant for low-watered seedlings (Fig. 5).
Plants grown in enhanced UV-A-plots had significantly higher
zeaxanthin content than control plants at predawn or than
UV-A + UV-B irradiated plants at midday (Table 3). Accordingly,
under reduced irrigation, UV-A-supplemented plants showed the
highest de-epoxidation state of the V-cycle (AZ/VAZ) at predawn
(Fig. 6). These plants also had the lowest foliar content of lutein
and lutein-5,6-epoxide (Fig. 6). At midday, UV-A-supplemented
leaves showed higher AZ/VAZ values, but only in comparison to
leaves under enhanced UV-A + UV-B (Table 3).
3.4. UV-absorbing compounds
Eight methanol-soluble compounds were detected. Two
belonged to the quercetin family (quercetin 3-O-glucuronide,
quercetin-O-glycoside) and two to the kaempferol family
(cis-trans-kaempferol
3-O-glucoside,
kaempferol
3,7-Odiglucoside) (Table 4). There were also three unidentified
compounds, named as C1, C2, and C3, with highly similar spectra
to that of quercetins, which were, thus, considered from the
quercetin family. Finally, another unidentified compound, C4, had a
spectrum with a high similarity to that of kaempferols and it
was considered from the kaempferol family (Table 4). Regarding
cell-wall compounds, only two were detected: p-coumaric (a
hydroxycinnamic acid) and one kaempferol derivative (Table 4).
Neither the total leaf content of phenols nor the total leaf
content of methanol-soluble (mainly located in vacuoles) and
methanol-insoluble (from the cell wall) UV-B-absorbing compounds (UAC280–315) or UV-absorbing compounds (UAC280–400)
varied in response to our experimental treatments or their
interaction (Table 4). The total amount of quercetins and
kaempferols was lower in plants exposed to enhanced UV-A or
enhanced UV-A + UV-B radiation than in control plants, although
the kaempferol content only differed significantly between UV-Asupplemented and control plants (Table 4). Among quercetins, the
leaf content of quercetin 3-O-glucuronide was reduced in UV-Aand UV-A + UV-B-supplemented plants compared to controls,
while the foliar amount of C1 was significantly lower only in
UV-A-supplemented plants (Table 4). Leaves exposed to enhancedUV-A radiation had also smaller amounts of cis-trans-kaempferol
3-O-glucoside than control plants (Table 4). Any of the two cellwall compounds detected responded significantly to the UV
radiation treatment (Table 4). The irrigation treatment did not
affect significantly the leaf content of phenols or UACs.
4. Discussion
4.1. Effects of enhanced UV radiation and low irrigation on plant
biomass and leaf gas exchange
Plant biomass accumulation is an integrated parameter used
commonly as an indicator of sensitivity to stressful conditions
(Smith et al., 2000). In our experiment, enhanced UV radiation
M. Bernal et al. / Environmental and Experimental Botany 109 (2015) 264–275
a)
A
A
a
a
A
2
b
1
b)
β-carotene (mg g DW-1)
0,35
a
A
0,28
A
ab
A
b
0,21
0,14
0,04
0,03
A
A
0,4
a
ab
A
b
0,2
b)
0,16
a
0,12
a
A
A
A
0,08
b
0,04
c)
c)
Ambient UV
Enhanced UV-A
Enhanced UV-A+UV-B
0,25
0,20
a
A
0,02
ab
A
0,01
Lutein-5,6-epoxide (mg g DW-1)
3
Lutein (mg g DW-1)
0,6
AZ/VAZ
Chlorophyll a+b (mg g DW-1)
a)
α -carotene (mg g DW-1)
271
Ambient UV
Enhanced UV-A
Enhanced UV-A+UV-B
b
A
0,15
A
A
0,10
A
ab
a
b
0,05
WW
LW
Irrigation treatment
Fig. 5. Predawn foliar content of chlorophyll a + b and a- and b-carotene for well(WW) and low-watered (LW) seedlings of Laurus nobilis grown under three UV
conditions. Values are means S.E. (N = 9). Different letters indicate statistically
significant differences (p 0.05) among UV conditions within each irrigation level
(upper and lower case letters for WW and LW plants, respectively).
(UV-A and UV-A + UV-B) positively affected the biomass accumulation of L. nobilis seedlings, indicating that this sclerophyllous
species has effective mechanisms for dealing with increased levels
of UV radiation. Our results also suggest that this UV-stimulation of
growth is mainly a response to enhanced UV-A radiation, since no
significant differences were found between plants grown under
enhanced UV-A radiation and those grown under enhanced
UV-A + UV-B radiation. Similar studies have also reported no effect
of UV-B radiation on plant biomass accumulation (de la Rosa et al.,
2001; Kostina et al., 2001; Hakala et al., 2002; Zaller et al., 2004;
Bassman and Robberecht, 2006; Wang et al., 2008), although
negative effects have been shown in many other studies, mainly on
crop species (Yuan et al., 2002; Zavala and Ravetta, 2002; Feng
et al., 2003; Gao et al., 2003; Zheng et al., 2003; Yao et al., 2006;
Kadur et al., 2007; Surabhi et al., 2009; Tsormpatsidis et al., 2010;
Yao and Liu, 2009). The effect of UV-A radiation on plant biomass
has received less attention than that of UV-B, but, like UV-B
radiation, this effect appears to be species-specific. Indeed, while
UV-A radiation negatively affected root biomass in Quercus robur
(Newsham et al., 1999) or total biomass in cucumber (Krizek et al.,
1997), it promoted overall growth in radish, probably due to an
WW
LW
LW
Irrigation treatment
Fig. 6. Predawn foliar content of lutein and lutein-5,6-epoxide and foliar deepoxidation state of the V-cycle (AZ/VAZ) for well- (WW) and low-watered (LW)
seedlings of Laurus nobilis under three UV conditions. Values are means S.E.
(N = 9). Different letters indicate statistically significant differences (p 0.05)
among UV conditions within each irrigation level (upper and lower case letters for
WW and LW plants, respectively). A, antheraxanthin; V, violaxanthin; Z, zeaxanthin.
increase in the foliar content of chlorophylls, the photosynthetic
activity, and the nitrogen metabolism (Tezuka et al., 1994).
In line with a previous study we performed using seedlings of
six Mediterranean species (Bernal et al., 2013), the possible
beneficial effect of UV-A supplementation on biomass accumulation in L. nobilis appears to occur principally when the plants are
growing under low water availability (Fig. 1). In fact, our results
suggest that plant exposure to enhanced UV-A radiation, may have
mitigated the negative impact of water deficit on leaf water status,
since while reduced irrigation decreased the leaf RWC of control
plants, it did not modify leaf RWC of plants grown under enhanced
UV-A or UV-A + UV-B (Fig. 2). Several previous studies have
reported an amelioration of water deficit in UV-B-irradiated plants
grown under low water availability (Feng et al., 2007; Schmidt
et al., 2000; Poulson et al., 2006; Nogués et al., 1998; Manetas et al.,
1997). However, in our study, the observed increase in leaf RWC in
response to enhanced UV radiation under water shortage (Fig. 2)
seems to be mediated mainly by UV-A radiation. In this case, the
improvement in water relations is not associated with a decrease in
leaf area or stomatal conductance (gs) (Fig. 3) (Feng et al., 2007;
Nogués et al., 1998; Poulson et al., 2006). Instead, it might be
272
M. Bernal et al. / Environmental and Experimental Botany 109 (2015) 264–275
Table 4
Overall means S.E. of total foliar phenolic content (mg AG equivalents g DM1), (A) methanol-soluble compounds (mg g DM1), and (B) methanol-insoluble cell-wall
compounds (mg g DM1) of Laurus nobilis seedlings grown under three different UV conditions (ambient UV, enhanced UV-A, and enhanced UV-A + UV-B, N = 24) and two
irrigation levels (WW, well-watered and LW, low-watered; N = 36). Different letters and values in boldface indicate statistical differences (p 0.05) in pairwise comparisons.
The interaction between UV radiation and irrigation treatments was not significant for any of the variables analysed.
UV treatment
Total phenols
–
(A) Methanol-soluble compounds
UAC280–315
UAC280–400
Quercetins
Quercetin 3-O-glucuronide
Quercetin-O-glycoside
C1 (Quercetin family)
C2 (Quercetin family)
C3 (Quercetin family)
Kaempferols
Cis-trans-kaempferol 3-O-glucoside
Kaempferol 3,7-O -diglucoside
C4 (Kaempferol family)
–
(B) Cell-wall compounds
UAC280–315
UAC280–400
p-coumaric
Kaempferol derivative
Irrigation treatment
Ambient
UV
Enhanced
UV-A
Enhanced
UV-A + UV-B
p-value
WW
LW
p-value
69.58 3.55
70.35 4.77
75.07 4.39
ns
72.22 3.91
71.04 3.02
ns
0.74 0.03
1.73 0.07
30.17 3.81
12.77 1.75
7.54 0.86
4.69 0.57
3.58 0.73
1.59 0.28
14.65 1.72
10.55 1.35
0.59 0.07
3.51 0.37
0.79 0.02
1.16 0.03
20.50 2.35 b
7.98 0.90b
5.67 0.80
2.61 0.38 b
3.06 0.55
1.17 0.15
11.16 1.30 b
7.55 0.85 b
0.41 0.05
3.20 0.61
0.75 0.02
1.15 0.04
22.10 1.89 b
8.86 0.77b
6.25 1.06
3.21 0.34 ab
2.58 0.30
1.20 0.09
12.58 1.38 ab
9.22 1.12 ab
0.46 0.04
2.89 0.29
ns
ns
0.01
0.01
ns
<0.01
ns
ns
0.05
0.03
ns
ns
0.75 0.02
1.19 0.03
24.95 2.58
10.01 1.05
6.88 0.86
3.65 0.44
2.99 0.48
1.41 0.19
12.91 1.38
9.50 1.13
0.49 0.04
2.91 0.24
0.77 0.02
1.13 0.03
23.65 2.18
9.78 1.06
6.11 0.61
3.37 0.33
3.16 0.42
1.23 0.12
12.73 1.04
8.75 0.68
0.49 0.05
3.49 0.44
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
0.57 0.01
1.39 0.02
0.47 0.02
0.72 0.02
0.59 0.02
1.43 0.04
0.46 0.01
0.71 0.03
ns
ns
ns
ns
0.56 0.02
1.36 0.06
0.49 0.02
0.74 0.02
a
a
a
a
a
0.58 0.01
1.42 0.02
0.43 0.02
0.67 0.042
0.60 0.01
1.45 0.03
0.48 0.01
0.74 0.03
ns
ns
ns
ns
UAC280–315, UV-B absorbing compounds; UAC280–400, UV absorbing compounds; ns, not significant.
explained, at least partially, by a greater leaf thickness (Table 2),
since thicker leaves have been often associated with an improvement in water relations (Gitz and Liu-Gitz, 2003; Ennajeh et al.,
2010). In addition, UV-A- and UV-A + UV-B-irradiated plants
tended to have, under low irrigation, higher leaf WUE values than
plants exposed to ambient UV levels, which resulted from
proportionally greater increases in leaf photosynthetic rates than
in gs (86% and 30%, respectively; Fig. 3). Hence, under low water
availability, improved WUE and leaf RWC in UV-irradiated plants,
compared to controls, might explain their higher biomass
production. As a consequence, our results suggest that future
increases in UV doses similar to the ones applied in this
experiment might benefit the growth of L. nobilis seedlings under
moderately drier conditions.
4.2. The role of photosynthetic pigments in the response of plants to
enhanced UV radiation and low watering
The xanthophyll V- and Lx-cycles have been associated with
thermal dissipation of excess light energy in the antenna of
photosystem II (García-Plazaola et al., 2007) and in fact, this has
been reported for L. nobilis in particular (Esteban et al., 2007, 2008).
The results from our study suggest that, under low water
availability, small increases in above ambient levels of UV-A
radiation might enhance the thermal dissipation of excess energy
(measured as NPQ) in this species (Fig. 4). This would be in
agreement with the higher levels of zeaxanthin (at predawn and
midday) and the higher AZ/VAZ values (at midday) found in UV-A
irradiated plants of L. nobilis (Table 3). Under reduced irrigation,
UV-A irradiated seedlings also showed the highest de-epoxidation
state of the V-cycle (AZ/VAZ) and the lowest leaf content in lutein5,6-epoxide at predawn (Fig. 6), which would support the idea that
these plants would require a higher thermal dissipation of light
energy in the antenna (García-Plazaola et al., 2002; Llorens et al.,
2002). At the same time, the observed reduction in the foliar
content of most of the main light-absorbing pigments in UV-Airradiated leaves (Table 3, Fig. 5) might have contributed to
avoiding the imbalance between light absorption and the energy
required for photosynthesis (Munné-Bosch and Alegre, 2000).
Nevertheless, taking into account, the low amount of UV-A
supplied by our lamps in relation to ambient levels, this point
clearly deserves further investigation.
Interestingly, we did not find significant differences between
controls and UV-A + UV-B-irradiated leaves in the NPQ, deepoxidation of the xanthophyll cycles or the amount of lightabsorbing pigments (Table 3 and Figs. 4–6). In the case of the deepoxidation of the V-cycle, this might be due to a UV-B-induced
inhibition of violaxanthin de-epoxidase, as has been observed in
isolated thylakoids and in intact leaves of pea (Pfündel et al., 1992).
Yang et al. (2007) reported higher levels of violaxanthin and lower
levels of zeaxanthin in leaves of wheat plants subjected to UV-A
plus UV-B radiation relative to those irradiated with UV-A
radiation only. At the same time, they found an increase in the
components of the cellular antioxidant system, such as superoxide
dismutase, catalase, ascorbate peroxidase, and glutathione reductase, in UV-A + UV-B-supplemented plants. These authors suggested that in UV-A + UV-B-irradiated plants the endogenous
antioxidant defense system is enhanced while in UV-A-irradiated
plants it is the thermal dissipation of light energy that is enhanced.
4.3. Response of specific phenolic compounds to enhanced UV
radiation and low irrigation
An increase in foliar UACs, especially phenols, is a common
plant response mechanism for dealing with increased UV radiation
(Julkunen-Tiitto et al., 2005; Caldwell et al., 2007). However, in the
present study, the experimental increases in UV radiation did not
change the total foliar content of UACs or phenolic compounds in L.
nobilis seedlings (Table 4), as was the case with previous studies on
other Mediterranean species (Bernal et al., 2013; Manetas, 1999;
Stephanou and Manetas, 1997). Despite the lack of observed UV
effects on the total amount of these compounds, changes in the
content of specific phenols upon UV exposure were found. Indeed,
plants of L. nobilis grown in enhanced UV-A plots had a lower
content of quercetin and kaempferol derivatives in the vacuoles
compared to control plants (Table 4). The presence of similar
M. Bernal et al. / Environmental and Experimental Botany 109 (2015) 264–275
amounts of these compounds in UV-A + UV-B- and UV-A-supplemented plants suggests that UV-B supplementation did not exert a
significant additional effect on the foliar accumulation of these
compounds (Table 4).
The greater need for energy dissipation in low-watered plants
grown in enhanced UV-A plots would suggest a possible photooxidative stress. However, these plants had the lowest levels of
quercetins and kaempferols which, especially in the case of
quercetins, are expected to increase under oxidative stress
(Agati et al., 2012; Pollastri and Tattini, 2011). Very few data are
available about the effect of UV-A radiation on the foliar amount of
phenols, but Wilson et al. (2001), in a controlled lab experiment
with Brassica napus, also reported a general down-regulation of
extractable flavonoids mediated by UV-A radiation affecting
quercetins more than kaempferols. They suggested that this
decrease could be a photobiological response involving activation/
inactivation of leaf photoreceptors, while rejecting the idea of
direct photochemical degradation. In our study, the photoprotective mechanisms detected in UV-A-irradiated leaves (i.e., a
reduction in the amount of light-harvesting pigments and an
increase in the dissipation of excess energy as heat) may have
lowered the production of ROS and, in consequence, the
requirement for antioxidant compounds. This would be consistent
with the significantly lower levels of leaf b-carotene found in these
plants both at predawn and midday (Table 3). Overall, it seems
evident that L. nobilis has the ability to use different mechanisms to
cope with and even to benefit from UV radiation increases.
5. Conclusions
L. nobilis plants produced more biomass when grown under
fluorescent lamps that provided above ambient levels of UV-A or
UV-A + UV-B radiation, especially under low water availability. This
was probably related to a UV-induced improvement of leaf water
status under water shortage, which seems to be a response to
enhanced UVA exposition. Our results also suggest that UV-Asupplemented plants grown under low water availability had an
excess of light, since they activated leaf photoprotective mechanisms, such as the de-epoxidation of the xanthophyll cycles for the
thermal dissipation of energy. Accordingly, these plants also had
lower foliar contents of light-harvesting pigments, which would
reduce the absorption of light by the antennae. Additional
exposure to UV-B radiation seemed to counteract UV-A-induced
effects on the studied photoprotective mechanisms without
altering the beneficial effects of this radiation on plant biomass
accumulation. Our results, therefore, point to different plant
responses to UV-A and UV-B radiation and highlight the
importance of taking UV-A radiation into account when investigating the effects of UV-B radiation on plants.
Acknowledgements
This research was supported by the following projects:
CGL2007-64583, CGC2010-17172, Consolider Ingenio Montes
(CSD2008-00040) and CGL2011-26977 funded by the Spanish
Government and by the SGR 2009-458 project funded by the
Catalan Government. We are also grateful to the Gavarres
Consortium for allowing us to perform the experiment in Can
Vilallonga.
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