Physiologia Plantarum 130: 495–510. 2007 Copyright ª Physiologia Plantarum 2007, ISSN 0031-9317
Photoprotection processes under water stress andrecovery in Mediterranean plants with differentgrowth forms and leaf habitsJeroni Galmesa,*, Anunciacion Abadıab, Josep Cifrea, Hipolito Medranoa and Jaume Flexasa
aGrup de Recerca en Biologia de les Plantes en Condicions Mediterranies, Universitat de les Illes Balears, Carretera de Valldemossa Km.7.5,
07122 Palma de Mallorca, SpainbDepartment of Plant Nutrition, Estacion Experimental de Aula Dei, CSIC, Zaragoza, Spain
Correspondence
*Corresponding author,
e-mail: [email protected]
Received 22 December 2006; revised 8
March 2007
doi: 10.1111/j.1399-3054.2007.00919.x
The response of photoprotection mechanisms to a short-term water stress
period followed by rewatering, to simulate common episodic water stress
periods occurring in Mediterranean areas, was studied in 10 potted plants
representative of different growth forms and leaf habits. During water stressand recovery, relative water content, stomatal conductance, leaf pigment
composition, electron transport rates, maximum quantum efficiency of PSII
photochemistry (Fv/Fm), thermal energy dissipation and photorespiration rates
(Pr) were determined. All the species analyzed proved to be strongly resistant to
photoinactivation of PSII under the imposed water stress conditions. The
responses of the analyzed parameters did not differ largely among species,
suggesting that Mediterranean plants have similar needs and capacity for
photoprotection under episodic water stress periods regardless of their growthform and leaf habit. A general pattern of photoprotection emerged, consisting
in maintenance or increase of Pr at mild stress and the increase of the thermal
energy dissipation at more severe stress. Adjustments in pigment pool sizes
were not an important short-term response to water stress. The increase of
thermal energy dissipation because of water stress depended mostly on the de-
epoxidation state of xanthophylls, although the slope and kinetics of such
relationship strongly differed among species, suggesting species-dependent
additional roles of de-epoxidated xanthophylls. Also, small decreases in Fv/Fm
at predawn during water stress were strongly correlated with maintained de-
epoxidation of the xanthophylls cycle, suggesting that a form of xanthophyll-
dependent sustained photoprotection was developed during short-term water
stress not only in evergreen but also in semideciduous and annual species.
Abbreviations – AN, net CO2 assimilation rate; DPS, de-epoxidation state; DPSMD, midday de-epoxidation state; DPSPD, predawn
de-epoxidation state; ETR, electron transport rate; Fm, maximum fluorescence; Fm#, steady-state maximum fluorescence yield;
Fo, background fluorescence signal; Fs, steady-state fluorescence signal; Fv/Fm, maximum quantum efficiency of PSII
photochemistry; gs, stomatal conductance; MiWS, mild water stress; MoWS, moderate water stress; NPQ, non-photochemical
quenching of chlorophyll fluorescence; Pr, photorespiration rate; RL, rate of non-photorespiratory CO2 evolution in the light; RW,
rewatering; RWCPD, relative water content at predawn; SeWS, severe water stress; VAZ, sum of violaxanthin, antheraxanthin and
zeaxanthin.
Physiol. Plant. 130, 2007 495
Introduction
Summer water deficit is considered the main environ-
mental constraint for plant growth and survival inMediterranean type ecosystems. In these environments,
natural vegetation has developed an array of adaptations
to drought, resulting in a high diversity of life habits and
growth forms. The resulting vegetation consists mostly of
deep-rooted evergreen sclerophyll trees and shrubs
which maintain green leaves during the summer drought
period, semideciduous shrubs which lose a part of their
leaves during summer, and geophytes and winter annualherbs which escape drought by finishing their annual
cycle before summer (Ehleringer and Mooney 1982). Low
soil water availability during summer is accompanied by
high temperature and excessive radiation, which imposes
a multiple stress to plants (Di Castri 1973). The
combination of these stresses can lead to photoinhibition
and photodamage of the photosynthetic apparatus, which
may result in decreased photosynthetic capacity and,eventually, in plant death (Chaves et al. 2002, Penuelas
et al. 2004). Because of this, and taking into account the
large variability of habitat microclimates in the Mediter-
ranean region, as well as the stochastic distribution of
rainfall, it is likely that Mediterranean plants may have
evolved a large diversity of photoprotective mechanisms
to cope with excess light, particularly during the summer
drought period.Many photoprotective mechanisms have been
described in higher plants (Bjorkman and Demmig-
Adams 1994, Niyogi 1999), including reducing light
absorption through leaf or chloroplast movements,
decreased Chl contents or reflective structures such as
hairs; regulation of energy dissipation through photo-
chemical (e.g. photorespiration) and non-photochemical
(e.g. safe thermal dissipation of excess absorbed lightenergy, via the xanthophyll cycle) mechanisms; scaveng-
ing of reactive oxygen species formed because of excess
light and repair and resynthesis of photodamaged
components of the photosynthetic apparatus (e.g. D1
protein). Many of these mechanisms have been described
in Mediterranean plants. For instance, steep leaf angles
have been shown as efficient structural photoprotective
features in perennial grasses like Stipa tenacissima(Valladares and Pugnaire 1999), semideciduous shrubs
like Cistus albidus and evergreen sclerophyll shrubs like
Arbutus unedo (Werner et al. 1999, 2001). Some semi-
deciduous shrubs present another mechanism to reduce
light absorption during summer, consisting of partial leaf
loss in parallel to a substantial loss of Chl in the remaining
leaves (Kyparissis et al. 1995, 2000; Munne-Bosch and
Alegre 2000a, 2000b). In Phlomis fruticosa, Chl lossduring summer is not accompanied by decreased
photochemical capacity, which suggests it as a photo-
protective feature (Kyparissis et al. 1995). In the tussock
grass S. tenacissima, which inhabits more arid environ-
ments than Phlomis, substantial loss of Chl is accompa-
nied by a large reduction in photochemical capacity and
a marked decrease in leaf water content, but leaves totallyrecover after autumn rainfalls. This has been interpreted
as a poikilohydric-type response allowing for a greater
tolerance to water shortage in the most extreme
Mediterranean environments (Balaguer et al. 2002).
Reduced light absorption through accumulation of red
carotenoids in leaf surfaces has also been recently
described as a particular photoprotective mechanism of
the evergreen shrub Buxus sempervirens (Hormaetxeet al. 2005). Mechanisms leading to reactive oxygen
scavenging and antioxidant protection have also been
described in Mediterranean plants, particularly in ever-
green and semideciduous shrubs. These mechanisms
include carotenoids (Munne-Bosch and Penuelas 2003),
isoprene (Affek and Yakir 2002), tocopherols (Munne-
Bosch and Penuelas 2004), diterpenes (Munne-Bosch
et al. 2001) and enzymatic antioxidants (Kyparissiset al. 1995).
Besides these mechanisms, thermal energy dissipation
in the pigment bed, associated with the so-called
xanthophyll cycle, is usually regarded as the most
important photoprotection mechanism in higher plants
(Demmig et al. 1987, Bjorkman and Demmig-Adams
1994). The first evidences that water stress increased de-
epoxidation of the xanthophyll cycle were in factdescribed in the Mediterranean evergreen sclerophylls
Nerium oleander (Demmig et al. 1988) and A. unedo
(Demmig-Adams et al. 1989). Since then, substantial
evidence has been accumulated for increased de-
epoxidation of the xanthophyll cycle during summer in
Mediterranean evergreens (Gulıas et al. 2002, Penuelas
et al. 2004), semideciduous (Munne-Bosch et al. 2003)
and perennial herbs (Balaguer et al. 2002). An increase inthe total xanthophyll pool during summer is also usually
observed (Garcıa-Plazaola et al. 1997, Faria et al. 1998),
although not in all the species (Munne-Bosch et al. 2003).
However, most of these studies have been focused on
evergreen shrubs and trees and semideciduous shrubs,
while much less information is available for semishrubs or
perennial herbs. On the other hand, most of these studies
have analyzed the variation of photoprotective mecha-nisms during the season. The short-term response (i.e.
days to weeks), which may be most relevant because of
the abundance of episodic drought periods in Mediterra-
nean areas, has been less evaluated, particularly in
relation to recovery after rewatering (RW). In the present
study, we assessed the relationship between the xantho-
phyll cycle and thermal dissipation and decreased
496 Physiol. Plant. 130, 2007
quantum efficiency of PSII during short-term water stress
and recovery in Mediterranean plants with different leaf
habits and growth forms. The objectives were (1) to study
how photoprotection responds to water stress in Medi-
terranean plants differing in growth forms, and (2) to
investigate the variability in the recovery of photo-protection and photoinhibition after RW.
Materials and methods
Plant material
Ten Mediterranean species naturally occurring in the
Balearic Islands, five of them endemic to these islands,were selected for this study (Table 1). Special care was
taken in the selection of the species, in order to include
taxons representing different growth forms and leaf
habits: two evergreen sclerophyll shrubs (Pistacia lentis-
cus and Hypericum balearicum), two evergreen sclero-
phyll semishrubs (Limonium gibertii and Limonium
magallufianum), three summer semideciduous shrubs
(Lavatera maritima, Phlomis italica and C. albidus), twoperennial herbs (Beta maritima ssp. maritima and Beta
maritima ssp. marcosii) and an annual herb (Diplotaxis
ibicensis). Seeds of each species were collected in the
field from natural populations and taken from several
parent plants to obtain a representative sample of
populations in the nature. Seeds were germinated on
filter paper moistened with deionized water in a con-
trolled environment (germination chamber, at 18�C indarkness). After germination and emergence of one true
leaf, 10 seedlings were transplanted into pots (25 l
volume, 40 cm height) containing a 40:40:20 mixture of
clay-calcareous soil, horticultural substrate (peat) and
pearlite (granulometry A13). Plants were grown outdoors
at the University of the Balearic Islands (Mallorca, Spain).
The experiment was performed in five rounds, each one
with one couple of species at the same time during thelate spring – early summer 2003 and 2004. Four weeks
before starting the experiment, plants were placed in
a controlled growth chamber with a 12-h photoperiod
(26�C day/20�C night) and a photon flux density at the top
of the leaves of about 600 mmol m22 s21. Plants were
daily irrigated with 50% Hoagland’s solution.
Measurements corresponding to control treatments
were made during the first day of the experiment, when allthe plants were well watered. Thereafter, irrigation was
stopped in five plants for each species. Pots were daily
weighed to determine the amount of water available for
plants with respect to that in control plants. Measure-
ments were made on days 4, 8 and 13–17 after water
withholding, when plants were subjected to mild water
stress (MiWS), moderate water stress (MoWS) and severe
water stress (SeWS) intensities, respectively. Each exper-
iment was stopped when the stomatal conductance (gs)
was close to zero. At this time, pots were irrigated at field
capacity, and measured the following day, considering it
the RW treatment. Control plants (C) were watered daily
to field capacity throughout the experiment and mea-sured every 5–6 days to ensure that they maintained
constant values of each parameter during the experiment.
Plant water status
Relative water content at predawn (RWCPD) was deter-
mined as follows: RWC ¼ (FW 2 DW)/(turgid weight 2
DW) � 100. Turgid weight was determined after placingthe samples in distilled water in darkness at 4�C to
minimize respiration losses, until they reached a constant
weight (full turgor, typically after 24 h). DW was obtained
after 48 h at 60�C in an oven. Four replicates per species
and treatment were obtained from different individuals.
Chl fluorescence measurements
Chl fluorescence parameters were measured on attached
leaves using a portable pulse amplitude modulation
fluorometer (PAM-2000, Walz, Effeltrich, Germany). For
each sampling time, treatment and species, four measure-
ments were made on different plants.
A measuring light of about 0.5 mmol photon m22 s21
was set at a frequency of 600 Hz to determine, atpredawn, the background fluorescence signal (Fo), the
maximum fluorescence (Fm) and the maximum quantum
efficiency of PSII photochemistry (Fv/Fm ¼ (Fm 2 Fo)/Fm).
At midday, steady-state fluorescence signal (Fs) was
measured on the same leaves with a photon flux density
around 1500 mmol m22 s21. To obtain the steady-state
maximum fluorescence yield (Fm#), saturation pulses of
about 10 000 mmol photon m22 s21 and 0.8 s durationwere applied. The Stern–Volmer non-photochemical
quenching of Chl fluorescence (NPQ) at midday was
calculated using the expression NPQ ¼ (Fm 2 Fm#)/Fm#.The PSII photochemical efficiency (Genty et al. 1989) was
then calculated as
DF=Fm# ¼
�Fm
#2Fs
�=Fm
#
and used for the calculation of the linear electrontransport rate (ETR) according to Krall and Edwards
(1992):
ETR ¼ DF=Fm# � PPFD � a � b;
where PPFD is the photosynthetically active photon flux
density, a is the leaf absorptance and b is a factor that
Physiol. Plant. 130, 2007 497
assumes equal distribution of energy between the twophotosystems (the actual factor has been described to be
between 0.4 and 0.6; Laisk and Loreto 1996). Leaf
absorptances were determined for all 10 species in 10
replicates on leaves of well-irrigated plants with a spec-
troradiometer coupled to an integration sphere (Uni-
Spec, PP-Systems, Amesbury, MA). A value of 0.84 was
obtained for all species, except for C. albidus and
P. italica, which presented leaf absorptance values of0.74 and 0.77, respectively. Potential changes in leaf
absorptance with water stress were not assessed but,
because changes in Chl content were small or non-
significant depending on the species, they were assumed
to be negligible, inducing no important biases in the
calculations of ETR.
Gas exchange measurements
Light-saturated net CO2 assimilation rates (AN) and gs
were measured at midmorning in one attached, fully
developed young leaf of four plants per species and treat-
ment with a gas exchange system (Li-6400, Li-Cor Inc.,
Lincoln, NE). Environmental conditions in the chamber
used for leaf measurements consisted in a photosynthetic
photon flux density of 1500 mmol m22 s21, a vapor pres-sure deficit of 1.0–1.5 kPa, an air temperature of 25�C and
an ambient CO2 concentration (Ca) of 400 mmol mol air21.
After inducing steady-state photosynthesis, four
photosynthesis response curves to varying substomatal
CO2 concentration (Ci) were performed per species
and treatment, and used to determine the rate of
Table 1. List of species considered with their growth form, family and a brief description. The number of plants used was 10 per species, and the age
differed because of the different phenology of the species selected. Plants of Pistacia lentiscus, Hypericum balearicum, Cistus albidus, Phlomis italica and
Lavatera maritima were 3 years old, plants of Limonium magallufianum and Limonium gibertii were 1.5 years old and plants of Diplotaxis ibicensis, Beta
maritima ssp. marcosii and B. maritima ssp. maritima were 6 months old at the onset of the experiments.
Growth form Species Code Family Description
Herbs B. maritima L. ssp. marcosii
A Juan and MB Crespo
MC Chenopodiaceae Perennial herb. Endemic of the
Balearic Islands, inhabiting a few small
islets subjected to strong saline spray.
B. maritima L. ssp. maritima MT Chenopodiaceae Perennial herb inhabiting coastal ecosystems.
Widespread in Mediterranean and
temperate climates.
D. ibicensis Pau DI Brassicaceae Annual herb, endemic of the Balearic Islands
and inhabiting a few coastal locations.
Semideciduous shrubs L. maritima Gouan LA Malvaceae Semideciduous shrub up to 2 m, densely
covered by hairs. Inhabits in
coastal locations.
P. italica L. PI Labiatae Semideciduous shrub up to 1 m, densely
covered by hairs. Endemic of the
Balearic Islands. The biggest populations
are found 500 m above the sea level,
where they coexist with C. albidus.
C. albidus L. CA Cistaceae Semideciduous shrub up to 1 m. Commonly
found in the Mediterranean garigue.
Its leaves are densely covered by hairs.
Woody evergreen
shrubs
H. balearicum L. HB Guttiferae Woody evergreen shrub up to 2 m, endemic
of the Balearic Islands. The biggest
populations are found in the garigue
500 m above the sea level, where
competes with P. lentiscus.
P. lentiscus L. PL Anacardiaceae Woody evergreen shrub up to 5 m,
commonly found in the Mediterranean
garigue.
Woody evergreen
semishrubs
L. magallufianum L. Llorens LM Plumbaginaceae Woody evergreen semishrub, in cushion-like
rosettes. Endemic of the Balearic Islands,
inhabiting just in one coastal marsh
located in Magalluf, Mallorca.
L. gibertii (Sennen) Sennen LG Plumbaginaceae Woody evergreen semishrub, in cushion-like
rosettes. Occurring in West Mediterranean
rocky and sandy coastal areas.
498 Physiol. Plant. 130, 2007
non-photorespiratory CO2 evolution in the light (RL) on
the same treatment, as in Grassi and Magnani (2005).
Photorespiration estimations
From combined gas exchange and Chl fluorescence
measurements, the photorespiration rate (Pr) was calcu-
lated according to Valentini et al. (1995). In their model,
they assumed that all the reducing power generated by
the electron transport chain is used for photosynthesis and
photorespiration, and that Chl fluorescence gives a reli-able estimate of the quantum yield of electron transport.
Thus, Pr can be solved from data of AN, RL and ETR,
and from the known stoichiometries of electron use
in photorespiration, as follows (Valentini et al. 1995):
Pr ¼ 1/12 [ETR 2 4 (AN 1 RL)].
Pigment analyses
Immediately after Chl fluorescence measurements (at pre-
dawn and midday), discs were punched from leaves of the
same plants showing the same orientation as those used for
fluorescence measurements and submersed into liquid
nitrogen. Four samples per treatment and species weretaken from different plants (four leaves per sample). Pig-
ments were extracted by grinding leaf tissue in a mortar with
acetone in the presence of sodium ascorbate. Pigments
were identified and quantified by high-performance liquid
chromatography according to Abadıa and Abadıa (1993)
with modifications as described in Larbi et al. (2004). The
de-epoxidation state (DPS) of the xanthophylls cycle was
calculated as (Z 1 0.5A)/(V 1 Z 1 A), where Z iszeaxanthin, A is anteraxanthin and V is violaxanthin.
Statistical analysis
Simple linear regression coefficients were calculated
using SPSS 12.0 software package (Anon 1990). A set ofsimple ANOVA were made to compare the different species
and treatments. Differences between means were re-
vealed by Duncan analyses (P< 0.05) performed with the
SPSS 12.0 software package.
For each treatment, a cluster analysis and a principal
component analysis were performed using STATGRAPHICS
PLUS 5.1 software package (Manugistics 1998) in order to
group both the species and parameters analyzed in a fewmore comprehensive variables.
Results
Plant water status
Leaf RWCPD decreased as water stress intensified
(Table 2). Under optimal conditions, RWCPD ranged
from 80.2% for D. ibicensis to 94.8% for P. lentiscus.
Under SeWS, RWCPD ranged from 37.9% for P. italica
to 69.5% for L. magallufianum. gs strongly differed
among species and growth forms, approximately in
a 10-fold range (Table 2). Under well-watered con-
ditions, L. maritima showed the highest gs (1.022 molH2O m22 s21), while P. lentiscus had the lowest
(0.122 mol H2O m22 s21). gs decreased in all the species
to values between 0 and 0.06 mol H2O m22 s21 as water
limitation increased.
Pigment composition under waterstress and recovery
Under well-watered conditions, leaf Chl content ex-
pressed on an area basis at midday was found to be
higher in the two Limonium species, with 794.7 and
736.2 mmol m22 for L. magallufianum and L. gibertii,
respectively (Fig. 1), while C. albidus presented the
lowest values (281.1 mmol m22). Increasing water stress
treatment influences on Chl largely depended on the
species. In some species (the two Beta, L. maritima andC. albidus), Chl was increased under SeWS with respect
to control plants (P < 0.05). In addition to this diversity
in the species response to water limitation, a high
variability in the intensity and the timing of the Chl
evolution because of water stress was also found. For
instance, the two Beta species, which increased their
Chl as water stress intensified, presented a different
pattern: B. maritima ssp. marcosii increased Chl atMoWS, while B. maritima ssp. maritima at SeWS.
Twenty-four hours after refilling pots at field capacity,
Chl evolution also depended strongly on the species
(Fig. 1). Hence, four species maintained similar Chl at
RW treatment when compared with SeWS, and the
remaining six species decreased Chl after RW (P <
0.05). It is remarkable that in B. maritima ssp. maritima
and P. italica, decreases of Chl after RW resulted invalues significantly lower than those measured under
well-watered conditions (P < 0.05).
The sum of violaxanthin, antheraxanthin and zeaxan-
thin (VAZ) per unit leaf area at midday under well-
watered conditions ranged from 11.2 mmol m22 for
C. albidus to 50.3 mmol m22 for L. maritima (Fig. 1). VAZ
per unit leaf area was affected by water stress only in
C. albidus, being significantly increased (P < 0.05). Asoccurred with Chl, refilling water at field capacity after
SeWS resulted in a specific pattern strongly dependent on
species.
Under control conditions, lutein content at midday
expressed on a Chl basis was found to be more similar
among species than other pigments, ranging between 94
and 127 mmol mol21 Chl. Lutein content on a Chl basis
Physiol. Plant. 130, 2007 499
was unaffected or enhanced by increasing water stress
intensity (Fig. 2). D. ibicensis, P. italica, C. albidus and
L. gibertii presented an increase in lutein content under
SeWS when compared with well-watered plants. Other
carotenoids such as neoxanthin did not show any specific
trend of response to water stress (data not shown).
VAZ content expressed on a Chl basis increased underSeWS at midday only in C. albidus and L. gibertii (P <
0.05), while for the remaining species it was found to be
unaffected (Fig. 2). Again, the intensity and timing of the
VAZ/Chl evolution because of increasing water stress
were highly species dependent.
Fig. 3 shows the evolution of violaxanthin, anther-
axanthin and zeaxanthin at midday throughout the
water stress experiment. Among well-watered plants,the woody evergreen shrubs, with about 65% of VAZ
pigments being violaxanthin, presented the lowest per-
centage of violaxanthin with respect to total VAZ pool.
On the contrary, in all the remaining species violax-
anthin accounted for approximately 90% of total VAZ
pool in control plants, except for B. maritima ssp.
marcosii with an intermediate behavior. In all the species,
except the two Limonium, violaxanthin content de-
creased with decreasing water availability (P < 0.05),
with a proportional increase of zeaxanthin (Fig. 3).
Antheraxanthin was kept at almost constant concentra-
tion through the entire experiment, and significantincreases because of water stress were only observed in
D. ibicensis, L. maritima and L. gibertii (P < 0.05). After
RW, all the species except L. magallufianum increased
violaxanthin and decreased zeaxanthin back to control
values.
All these changes in xanthophylls composition induced
similar trends in the DPS of xanthophylls. Both at predawn
and midday, DPS was largely increased in parallel withincreasing water stress intensity (data not shown). How-
ever, large differences were obtained in DPS among
species. For instance, under SeWS midday de-epoxydation
state (DPSMD) ranged from 0.1 in L. magallufianum to more
than 0.6 in H. balearicum and B. maritima ssp. marcosii,
Table 2. RWCPD and gs for the 10 selected species under different treatments: control (C), MiWS, MoWS, SeWS and RW. Values are means� SE of four
replicates per species and treatment.
C MiWS MoWS SeWS RW
Beta maritima ssp. marcosii
RWCPD 85.1 � 3.8 87.3 � 2.1 78.0 � 1.7 51.1 � 5.7 86.7 � 2.0
gs 0.450 � 0.017 0.510 � 0.044 0.163 � 0.025 0.009 � 0.004 0.421 � 0.066
B. maritima ssp. maritima
RWCPD 83.0 � 0.9 80.2 � 4.1 79.0 � 2.5 51.0 � 3.5 84.7 � 1.9
gs 0.704 � 0.087 0.591 � 0.063 0.295 � 0.059 0.008 � 0.002 0.431 � 0.129
Diplotaxis ibicensis
RWCPD 80.2 � 1.3 70.4 � 1.4 67.5 � 3.2 62.3 � 7.2 79.9 � 2.8
gs 0.510 � 0.035 0.377 � 0.020 0.160 � 0.022 0.059 � 0.012 0.280 � 0.012
Lavatera maritima
RWCPD 86.6 � 2.3 82.2 � 2.5 73.4 � 3.5 54.8 � 5.5 80.6 � 3.2
gs 1.022 � 0.076 0.755 � 0.089 0.215 � 0.027 0.052 � 0.010 0.691 � 0.076
Phlomis italica
RWCPD 83.6 � 1.5 80.0 � 0.8 75.6 � 0.8 37.9 � 3.0 74.4 � 1.6
gs 0.357 � 0.041 0.281 � 0.062 0.065 � 0.012 0.016 � 0.001 0.111 � 0.025
Cistus albidus
RWCPD 85.7 � 4.3 88.3 � 4.3 79.5 � 4.0 46.0 � 6.1 70.2 � 1.5
gs 0.318 � 0.037 0.206 � 0.050 0.104 � 0.040 0.022 � 0.004 0.087 � 0.025
Hypericum balearicum
RWCPD 91.2 � 1.3 93.1 � 1.1 89.9 � 1.3 48.7 � 5.2 85.5 � 2.4
gs 0.330 � 0.025 0.299 � 0.011 0.150 � 0.026 0.023 � 0.004 0.045 � 0.005
Pistacia lentiscus
RWCPD 94.8 � 0.5 87.5 � 3.3 86.1 � 3.8 53.7 � 4.9 81.2 � 5.4
gs 0.122 � 0.020 0.110 � 0.015 0.075 � 0.012 0.014 � 0.002 0.021 � 0.004
Limonium magallufianum
RWCPD 88.9 � 0.5 85.8 � 2.0 80.3 � 2.3 69.5 � 1.6 87.0 � 2.7
gs 0.246 � 0.016 0.114 � 0.013 0.054 � 0.007 0.017 � 0.005 0.086 � 0.010
Limonium gibertii
RWCPD 90.6 � 0.9 88.3 � 1.9 77.6 � 0.8 66.8 � 5.1 87.7 � 4.2
gs 0.187 � 0.021 0.153 � 0.032 0.067 � 0.013 0.029 � 0.007 0.052 � 0.006
500 Physiol. Plant. 130, 2007
P. italica and H. balearicum. After RW, the extent of
recovery of DPSMD was highly species dependent, from no
recovery (D. ibicensis) to total recovery (L. gibertii).
Pigment composition at predawn (not shown) fol-
lowed a pattern similar to that observed at midday. In
fact, correlations among values measured at midday
and predawn were highly significant (P < 0.01) for all
the pigments considered in the study. As expected, for
all the species, VAZ pool was always in a highly
epoxidated state at predawn for most treatments. This
was because of a lower concentration of zeaxanthin at
expenses of an increase in the violaxanthin content,while the anteraxanthin content did not change
10
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Chl
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Chl
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600
700
800
VA
Z (
µmol
m–2
) V
AZ
(µm
ol m
–2)
VA
Z (
µmol
m–2
) V
AZ
(µm
ol m
–2)
VA
Z (
µmol
m–2
)
10
20
30
40
50
60
Chl
a +
b (
µmol
m–2
)
300
400
500
600
700
800
10
20
30
40
50
60
Treatment
C
MiW
S
MoW
S
SeW
S
RW
Chl
a +
b (
µmol
m–2
)
300
400
500
600
700
800
Treatment
C
MiW
S
MoW
S
SeW
S
RW
10
20
30
40
50
60
B. maritima ssp. marcosiiHE
B. maritima ssp. maritimaHE
D. ibicensisHE
L. maritimaSDS
P. italicaSDS
C. albidusSDS
H. balearicumWES
P. lentiscusWES
L. magallufianumWESS
L. gibertiiWESS
a aab
bc c aabab
bab
ab b bc
c
aaa
aa
aa
ab
b
a
a
a
ababb
aa
aa
aa
b
babab
a
ab
b bb
aaa
aab
bbb
aa
abb
b b
aa
aaa
a aab
ab b
a ab
b
aba
a a a a
aa a a
a
a
aabb
a
a
aa
aa
aa
aa
a
Fig. 1. Total Chl content (Chl a 1 b, d) and the sum of violaxanthin,
antheraxanthin and zeaxanthin (VAZ, s), expressed in mmol m22, at
midday under different treatments: control (C), MiWS, MoWS, SeWS and
RW. Values are means � SE of four replicates per species and treatment.
Different letters denote statistical differences by Duncan test (P < 0.05)
among treatments for each parameter. Growth form abbreviations:
HE, herbs; SDS, semideciduous shrubs; WES, woody evergreen shrubs;
WESS, woody evergreen semishrubs.
Fig. 2. Lutein (d) and the VAZ (s) at midday, expressed in mmol mol21
Chl, at midday under different treatments: control (C), MiWS, MoWS,
SeWS and RW. Values are means � SE of four replicates per species
and treatment. Different letters denote statistical differences by Duncan
test (P < 0.05) among treatments for each parameter. Growth form
abbreviations: HE, herbs; SDS, semideciduous shrubs; WES, woody
evergreen shrubs; WESS, woody evergreen semishrubs.
Physiol. Plant. 130, 2007 501
significantly. Finally, variations of Chl and VAZ con-
centrations from predawn to midday, when occurred,
were species specific and no-general pattern was
observed.
Photoprotection and photoinhibitionunder water stress and recovery
As water stress intensified, Pr was kept at control values or
increased, depending on the species, and only at MoWS
to SeWS did photorespiration decline (Fig. 4). NPQ
0
20
40
60
80
V, A
, Z (
mm
ol m
ol–1
Chl
)V
, A, Z
(m
mol
mol
–1 C
hl)
V, A
, Z (
mm
ol m
ol–1
Chl
)V
, A, Z
(m
mol
mol
–1 C
hl)
V, A
, Z (
mm
ol m
ol–1
Chl
)
V, A
, Z (
mm
ol m
ol–1
Chl
)V
, A, Z
(m
mol
mol
–1 C
hl)
V, A
, Z (
mm
ol m
ol–1
Chl
)V
, A, Z
(m
mol
mol
–1 C
hl)
V, A
, Z (
mm
ol m
ol–1
Chl
)
0
20
40
60
80
0
20
40
60
80
0
20
40
60
80
B. maritima ssp. marcosiiHE
B. maritima ssp. maritimaHE
D. ibicensisHE
L. maritimaSDS
0
20
40
60
80
0
20
40
60
80
0
20
40
60
80
0
20
40
60
80
Treatment
C
MiW
S
MoW
S
SeW
S
RW
0
20
40
60
80
Treatment
C
MiW
S
MoW
S
SeW
S
RW
0
20
40
60
80
P. italicaSDS
C. albidusSDS
H. balearicumWES
P. lentiscusWES
L. magallufianumWESS
L. gibertiiWESS
a a
bb
c
aaa
aa
aab
bc
c
ab
aab
bc
cdd
a
b
abaa
b
b
b
aa
cc
baa
a
bb
b
b
ccb
aab
a
bc
b
bc
bab
bca
d
abcbca
a
d
b
aaab
b
ab
cc c
bbaa
a
cbbc
b
c b
abaa
b babaa
a
c
b
cabc
a
b
aa
ab
c
abab b
a
a
b
bcc
c
b ba
abab
c
b
ababa
a
aa
aa
a baaa
a
b
aaa
a
b
aaa
c
ababa
c abc
aba
Fig. 3. Violaxanthin (V, d), antheraxanthin (A, s) and zeaxanthin (Z, ;)
at midday, expressed in mmol mol21 Chl, under different treatments:
control (C), MiWS, MoWS, SeWS and RW. Values are means � SE of
four replicates per species and treatment. Different letters denote
statistical differences by Duncan test (P < 0.05) among treatments
for each pigment. Growth form abbreviations: HE, herbs; SDS, semi-
deciduous shrubs; WES, woody evergreen shrubs; WESS, woody
evergreen semishrubs.
Pr (
% c
ontr
ol)
Pr (
% c
ontr
ol)
Pr (
% c
ontr
ol)
Pr (
% c
ontr
ol)
Pr (
% c
ontr
ol)
20406080
100120140160
NP
Q
1.0
1.5
2.0
2.5
3.0
3.5
20406080
100120140160
NP
Q
1.0
1.5
2.0
2.5
3.0
3.5
Treatment
C
MiW
S
MoW
S
SeW
S
RW
20406080
100120140160
Treatment
C
MiW
S
MoW
S
SeW
S
RW
NP
Q
1.0
1.5
2.0
2.5
3.0
3.5
20406080
100120140160
NP
Q
1.0
1.5
2.0
2.5
3.0
3.5
20406080
100120140160
NP
Q
1.0
1.5
2.0
2.5
3.0
3.5
B. maritima ssp. marcosiiHE
B. maritima ssp. maritimaHE
D. ibicensisHE
L. maritimaSDS
P. italicaSDS
C. albidusSDS
H. balearicumWES
P. lentiscusWES
L. magallufianumWESS
L. gibertiiWESS
a a
b
bc c
a
bb
b
c
a
b bb
c
a a aa
b
ab b
cc
c
c
b
a a
a
bb
b
ca
ab
a
b
a
b
b
bcc
aa
cd
bc
d
ab
b
b b
c
a
a
b
bcb
a
a
b
a
b
a
b
b
b
c
a
b
b
a
b
a
bb
a
bb
abab ab
b
a
b
a
abb
aa
c
bb
d
a
c
bcab
d
Fig. 4. Pr (expressed as % in respect to control treatment values, d) and
midday NPQ (s) under different treatments: control (C), MiWS, MoWS,
SeWS and RW. Values are means � SE of four replicates per species
and treatment. Different letters denote statistical differences by Duncan
test (P < 0.05) among treatments for each parameter. Growth form
abbreviations: HE, herbs; SDS, semideciduous shrubs; WES, woody
evergreen shrubs; WESS, woody evergreen semishrubs.
502 Physiol. Plant. 130, 2007
increased progressively as water stress intensified, par-
ticularly at MoWS to SeWS (Fig. 4), while it was
negatively correlated with Pr (r ¼ 20.476, P < 0.01).
Both Pr and NPQ correlated with soil water availability
(r ¼ 0.386, P < 0.01, and r ¼ 20.591, P < 0.01,
respectively). The maximum NPQ values reached understress were similar in all species (between 2.5 and 3.5).
After RW, all the species except C. albidus showed some
relaxation of NPQ, but only in B. maritima ssp. maritima,
H. balearicum and P. lentiscus the relaxation was
complete (Fig. 4).
By contrast, the Fv/Fm, measured at predawn, followed
a pattern that differed among species (Fig. 5). The lowest
values of maximum Fv/Fm were generally higher than0.75, and only in P. lentiscus decreased to 0.60. All the
species except L. magallufianum progressively decreased
Fv/Fm as water stress intensified and presented signi-
ficantly lower values at SeWS with respect to control
(P < 0.05). The semideciduous shrubs L. maritima and
P. italica showed significant decreases at MoWS (P <
0.05). In the other species, Fv/Fm was maintained at
control values or declined only slightly (and often non-significantly) from MiWS to MoWS and declined to
some extent at SeWS. The only exception was
L. magallufianum, which maintained high values through
the entire experiment. A large variability was also
observed in the recovery of Fv/Fm after RW, from total
(L. maritima) or near total (H. balearicum, C. albidus) to
almost no recovery (D. ibicensis, P. lentiscus, B. maritima
ssp. marcosii). Finally, in four species (B. maritima ssp.maritima, the two Limonium and P. italica), there was
some decline of Fv/Fm after RW.
In well-watered plants, the rate of linear ETR ranged
from 294 mmol e2 m22 s21 for L. maritima to 122 mmol
e2 m22 s21 C. albidus (Fig. 5). ETR decreased signifi-
cantly in all species because of water stress imposition,
with the lowest values observed under SeWS, where
P. lentiscus presented the lowest ETR, with 35 mmol e2 m22
s21, andL.maritima thehighest,with167mmole2 m22 s21.
After RW all species increased ETR, but the extent of such
recovery was a species-specific response and did not
depend on the extent of previous decrease in ETR.
Discussion
Pigment composition under waterstress and recovery
Although Chl loss has been considered a negative con-
sequence of stress, decreased Chl content has also beendescribed in some Mediterranean species as a regulatory
mechanism to reduce the amount of photons absorbed
by leaves, conferring some degree of photoprotection
under drought (Balaguer et al. 2002, Kyparissis et al.2000, Munne-Bosch and Alegre 2000). However, in the
present study, leaf Chl content was generally unaffected
by water stress, with a few exceptions (Fig. 1). This
suggests that adjusting Chl may not be a major photo-
protective response to short-term water stress or that the
presence of such response is a strong species-dependent
Fv/
Fm
Fv/
Fm
Fv/
Fm
Fv/
Fm
Fv/
Fm
0.55
0.60
0.65
0.70
0.75
0.80
0.85
50
100
150
200
250
300
0.55
0.60
0.65
0.70
0.75
0.80
0.85
50
100
150
200
250
300
Treatment
C
MiW
S
MoW
S
SeW
S
RW
0.55
0.60
0.65
0.70
0.75
0.80
0.85
Treatment
C
MiW
S
MoW
S
SeW
S
RW
ET
R (
µmol
e-m
–2 s
–1)
ET
R (
µmol
e-m
–2 s
–1)
ET
R (
µmol
e-m
–2 s
–1)
ET
R (
µmol
e-m
–2 s
–1)
ET
R (
µmol
e-m
–2 s
–1)
50
100
150
200
250
300
0.55
0.60
0.65
0.70
0.75
0.80
0.85
50
100
150
200
250
300
0.55
0.60
0.65
0.70
0.75
0.80
0.85
50
100
150
200
250
300
B. maritima ssp. marcosiiHE
B. maritima ssp. maritimaHE
D. ibicensisHE
L. maritimaSDS
P. italicaSDS
C. albidusSDS
H. balearicumWES
P. lentiscusWES
L. magallufianumWESS
L. gibertiiWESS
ab
b
b
c
a aabbb
ab
bb
c abbcbc c
aa
b
aaa a
bbb
a
b
bbb
aab
bc
bcc
a
b bcc
aabbcc
aa a
b b
aab ababb
a
bbb
c
a abbc c c
a a
b b b a a
bbb
aabab
bb
aabababb
aab
b
cc
aab
bc
cc
Fig. 5. Fv/Fm of PSII measured at predawn (d) and the linear ETR (s)
under different treatments: control (C), MiWS, MoWS, SeWS and RW.
Values are means � SE of four replicates per species and treatment.
Different letters denote statistical differences by Duncan test (P < 0.05)
among treatments for each parameter. Growth form abbreviations:
HE, herbs; SDS, semideciduous shrubs; WES, woody evergreen shrubs;
WESS, woody evergreen semishrubs.
Physiol. Plant. 130, 2007 503
feature. This fact seems to be in accordance with previous
studies that questioned the role of changes in Chl content
in regulating light interception (Bjorkman and Demmig-
Adams 1987).
The VAZ per unit leaf area has been shown to increase
(Garcıa-Plazaola et al. 1997), remain constant (Kyparissiset al. 1995, Munne-Bosch et al. 2003) or even decrease
(Balaguer et al. 2002, Munne-Bosch and Penuelas 2003)
in different Mediterranean species from spring to late
summer. Our results seem to support those works
suggesting that VAZ per unit leaf area is not significantly
affected by water stress (Fig. 1), except in C. albidus.
Furthermore, in accordance with previous reports in
Mediterranean plants (Martınez-Ferri et al. 2000) andtropical evergreens (Demmig-Adams and Adams 2006),
no inverse relationship between VAZ size and photosyn-
thetic capacity among species was found. Increasing VAZ
pool per Chl may be another mechanism to increase
photoprotection capacity in leaves, and it has been
shown to generally occur during summer in both
evergreen sclerophylls and semideciduous shrubs (Faria
et al. 1998, Kyparissis et al. 1995, 2000). In short-termwater stress experiments, VAZ/Chl ratio has been shown
to increase in the evergreen sclerophylls A. unedo
(Munne-Bosch and Penuelas 2004) and Phillyrea angus-
tifolia (Munne-Bosch and Penuelas 2003, Penuelas et al.
2004). In the present study, none of the two woody
evergreen shrubs analyzed (P. lentiscus and H. balear-
icum) presented such an increase (Fig. 2), suggesting that
this may not be a specific feature of evergreen sclerophyllspecies. An increased VAZ/Chl ratio in response to water
stress, accompanied by increases in the lutein content,
was observed only in three of the species analyzed
(Fig. 2), an evergreen semishrub (L. gibertii) and two
semideciduous shrubs (C. albidus and P. italica).
Therefore, in the species analyzed neither Chl nor total
VAZ pool adjustments seemed to be important responses
to short-term water stress. However, changes in thede-epoxydation state of the xanthophylls cycle were
a general fate in all the species (Fig. 3). In general, water
stress activated the de-epoxidation of violaxanthin to
zeaxanthin, while antheraxanthin was kept at more
constant concentration through the entire experiment
(Fig. 3). Zeaxanthin formation is related to the dissipation
of excess absorbed light as heat, as indicated by the strong
correlations found between the DPS of the xanthophyllscycle and NPQ (as discussed in the next section).
Photoprotection and photoinhibition underwater stress and recovery
When photosynthesis progressively declines with
drought, photorespiration and thermal energy dissipation
are regarded as the most important photoprotective
mechanisms leading to dissipation of excess absorbed
light (Powles and Osmond 1978, Demmig et al. 1988,
Flexas and Medrano 2002). The importance of alternative
electron sinks, such as the Mehler-ascorbate peroxidase
reaction, has been shown to be minor both under well-watered conditions and drought in other Mediterranean
species (Flexas and Medrano 2002). In the present study,
photorespiration declined when water stress became
MoWS to SeWS, concomitantly with increases in the
NPQ, an indicator of thermal energy dissipation in the
pigment bed (Bjorkman and Demmig-Adams 1994)
(Fig. 4). These results demonstrate that, as already shown
for some Mediterranean evergreen sclerophylls (Garcıa-Plazaola et al. 1997, Gulıas et al. 2002), maintaining
photorespiration and increasing NPQ are common
responses to water stress in other Mediterranean species,
including herbs and semideciduous shrubs. Remarkably,
most of the species increased the photorespiratory oxygen
metabolism from control to MiWS, in accordance with
Flexas and Medrano (2002). Bjorkman and Demmig-
Adams (1994) already pointed that the rate of photores-piration and hence its contribution to energy dissipation
would be expected to be greater in temporarily water-
stressed leaves in which the intrinsic photosynthetic
capacity remained unchanged and the decline in AN is
mainly because of a decrease in intercellular CO2
pressure, as occurred in the present study (data not
shown). Role of the photorespiratory oxygen metabolism
in photoprotection has been largely described (Flexas andMendrano 2002, Niyogi 1999). Remarkably, the only
species that did not present the initial increase at MiWS
was L. gibertii, which has been shown to present the
highest value of Rubisco specificity for CO2 up to now
described in higher plants (Galmes et al. 2005).
As usually described in Mediterranean (Damesin and
Rambal 1995, Valladares et al. 2005) as well as in non-
Mediterranean species (Flexas et al. 2004), decreasesin Fv/Fm were generally small (except for P. lentiscus)
although significant, indicating only a minor photo-
inhibitory effect of water stress. Although the maximum
extent of Fv/Fm decline did not differ among growth forms,
there was a certain effect of growth form in the pattern of
Fv/Fm response to water stress because the semideciduous
species declined Fv/Fm progressively from early stages of
stress, while in other groups it declined only at SeWS. Thiswater stress-induced decline in Fv/Fm was not accompa-
nied by decreased Chl content (Fig. 1), and hence may
not be associated with the photoprotective mechanism
consisting of decreasing light absorption, as described for
P. fruticosa or S. tenacissima (Balaguer et al. 2002,
Kyparissis et al. 1995). Although the semideciduous spe-
cies included in this study provide several morphological
504 Physiol. Plant. 130, 2007
adaptations, e.g. leaf trichomes, high leaf reflectance,
these were not sufficient to prevent some decrease of Fv/
Fm in plants subjected to stress. Different mechanisms
may operate to decrease Fv/Fm, depending on the species.
For instance, in L. maritima, decreasing Fv/Fm was
paralleled by a progressive increase of Fo (data notshown), therefore suggesting progressive photoinactiva-
tion of PSII centers from early water stress stages. By
contrast, Fo progressively declined in P. italica and
C. albidus, suggesting that decreased Fv/Fm was reflecting
sustained photoprotection in these two species. Alterna-
tively, decreased Fv/Fm could be reflecting other causes,
such as changes in PSI fluorescence, uncoupling of
external antennae, etc. Whatever the reason, decreases inFv/Fm were low and did not limit ETR (Fig. 5).
The extent of recovery of Fv/Fm was not dependent
on plant growth form. For instance, among the
evergreens, recovery was null in P. lentiscus while
almost complete in H. balearicum. Also, the extent of
recovery did not depend on the maximum extent of
Fv/Fm depression achieved during water stress. For
instance, recovery was complete in L. maritima andnull in D. ibicensis, while both species had achieved
a similar Fv/Fm. In four species (B. maritima ssp.
maritima, the two Limonium and P. italica), there was
some decline of Fv/Fm after RW, which in three of
them was accompanied by decreased chlorophyll but
not xanthophyll content (Fig. 1). This has been already
observed after water stress in Vigna unguiculata (De
Souza et al. 2004) and after photoinhibitory experi-ments in different species (J. Flexas, J. Galmes, and
H. Medrano, Universitat de les Illes Balears, Palma,
unpublished data). Probably, this effect may be related
to some membrane damage caused by RW, or it may
be because of the fact that recovery of PSII after
photoinhibition requires degradation and de novo syn-
thesis of damaged components, particularly D1 pro-
tein (Aro et al. 1994). Assuming that this is a generalprocess that may occur in all species, differences in
the observed behavior of Fv/Fm at a fixed time (24 h)
after RW may reflect interspecific differences in the
velocity with which they can recover PSII. In this sense,
L. maritima would be the species with the fastest
capacity for recovery, followed by H. balearicum and
C. albidus. The four species showing a decline of Fv/Fm
during recovery measurements may have an intermedi-ate speed, having already started the process 24 h after
RW, while those showing no change (D. ibicensis,
P. lentiscus, B. maritima ssp. marcosii) may be re-
garded as having the lowest capacity and/or velocity
for recovery. It is remarkable that also in species
showing no water stress-induced decline of Fv/Fm, such
as L. magallufianum, Fv/Fm was decreased after RW.
General pattern of photoprotective responses tostomatal closure
Despite the observed differences between species in
photoprotection and photoinhibition response to water
stress, they all respond to a general pattern described for
C3 plants when gs is used as a reference for water stress
intensity (Flexas and Medrano 2002, Flexas et al. 2004).
This pattern is characterized by two phases of response to
water stress, a first phase corresponding to gs declining
from a maximum to about 0.15–0.20 mol H2O m22 s21,and a second phase corresponding to further decreases in
gs (Fig. 6). During the first phase, photorespiration acts as
a major photoprotective mechanism, being kept at
control values or even increased (Fig. 6A), while NPQ
increases only slightly (Fig. 6B) and Fv/Fm is maintained
above 0.8 (Fig. 6C), indicating little or no photoinhi-
bition. During the second phase, photorespiration
decreases (Fig. 6A) and NPQ largely increases (Fig. 6B),suggesting that thermal dissipation becomes the major
photoprotective mechanism during this phase. In this
phase, decreases of Fv/Fm eventually occur, to an extent
that largely differs among species (Fig. 6C). The decline of
Fv/Fm under severe water stress may be understood as
a consequence of water stress-induced photosynthesis
decline rather than its cause because ETR (Fig. 5) and net
photosynthesis (not shown) start to decline well beforeFv/Fm and achieve much larger reductions. Another
indication that water stress-induced variations in Fv/Fm
did not limit photosynthesis comes from the fact that
despite Fv/Fm was further reduced after RW in four
species, ETR and net photosynthesis simultaneously
recovered by about 50% in these species, as well as in
those showing no Fv/Fm recovery except P. lentiscus
(Fig. 5 and data not shown). These results are consistentwith the fact that leaves present a much larger PSII
concentration than needed for photosynthesis, so that
up to 50% of PSII units can be damaged before any effect
is detectable in photosynthesis (Lee et al. 1999).
Relationship between thermal dissipation andpigment composition
Highly significant correlations were found between
DPSMD and NPQ, which suggest that a large part of the
NPQ is related to DPS in Mediterranean species (Fig. 7).However, the slope and kinetics of such relationship
strongly differed among species, suggesting species-
dependent additional roles of de-epoxidated xantho-
phylls (Demming-Adams and Adams 2006). PSII
efficiency did not return to optimum values before dawn
under SeWS in any species. However, the significant
correlations observed between predawn de-epoxidation
Physiol. Plant. 130, 2007 505
state (DPSPD) and Fv/Fm before dawn for most of the
species (Fig. 8) suggest that the low PSII was probably the
result of sustained increases in the DPS of the xanthophyll
cycle rather than a result of photoinhibitory damage to the
leaves. This may reflect one of the two forms of thezeaxanthin-related sustained photoprotection forms,
described in evergreen species (Demming-Adams and
Adams 2006). Remarkably, the present results suggestthat this form of sustained photoprotection may also
occur in some Mediterranean semideciduous shrubs,
perennials and annuals. As already shown for the
relationship between NPQ and DPSMD, the slope of the
relationship between Fv/Fm and DPSPD was also depen-
dent on the species, with no apparent relation to species
growth forms (Fig. 8). It is also worth nothing that in both
Fig. 6. Relationship between gs and (A) Pr (expressed as % in respect
to control treatment values), (B) midday NPQ and (C) Fv/Fm. Values are
means � SE of four replicates per species and treatment. RW values were
not included. Symbols and species are as follows: d, Diplotaxis ibicensis;
s, Beta maritima ssp. marcosii; n, Beta maritima ssp. maritima; h,
Limonium magallufianum; :, Limonium gibertii; n, Phlomis italica; ;,
Lavatera maritima; ,, Cistus albidus; ¤, Pistacia lentiscus; ), Hypericum
balearicum.
DPSMDDPSMD
0.2 0.4 0.6 0.8 0.2 0.4 0.6 0.8
1.0
1.5
2.0
2.5
3.0
3.5
1.0
1.5
2.0
2.5
3.0
3.5
1.0
1.5
2.0
2.5
3.0
3.5
1.0
1.5
2.0
2.5
3.0
3.5
NP
QN
PQ
NP
QN
PQ
NP
Q
NP
QN
PQ
NP
QN
PQ
NP
Q
1.0
1.5
2.0
2.5
3.0
3.5
1.0
1.5
2.0
2.5
3.0
3.5
1.0
1.5
2.0
2.5
3.0
3.5
1.0
1.5
2.0
2.5
3.0
3.5
1.0
1.5
2.0
2.5
3.0
3.5
1.0
1.5
2.0
2.5
3.0
3.5
B. vulgaris ssp. marcosiir2 = 0.941
B. vulgaris ssp. maritimar2 = 0.549
D. ibicensisr2 = 0.672
L. maritimar2 = 0.990
P. italicar2 = 0.903
C. albidusr2 = 0.971
H. balearicumr2 = 0.718
P. lentiscusr2 = 0.051
L. magallufianumr2 = 0.925
L. gibertiir2 = 0.980
Fig. 7. Relationship between the midday NPQ and the DPSMD for the five
treatments studied. Regression coefficients are shown for each one of the
species. Measurements corresponding to RW treatment are indicated by
s and were not considered for the regression adjustment. Values are
means � SE of four replicates per species and treatment.
506 Physiol. Plant. 130, 2007
relationships, the value after RW does not always fit the
same regression line described for the remaining treat-
ments, a phenomenon that may deserve further attention
in future studies.
Differences in photoprotection among growthforms and evolutionary groups
Five of the 10 species included in the present work were
endemic of the Balearic Islands. The geographically
limited distribution of the endemic species may be because
of underlying negative ecophysiological traits that impede
these species becoming more widespread. In other insular
systems with a high percentage of endemicity, native
species have been shown to exhibit greater photoinhibition
and lower performance than invasive species whensubjected to high light levels (Durand and Goldstein
2001, Yamashita et al. 2000). Although no previous
attempts exist in comparing photoinhibition and photo-
protective strategies between endemic and non-endemic
species in the Mediterranean basin, Gulıas et al. (2003)
showed that endemic species presented in general a 20%
lowerphotosyntheticcapacity when comparedwith widely
distributed species. In the present work, a set of ANOVA’s wasmade to investigate possible differences in photoprotection
mechanisms and photoinhibition processes between
endemic and non-endemic species. Differences were
examined for each parameter analyzed and considering
all treatments and sampling time (i.e. predawn and
midday). Nevertheless, differences between both evolu-
tionary groups were only significant (P< 0.05) for lutein, b-
carotene and total xanthophyll content under MoWS atmidday. Therefore, at least for the species included in the
present survey, differences in the photoprotection mecha-
nisms are not the main cause that limits distribution and
competitiveness of endemic species in the Balearic Islands.
A series of cluster analysis of the species considered in
the present survey was performed for each treatment (data
not shown). Such analysis, which included the main
parameters measured at midday, reflects the existence ofa continuum of behavior in response to water stress that is
independent of growth form. Effectively, in any of the
treatments, no clear grouping of species according to
their growth form was observed.
Once it has been shown that no important differences
among growth forms could be established, the next step
was to summarize the general variance in few compo-
nents. In this way, the principal component analysis of thedifferent variables measured at midday distinguished
three main groups explaining approximately 70% of total
variance. The first component (approximately 40% of
total variance) was formed by all pigments except those
representing de-epoxidated states, i.e. carotenes, chlor-
ophylls, lutein epoxide, neoxanthin and violaxanthin
content. The second component (approximately 20% of
total variance) was formed by pigments and indexesreflecting the de-epoxidated stated of the xanthophylls
cycle (i.e. DPS, antheraxanthin and zeaxanthin) and
treatment. Finally, the third component (approximately
10% of total variance) was formed by ETR, NPQ and Pr.
The remaining components, which included species,
growth form and evolutionary history, accounted for less
than 7% of total variance.
0.1 0.2 0.3 0.4
0.720.740.760.780.800.820.84
DPSPD DPSPD
0.1 0.2 0.3 0.4
0.720.740.760.780.800.820.84
0.55
0.60
0.65
0.70
0.75
0.80
0.720.740.760.780.800.820.84
0.720.740.760.780.800.82
0.84
0.720.740.760.780.800.820.84
0.720.740.760.780.800.820.84
0.720.740.760.780.800.820.84
0.720.740.760.780.800.820.84
Fv/
Fm
Fv/
Fm
Fv/
Fm
Fv/
Fm
Fv/
Fm
Fv/
Fm
Fv/
Fm
Fv/
Fm
Fv/
Fm
Fv/
Fm
0.720.740.760.780.800.820.84
B. maritima ssp. marcosiir2 = 0.323
B. maritima ssp. maritimar2 = 0.776
D. ibicensisr2 = 0.887
L. maritimar2 = 0.560
P. italicar2 = 0.584
C. albidusr2 = 0.565
H. balearicumr2 = 0.611
P. lentiscusr2 = 0.937
L. magallufianumr2 = 0.268
L. gibertiir2 = 0.970
Fig. 8. Relationship between the Fv/Fm measured at predawn and DPSPD
of the xanthophyll cycle, for the five treatments studied. Regression
coefficients are shown for each one of the species. Measurements
corresponding to RW treatment are indicated by s and were not
considered for the regression adjustment. Values are means � SE of four
replicates per species and treatment. Note the different y-axis scale for
Pistacia lentiscus.
Physiol. Plant. 130, 2007 507
To further confirm the no existence of clear differences
among growth forms, species were plotted against the
two main principal components (Fig. 9). Such analysis, in
addition, allowed visualizing how the different species
are differentially distributed vs these principal compo-
nents, and how such distribution varied according to thewater stress treatments.
Concluding remarks
The present study shows that Mediterranean plants,
regardless of their growth form, are substantially resistant
to water stress-induced photoinhibition. However, al-
though all these species achieve photoprotection by
a combination of photochemical (photorespiration) and
non-photochemical (thermal dissipation) mechanisms,
the mechanisms and/or pigments involved in the latter
may differ among species, in a manner that is indepen-dent of the plant growth form. Similarly, the velocity of
PSII recovery from photoinhibition or sustained photo-
protection also differs among species.
These features may reflect adaptations to particular
environments and are in agreement with the different
species distribution. For instance, a very effective photo-
protection under water stress may be of adaptive valuesfor Limonium, C. albidus or P. italica because they all
inhabit sun-exposed areas, while being shallow-rooted
species that retain some green leaves in summer.
Therefore, they may have the capacity to respond to
frequent short episodes of drought in addition to the long
summer drought period. An alternative adaptation for
a species inhabiting similar areas, such as L. maritima,
may be to possess a high plasticity, which includes a highcapacity for rapid recovery. By contrast, the species
belonging to other growth form groups may have to
endure less frequent periods of combined drought and
high light intensity, the herbs because they do not retain
leaves during summer and the large woody perennials
because they use to live under the shade of adult plants
when young, and be deep-rooted when adult. Heteroge-
neity in the ecological performance of Mediterraneanspecies may be a consequence of the functional
complexity of Mediterranean ecosystems and likely
reflects the fact that any species in this environment has
to endure temporary drought periods, which has lead to
an array of different adaptive strategies.
Acknowledgements – Dr M Ribas-Carbo is acknowledged
for his helpful comments on a previous version of the
manuscript and grammatical corrections. This work was partly
funded by Projects REN2001-3506-CO2-O2 and BFU2005-
03102/BFI (Plan Nacional, Spain).
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