photoprotection processes under water stress and recovery in

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


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.


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


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


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

a +

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)

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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.


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



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. 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



D. ibicensisHE

L. maritimaSDS

P. italicaSDS

C. albidusSDS

H. balearicumWES

P. lentiscusWES


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.


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



D. ibicensisHE

L. maritimaSDS

P. italicaSDS

C. albidusSDS

H. balearicumWES

P. lentiscusWES


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.


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


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


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.


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.


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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–5 –4 –3 –2 –1 0 1 2 3 4 5

Com

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nt 2

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