- Email: [email protected]
Non-invasive tools to estimate stress-induced changes in photosynthetic performance in plants inhabiting Mediterranean areas
Accepted Manuscript Title: Non-invasive tools to estimate stress-induced changes in photosynthetic performance in plants inhabiting Mediterranean areas Author: Lucia Guidi Angeles Calatayud PII: DOI: Reference:
S0098-8472(13)00218-9 http://dx.doi.org/doi:10.1016/j.envexpbot.2013.12.007 EEB 2727
To appear in:
Environmental and Experimental Botany
Received date: Revised date: Accepted date:
29-5-2013 27-11-2013 9-12-2013
Please cite this article as: Guidi, L., Calatayud, A.,Non-invasive tools to estimate stress-induced changes in photosynthetic performance in plants inhabiting Mediterranean areas, Environmental and Experimental Botany (2013), http://dx.doi.org/10.1016/j.envexpbot.2013.12.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
RS
TR
T LAS OP R LO CH
ES
S
FLUORESCENCE
TEM PER • WIN ATURE T • SUM ER MER
us
HEAT
cr
WA TE
ip t
Graphical Abstract (for review)
chl a*
chl a*
XANTHOPHYLL CYCLE
2e-
PH
2e-
an
OT OC
HE
M
SALINITY
M
ele ct ro n ch tran ain sp or
IST
t
chl a
d
Ac ce pt e ITY HT ANT Y G I L • QU LIT UA •Q
NADPH
LHCI
PS I ATP
PS II H2O
CALVIN CYCLE
chl a
LHCII
TS UTAN POLL ONE S • OZ Y METAL V A E •H
NADP+ + H+
RY
2H+ + 1/2 O2
ROS H2O2 O2.OH.-
OXIDATIVE DAMAGE
GE
N HA
EC AT LIM
C
Page 1 of 53
*Research Highlights
ip t
cr
us an
M
d
te
Mediterranean environment is characterized by rainy winters and long hot summers, with high irradiance and little or no precipitation Chlorophyll fluorescence and gas exchange are non-invasive, rapid, and inexpensive techniques for measuring photosynthesis in leaves In the Mediterranean environment, plant species are continuously subjected to various abiotic stresses such as light intensity, drought, extreme temperature, air pollutants, etc. These environmental factors adversely affect plant development and damage to the photosystem is the first manifestation of plant response to abiotic stress. In the review, the effects of various abiotic stresses on the photochemistry of Mediterranean plant species are reported using Chl a fluorescence and gas exchange.
Ac ce p
Page 2 of 53
*Manuscript
Non-invasive tools to estimate stress-induced changes in photosynthetic performance in plants inhabiting Mediterranean areas Lucia Guidi1, Angeles Calatayud2
ip t
Department of Agriculture, Food and Environment, University of Pisa, Via del Borghetto 80 -
56124 Pisa (Italy) 2
Instituto Valenciano de Investigaciones Agrarias (IVIA), Department of Horticulture, Ctra.
us
Moncada-Naquera km. 4.5, 46113 Moncada, Valencia, Spain
cr
1
an
Corresponding author: Lucia Guidi, Department of Agriculture, Food and Environment,
d
502216630, Email: [email protected]
M
University of Pisa, Via del Borghetto 80 -56124 Pisa (Italy), Phone: +39 502216613, Fax: +39
Ac ce p
te
Contents 1. Abiotic stresses imposed by the Mediterranean climate measurements 2. What is chlorophyll a fluorescence? 2.1 Slow chlorophyll a fluorescence induction kinetic 2.2 Fast chlorophyll fluorescence or direct fluorescence: OJIP transient 2.3 Seeing is believing: Chlorophyll a fluorescence imaging 2.4 Relation between Chlorophyll a fluorescence and gas exchange: physiological implications 3. Main abiotic stresses in Mediterranean climate 3.1. High solar irradiance 3.2. Temperature stress 3.3. Water stress 3.4. Salinity 3.5. Ozone 3.6. Climate change 4. Conclusion Acknowledgments References
3 4 5 6 7 8 8 8 13 16 19 22 25 26 28 28
Abstract 1
Page 3 of 53
In Mediterranean areas, plants are concomitantly exposed to various abiotic stresses such as light intensity, water deficit, extremes in air temperature, air pollutants, etc. These environmental pressures adversely affect plant development. Changes in photosystem
ip t
activity are an early response of plants to abiotic stresses. Therefore, chlorophyll (Chl) fluorescence and gas exchange, two non-invasive, rapid and inexpensive techniques for
cr
measuring photosynthesis in leaves, have been widely used by plant ecophysiologists to
us
analyze plant responses to stressful conditions. Chl a fluorescence and gas exchange parameters can be indeed used to evaluate changes in photochemical and non-
an
photochemical processes in photosystems associated with electron transport, CO2 fixation, and heat dissipation.
M
In this review, we focus our analysis on the effects of different abiotic stresses on the
d
photochemistry of Mediterranean plants using Chl a fluorescence and gas exchange
te
measurements. Since changes in photosynthetic parameters are observed in the absence of visual injuries, these methodologies constitute fundamental tools to predict and evaluate the
Ac ce p
extent to which abiotic stresses damage photosynthesis.
Key words: abiotic stress, chlorophyll a fluorescence, climate change, gas exchange, oxidative stress, photosynthesis
Abbreviations: NO: non-regulated heat dissipation (a loss process due to PSII inactivity); NPQ: regulated heat dissipation (a protective loss process); PSII: actual quantum yield of PSII photochemistry; CFI: chlorophyll fluorescence imaging; Chl: chlorophyll; F0, Fv and Fm: 2
Page 4 of 53
minimum, variable and maximum Chl fluorescence in dark adapted conditions; F 0’ and Fm’: minimum and maximum Chl fluorescence in light conditions; Fs: Chl fluorescence in steady state conditions; Fv/Fm: maximum quantum yield of PSII photochemistry; g m: CO2 mesophyll
ip t
conductance; gs: water vapor stomatal conductance ; LHC: light harvesting complex; Lut: lutein; NPQ: non-photochemical quenching; PAM: pulse amplitude modulate; PQ:
cr
plastoquinone; PSI and PSII: photosystem I and II; QA: primary quinone acceptor of
us
photosystem II; qL: photochemical quenching coefficient in the lake model; qp and qN: photochemical and non-photochemical quenching coefficients; ROS: reactive oxygen species; ribulose-1,5-bisphosphate
carboxylase/oxygenase;
an
Rubisco:
VAZ:
violaxanthin,
M
antheraxanthin, zeaxanthin cycle; Zea: zeaxanthin
d
1. Abiotic stresses imposed by the Mediterranean climate
te
The Mediterranean area is characterized by rainy autumns-winters, and long warm summers, during which sunlight irradiance is high and water availability is low. Thus,
Ac ce p
environmental stresses Mediterranean plants face during summer season are mostly drought, heat and high sunlight irradiance (Sánchez-Gómez et al., 2006; Galmés et al., 2007). Mediterranean species may also suffer from anthropogenic factors, such as increased tropospheric ozone (O3) concentrations and high UV-B radiation (Krupa, 2000). The Mediterranean basin is also expected to be more strongly affected than other areas worldwide from by global climate change (IPCC, 2007). Common characteristics of Mediterranean plants are an evergreen and sclerophylls habitus. Sclerophylls consist of coriaceous leaves, with 2–3 well-packed palisade layers, thick both cuticle and cell wall, high density of small stomata (De Lillis, 1991). However, species 3
Page 5 of 53
that co-occur in the very same Mediterranean habitats greatly differ in their eco-physiological traits as well as for their responses (and tolerance) to different stresses (Flexas et al., 2013). In summary, the Mediterranean climate exposes plants to multiple and uncorrelated
serious challenge for plants inhabiting Mediterranean areas.
ip t
stresses, their effects being currently exacerbated by global change. This represents a
cr
Chl a fluorescence and gas exchange techniques are fundamental to analyze the effect
us
of changes in environmental conditions on photosynthetic performance in Mediterranean species, and, hence on the overall agro-forest system. Here we offer an overview of basic
an
knowledge and application of chlorophyll fluorescence-based techniques for ecophysiological
d
2. What is chlorophyll a fluorescence?
M
analyses.
te
When Chl absorbs light energy, it remains in an excited state (first excited singlet) for just a very short period (usually nanoseconds). Excitation energy can be lost from this excited
Ac ce p
singlet state through releasing heat and/or fluorescence of red- far-red light, or through energy transfer to an adjacent pigment by inductive resonance There are different techniques for measuring Chl a fluorescence. In this review Pulse Amplitude Modulate (PAM) Chl fluorescence technologist and its physiological implications are tackled.
2.1. Slow chlorophyll a fluorescence induction kinetic The slow Chl a fluorescence induction kinetics includes transitions of the photosynthetic apparatus from dark-adapted to light-adapted state. The sample is illuminated 4
Page 6 of 53
with a low-energy modulated light (< 10 µmol m -2s-1) and fluorescence increases to a background fluorescence value that represents the minimum fluorescence level, namely F 0. The illumination of the sample with a saturating pulse of light (< 10000 µmol m -2s-1) then
ip t
induces an increase of Chl fluorescence yield (Fm level or maximal fluorescence). The fluorescence then decreases to reach a steady-state level (Fs). Subsequently, saturation
cr
pulse during actinic light induction kinetics enables F0’ to be reached (turning off the actinic
us
light in the presence of far-red light) and Fm’ values that are respectively the minimum and maximum fluorescence yield in the light. The information obtained from Chl a fluorescence
an
induction kinetics can be decoded using a set of Chl a fluorescence parameters mostly defined over the last four decades.
M
The maximum quantum yield of PSII photochemistry [Fv/Fm = (Fm-F0)/Fm], is a useful
d
indicator of the leaf potential photosynthetic capacity (Kitajima and Butler, 1975)
te
Effective (or actual) quantum yield of photochemistry energy conversion in PSII [PSII = (Fm’-Fs)/Fm’]. This parameter measures the proportion of light absorbed by chlorophyll
Ac ce p
associated with PSII that is actually used in photochemistry (Maxwell and Johnson, 2000). PSII is closely related with the quantum yield of the non-cyclic electron transport rate (Genty et al., 1989).
Photochemical quenching of variable Chl fluorescence yield (qP or qL). According to the ‘puddle’ model (each PSII centre possesses its own independent antenna system), photochemical quenching is defined as qP = (Fm’-Fs)/(Fm’-F0’) (Schreiber et al., 1986). The qL coefficient is the photochemical quenching based on the ‘lake’ model [a photosynthetic unit may consist of a relatively larger number of reaction centres, embedded in a common matrix of antenna, with a high connectivity of PSII units, and where all open reaction centres 5
Page 7 of 53
compete for excitation in the pigment bed (Kramer et al., 2004)] is calculated as qL
=
(Fm’-
Fs)/(Fm’-F’0) x F0’/Fs = qP x F0’/Fs. qP and qL quantify the photochemical capacity of PSII in light-adapted state (Schreiber et al., 1986).
ip t
Non-photochemical quenching (qN, NPQ and NPQ). The parameter NPQ = [(Fm-Fm’)/Fm’] (Schreiber et al., 1986; Bilger and Björkman, 1991) is derived from the Stern-Volmer equation
cr
based on the ‘puddle’ model. NPQ and qN are mostly used to indicate the excess of radiant
us
energy dissipated as heat in PSII. Estimation of qN requires measurements of F0’ being qN = 1-(Fm’-F0)/(Fm-F0). The quantum yield of regulated non-photochemical energy loss in PSII, i.e.
an
NPQ in the ‘lake’ antenna model, represents the yield of heat dissipation trough down-
M
regulated mechanisms (Kramer et al., 2004). NPQ is complementary to both PSII and quantum yield of non-regulated energy dissipated in PSII, i.e. NO [NO= 1/(NPQ+1+qL(Fm/F0-
te
d
1)], being calculated as NPQ = 1- PSII -NO.
Ac ce p
2.2. Fast chlorophyll fluorescence or direct fluorescence: OJIP transient Transient Chl fluorescence (dark adapted to light state) is based on the so-called OIJP fast induction (Strasser et al., 2004). The OJIP transient reflects an accumulation of reduced QA, as the net result of QA reduction by PSII and QA- reoxidation by PSI. Upon sample irradiation with a saturating-continuous light after dark-adaptation, a rise of Chl fluorescence yield is induced from the initial value, F0 (point O), to a maximum fluorescence level, Fp (point P). The time to reach Fp is usually between 0.3-1s depending on samples and saturated light intensity. Two inflection points are observed between O and P, and are labelled as points J and I.
6
Page 8 of 53
The first photochemical phase (the O-J rise) is mostly related to the reduction of QA, when the rise in fluorescence strongly depends on the number of absorbed photons. At the end of this phase, QA is almost completely reduced (Stirbet and Govindjee, 2012). The
ip t
thermal phase (the J-I-P rise) then occurs. The J-I phase represents the reduction of the PQpool by PSII and inflection point I is achieved when the oxidation rate of the PQ-pool is close
cr
to its maximum. The I-P phase represents the complete reduction of electron acceptors,
us
NADP+ and ferredoxin, to PSI. During the thermal phase (J-I-P), QA continues to be photoreduced, until the fluorescence reaches its maximum yield (Fp=Fm). In parallel, the PQ-pool is
an
reduced and a trans-membrane pH gradient starts to build (Stirbet and Govindjee, 2012).
M
2.3. Seeing is believing: Chlorophyll a fluorescence imaging
d
Chl a fluorescence measurement is on single point measurements on the leaf; but
te
heterogeneity in photosynthesis is frequent in the leaf, thus methodology may be source of errors. The development of Chl fluorescence imaging (CFI) overcomes this problem, still
Ac ce p
maintaining advantages of non-imaging Chl fluorescence (Gorbe and Calatayud, 2012). CFI is based on the same PAM technology and enables the achievement of identical fluorescence intensity levels as non-imaging PAM Chl fluorescence: F0, Fm, Fm’ and Fs (Nedbal and Whitmarsh, 2004).
Fluorescence imaging reveals a wide range of sample characteristics, including spatial variations due to differences in physiological states, which may frequently occur in stressed plants. Moreover, CFI can screen a large number of plants simultaneously, and it is rapid, non-destructive and relatively inexpensive.
7
Page 9 of 53
2.4. Relation between Chlorophyll a fluorescence and gas exchange: physiological implications The combination of gas exchange with Chl a fluorescence gives insights into
photosynthesis
parameters.
Both
techniques
indeed
ip t
photosynthetic regulation and provide opportunities to estimate a wide range of estimate
mesophyll
diffusion
cr
conductance (gm) (Bongi and Loreto, 1989; Evans and von Caemmerer, 1996), the relative
us
CO2/O2 specificity factor for Ribulose-1,5bis-phosphate carboxylase/oxygenase (Rubisco) (Peterson, 1989), as well as the proportion of photon flux density absorbed by photosynthetic
an
pigments channeled towards PSII (Makino et al., 2002). For detailed reviews on different parameters estimable through a combination of gas exchange and Chl fluorescence
d
M
measurements see Laisk and Loreto (1996), Laisk et al. (2002) and Yin et al. (2011).
3.1 High solar irradiance
te
3. Main abiotic stresses in Mediterranean climate
Ac ce p
Light provides power necessary for photosynthesis reactions, but in the Mediterranean area sunlight irradiance is often a stress agent (Flexas et al., 2013). The mechanisms by which - both at leaf or chloroplast level - Mediterranean plants respond to different light environments do not differ from those adopted by plants from other biomes and include (Takahashi and Badger, 2011): -
leaf and chloroplast movement to reduce absorption of excess light (Davis et al., 2011; Terashima et al., 2011);
-
light screening e.g. driven by phenolics in epidermal cells (Hernández and Van Breusegem, 2010; Agati et al., 2011; Hernández et al., 2011); 8
Page 10 of 53
-
up-regulation of antioxidant defences aimed at scavenging reactive oxygen species (ROS) (Ballottari et al., 2012; Murata et al., 2012)
-
thermal
dissipation of excess light energy (de Bianchi et al., 2010; Johnson and
-
ip t
Ruban, 2011; Johnson et al., 2011);
cyclic electron transport via PSII: appropriate rates of this pathway would ensure the
cr
maintenance of the transthylakoidal proton gradient required for photophosphorylation
us
and dissipation of the excess energy at PSII level. Defects in this pathway result in the increased sensitivity of PSII to photoinhibition (Takahashi et al., 2009; Rochaix, 2011;
-
an
Murata et al., 2012);
enhancement of photorespiration and/or Mehler pathway, in which oxygen represents
M
an alternative electron acceptor that prevents over-reduction and photo-inactivation of
d
PSII (Bauwe et al., 2010; Kangasjärvi et al., 2012).
te
Leaf plasticity is crucial for the acclimation of plants to different light conditions, as encountered in the Mediterranean area (Valladares et al., 2000). However, this matter is still
Ac ce p
controversial. Changes in leaf inclination have been observed in Mediterranean evergreen sclerophylls, thus contributing to maintain higher Fv/Fm ratio as compared with horizontal leaves (Werner et al., 2002). Vaz et al. (2011) reported that leaf structural plasticity in two oak species (Quercus ilex and Quercus suber) is a stronger determinant for acclimation to high light irradiance than biochemical and physiological plasticity. On the contrary, Valladares et al. (2002) reported that greater photo-tolerance is linked to enhanced physiological plasticity, mostly on photosynthesis-related traits. Mediterranean ecosystem is dominated by evergreen sclerophylls and malacophyllous species, which strongly differ in their physiological and structural adaptations to cope with 9
Page 11 of 53
light stress factors. Semi-deciduous species reduce foliage area, thus restricting their growth during favorable periods, thus avoiding photo-inhibition. In contrast, evergreen sclerophylls tolerate stress conditions with intact green leaves, by displaying a variety of physiological
ip t
adaptations, such as small and thick leaves, with very thick cuticles and deep root stems (Mooney, 1981). Evergreen schlerophylls have also several adaptive mechanisms, such as
cr
low photosynthetic rate effective stomatal control, thus reducing photosynthetic rate during
us
periods of excess light stress (e.g., water stress coupled with high sunlight irradiance during summer).
an
Carotenoids play a major role in photo-protection due to their capacity of deactivating triplet Chl (3Chl*) and singlet oxygen (1O2). Xanthophylls are involved either directly or
M
indirectly in non-photochemical quenching of excess light energy dissipated in PSII antenna.
d
Recent evidence suggests that the photoprotective role of lutein (Lut) is mostly due to
te
deactivation of 3Chl*; whereas zeaxanthin (Zea) is a major player in the deactivation of excited singlet 1Chl* and thus in NPQ. Nonetheless, the exact role of Zea in NPQ is still questionable
Ac ce p
(Horton et al., 2005; Avenson et al., 2008; Jahns and Holzwarth, 2012). It is known that Zea contributes to all qN mechanisms – with the exception of quenching associated with state transitions. Additionally, overwhelming evidence supports that Zea serves important functions as an antioxidant in the lipid phase of the membrane. The sustained depression of Fv/Fm to values near zero concomitant with the reduction of the photosynthetic capacity in highly Zea-accumulating over-wintering evergreens suggests that strong reduction of PSII efficiency does not always indicate undesirable photoinhibition, but may represent a powerful photoprotective mechanism to reduce ROS (Adams et al., 2006). These findings underline the important photoprotective role of Zea under a large 10
Page 12 of 53
variety of photo-oxidative and photo-inhibitory conditions (Jahns and Holzwarth, 2012). This role of xanthophyll is well documented also in Mediterranean species. Recently, three functional pools of Zea with very different roles in photo-protection in Quercus coccifera
ip t
(Peguero-Pina et al., 2013) have been identified: (i) a background pool that is essentially present at predawn in unquenched state; (ii) a second pool that increases after ca. 30-90 s
cr
following high light conditions and contributes strongly to NPQ; and (iii) a third pool that forms
us
on a longer time scale, and little affects NPQ. Authors suggest that ΔpH is crucial for NPQ induction and relaxation in Quercus coccifera during light transitions, but only a minor fraction
an
of Zea is associated with quenching. Zea has been proposed to participate in photo-protection behaving as an antioxidant.
M
In the Mediterranean area another potential environmental constraint for plants is
d
represented by high fluxes of UV-B radiation (McKenzie et al., 2007). An excess of UV
te
radiation (both UV-B and UV-A) can generate ROS, thus resulting in cellular damage (Jansen et al., 1998), unless plants have developed protective mechanisms against shortwave solar
Ac ce p
radiation. Leaves represent most sensitive organs to UV-B radiation, and several review articles have discussed about biochemical, morphological, and anatomical changes operating at leaf level in response to UV-B (Caldwell et al., 2007; Morales et al., 2010; Paul et al., 2012).
Realistic UV-B studies have little impact on plant growth (Ballaré et al., 2011). Fv/Fm and PSII were unaffected by high UV-B radiation (32-40 kJ m-2d-1) when sunlight irradiance exceeded 400-500 μmol m−2s−1 (Nogués and Baker, 1995). In a field UV-exclusion experiment, the performance of Ligustrum vulgare was mostly affected by visible than by UVwavelengths (Guidi et al. (2011) (Figure 1). 11
Page 13 of 53
It is likely that detrimental effects of UV-B radiation in Mediterranean areas occur when plants are concomitantly exposed to a wide range of stress agents, such as pollutants, nutrient deficiency, drought, high light, and excess soil salinity (Agrawal and Rathore, 2007;
ip t
Guidi et al., 2011; Verdaguer et al., 2012; Barnes et al., 2013). Mediterranean plants have developed effective mechanisms of protection against UV-B stress, such as their inherently
cr
high antioxidant system (Prado et al., 2012) and the biosynthesis of UV-absorbing
us
compounds, such as flavonoids (Agati and Tattini, 2010; Agati et al., 2011).
In conclusion, Mediterranean plants exhibit plastic responses to high sunlight at both
an
morphological and physiological levels. Acclimation to irradiance also involves changes in leaf chemical composition to reduce light absorption in leaves exposed to sunlight, and to
M
increase the excitation energy absorbed in shade leaves. All the adjustment observed enable
d
more efficient carbon assimilation in plants exposed to sunshine, while shaded leaves
te
increase the surface area and nitrogen investment for light capture. Different factors play a decisive role including: (i) leaf position: leaves in a southern part of the canopy that receive
Ac ce p
plenty of sunlight are the most photo-inhibited; (ii) the season: Mediterranean plants suffer photo-inhibition during clear sunny days; and (iii) inter/intra-specific differences. In general, evergreen sclerophyllous Mediterranean species are characterized by a high tolerance towards excessive sunlight and physiological changes play the preeminent role. A dynamic short-term photo-inhibition occurs and results in a reversible diurnal decline in F v/Fm. This dcline frequently is accompanied by a pronounced increase in Zea which is able to mediate the de-excitation process of excited Chl from the PSII reaction centers – and probably protect thylakoid membranes from the negative effects of accumulated excitation energy. Semi-
12
Page 14 of 53
deciduous Mediterranean species show a different strategy with pronounced structural changes aimed at reducing light interception (Niinemets, 2010).
ip t
3.2 Temperature stress
Extremes in high temperature and drought are considered major constraints for plants
cr
during the Mediterranean summer. However, in mountainous areas or/and in Mediterranean
us
continental areas, cold or freezing temperatures in winter may also impact plant survival and growth.
an
Broadly, high temperatures affect the photosynthetic apparatus through inactivation of photosystems and molecular alterations of thylakoid membranes, and decreasing CO 2
M
assimilation and electron transport rate Photosynthesis is highly sensitive to high
d
temperature, and PSII is considered as the most heat-sensitive component of the
te
photosynthetic apparatus (Berry and Bjorkman, 1980; Dias et al., 2011). Consequently, reductions in Fv/Fm in Mediterranean species frequently occur during summer. Studies in
Ac ce p
three dominant species typical of Mediterranean shrub land during seven years (Erica multiflora, Globularia alypum and Pinus halepensis) showed decreases in Fv/Fm ratio up to 0.67-0.69 indicating restriction in carbon gain (Prieto et al., 2009). Similar results were observed in Quercus ilex and Phyllyrea latifolia (Ogaya et al., 2011) in which Fv/Fm values were highly dependent on both air temperature and genotypes: F v/Fm ratio was higher in Phyllirea latifolia than in Quercus ilex. These findings conform to Q. ilex and Phillyrea latifolia typically inhabiting sub-humid or dry Mediterranean areas, respectively. Studies on the effects of high temperatures are performed on Ceratonia siliqua, a Mediterranean sclerophyllous evergreen tree (Osório et al., 2011). Plants were subjected to two thermal regimes: moderate 13
Page 15 of 53
(25/18ºC) or high (32/21ºC) temperatures. The reduction in photosynthetic rate was 33% at 25/18º C whereas at higher temperatures it was 84%. In spite of this, the electron transport rate was not affected suggesting that non-regulated quenching mechanisms (e.g..
ip t
photorespiration and/or Mehler reaction) tend to down-regulate PSII activity photoprotecting it under high temperature, with the xanthophyll cycle-mediated thermal dissipation playing
cr
possibly a much less important role.
us
An interesting study case of the mechanisms of photoprotection operating in Quercus suber, Quercus ilex, Olea europaea and Eucalyptus globulus during summer has been
an
conducted by Faria et al. (1998) in Portugal. Authors reported that stomatal closure mostly controlled daily variations in carbon assimilation over the whole-period. Nonetheless, decline
M
in photochemical yield of photosystem II (PSII) was observed as plants suffered from severe
d
excess light stress. This resulted from high temperature-induced stomatal closure, which in
te
turn strongly depressed net carbon gain. Consistently, all species displayed an increased need of thermal dissipation of excess photon energy, which was sustained by stimulation of
Ac ce p
xanthophyll cycle. Net photosynthesis was lowest in Olea europaea: this was paralleled with highest both carotenoid to chlorophyll ratio and rates of violaxanthin de-epoxidation. This allowed in an increase in thermal dissipation of the excess radiant energy, but led to rather small values of light utilization in photochemistry (it averaged from 31% in the morning and 29% in the evening). In contrast, in Eucalyptus globulus photosynthetic rates were highest, thermal dissipation of absorbed radiation lowest and, values of light utilization in photochemistry were approximately 60%. These findings show from one hand, highly coordinated response mechanisms involving stomatal control photosynthetic capacity, and biochemical adjustments and from the other hand largely different physiological-biochemical 14
Page 16 of 53
responses to the same environmental constrain adopted by Mediterranean plants. This is in some relation with the high level of biodiversity usually observed in Mediterranean ecosystems.
ip t
Photochemical efficiency of PSII attributable to an impairment of LHC (Oliveira and Peñuelas, 2000) can also occur in forest vegetation in continental Mediterranean areas, as
cr
usually observed during winter. Photo-inhibition is particularly severe in sunny days following
us
overnight frosts, as frequently occurs during Mediterranean winter. Garcia-Plazaola et al. (1999) reported three different strategies to cope with the winter stress in Mediterranean
an
evergreens. The first showed by some Mediterranean sclerophyllus in which reversible reductions of PSII efficiency (i.e. reduction in Fv/Fm ratio) related to the activity of the
M
xanthophyll cycle allowed to an adaptation of the photosynthetic apparatus to winter stress.
d
Among these species, Q. ilex showed the higher ability to adapt its photochemical efficiency
te
and this can be explained by its wide altitudinal distribution. A second type of photo-protection is showed by malacophyllous semideciduous Cistus species which maintained a higher
Ac ce p
photosynthetic rate dissipating a large amount of excitation energy by carbon assimilation and very low down-regulation of PSII efficiency under moderate stress conditions. Under more stressful conditions, these species lost their leaves as reported also by Oliveira and Peñuelas (2004). Finally, the third way of adaptation is showed by Boxus sempervirens that contains high amount of ascorbate and tocopherol and a high carotenoid/Chl and Zea/Chl ratios. In Boxus photochemical efficiency of PSII was highly reduced because of the loss of D1 protein and/or the retention of de-epoxidized xanthophylls. In Mediterranean plants, response mechanisms to low or high temperatures do not substantially differ. Silva-Cancino et al. (2012) used CFI to analyze species-specific 15
Page 17 of 53
responses to cope with winter stress in evergreens: Buxus semprevirens, Cistus albidus and Arctostaphylus uva-ursi. In Buxus semprevirens a decrease in Fv/Fm over winter, was accompanied by a massive accumulation of red pigments in sub epidermal cells. Recovery
ip t
from winter photo-inhibition was characterized by leaf re-greening and decrease in VAZ pool pigments (Silva-Cancino et al., 2012). Photosynthetic decreases, as well as reductions in
cr
photochemical PSII activity at extreme temperatures, are often regulatory mechanisms
us
associated with photo-protective strategies that avoid irreversible damage to photosystem by so-called ‘dynamic’ photo-inhibition. These strategies may be reversed when environmental
an
conditions become favorable for growth (Larcher, 2000).
M
3.3 Water stress
d
The scarcity of soil water is likely the major environmental constrain for plants inhabiting
te
Mediterranean areas. Photosynthetic CO2 assimilation in Mediterranean species is impaired by drought in different ways, but stomatal closure represents is primarily involved in drought-
Ac ce p
induced depression of carbon assimilation (Chaves et al., 2002). Mesophyll conductance to CO2 (gm) has been reported that in drought-adapted species diffusional limitations account for most of the observed water stress-induced depression in photosynthesis. gm has long been reported to depend on leaf and cell anatomy (Laisk et al., 1970; Nobel et al., 1975), but recently aquaporins have been reported to greatly affect gm. Aquaporins effectively regulate CO2, not only water diffusion through membranes (Heckwolf et al., 2011). Details about the key role of gm in photosynthesis have been recently reviewed (Flexas et al., 2012; Griffiths and Helliker, 2013).
16
Page 18 of 53
Overall, Mediterranean evergreens display low responsiveness to environmental changes: they have phenotypic stability and conservative resource-use strategy (Valladares et al., 2000). Conversely, Quercus coccifera and Pinus halepensis show a high plastic
ip t
response of gas exchange to drought, in terms of stomatal control, and hence in net CO2 assimilation and transpiration rates. However, these species have low plasticity of leaf
cr
photochemistry. Photochemical efficiency was little affected during drought stress, as
us
physiological and biochemical features, including photo-respiration, pigments and antioxidant defenses mostly varied during stress episodes (Baquedano et al., 2008).
an
As already mentioned, drought stress occurs in concomitance with high sunlight irradiance during summer in Mediterranean areas. Balancing light capture and energy use is
M
therefore a great challenge for Mediterranean plants under drought stress, with dramatic
d
consequences on the performance of the photosynthetic (Chaves et al., 2009). Drought-
te
induced restriction of CO2 assimilation ultimately results in a massive accumulation of ROS and, hence, in oxidative damage. However, Mediterranean plants have evolved strategies
Ac ce p
and are equipped with photoprotective mechanisms such as xanthophyll cycle pigments (Munné-Bosch and Peñuelas, 2003; Galmes et al., 2007) and antioxidants (enzymatic and non-enzymatic) (Munné-Bosch and Peñuelas, 2003; Melgar et al., 2009). Sunlight irradiance strongly modulates plant responses to water deficit (Fini et al., 2012). Guidi et al. (2008) have shown that in Ligustrum vulgare under mild drought, net photosynthesis decreased more in plants growing under full sun than in plants growing under partial shading, as consequence of greater reductions in Fv/Fm, PSII and gs. Nonetheless, oxidative stress and damage were lower in sun than in shade plants, as in sun plants antioxidant defenses were more active than in shade plants. 17
Page 19 of 53
A drought-avoidance strategy has been identified as a general feature of evergreen oaks inhabiting xeric sites and is characterized by low leaf area per shoot volume ratio, strong stomatal control, moderate leaf osmotic potential, and high relative water content. Deciduous
ip t
species usually show a drought-tolerant behavior mediated by phenol-morphological features such as short leaf lifetimes, or leaf pubescence (Oliveira and Peñuelas, 2004). A comparative
cr
study in Quercus ilex (evergreen) vs Q. robur (deciduous) revealed different photosynthetic
us
strategies to cope with severe water stress (Koller et al., 2013). Both oak taxa decreased CO 2 assimilation rate at saturating light due to stomatal closure and Fv/Fm ratio decreased
an
significantly only in Q. ilex while remained unaffected in Q. robur. Chl fluorescence measurements shown that in the former species there were a high amount of non-functional
M
PSII reaction centers not involved in electron transport but that dissipated excitation energy
d
independently from the xanthophyll cycle (no changes in NPQ). Conversely, in Q. robur the
te
unaffected photoactive reaction centers under water stress together with the strongly decreased in photosynthetic rate enforced the strong decrease in qP and the increase in NPQ
Ac ce p
under actinic light. In this species additionally and/or alternatively, dissociation of PSII antennae from the reaction centers and emission of absorbed light through heat dissipation by the antenna carotenoids could represent an option to release electron pressure in the electron transport chain.
In conclusion, the alterations that occur in response to drought in Mediterranean plants include the restriction of CO2 diffusion to the chloroplast, as well as metabolic changes, and the modulation of expression of photosynthesis-related genes. However, the involvement of the different mechanisms is strictly related to the experimental conditions, the different species and the severity of imposed stress. 18
Page 20 of 53
3.4. Salinity Soil and water salinity are major environmental challenges for Mediterranean plants. Salinity detrimentally affects the performance of plants through potential toxicity driven by
ip t
specific ions and a general constrain due to osmotic unbalance (Munns and Tester, 2008). High concentration of salts in the soil reduces a plant’s ability to extract water from the soil
cr
thus resulting in water stress.
us
Physiological and biochemical processes fundamental for plant metabolism, such as photosynthesis and respiration, are highly sensitive to excess of salts (Munns, 1993; Stepien
an
and Johnson, 2009; Duarte et al., 2013).Early responses to salinity do not markedly differ from responses to drought stress (Munns, 2002), particularly in Mediterranean plants (Chaves
M
et al., 2009).
d
Biomass reduction (Degl’Innocenti et al., 2009; Jaoudé et al., 2012; Cocozza et al.,
te
2013), the first visible effect induced by salt stress, is caused by the increased energy consumption for various tolerance mechanisms, e.g. for synthesizing compatible organic
Ac ce p
solutes and proteins (Gucci and Tattini, 1997; Ben Hassine et al., 2009; Chen and Jiang, 2010; Rajavindran and Natarajan, 2012; Gill et al., 2013). Notably, salt tolerance greatly differs in co-occurring Mediterranean plants. Three Mediterranean evergreens differ greatly in their strategies for salt allocation: the ‘saltexcluders’ Olea europaea and Phyllirea latifolia (both Oleaceae); and Pistacia lentiscus, which, instead, mostly uses Na+ and Cl- for osmotic adjustment. Both Oleaceae spp. undergo severe leaf dehydration, and reduce net photosynthesis and whole-plant growth to a significantly greater degree than Pistacia lentiscus (Tattini et al., 2009). These contrasting strategies to manage the allocation of potentially toxic ions in sensitive leaf organs were 19
Page 21 of 53
reflected in markedly different biochemical adjustments. These included the activation of violaxanthin cycle pigments and antioxidant defences, aimed at avoiding and countering saltinduced reductions in the use of radiant energy to photosynthesis. In salt-excluding O.
ip t
europaea and P. latifolia leaf biochemistry was greatly altered as compared with P. lentiscus to minimize oxidative load (Tattini et al., 2009).
cr
Olea europea, is tolerant to salinity stress (Therios and Misopolinos, 1988; Gucci and
us
Tattini, 1997). Survival and not growth performance follows response mechanisms operating in olive plants in response to salinity stress, as occurs in most Mediterranean (Munns and
an
Tester, 2008; Cimato et al., 2010). These mechanisms are based mostly in reducing transpiration and hence water mass flow-transport of potentially toxic ions to sensitive shoot
M
organs. This imposes severe leaf dehydration and steep reduction in gas exchange
d
performance during salt stress, but preserves young leaves from massive accumulation of
te
toxic ions. Interestingly, reduction in transpiration rate and CO2 photo-assimilation are adaptive mechanisms to salinity stress in olive and other salt-excluding evergreens (Tattini et
Ac ce p
al., 2002; Cimato et al., 2010).
During drought in the Mediterranean area, salinity stress occurs in concomitance with high sunlight irradiance (Chaves et al., 2003). In olive, Melgar et al. (2009) found that sunexposed leaves had higher Na+, Cl− and mannitol content than shaded leaves. The high concentration of VAZ-cycle pigments in sun-exposed leaves suggests that Zea may protect the chloroplast from photo-oxidative damage through ROS scavenging rather than dissipating excess excitation energy via NPQ. In fact recently it has been reported that Zea plays a significant role as chloroplastic antioxidant, specifically protecting highly unsaturated chloroplast membranes from photo-oxidation (Havaux and Niyogi, 1999; Havaux et al., 2000). 20
Page 22 of 53
Given that most of the light-related increase in the VAZ pool resulted in increases in the ‘free’ VAZ, membrane protection from peroxidation may be the primary role of light-related enhancements of VAZ pool size. Beside zeaxanthin playing a role in NPQ and acting as an
ip t
antioxidant, the xanthophyll cycle may also be involved in the protection of plants from stress (Havaux and Tardy, 1996). In addition, the results obtained from Melgar et al. (2009) suggest
cr
that when olive was subjected to salinity under partial shading, the antioxidant defense
us
system may be ineffective to counter salt-induced oxidative damage (Remorini et al., 2009). Interestingly, Melgar et al. (2009) found a steep decline in midday Fv/Fm because of high
an
sunlight in control plants (from 0.826 in shade to 0.794 in sun-exposed leaves), whereas the midday Fv/Fm value was greater in salt-treated sun-exposed leaves (0.822) than in
M
corresponding shaded leaves (0.808) (Figure 2). The authors found also a light-induced
d
increase in the carotenoid to Chltot ratio, which mostly regarded the relative concentration of
te
violaxanthin-cycle pigments and concluded that xanthophyll in addition to their contribute to protect PSII photochemistry from irreversible photo-damage via NP quenching, likely served
Ac ce p
important antioxidant functions. Zeaxanthin is unlikely to participate in the thermal dissipation of excess excitation energy in the chloroplast, when the concentration of violaxanthin-cycle pigment exceeds 50 mmol mol−1 Chltot, as suggested by Havaux and Niyogi (1999). To conclude salinity represent a serious constrain in Mediterranean species that show integrated anatomical, physiological and biochemical adjustment to survive in this environment. Photosynthesis is certainly the principal target of salinity and Mediterranean species showed different mechanisms aimed to contrast the salt-induced oxidative load through photo-protective mechanisms.
21
Page 23 of 53
3.5. Ozone Ozone (O3) is one of the most important pollutants in the Mediterranean area, the concentration of which increases because of high sunlight irradiance high temperatures, and
ip t
recirculation processes of polluted air masses (Sanz et al., 2007). As a consequence, the Mediterranean area suffers from critical level of this photochemical oxidant. In fact, levels of
us
protection (Gimeno et al., 1999; Calatayud and Bareno, 2001).
cr
O3 during spring and summer exceed UN-ECE critical level guidelines for vegetation
Ozone affects growth, photosynthetic performance and induces oxidative damage in
an
Mediterranean plants (Ribas et al., 2005; Velikova et al., 2005; Bussotti, 2008) even though evergreen broadleaves seems to be quite O3-tolerant (Manes et al., 1998; Calatayud et al.,
M
2010). Indeed, Pinus halepensis and Pinus pinea are sensitive to O3 (Nali et al., 2004).
d
Furthermore, intra-specific variability in O3-sensitivity has been frequently observed.
te
Differential response of three cabbage varieties to O3 has been reported (Table 1; Calatayud et al., 2002). However, Bussotti et al. (2011) analyzing the effect of O3 on Fv/Fm in woody
Ac ce p
plants, found that O3 did not affect Fv/Fm in 48% of examined experiments. Furtheremore, Calatayud et al. (2007) observed no significant effects of ozone on Fv/Fm in four maple species (Acer campestre, A. opalus, A. monspessulanum and A. pseudoplatanus), which are important components in humid Mediterranean regions, A. pseudoplatanus and A. opalus were most sensitive as revealed by O3-induced reductions in PSII and in the quantum efficiency of excitation capture by oxidized reaction centers in PSII (exc) and in qP.. These findings were consistent with a decrease in CO2 assimilation because of ozone treatment. (Calatayud et al., 2007). Studies in Pinus uncinata, the dominant specie in subalpine Pyrenees forest (Díaz-de-Quijano et al., 2012), did not detect significant changes in Fv/Fm 22
Page 24 of 53
ratio and Chl content under 1.8 x O3 ambient. Changes conversely were observed on photosynthetic rate and stomatal conductance, higher in O 3-treated plants as compared to the controls. Thus, the surplus of CO2 photoassimilation is supposed to represent an investment
ip t
of the assimilate carbon towards the pathways for the synthesis of antioxidants (Diaz-deQujano et al., 2012).
cr
It is known as a large part of diffusive resistance to O3 relies in the stomata and that
us
the different sensitivity to this pollutant could be explained by differences in leaf conductance (Reich, 1987). Stomata are also the way by which O3 entries into plants and so usually an O3-
an
induced reduction in stomata is significantly coupled with a reduction in CO2 (Weber et al., 1993; Clark et al., 1996).
M
It is difficult to compare the results from various research studies; in fact, plant
d
response depends on experimental conditions, age of the leaves and heterogeneity inside the
te
leaf, evergreen vs deciduous species and sensitivity to air pollutants. For example, Quercus ilex is tolerant to on both short-term and long-term basis. (Velikova et al., 2005; Calatayud et
Ac ce p
al., 2011; Mereu et al., 2011). All authors did found negligible effects of ozone on gas exchange and Chl fluorescence parameters. Nonetheless, aged leaves were more sensitive than one-year-old leaves, as the result of lower stomatal conductance and hence reduced O3 uptake (Velikova et al., 2005). Significant reduction in photosynthetic rate, stomatal conductance, Rubisco carboxylation efficiency, ribulose-1.5-bisphosphate regeneration capacity, Fv/Fm, PSII and qP were instead observed in deciduous Mediterranean species, such Pistacia terebinthus and Viburnum lantana. In contrast evergreen relatives, i.e. Pistacia lentiscus and Viburnum tinus were tolerant to ozone (Calatayud et al., 2010). Ozonetolerance in evergreen species was mostly related with the activation of antioxidant defenses, 23
Page 25 of 53
possibly as a consequence of minor reduction in gas exchange performance as compared with their deciduous relatives (Fini et al., 2012). Plant species and cultivars exhibit a wide range of O3 sensitivity evident a s heritable
ip t
characteristics related to identifiable metabolic and molecular processes that affect sensitivity to O3 (Fiscus et al., 2005). Overall, plant response to O3 seems mostly mediated by stomatal
cr
conductance (see Fiscus et al., 2005) and the mechanisms that determine plants sensitivity or
us
tolerance are not clearly understood. However, plants with similar gs largely differ in O3tolerances (Calatayud et al., 2010). It has been suggested that an integrated network of
an
antioxidant defenses, which includes antioxidant enzymes and low molecular-weight antioxidants (ascorbate, glutathione or -tocopherol), not only stomatal aperture contribute to
M
ozone tolerance in a wide range of species (Nali et al., 2004).
d
Tolerance to ozone in Mediterranean evergreens has been suggested to depend on
te
their sclerophylly, which translates into low gas exchange capacity, but also on a ‘constitutively’ effective pool of antioxidant defenses (Manes et al., 1998; Nali et al., 2004). It
Ac ce p
has been reported that more than one mechanism is involved in the tolerance to O 3 (Guidi et al., 2010). Such tolerance might overlap with their level of tolerance to other environmental constraints in Mediterranean area such as for example water stress and is related to the high constitutive levels, and/or O3-induced increases in antioxidants.
3.6 Climate change Models of global climate change predict a further 1.8-4°C warming by 2100 (Mc Arthy et al., 2001) and changing patterns of rainfall as a result of rising atmospheric CO 2 concentration and other greenhouse gases. Climate change could be particularly important in 24
Page 26 of 53
the Mediterranean basin (Christensen et al., 2007), affecting vegetation structure and plant productivity. Elevated CO2 concentration, increases in air temperature, and drought represent the main environmental constraints on crop and forest ecosystems. A prediction for the
ip t
effects of climate change on plants takes into account of these variables as well as their interactions. However, such a comprehensive picture for the effects of multiple stresses on
cr
plants, including those inhabiting the Mediterranean area has not been rarely drawn. In Pinus
us
taeda (Wertin et al., 2010) the combined effect of elevated CO2 (700 nL L-1), air temperature (+2ºC above air temperature), and water stress (25% respect to well watered conditions ) did
an
not affect photosynthesis and Fv/Fm while steeply decreasing the gs (-60%). WUE increased markedly, conforming to current model in which CO2 decreases stomatal conductance with
M
little effect on CO2 assimilation rate.
d
In general terms, combined drought and elevated temperature had more detrimental
te
effects than either stress alone under ambient CO2 concentration. Elevated CO2 mitigated the degree of change in photosynthesis parameters under heat stress and drought, at least for
Ac ce p
short-term exposures where photosynthetic activity can be stimulated or maintained. Photosynthetic down-regulation has been proposed to explain the acclimation to climate change (see in Aranjuelo et al., 2005; Erice et al., 2006) under middle and long-term exposure. Down-regulation can be caused by non-stomatal and/or stomatal limitations. Nonstomatal limitations are proposed to explain the decrease in PSII efficiency as a result of the accumulation of inactive PSII reaction centres, and the decrease in LHCs (Kalina et al., 1997) and/or a reduced carboxylation efficiency (Long et al, 2004), or a reduced amount/activity of Rubisco. Moreover, safe energy dissipation mediated by non-photochemical processes modulated by xanthophylls cycle has occurred and this is combined with an effective 25
Page 27 of 53
antioxidant system. Stomatal limitations explain the decrease in photosynthesis because the depletion of the intercellular CO2 concentration. The protecting effect of elevated CO2 against water stress under high temperature is associated with mechanisms for maintaining higher
ip t
relative water content and turgor potential with a higher WUE and osmotic adjustment (Tuba and Lichtenthaler 2007). Many other studies suggest that photosynthetic down-regulation
cr
results from an insufficient sink capacity (Ainsworth et al., 2004). Plants grown under elevated
us
CO2 concentration have a greater carbohydrate content in leaves than leaves of plants grown under ambient CO2 (Geiger et al., 1999). The carbohydrates accumulate and the gene
an
expression for photosynthetic apparatus is suppressed through the possible increase in hexose cycling within the leaves – resulting in decreased photosynthetic capacity and a
M
notable decrease in the amount of Rubisco (Erice et al., 2006).
d
The down-regulated photosynthetic response to climate change depends on genetic,
te
relative availability of the other co-limiting environmental factors, and on the degree of pressure of each stress (CO2, water, and temperature) on the ecosystem (Aranjuelo et al.,
Ac ce p
2005). Plant adaptation to climate change will depend on the buffer capacity to maintain metabolic functions and the interaction between the soil-plant-atmosphere system and this constitutes a complex network.
4. Conclusions
The Mediterranean ecosystem is complex and many abiotic environmental factors can affect plant species simultaneously. The large number of rare species that have survived for tens to hundreds of thousands of years enables us to accumulate knowledge on the physiological, cellular, and molecular responses of plants to these environmental constraints 26
Page 28 of 53
(drought, salinity, high light, high temperature, etc.) including the signaling of events occurring under these stresses. To further understand the complexity of plant response to stresses in the Mediterranean area, including the effects on photosynthesis, it is necessary to strengthen
ip t
multilevel genomic and physiological studies, and cover the differing intensities and timing of the imposition of stresses in genotypes with differing sensitivities to stress.
cr
Clearly, many difficulties occur in these studies. In addition to the difficulties posed by
us
the experimental conditions (length of time exposure, field as compared to greenhouse conditions, species utilized, etc.) there is the added difficulty of studying the interaction among
an
different stresses. Drought and salinity represent the most important stresses in the Mediterranean area but plant response is also linked to the intensity and quality of light.
M
Photosynthesis is limited in Mediterranean plant species by environmental constraints
d
either direct (as the diffusion limitations through the stomata and the mesophyll and the
te
alterations in photosynthetic metabolism) or secondary, such as the oxidative stress arising from the superimposition of multiple stresses. Gas exchange and chlorophyll fluorescence
Ac ce p
permit to collect a large pool of data regarding photosynthetic process in Mediterranean species subjected to many environmental stresses. These non-invasive methodologies give important information on the functionality of photosynthetic apparatus that combines with other physiological, biochemical and molecular studies. Furthermore, they sometimes permit, to understand the mechanisms on the basis of the plant response. In spite of the reliability of Chl fluorescence and gas exchange techniques for early stress diagnostic, they have some weakness: there is a variety of equipment and differing measuring protocols, various stress-dose applications and parameters make comparison among experiments difficult. These discordances should be overcome by standardizing and 27
Page 29 of 53
defining common protocols so that the experiments can be reproducible and compared. In addition, in field experiments it is difficult to discover the type of stress that is affecting the plant through Chl fluorescence and gas exchange. In our opinion, the effects induced by
ip t
abiotic stresses on Mediterranean plants are similar to those induced in plants from other biomes.
cr
In view of above, Chl fluorescence and gas exchange are useful approaches but until
us
now they have not suitable for understanding the mechanisms on the basis of plant
an
responses in Mediterranean area.
Acknowledgments
M
The authors thank Dr. Marco Landi and phD-Student Consuelo Penella for their contribution
d
in this review. This work has been financed by INIA (Spain) through project RTA2010-00038-
Ac ce p
(Fondi di Ateneo 2007-2012).
te
C03-01 and the European Regional Development Fund (ERDF) and by University of Pisa
References
Adams, W.W.III, Demmig-Adams, B., 2004. Chlorophyll fluorescence as a tool to monitor plant response to the environment. In: Papageorgiou, G.C., Govindjee (Eds.), Chlorophyll a Fluorescence: A Probe of Photosynthesis. Springer, Dordrecht, pp. 583604. Agati, G., Biricolti, S., Guidi, L., Ferrini, F., Fini, A., Tattini, M., 2011. The biosynthesis of flavonoids is enhanced similarly by UV radiation and root zone salinity in L. vulgare leaves. Journal of Plant Physiology 168, 204-212. 28
Page 30 of 53
Agati, G., Tattini, M., 2010. Multiple functional roles of flavonoids in photoprotection. New Phytologist 186, 786-793. Agrawal, S.B., Rathore, D., 2007. Changes in oxidative stress defense system in wheat
ip t
(Triticum aestivum L.) and mung bean (Vigna radiata L.) cultivars grown with and without mineral nutrients and irradiated with supplemental ultraviolet-B. Environmental and
cr
Experimental Botany 59, 21-33.
us
Ainsworth, E.A., Rogers, A., Nelson, R., Long, S.P., 2004. Testing the ‘source-sink’ hypothesis of down-regulation of photosynthesis in elevated [CO2] in the field with a
an
single gene substitution in Glycine max. Agricultural and Forest Meteorology 122, 85-94. Aranjuelo, I., Pérez, P., Hernández, L., Irigoyen, J.J., Zita, C., Martínez-Carrasco, R.,
M
Sánchez Díaz, M., 2005. The response of nodulated alfalfa to water supply,
te
123, 348-358
d
temperature, and elevated CO2: photosynthetic down regulation. Physiologia Plantarum
Avenson, T.J., Ahn, T.K., Zigmantas, D., Niyogi, K.K., Li, Z., Ballottari, M., Bassi, R., Fleming,
Ac ce p
G.R., 2008. Zeaxanthin radical cation formation in minor light-harvesting complexes of higher plant antenna. Journal of Biological Chemistry 283, 3550-3558. Ballaré, C.L., Caldwell, M.M., Flint, S.D., Robinson, S.A., Bornman, J.F., 2011. Effects of solar ultraviolet radiation on terrestrial ecosystems. Patterns, mechanisms, and interactions with climate change. Photochemical and Photobiological Science 10, 226241. Ballottari, M., Girardon, J., Dall'Osto, L., Bassi, R., 2012. Evolution and functional properties of Photosystem II light harvesting complexes in eukaryotes. Biochimica et Biophysica Acta (BBA) - Bioenergetic 1817, 143-157. 29
Page 31 of 53
Baquedano, F.J., Valladares, F., Castillo, F.J., 2008. Phenotypic plasticity blurs ecotypic divergence in the response of Quercus coccifera and Pinus halepensis to water stress. European Journal of Forest Research 127, 495-506.
ip t
Barnes, P.W., Kersting, A.R. Flint, S.D., Beyschlag, W., Ryel, R.J., 2013. Adjustments in epidermal UV-transmittance of leaves in sun-shade transitions. Physiologia Plantarum
cr
149, 200-213.
us
Bauwe, H., Hagemann, M., Fernie, A.R., 2010. Photorespiration: players, partners and origin. Trends in Plant Science 15, 330-336.
an
Ben Hassine, A., Ghanem, M.E., Bouzid, S., Lutts, S., 2009. Abscisic acid has contrasting effects on salt excretion and polyamine concentrations of an inland and a coastal
M
population of the Mediterranean xero-halophyte species Atriplex halimus. Annals of
d
Botany 104, 925-936.
te
Berry, J., Bjorkman, O., 1980. Photosynthetic response and adaptation to temperature in higher plants. Annual Review of Plant Physiology 31, 491-543,
Ac ce p
Bilger, W., Björkman, O., 1991. Temperature dependence of violaxanthin de-epoxidation and non-photochemical fluorescence quenching in intact leaves of Gossypium hirsutum L. and Malva parviflora L. Planta 184, 226-234. Bongi, G., Loreto, F., 1989. Gas-exchange properties of salted stressed olive (Olea europea L.) leaves. Plant Physiology 90, 1408-1416. Bussotti, F. 2008. Functional leaf traits, plant communities and acclimation processes in relation to oxidative stress in trees: a critical overview. Global Change Biology 14, 27272739.
30
Page 32 of 53
Bussotti, F., Desotgiu, R., Cascio, C., Pollastrini, M., Gravano, E., Gerosa, G., Marzuoli, R., Nali, C., Lorenzini, G., Salvatori, E., Manes, F., Schaub, M., Strasser, R.J., 2011. Ozone stress in woody plants assessed with chlorophyll a fluorescence. A critical reassessment
ip t
of existing data. Environmental and Experimental Botany 73, 19-30.
Calatayud, A., Alvarado, J.W., Barreno, E., 2002. Differences in ozone sensitivity in three
cr
varieties of cabbage (Brassica oleracea L.) in the rural Mediterranean area. Journal
us
Plant Physiology 159, 863-868.
Calatayud, A., Barreno, E., 2001. Chlorophyll a fluorescence, antioxidant enzymes and lipid
an
peroxidation in tomato in response to ozone and benomyl. Environmental Pollution 115, 283-289.
M
Calatayud, V., Cerveró, J., Calvo, E., García-Breijo, F.-J., Reig-Armiñana, J., Sanz, M.J.,
d
2011. Responses of evergreen and deciduous Quercus species to enhanced ozone
te
levels. Environmental Pollution 159, 55–63. Calatayud, V., Cerveró, J., Sanz, M.J., 2007. Foliar, physiological and growth responses of
Ac ce p
four maple species exposed to ozone. Water, Air and Soil Pollution 185, 239-254. Calatayud, V., Marco, F., Cerveró, J., Sánchez-Peña, G., Sanz, M.J., 2010. Contrasting ozone sensitivity in related evergreen and deciduous shrubs. Environmental Pollution 158, 3580–3587.
Caldwell, M.M., Bornman, J.F., Ballaré, C.L., Flint, S.D., Kulandaivelu, G., 2007. Terrestrial ecosystems, increased solar ultraviolet radiation, and interactions with other climate change factors. Photochemical and Photobiological Science 6, 252-266.
31
Page 33 of 53
Chaves, M M., Pereira, J.S., Maroco, J., Rodrigues, M.L., Ricardo, C.P.P.,. Osório, M.L., Carvalho, I., Faria, T., Pinheiro, C., 2002. How plants cope with water stress in the field? Photosynthesis and growth. Annals of Botany 89, 907-916.
ip t
Chaves, M.M., Flexas, J., Pinheiro, C., 2009. Photosynthesis under drought and salt stress: regulation mechanisms from whole plant to cell. Annals of Botany 103, 551-560.
cr
Chaves, M.M., Maroco, J., Pereira, J.S., 2003. Understanding plant responses to drought –
us
from genes to whole plant. Functional Plant Biology 30, 239-264.
Chen, H., Jiang, J.-G., 2010. Osmotic adjustment and plant adaptation to environmental
an
changes related to drought and salinity. Environmental Reviews 18(NA), 309-319. Christensen, J., Carter, T., Rummukainen, M., Amanatidis, G., 2007. Evaluating the
M
performance and utility of regional climate models: the PRUDENCE project. Climatic
d
Change 81, 1–6.
te
Cimato, A., Castelli, S., Tattini, M., Traversi, M.L., 2010. An ecophysiological analysis of salinity tolerance in olive. Environmental and Experimental Botany 68, 214-221.
Ac ce p
Clark, C.S., Weber, J.A., Lee, E.H., Hogsett, W.E. 1996. Reductions in gas exchange of Populus tremuloides caused by leaf ageing and ozone exposure. Canadian Journal of Forest Research 26 1384-1391. Cocozza, C., Pulvento, C., Lavini, A., Riccardi, M., d'Andria, R., Tognetti, R., 2013. Effects of increasing salinity stress and decreasing water availability on ecophysiological traits of quinoa (Chenopodium quinoa Willd.) grown in a Mediterranean-type agroecosystem. Journal of Agronomy and Crop Science (in press). DOI: 10.1111/jac.12012
32
Page 34 of 53
Davis, P.A., Caylor, S., Whippo, C.W., Hangarter R.P., 2011. Changes in leaf optical properties associated with light-dependent chloroplast movements. Plant, Cell & Environment 34, 2047-2059.
ip t
de Bianchi, S., Ballottari, M., Dall’Osto, L., Bassi, R., 2010. Regulation of plant light harvesting by thermal dissipation of excess energy. Biochemical Society Transaction 38, 651-660.
cr
De Lillis, M., 1991. An ecomorphological study of the evergreen leaf. Braun-Blanquetia 7: 1-
us
127.
Degl’Innocenti, E., Hafsi, C., Guidi, L., Navari-Izzo, F., 2009. The effect of salinity on
an
photosynthetic activity in potassium-deficient barley species. Journal of Plant Physiology 166, 1968-1981.
M
Adams, W.W. , Zarter, C.R., Mueh, K.E., Amiard, V., Demmig-Adams, B., 2006. Energy
d
dissipation and photoinhibition: a continuum of photoprotection. In: Demmig-Adams, B.,
te
Adams, W.W., Mattoo, A.K. (Ed.), Photoprotection, Photoinhibition, Gene Regulation, and Environment. Springer, Dordrecht, pp. 49-64.
Ac ce p
Díaz-de-Quijano, M., Schaub, M., Bassin, S., Volk, M., Peñuelas, J., 2012. Ozone visible symptoms and reduced root biomass in the subalpine species Pinus uncinata after two years of free-air ozone fumigation. Environmental Pollution 169, 250-257. Dias, A.S., Semedo, J., Ramalho, J.C., Lidon, F.C., 2011. Bread and durum wheat under heat stress: a comparative study on the photosynthetic performance. Journal of Agronomy and Crop Science 197, 50-56. Duarte, B. Santos, D., Marques, J.C., Caçador, I. 2013. Ecophysiological adaptations of two halophytes to salt stress: Photosynthesis, PS II photochemistry and anti-oxidant
33
Page 35 of 53
feedback – Implications for resilience in climate change. Plant Physiology and Biochemistry 67, 178-188. Erice, G., Irigoyen, J.J., Pérez, P., Martínez-Carrasco, R., Sánchez-Díaz, M. 2006. Effect of
a cutting regrowth cycle. Physiologia Plantarum 126, 458-468.
ip t
elevated CO2, temperature and drought on photosynthesis and nodulated alfalfa during
cr
Evans, J.R., von Caemmerer, S., 1996. Carbon dioxide diffusion inside leaves. Plant
us
Physiology 110, 339-346.
Faria, T., Silvério, D., Breia, E., Cabral, R., Abadia, A., Abadia, J., Pereira, J.S., Chaves, M.M.
an
1998. Differences in the response of carbon assimilation to summer stress (water deficits, high light and temperature) in four Mediterranean tree species. Physiologia
M
Plantarum 102, 419-428.
d
Fini, A., Guidi, L., Ferrini, F., Brunetti, C., Di Ferdinando, M., Biricolti, S., Pollastri, S.,
te
Calamai, L., Tattini, M., 2012. Drought stress has contrasting effects on antioxidant enzymes activity and phenylpropanoid biosynthesis in Fraxinus ornus leaves: An excess
Ac ce p
light stress affair? Journal of Plant Physiology 169, 929-939. Fiscus, E.L., Booker, F.L., Burkey, K.O., 2005. Crop responses to ozone: uptake, modes of action, carbon assimilation and partitioning. Plant, Cell & Environment 28, 997-1011. Flexas, J., Barbour, M.M., Brendel, O., Cabrera, H.M., Carriquí, M., Díaz-Espejo, A., Douthe, C., Dreyer, E., Ferrio, J.P., Gago, J., Gallé, A., Galmés, J., Kodama, N., Medrano, H., Niinemets, U., Peguero-Pina, J.J., Pou, A., Ribas-Carbó, M., Tomás, M., Tosens, T., Warren, C.R., 2012. Mesophyll diffusion conductance to CO2: An unappreciated central player in photosynthesis. Plant Science 193–194, 70-84.
34
Page 36 of 53
Flexas, J., Diaz-Espejo, A., Gago, J., Gallé, A., Galmés, J., Gulías, J., Medrano, H., 2013. Photosynthetic limitations in Mediterranean plants: A review. Environmental and Experimental Botany in press (http://dx.doi.org/10.1016/j.envexpbot.2013.09.002).
ip t
Galmés, J., Medrano, H., Flexas J., 2007. Photosynthetic limitations in response to water stress and recovery in Mediterranean plants with different growth forms. New Phytologist
cr
175, 81-93.
us
García-Plazaola, J.I., Artetxe, U., Duñabeitia, M.K., Becerril, J.M., 1999. Role of photoprotective systems of Holm-Oak (Quercus ilex) in the adaptation to winter
an
conditions. Journal of Plant Physiology 155, 625–630.
Geiger, M., Haake, V., Ludewig, F., Sonnewald, U., Stitt, M., 1999. The nitrate and
M
ammonium nitrate supply have a major influence on the response of photosynthesis,
d
carbon metabolism, nitrogen metabolism and growth to elevated carbon dioxide in
te
tobacco. Plant Cell & Environment 22, 1177-1199. Genty, B., Briantais, J.M., Baker, N.R., 1989. The relationship between the quantum yield of
Ac ce p
photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochimica et Biophysica Acta 990, 87–92.
Gill, R., Boscaiu, M., Lull, C., Bautista, I., Lidan, A., Vicente, O., 2013. Are soluble carbohydrates ecologically relevant for salt tolerance in halophytes? Functional Plant Biology (in press) DOI: 10.1007/s11103-013-0031-6. Gimeno, B.S., Bermejo, V., Reinert, R.A., Zheng, Barnes, J.D., 1999. Adverse effects of ambient ozone on watermelon yield and physiology at a rural site in Eastern Spain. New Phytologist 144, 245–260.
35
Page 37 of 53
Gorbe, E., Calatayud, A., 2012. Applications of chlorophyll fluorescence imaging technique in horticultural research: A review. Scientia Horticulturae 138, 24–35. Govindjee, 1995. Sixty-three years since Kautsky: chlorophyll a fluorescence. Australian
ip t
Journal of Plant Physiology 22, 131-160.
Griffiths, H., Helliker, B.R., 2013. Mesophyll conductance: internal insights of leaf carbon
cr
exchange. Plant, Cell & Environment 36, 733-735.
us
Gucci, R., Tattini, M., 1997. Salinity tolerance in olive. Horticultural Review 17, 177-204. Guidi, L., Degl’Innocenti, E., Remorini, D., Biricolti, S., Fini, A., Ferrini, F., Nicese, F.P.,
an
Tattini, M., 2011. The impact of UV-radiation on the physiology and biochemistry of
Experimental Botany 70, 88-95.
M
Ligustrum vulgare exposed to different visible-light irradiance. Environmental and
d
Guidi, L., Degl’innocenti, E., Remorini, D., Massai, R., Tattini, M., 2008. Interactions of water
te
stress and solar irradiance on the physiology and biochemistry of Ligustrum vulgare. Tree Physiology 28, 873–883.
Ac ce p
Guidi, L., Degl’Innocenti, E., Giordano, C., Biricolti, S., Tattini, M. 2010. Ozone tolerance in Phaseolus vulgaris depends on more than one mechanism. Environmental Pollution 158, 3164-3171.
Havaux, M., Niyogi, K.K., 1999. The violaxanthin cycle protects plants from photooxidative damage by more than one mechanism. PNAS 96, 8762-8767. Havaux, M., Bonfils J.-P., Lütz C., Niyogi K.K., 2000. Photodamage of the photosynthetic apparatus and its dependence on the leaf developmental stage in the npq1 Arabidopsis mutant deficient in the xanthophyll-cycle enzyme violaxanthin deepoxidase. Plant Physiology 124, 273-284. 36
Page 38 of 53
Havaux, M., Tardy, F. 1996. Temperature-dependent adjustment of the thermal stability of photosystem II in vivo: possible involvement of xanthophyll-cycle pigments. Planta 198, 324-333.
ip t
Heckwolf, M., Pater, D., Hanson, D.T., Kaldenhoff, R., 2011. The Arabidopsis thaliana aquaporin AtPIP1;2 is a physiologically relevant CO2 transport facilitator. Plant Journal
cr
67, 795-804.
us
Hernández, I. Alegre, L., Munné-Bosch, S., 2011. Plant aging and excess light enhance flavan-3-ol content in Cistus clusii. Journal of Plant Physiology 168, 96-102.
an
Hernández, I., Van Breusegem, F., 2010. Opinion on the possible role of flavonoids as energy escape valves: Novel tools for nature's Swiss army knife? Plant Science 179, 297-301.
M
Horton, P., Wentworth, M., Ruban, A., 2005. Control of the light harvesting function of
te
FEBS Letters 579, 4201-4206.
d
chloroplast membranes: the LHCII-aggregation model for non-photochemical quenching.
IPCC. 2007. Summary for policymakers In: Solomon, S., Qin, D., Manning, M., Chen, Z.,
Ac ce p
Marquis, M., Averyt, K.B., Tignor, M., Miller, H.L. (Eds.), Climate change 2007: the physical science basis. Contribution of working group I to the fourth assessment report of the intergovernmental panel on climate change. Cambridge University Press, Cambridge, pp. 747-846.
Jahns, P., Holzwarth, A.R., 2012. The role of the xanthophyll cycle and of lutein in photoprotection of photosystem II. Biochimica et Biophysica Acta 1817, 182-193. Jansen, M.A.K., Gaba, V., Greenberg, B.M., 1998. Higher plants and UV-B radiation: balancing damage, repair and acclimation. Trends in Plant Science 3, 131-135.
37
Page 39 of 53
Jaoudé, R.A., de Dato, G., De Angelis, P., 2012. Photosynthetic and wood anatomical responses of Tamarix africana Poiret to water level reduction after short-term fresh- and saline-water flooding. Ecological Research 27, 857-866.
ip t
Johnson, M.P., Goral, T.K., Duffy, C.D.P., Brain, A.P.R. Mullineaux, C.W., Ruban, A.V., 2011. Photoprotective energy dissipation involves the reorganization of Photosystem II light-
cr
harvesting complexes in the grana membranes of spinach chloroplasts. The Plant Cell
us
23, 1468-1479.
Johnson, M.P., Ruban, A.V., 2011. Restoration of rapidly reversible photoprotective energy
an
dissipation in the absence of PsbS protein by enhanced ΔpH. The Journal of Biological Chemistry 286, 19973-19981.
M
Kalina, J., Cajanek, M., Spunda, V., Marck, M.V., 1997. Changes of the primary
d
photosynthetic reaction of Norway sprunce under elevated CO 2. In: Mohren, G.M.J.,
te
Sabaté, S., (Eds.), Impacts of Global Change on Tree Physiology and Forest Ecosystems. Kluwer Academic Publishers, Dordrecht, pp. 59-69.
Ac ce p
Kangasjärvi, S., Neukermans, J., Li, S., Aro, E.-M., Noctor, G., 2012. Photosynthesis, photorespiration, and light signalling in defence responses. Journal of Experimental Botany 63, 1619-1636.
Kitajima, M., Butler, W.L., 1975. Quenching of chlorophyll fluorescence and primary photochemistry in chloroplasts by dibromothymoquinone. Biochimica et Biophysica Acta 376,105 -115. Koller, S., Holland, V., Brűggermann, W., 2013. Effects of drought stress on the evergreen Quercus ilex L., the deciduous Q. robur and their hybrid Q. x turneri Wild. Photosynthetica 51, 574-582 . 38
Page 40 of 53
Kramer, D.M. Johnson, G. Kiirats, O. Edwards G.E., 2004. New fluorescence parameters for the determination of QA redox state and excitation energy fluxes. Photosynthesis Research 79, 209-218.
ip t
Krupa S.V., 2000. Ultraviolet-B radiation, ozone and plant biology. Environmental Pollution 110, 193-194.
chlorophyll
fluorescence
(Ribulose-1,5-Bisphosphate
carboxylase/oxygenase
us
and
cr
Laisk, A., Loreto, F., 1996. Determining photosynthetic parameters from leaf CO2 exchange
specificity factor, dark respiration in the light, excitation distribution between
an
photosystems, alternative electron transport rate, and mesophyll diffusion resistance. Plant Physiology 110, 903-912.
M
Laisk, A., Oja, V., Rahi, M., 1970. Diffusion resistances as related to the leaf anatomy.
d
Fiziologiya Rastenii 17, 40-48.
te
Laisk, A., Oja, V., Rasulov, B., Rämma, H., Eichelmann, H., Kasparova, I., Pettai, H., Padu, E., Vapaavuori, E., 2002. A computer-operated routine of gas exchange and optical
Ac ce p
measurements to diagnose photosynthetic apparatus in leaves. Plant, Cell & Environment 25, 923-943.
Larcher, W., 2000. Temperature stress and survival ability of Mediterranean sclerophyllous plants. Plant Biosystems 134, 279–295. Leshem, Y.Y., Kuiper, P.J.C. 1996. Is there a gas (general adaptation syndrome) response to various types of environmental stress? Biologia Plantarum 38, 1-18. Long. S.P., Ainsworth, E.A., Rogers, A., Ort, D.R., 2004. Rising atmospheric carbon dioxide: Plant face the future. Annual Review of Plant Biology 55, 591-628.
39
Page 41 of 53
Makino, A.,Miyake, C., Yokota, A., 2002. Physiological functions of the water–water cycle (Mehler reactions) and the cyclic electron flow around PSI in rice leaves. Plant and Cell Physiology 43, 1017-1026.
ip t
Manes, F., Vitale, M., Donato, E., Paoletti, E. 1998. O3 and O3+CO2 effects on a Mediterranean evergreen broadleaf tree, holm oak (Quercus ilex L.). Chemosphere 36,
cr
801-806.
us
Maxwell, K., Johnson, G.N., 2000. Chlorophyll fluorescence-a practical guide. Journal of Experimental Botany 51, 659–68.
an
McCarthy, J.J., Canziani, O., Leary, N.A., Dokken, D.J., White, K.S., 2001. Climate Change 2001: Impacts, Adaptation and Vulnerability. IPCC Working Group II, Cambridge
M
University Press, Cambridge.
d
McKenzie, R.L., Aucamp, P.J., Bais, A.F., Björn, L.O., Ilyas, M., 2007. Changes in
te
biologically-active ultraviolet radiation reaching the Earth's surface. Photochemistry and Photobiology Science 6, 218-231.
Ac ce p
Melgar, J.C., Guidi, L., Remorini, D., Agati, G., Degl’innocenti, E., Castelli, S., Baratto, M.C., Faraloni, C., Tattini, M., 2009. Antioxidant defences and oxidative damage in salt-treated olive plants under contrasting sunlight irradiance. Tree Physiology 29, 1187-1198. Mereu, S., Gerosa, G., Marzuoli, R., Fusaro, L., Salvatori, E., Finco, A., Spano, D., Manes, F. (2011). Gas exchange and JIP-test parameters of two Mediterranean maquis species are affected by sea spray and ozone interaction. Environmental and Experimental Botany 73, 80-88.
40
Page 42 of 53
Mooney, H.A., 1981. Primary production in Mediterranean-climate regions. In: Di Castri, F., Goodall, D.W., Specht, R.L. (Ed.), Mediterranean-type Shrublands. Ecosystems of the World 11. Elsevier Scientific Publishing, Amsterdam, pp. 249-256.
ip t
Morales, L.O., Tegelberg, R., Brosché, M., Keinänen, M., Lindfors, A., Aphalo, P.J., 2010.
in Betula pendula leaves. Tree Physiology 30, 923-934.
cr
Effects of solar UV-A and UV-B radiation on gene expression and phenolic accumulation
us
Munné-Bosch, S., Peñuelas, J. 2013. Photo- and antioxidative protection during summer leaf senescence in Pistacia lentiscus L. grown under Mediterranean field conditions. Annals
an
of Botany 92, 385-391.
Munns, R., 1993. Physiological processes limiting plant growth in saline soils: some dogmas
M
and hypotheses. Plant, Cell & Environment 16, 15-24.
te
25, 239-250.
d
Munns, R., 2002. Comparative physiology of salt and water stress. Plant, Cell & Environment
Munns, R., Tester, M., 2008. Mechanisms of salinity tolerance. Annual Review of Plant
Ac ce p
Biology 59, 651-681.
Murata, N., Allakhverdiev, S.I., Nishiyama, Y., 2012. The mechanism of photoinhibition in vivo: Re-evaluation of the roles of catalase, α-tocopherol, non-photochemical quenching, and electron transport. Biochimica et Biophysica Acta (BBA) - Bioenergetics 1817, 11271133.
Nali, C., Paoletti, E., Marabottini, R., Della Rocca, G, Lorenzini, G., Paolacci, A.R., Ciaffi, M., Badiani, M., 2004. Ecophysiological and biochemical strategies of response to ozone in Mediterranean evergreen broadleaf species. Atmospheric Environment 38, 2247-2257.
41
Page 43 of 53
Nedbal, L., Whitmarsh, J., 2004. Chlorophyll fluorescence imaging of leaves and fruits. In: Govindjee,
Papageorgiou,
G.,
Ed.,
Chlorophyll
fluorescence:
A
signature
of
photosynthesis. Kluwer Academic Publishers., Dordrecht, The Netherlands, pp. 389–
ip t
407.
Niinemets, U., 2010. A review of light interception in plant strands from leaf to canopy in
cr
different plant functional types and in species with varying shade tolerance. Ecological
us
Research 25, 693-714.
Nobel, P.S., Zaragoza, L.T., Smith, W.K., 1975. Relation between mesophyll surface area,
an
photosynthetic rate, and illumination level during development for leaves of Plectranthus parviflorus Henckel. Plant Physiology 55, 1067-1070.
M
Nogués, S., Baker, N.R., 1995. Evaluation of the role of damage to photosystem II in the
te
Environment 18, 781-787.
d
inhibition of CO2 assimilation in pea leaves on exposure to UV-B. Plant, Cell &
Ogaya, R., Peñuelas, J., Asensio, D., Llusià, J., 2011. Chlorophyll fluorescence responses to
Ac ce p
temperature and water availability in two co-dominant Mediterranean shrub and tree species in a long-term field experiment simulating climate change. Environmental and Experimental Botany 73, 89–93. Oliveira, G., Peñuelas, J., 2000. Comparative photochemical and phenomorphological responses to winter stress of an evergreen (Quercus ilex L.) and a semi-deciduous (Cistus albidus). Acta Oecologica 21, 97–107. Oliveira, G., Peñuelas, J., 2004. The effect of winter cold stress on photosynthesis and photochemical efficiency of PSII of two Mediterranean woody species - Cistus albidus and Quercus ilex. Plant Ecology 175, 179-191. 42
Page 44 of 53
Osório, M.L., Osório, J., Vieira, A.C., Gonçalves, S., Romano, A., 2011. Influence of enhanced temperature on photosynthesis, photooxidative damage, and antioxidant strategies in Ceratonia siliqua L. seedlings subjected to water deficit and rewatering.
ip t
Photosynthetica 49, 3–12.
Paul, N.D., Moore, J.P., McPherson, M., Lambourne, C., Croft, P., Heaton, J.C., Wargent,
cr
J.J., 2012. Ecological responses to UV radiation: interactions between the biological
us
effects of UV on plants and on associated organisms. Physiologia Plantarum 145, 565581.
an
Peguero-Pina, J.J., Gil-Pelegrín, E., Morales, F., 2013. Three pools of zeaxanthin in Quercus coccifera leaves during light transitions with different roles in rapidly reversible
M
photoprotective energy dissipation and photoprotection. Journal of Experimental Botany
d
64, 1649-1661.
te
Peterson, R.B., 1989. Partitioning of noncyclic photosynthetic electron transport to O2dependent dissipative processes as probed by fluorescence and CO 2 exchange. Plant
Ac ce p
Physiology 90, 1322-1328.
Prado, F.E., Rosa, M., Prado, C., Podazza, G., Interdonato, R., González, J.A., Hilal, M., 2012. UV-B radiation, its effects and defense mechanisms in terrestrial plants. In: Ahmad, P., Prasad, M.N.V. (Eds.), Environmental Adaptations and Stress Tolerance of Plants in the Era of Climate Change. Springer, Dordrecht, pp. 57-83. Prieto, P., Peñuelas, J., Llusià, J., Asensio, D., Estiarte, M., 2009. Effects of long-term experimental night-time warming and drought on photosynthesis, Fv/Fm and stomatal conductance in the dominant species of a Mediterranean shrubland. Acta Physiologiae Plantarum 31, 729–739. 43
Page 45 of 53
Rajaravindran, M., Natarajan, S., 2012. Effect of NaCl stress on biochemical and enzymes changes of the halophyte Suaeda maritime Dum. International Journal of Research in Plant Science 2, 1-7.
ip t
Reich, P.B., 1987. Quantifying plant response to ozone: a unifying theory. Tree Physiology 3, 63-91.
cr
Remorini, D., Melgar, J.C., Guidi, L., Degl’Innocenti, E., Castelli, S., Traversi, M.L., Massai,
us
R., Tattini, M., 2009. Interaction effects of root-zone salinity and solar irradiance on the physiology and biochemistry of Olea europaea. Environmental and Experimental Botany
an
65, 210-219.
Ribas, A., Peñuelas, J., Elvira, S., Gimeno, B.S. 2005. Ozone exposure induces the activation
M
of leaf senescence-related processes and morphological and growth changes in
d
seedlings of Mediterranean tree species. Environmental Pollution 134, 291-300.
te
Rochaix, J.-D., 2011. Regulation of photosynthetic electron transport. Biochimica et Biophysica Acta - Bioenergetics 1807, 375-383.
Ac ce p
Sánchez-Gómez, D., Valladares, F., Zavala, M.A., 2006. Performance of seedlings of Mediterranean woody species under experimental gradients of irradiance and water availability: trade-offs and evidence for niche differentiation. New Phytologist 170, 795806.
Sanz, M.J., Calatayud, V., Sánchez-Peña, G., 2007. Measures of ozone concentrations using passive sampling in forests of South Western. Environmental Pollution 145, 620–628. Schreiber, U., Schliwa, U., Bilger, W., 1986. Continuous recording of photochemical and nonphotochemical chlorophyll fluorescence quenching with a new type of modulation fluorometer. Photosynthesis Research 10, 51–62. 44
Page 46 of 53
Silva-Cancino, M.C., Esteban, R., Artetxe, U., Plazaola, J.I.G., 2012. Patterns of spatiotemporal distribution of winter chronic photoinhibition in leaves of three evergreen Mediterranean species with contrasting acclimation responses. Physiologia Plantarum
ip t
144, 289–301.
Stepien, P., Johnson, G.N., 2009. Contrasting responses of photosynthesis to salt stress in
cr
the glycophyte Arabidopsis and the halophyte Thellungiella: role of the plastid terminal
us
oxidase as an alternative electron sink. Plant Physiology 149, 1154-1165. Stirbet, A., Govindjee, 2012. Chlorophyll a fluorescence induction: a personal perspective of
an
the thermal phase, the J-I-P rise. Photosynthesis Research 113, 15-61. Strasser, R.J., Srivastava, A., Tsimilli-Michael, M., 2004. Analysis of fluorescence transient.
M
In: Papageorgiou, G.C., Govindjee, (Eds.) Chlorophyll a Fluorescence: A signature of
te
321-362.
d
photosynthesis. Advances in Photosynthesis and Respiration. Springer, Dordrecht, pp.
Takahashi, S., Badger, M.R. 2011. Photoprotection in plants: a new light on photosystem II
Ac ce p
damage. Trends in Plant Science 16, 53-60. Takahashi, S., Milward, S.E., Fan, D.-Y., Chow, W.S., Badger, M.R., 2009. How does cyclic electron flow Alleviate photoinhibition in Arabidopsis? Plant Physiology 149, 1560-1567. Tattini, M., Montagni, G., Traversi M.L., 2002. Gas exchange, water relations and osmotic adjustment in Phillyrea latifolia grown at various salinity concentrations. Tree Physiology 22, 403-412. Tattini, M., Traversi, M.L., Castelli, S. Biricolti, S., Guidi, L. Massai, R., 2009. Contrasting response mechanisms to root-zone salinity in three co-occurring Mediterranean woody
45
Page 47 of 53
evergreens: a physiological and biochemical study. Functional Plant Biology 36, 551563. Terashima, I., Hanba, I.T., Tholen, D., Niinemets, U., 2011. Leaf functional anatomy in
ip t
relation to photosynthesis. Plant Physiology 155, 108-116.
Therios, I.N. Misopolinos, N.D., 1988. Genotypic response to sodium chloride salinity of four
cr
major olive cultivars (Olea europaea L.). Plant and Soil 106, 105-111.
us
Tuba Z, Lichtenthaler H. K., 2007. Long-term acclimation of plants to elevated CO2 and its interaction with stresses. Annals of the New York Academy of Sciences 1113, 135-46.
an
Valladares, F., Chico, J., Aranda, I., Balaguer, L., Dizengremel, P., Manrique, E., Dreyer, E. 2002. The greater seedling high-light tolerance of Quercus robur over Fagus sylvatica is
M
linked to a greater physiological plasticity. Trees 16, 395-403.
d
Valladares, F., Martinez-Ferri, E., Balaguer, L., Perez-Corona, E., Manrique E., 2000. Low
te
leaf-level response to light and nutrients in Mediterranean evergreen oaks: a conservative resource-use strategy? New Phytologist 148, 79-91.
Ac ce p
Vaz, M., Maroco, J., Ribeiro, N., Gazarini, L.C., Pereira, J.S., Chaves, M.M., 2011. Leaf-level responses to light in two co-occurring Quercus (Quercus ilex and Quercus suber): leaf structure, chemical composition and photosynthesis. Agroforestry Systems 82, 173-181. Velikova, V., Tsonev, T., Pinelli, P., Alessio, G.A., Loreto, F., 2005. Localized ozone fumigation system for studying ozone effects on photosynthesis, respiration, electron transport rate and isoprene emission in field-grown Mediterranean oak species. Tree Physiology 12, 1523-1532. Verdaguer, D., Llorens, L., Bernal, M., Badosa, J., 2012. Photomorphogenic effects of UVB and UVA radiation on leaves of six Mediterranean sclerophyllous woody species 46
Page 48 of 53
subjected to two different watering regimes at the seedling stage. Environmental and Experimental Botany 79, 66-75. Weber, J.A., Scott Clark, C., Hogsett, W.E. 1993. Analysis of the relationships among O3
ip t
uptake, conductance, and photosynthesis in needles of Pinus ponderosa. Tree Physiology 13, 157-172.
cr
Werner, C., Correia, O., Beyschlag, W. 2002. Characteristic patterns of chronic and dynamic
us
photoinhibition of different functional groups in a Mediterranean ecosystem. Functional Plant Biology 29, 999-1011-
an
Wertin, T.M., Mcguire, M.A., Teskey, R.O., 2010. The influence of elevated temperature, elevated atmospheric CO2 concentration and water stress on net photosynthesis of
M
loblolly pine (Pinus taeda L.) at northern, central and southern sites in its native range.
d
Global Change Biology 16, 2089–2103.
te
Yin, X., Sun, Z., Struik, P.C., Gu, J. 2011. Evaluating a new method to estimate the rate of leaf respiration in the light by analysis of combined gas exchange and chlorophyll
Ac ce p
fluorescence measurements. Journal of Experimental Botany 62, 3489-3499.
47
Page 49 of 53
Table 1. Changes in dark-adapted F0, Fm and Fv/Fm in cabbage leaves at the end of the growing station (30 days in the field) with charcoal-filtered air (CFA), non-filtered air (NFA) and NFA+O3 (63 nL L-1) treatments. Values are the means of ten samples. For comparison of means, variance analysis (ANOVA) followed by the least significance
ip t
differences (LSD) test, calculated at 95% confidence level, were performed. Values followed by the same letter indicate no significant differences (from Calatayud et al.,
Caramba F0
Fm
Sentinel Fv/Fm
F0
Fm
Othelo
us
Treatment
cr
2002).
Fv/Fm
F0
Fm
Fv/Fm
0.236a 1.116a 0.789a 0.236a
1.127a 0.790a 0.248a 1.203a 0.794a
NFA
0.242ab 0.955b 0.743b 0.241ab 1.052ab 0.770a 0.256a 1.220a 0.789a
NFA+O3
0.257b 0.793c 0.675c 0.255b
M
an
CFA
Ac ce p
te
d
0.870b 0.705b 0.226b 1.045b 0.780a
48
Page 50 of 53
Figure legend Figure 1. Maximum (Fv/Fm, A) and actual (ΦPSII, B) quantum efficiency of PSII photochemistry, non-photochemical quenching (qNP, C), and excitation pressure on PSII
ip t
(1−qP, D) in leaves of Ligustrum vulgare exposed to 35% (grey bars) or 100% sunlight irradiance (open bars) in the absence or in the presence (hutched bars) of UV-radiation. Data
cr
is mean±standard deviation (n=5), and at each sampling date, when not accompanied by the
us
same letter, significantly differ for P<0.05 using a least significant difference (LSD) test (from Guidi et al., 2011).
an
Figure 2. Time course of maximum quantum efficiency of PSII photochemistry (Fv/Fm) in leaves of O. europaea plants exposed to 15% (circles, shade) or 100% sunlight (circles, sun)
M
and supplied with 0 (open symbols) or 125 mM NaCl (closed symbols). Measurements were conducted at midday, after 7, 14 and 35 days of treatment. Summary of the results of a three-
d
way ANOVA (total error df = 59) for variation in Fv/Fm, with light (L), salt (S) and treatment
te
period (t) as fixed factors, with their interaction factors (L×S; L×t; S×t, L×S×t) is shown.
Ac ce p
**P<0.001 and data are mean±SD, n=4 (from Melgar et al., 2009).
49
Page 51 of 53
M
an
us
cr
ip t
Figure 1
Ac
ce pt
ed
Figure 1.
Page 52 of 53
Ac
ce pt
ed
M
an
us
cr
ip t
Figure 2
Page 53 of 53
S0098-8472(13)00218-9 http://dx.doi.org/doi:10.1016/j.envexpbot.2013.12.007 EEB 2727
To appear in:
Environmental and Experimental Botany
Received date: Revised date: Accepted date:
29-5-2013 27-11-2013 9-12-2013
Please cite this article as: Guidi, L., Calatayud, A.,Non-invasive tools to estimate stress-induced changes in photosynthetic performance in plants inhabiting Mediterranean areas, Environmental and Experimental Botany (2013), http://dx.doi.org/10.1016/j.envexpbot.2013.12.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
RS
TR
T LAS OP R LO CH
ES
S
FLUORESCENCE
TEM PER • WIN ATURE T • SUM ER MER
us
HEAT
cr
WA TE
ip t
Graphical Abstract (for review)
chl a*
chl a*
XANTHOPHYLL CYCLE
2e-
PH
2e-
an
OT OC
HE
M
SALINITY
M
ele ct ro n ch tran ain sp or
IST
t
chl a
d
Ac ce pt e ITY HT ANT Y G I L • QU LIT UA •Q
NADPH
LHCI
PS I ATP
PS II H2O
CALVIN CYCLE
chl a
LHCII
TS UTAN POLL ONE S • OZ Y METAL V A E •H
NADP+ + H+
RY
2H+ + 1/2 O2
ROS H2O2 O2.OH.-
OXIDATIVE DAMAGE
GE
N HA
EC AT LIM
C
Page 1 of 53
*Research Highlights
ip t
cr
us an
M
d
te
Mediterranean environment is characterized by rainy winters and long hot summers, with high irradiance and little or no precipitation Chlorophyll fluorescence and gas exchange are non-invasive, rapid, and inexpensive techniques for measuring photosynthesis in leaves In the Mediterranean environment, plant species are continuously subjected to various abiotic stresses such as light intensity, drought, extreme temperature, air pollutants, etc. These environmental factors adversely affect plant development and damage to the photosystem is the first manifestation of plant response to abiotic stress. In the review, the effects of various abiotic stresses on the photochemistry of Mediterranean plant species are reported using Chl a fluorescence and gas exchange.
Ac ce p
Page 2 of 53
*Manuscript
Non-invasive tools to estimate stress-induced changes in photosynthetic performance in plants inhabiting Mediterranean areas Lucia Guidi1, Angeles Calatayud2
ip t
Department of Agriculture, Food and Environment, University of Pisa, Via del Borghetto 80 -
56124 Pisa (Italy) 2
Instituto Valenciano de Investigaciones Agrarias (IVIA), Department of Horticulture, Ctra.
us
Moncada-Naquera km. 4.5, 46113 Moncada, Valencia, Spain
cr
1
an
Corresponding author: Lucia Guidi, Department of Agriculture, Food and Environment,
d
502216630, Email: [email protected]
M
University of Pisa, Via del Borghetto 80 -56124 Pisa (Italy), Phone: +39 502216613, Fax: +39
Ac ce p
te
Contents 1. Abiotic stresses imposed by the Mediterranean climate measurements 2. What is chlorophyll a fluorescence? 2.1 Slow chlorophyll a fluorescence induction kinetic 2.2 Fast chlorophyll fluorescence or direct fluorescence: OJIP transient 2.3 Seeing is believing: Chlorophyll a fluorescence imaging 2.4 Relation between Chlorophyll a fluorescence and gas exchange: physiological implications 3. Main abiotic stresses in Mediterranean climate 3.1. High solar irradiance 3.2. Temperature stress 3.3. Water stress 3.4. Salinity 3.5. Ozone 3.6. Climate change 4. Conclusion Acknowledgments References
3 4 5 6 7 8 8 8 13 16 19 22 25 26 28 28
Abstract 1
Page 3 of 53
In Mediterranean areas, plants are concomitantly exposed to various abiotic stresses such as light intensity, water deficit, extremes in air temperature, air pollutants, etc. These environmental pressures adversely affect plant development. Changes in photosystem
ip t
activity are an early response of plants to abiotic stresses. Therefore, chlorophyll (Chl) fluorescence and gas exchange, two non-invasive, rapid and inexpensive techniques for
cr
measuring photosynthesis in leaves, have been widely used by plant ecophysiologists to
us
analyze plant responses to stressful conditions. Chl a fluorescence and gas exchange parameters can be indeed used to evaluate changes in photochemical and non-
an
photochemical processes in photosystems associated with electron transport, CO2 fixation, and heat dissipation.
M
In this review, we focus our analysis on the effects of different abiotic stresses on the
d
photochemistry of Mediterranean plants using Chl a fluorescence and gas exchange
te
measurements. Since changes in photosynthetic parameters are observed in the absence of visual injuries, these methodologies constitute fundamental tools to predict and evaluate the
Ac ce p
extent to which abiotic stresses damage photosynthesis.
Key words: abiotic stress, chlorophyll a fluorescence, climate change, gas exchange, oxidative stress, photosynthesis
Abbreviations: NO: non-regulated heat dissipation (a loss process due to PSII inactivity); NPQ: regulated heat dissipation (a protective loss process); PSII: actual quantum yield of PSII photochemistry; CFI: chlorophyll fluorescence imaging; Chl: chlorophyll; F0, Fv and Fm: 2
Page 4 of 53
minimum, variable and maximum Chl fluorescence in dark adapted conditions; F 0’ and Fm’: minimum and maximum Chl fluorescence in light conditions; Fs: Chl fluorescence in steady state conditions; Fv/Fm: maximum quantum yield of PSII photochemistry; g m: CO2 mesophyll
ip t
conductance; gs: water vapor stomatal conductance ; LHC: light harvesting complex; Lut: lutein; NPQ: non-photochemical quenching; PAM: pulse amplitude modulate; PQ:
cr
plastoquinone; PSI and PSII: photosystem I and II; QA: primary quinone acceptor of
us
photosystem II; qL: photochemical quenching coefficient in the lake model; qp and qN: photochemical and non-photochemical quenching coefficients; ROS: reactive oxygen species; ribulose-1,5-bisphosphate
carboxylase/oxygenase;
an
Rubisco:
VAZ:
violaxanthin,
M
antheraxanthin, zeaxanthin cycle; Zea: zeaxanthin
d
1. Abiotic stresses imposed by the Mediterranean climate
te
The Mediterranean area is characterized by rainy autumns-winters, and long warm summers, during which sunlight irradiance is high and water availability is low. Thus,
Ac ce p
environmental stresses Mediterranean plants face during summer season are mostly drought, heat and high sunlight irradiance (Sánchez-Gómez et al., 2006; Galmés et al., 2007). Mediterranean species may also suffer from anthropogenic factors, such as increased tropospheric ozone (O3) concentrations and high UV-B radiation (Krupa, 2000). The Mediterranean basin is also expected to be more strongly affected than other areas worldwide from by global climate change (IPCC, 2007). Common characteristics of Mediterranean plants are an evergreen and sclerophylls habitus. Sclerophylls consist of coriaceous leaves, with 2–3 well-packed palisade layers, thick both cuticle and cell wall, high density of small stomata (De Lillis, 1991). However, species 3
Page 5 of 53
that co-occur in the very same Mediterranean habitats greatly differ in their eco-physiological traits as well as for their responses (and tolerance) to different stresses (Flexas et al., 2013). In summary, the Mediterranean climate exposes plants to multiple and uncorrelated
serious challenge for plants inhabiting Mediterranean areas.
ip t
stresses, their effects being currently exacerbated by global change. This represents a
cr
Chl a fluorescence and gas exchange techniques are fundamental to analyze the effect
us
of changes in environmental conditions on photosynthetic performance in Mediterranean species, and, hence on the overall agro-forest system. Here we offer an overview of basic
an
knowledge and application of chlorophyll fluorescence-based techniques for ecophysiological
d
2. What is chlorophyll a fluorescence?
M
analyses.
te
When Chl absorbs light energy, it remains in an excited state (first excited singlet) for just a very short period (usually nanoseconds). Excitation energy can be lost from this excited
Ac ce p
singlet state through releasing heat and/or fluorescence of red- far-red light, or through energy transfer to an adjacent pigment by inductive resonance There are different techniques for measuring Chl a fluorescence. In this review Pulse Amplitude Modulate (PAM) Chl fluorescence technologist and its physiological implications are tackled.
2.1. Slow chlorophyll a fluorescence induction kinetic The slow Chl a fluorescence induction kinetics includes transitions of the photosynthetic apparatus from dark-adapted to light-adapted state. The sample is illuminated 4
Page 6 of 53
with a low-energy modulated light (< 10 µmol m -2s-1) and fluorescence increases to a background fluorescence value that represents the minimum fluorescence level, namely F 0. The illumination of the sample with a saturating pulse of light (< 10000 µmol m -2s-1) then
ip t
induces an increase of Chl fluorescence yield (Fm level or maximal fluorescence). The fluorescence then decreases to reach a steady-state level (Fs). Subsequently, saturation
cr
pulse during actinic light induction kinetics enables F0’ to be reached (turning off the actinic
us
light in the presence of far-red light) and Fm’ values that are respectively the minimum and maximum fluorescence yield in the light. The information obtained from Chl a fluorescence
an
induction kinetics can be decoded using a set of Chl a fluorescence parameters mostly defined over the last four decades.
M
The maximum quantum yield of PSII photochemistry [Fv/Fm = (Fm-F0)/Fm], is a useful
d
indicator of the leaf potential photosynthetic capacity (Kitajima and Butler, 1975)
te
Effective (or actual) quantum yield of photochemistry energy conversion in PSII [PSII = (Fm’-Fs)/Fm’]. This parameter measures the proportion of light absorbed by chlorophyll
Ac ce p
associated with PSII that is actually used in photochemistry (Maxwell and Johnson, 2000). PSII is closely related with the quantum yield of the non-cyclic electron transport rate (Genty et al., 1989).
Photochemical quenching of variable Chl fluorescence yield (qP or qL). According to the ‘puddle’ model (each PSII centre possesses its own independent antenna system), photochemical quenching is defined as qP = (Fm’-Fs)/(Fm’-F0’) (Schreiber et al., 1986). The qL coefficient is the photochemical quenching based on the ‘lake’ model [a photosynthetic unit may consist of a relatively larger number of reaction centres, embedded in a common matrix of antenna, with a high connectivity of PSII units, and where all open reaction centres 5
Page 7 of 53
compete for excitation in the pigment bed (Kramer et al., 2004)] is calculated as qL
=
(Fm’-
Fs)/(Fm’-F’0) x F0’/Fs = qP x F0’/Fs. qP and qL quantify the photochemical capacity of PSII in light-adapted state (Schreiber et al., 1986).
ip t
Non-photochemical quenching (qN, NPQ and NPQ). The parameter NPQ = [(Fm-Fm’)/Fm’] (Schreiber et al., 1986; Bilger and Björkman, 1991) is derived from the Stern-Volmer equation
cr
based on the ‘puddle’ model. NPQ and qN are mostly used to indicate the excess of radiant
us
energy dissipated as heat in PSII. Estimation of qN requires measurements of F0’ being qN = 1-(Fm’-F0)/(Fm-F0). The quantum yield of regulated non-photochemical energy loss in PSII, i.e.
an
NPQ in the ‘lake’ antenna model, represents the yield of heat dissipation trough down-
M
regulated mechanisms (Kramer et al., 2004). NPQ is complementary to both PSII and quantum yield of non-regulated energy dissipated in PSII, i.e. NO [NO= 1/(NPQ+1+qL(Fm/F0-
te
d
1)], being calculated as NPQ = 1- PSII -NO.
Ac ce p
2.2. Fast chlorophyll fluorescence or direct fluorescence: OJIP transient Transient Chl fluorescence (dark adapted to light state) is based on the so-called OIJP fast induction (Strasser et al., 2004). The OJIP transient reflects an accumulation of reduced QA, as the net result of QA reduction by PSII and QA- reoxidation by PSI. Upon sample irradiation with a saturating-continuous light after dark-adaptation, a rise of Chl fluorescence yield is induced from the initial value, F0 (point O), to a maximum fluorescence level, Fp (point P). The time to reach Fp is usually between 0.3-1s depending on samples and saturated light intensity. Two inflection points are observed between O and P, and are labelled as points J and I.
6
Page 8 of 53
The first photochemical phase (the O-J rise) is mostly related to the reduction of QA, when the rise in fluorescence strongly depends on the number of absorbed photons. At the end of this phase, QA is almost completely reduced (Stirbet and Govindjee, 2012). The
ip t
thermal phase (the J-I-P rise) then occurs. The J-I phase represents the reduction of the PQpool by PSII and inflection point I is achieved when the oxidation rate of the PQ-pool is close
cr
to its maximum. The I-P phase represents the complete reduction of electron acceptors,
us
NADP+ and ferredoxin, to PSI. During the thermal phase (J-I-P), QA continues to be photoreduced, until the fluorescence reaches its maximum yield (Fp=Fm). In parallel, the PQ-pool is
an
reduced and a trans-membrane pH gradient starts to build (Stirbet and Govindjee, 2012).
M
2.3. Seeing is believing: Chlorophyll a fluorescence imaging
d
Chl a fluorescence measurement is on single point measurements on the leaf; but
te
heterogeneity in photosynthesis is frequent in the leaf, thus methodology may be source of errors. The development of Chl fluorescence imaging (CFI) overcomes this problem, still
Ac ce p
maintaining advantages of non-imaging Chl fluorescence (Gorbe and Calatayud, 2012). CFI is based on the same PAM technology and enables the achievement of identical fluorescence intensity levels as non-imaging PAM Chl fluorescence: F0, Fm, Fm’ and Fs (Nedbal and Whitmarsh, 2004).
Fluorescence imaging reveals a wide range of sample characteristics, including spatial variations due to differences in physiological states, which may frequently occur in stressed plants. Moreover, CFI can screen a large number of plants simultaneously, and it is rapid, non-destructive and relatively inexpensive.
7
Page 9 of 53
2.4. Relation between Chlorophyll a fluorescence and gas exchange: physiological implications The combination of gas exchange with Chl a fluorescence gives insights into
photosynthesis
parameters.
Both
techniques
indeed
ip t
photosynthetic regulation and provide opportunities to estimate a wide range of estimate
mesophyll
diffusion
cr
conductance (gm) (Bongi and Loreto, 1989; Evans and von Caemmerer, 1996), the relative
us
CO2/O2 specificity factor for Ribulose-1,5bis-phosphate carboxylase/oxygenase (Rubisco) (Peterson, 1989), as well as the proportion of photon flux density absorbed by photosynthetic
an
pigments channeled towards PSII (Makino et al., 2002). For detailed reviews on different parameters estimable through a combination of gas exchange and Chl fluorescence
d
M
measurements see Laisk and Loreto (1996), Laisk et al. (2002) and Yin et al. (2011).
3.1 High solar irradiance
te
3. Main abiotic stresses in Mediterranean climate
Ac ce p
Light provides power necessary for photosynthesis reactions, but in the Mediterranean area sunlight irradiance is often a stress agent (Flexas et al., 2013). The mechanisms by which - both at leaf or chloroplast level - Mediterranean plants respond to different light environments do not differ from those adopted by plants from other biomes and include (Takahashi and Badger, 2011): -
leaf and chloroplast movement to reduce absorption of excess light (Davis et al., 2011; Terashima et al., 2011);
-
light screening e.g. driven by phenolics in epidermal cells (Hernández and Van Breusegem, 2010; Agati et al., 2011; Hernández et al., 2011); 8
Page 10 of 53
-
up-regulation of antioxidant defences aimed at scavenging reactive oxygen species (ROS) (Ballottari et al., 2012; Murata et al., 2012)
-
thermal
dissipation of excess light energy (de Bianchi et al., 2010; Johnson and
-
ip t
Ruban, 2011; Johnson et al., 2011);
cyclic electron transport via PSII: appropriate rates of this pathway would ensure the
cr
maintenance of the transthylakoidal proton gradient required for photophosphorylation
us
and dissipation of the excess energy at PSII level. Defects in this pathway result in the increased sensitivity of PSII to photoinhibition (Takahashi et al., 2009; Rochaix, 2011;
-
an
Murata et al., 2012);
enhancement of photorespiration and/or Mehler pathway, in which oxygen represents
M
an alternative electron acceptor that prevents over-reduction and photo-inactivation of
d
PSII (Bauwe et al., 2010; Kangasjärvi et al., 2012).
te
Leaf plasticity is crucial for the acclimation of plants to different light conditions, as encountered in the Mediterranean area (Valladares et al., 2000). However, this matter is still
Ac ce p
controversial. Changes in leaf inclination have been observed in Mediterranean evergreen sclerophylls, thus contributing to maintain higher Fv/Fm ratio as compared with horizontal leaves (Werner et al., 2002). Vaz et al. (2011) reported that leaf structural plasticity in two oak species (Quercus ilex and Quercus suber) is a stronger determinant for acclimation to high light irradiance than biochemical and physiological plasticity. On the contrary, Valladares et al. (2002) reported that greater photo-tolerance is linked to enhanced physiological plasticity, mostly on photosynthesis-related traits. Mediterranean ecosystem is dominated by evergreen sclerophylls and malacophyllous species, which strongly differ in their physiological and structural adaptations to cope with 9
Page 11 of 53
light stress factors. Semi-deciduous species reduce foliage area, thus restricting their growth during favorable periods, thus avoiding photo-inhibition. In contrast, evergreen sclerophylls tolerate stress conditions with intact green leaves, by displaying a variety of physiological
ip t
adaptations, such as small and thick leaves, with very thick cuticles and deep root stems (Mooney, 1981). Evergreen schlerophylls have also several adaptive mechanisms, such as
cr
low photosynthetic rate effective stomatal control, thus reducing photosynthetic rate during
us
periods of excess light stress (e.g., water stress coupled with high sunlight irradiance during summer).
an
Carotenoids play a major role in photo-protection due to their capacity of deactivating triplet Chl (3Chl*) and singlet oxygen (1O2). Xanthophylls are involved either directly or
M
indirectly in non-photochemical quenching of excess light energy dissipated in PSII antenna.
d
Recent evidence suggests that the photoprotective role of lutein (Lut) is mostly due to
te
deactivation of 3Chl*; whereas zeaxanthin (Zea) is a major player in the deactivation of excited singlet 1Chl* and thus in NPQ. Nonetheless, the exact role of Zea in NPQ is still questionable
Ac ce p
(Horton et al., 2005; Avenson et al., 2008; Jahns and Holzwarth, 2012). It is known that Zea contributes to all qN mechanisms – with the exception of quenching associated with state transitions. Additionally, overwhelming evidence supports that Zea serves important functions as an antioxidant in the lipid phase of the membrane. The sustained depression of Fv/Fm to values near zero concomitant with the reduction of the photosynthetic capacity in highly Zea-accumulating over-wintering evergreens suggests that strong reduction of PSII efficiency does not always indicate undesirable photoinhibition, but may represent a powerful photoprotective mechanism to reduce ROS (Adams et al., 2006). These findings underline the important photoprotective role of Zea under a large 10
Page 12 of 53
variety of photo-oxidative and photo-inhibitory conditions (Jahns and Holzwarth, 2012). This role of xanthophyll is well documented also in Mediterranean species. Recently, three functional pools of Zea with very different roles in photo-protection in Quercus coccifera
ip t
(Peguero-Pina et al., 2013) have been identified: (i) a background pool that is essentially present at predawn in unquenched state; (ii) a second pool that increases after ca. 30-90 s
cr
following high light conditions and contributes strongly to NPQ; and (iii) a third pool that forms
us
on a longer time scale, and little affects NPQ. Authors suggest that ΔpH is crucial for NPQ induction and relaxation in Quercus coccifera during light transitions, but only a minor fraction
an
of Zea is associated with quenching. Zea has been proposed to participate in photo-protection behaving as an antioxidant.
M
In the Mediterranean area another potential environmental constraint for plants is
d
represented by high fluxes of UV-B radiation (McKenzie et al., 2007). An excess of UV
te
radiation (both UV-B and UV-A) can generate ROS, thus resulting in cellular damage (Jansen et al., 1998), unless plants have developed protective mechanisms against shortwave solar
Ac ce p
radiation. Leaves represent most sensitive organs to UV-B radiation, and several review articles have discussed about biochemical, morphological, and anatomical changes operating at leaf level in response to UV-B (Caldwell et al., 2007; Morales et al., 2010; Paul et al., 2012).
Realistic UV-B studies have little impact on plant growth (Ballaré et al., 2011). Fv/Fm and PSII were unaffected by high UV-B radiation (32-40 kJ m-2d-1) when sunlight irradiance exceeded 400-500 μmol m−2s−1 (Nogués and Baker, 1995). In a field UV-exclusion experiment, the performance of Ligustrum vulgare was mostly affected by visible than by UVwavelengths (Guidi et al. (2011) (Figure 1). 11
Page 13 of 53
It is likely that detrimental effects of UV-B radiation in Mediterranean areas occur when plants are concomitantly exposed to a wide range of stress agents, such as pollutants, nutrient deficiency, drought, high light, and excess soil salinity (Agrawal and Rathore, 2007;
ip t
Guidi et al., 2011; Verdaguer et al., 2012; Barnes et al., 2013). Mediterranean plants have developed effective mechanisms of protection against UV-B stress, such as their inherently
cr
high antioxidant system (Prado et al., 2012) and the biosynthesis of UV-absorbing
us
compounds, such as flavonoids (Agati and Tattini, 2010; Agati et al., 2011).
In conclusion, Mediterranean plants exhibit plastic responses to high sunlight at both
an
morphological and physiological levels. Acclimation to irradiance also involves changes in leaf chemical composition to reduce light absorption in leaves exposed to sunlight, and to
M
increase the excitation energy absorbed in shade leaves. All the adjustment observed enable
d
more efficient carbon assimilation in plants exposed to sunshine, while shaded leaves
te
increase the surface area and nitrogen investment for light capture. Different factors play a decisive role including: (i) leaf position: leaves in a southern part of the canopy that receive
Ac ce p
plenty of sunlight are the most photo-inhibited; (ii) the season: Mediterranean plants suffer photo-inhibition during clear sunny days; and (iii) inter/intra-specific differences. In general, evergreen sclerophyllous Mediterranean species are characterized by a high tolerance towards excessive sunlight and physiological changes play the preeminent role. A dynamic short-term photo-inhibition occurs and results in a reversible diurnal decline in F v/Fm. This dcline frequently is accompanied by a pronounced increase in Zea which is able to mediate the de-excitation process of excited Chl from the PSII reaction centers – and probably protect thylakoid membranes from the negative effects of accumulated excitation energy. Semi-
12
Page 14 of 53
deciduous Mediterranean species show a different strategy with pronounced structural changes aimed at reducing light interception (Niinemets, 2010).
ip t
3.2 Temperature stress
Extremes in high temperature and drought are considered major constraints for plants
cr
during the Mediterranean summer. However, in mountainous areas or/and in Mediterranean
us
continental areas, cold or freezing temperatures in winter may also impact plant survival and growth.
an
Broadly, high temperatures affect the photosynthetic apparatus through inactivation of photosystems and molecular alterations of thylakoid membranes, and decreasing CO 2
M
assimilation and electron transport rate Photosynthesis is highly sensitive to high
d
temperature, and PSII is considered as the most heat-sensitive component of the
te
photosynthetic apparatus (Berry and Bjorkman, 1980; Dias et al., 2011). Consequently, reductions in Fv/Fm in Mediterranean species frequently occur during summer. Studies in
Ac ce p
three dominant species typical of Mediterranean shrub land during seven years (Erica multiflora, Globularia alypum and Pinus halepensis) showed decreases in Fv/Fm ratio up to 0.67-0.69 indicating restriction in carbon gain (Prieto et al., 2009). Similar results were observed in Quercus ilex and Phyllyrea latifolia (Ogaya et al., 2011) in which Fv/Fm values were highly dependent on both air temperature and genotypes: F v/Fm ratio was higher in Phyllirea latifolia than in Quercus ilex. These findings conform to Q. ilex and Phillyrea latifolia typically inhabiting sub-humid or dry Mediterranean areas, respectively. Studies on the effects of high temperatures are performed on Ceratonia siliqua, a Mediterranean sclerophyllous evergreen tree (Osório et al., 2011). Plants were subjected to two thermal regimes: moderate 13
Page 15 of 53
(25/18ºC) or high (32/21ºC) temperatures. The reduction in photosynthetic rate was 33% at 25/18º C whereas at higher temperatures it was 84%. In spite of this, the electron transport rate was not affected suggesting that non-regulated quenching mechanisms (e.g..
ip t
photorespiration and/or Mehler reaction) tend to down-regulate PSII activity photoprotecting it under high temperature, with the xanthophyll cycle-mediated thermal dissipation playing
cr
possibly a much less important role.
us
An interesting study case of the mechanisms of photoprotection operating in Quercus suber, Quercus ilex, Olea europaea and Eucalyptus globulus during summer has been
an
conducted by Faria et al. (1998) in Portugal. Authors reported that stomatal closure mostly controlled daily variations in carbon assimilation over the whole-period. Nonetheless, decline
M
in photochemical yield of photosystem II (PSII) was observed as plants suffered from severe
d
excess light stress. This resulted from high temperature-induced stomatal closure, which in
te
turn strongly depressed net carbon gain. Consistently, all species displayed an increased need of thermal dissipation of excess photon energy, which was sustained by stimulation of
Ac ce p
xanthophyll cycle. Net photosynthesis was lowest in Olea europaea: this was paralleled with highest both carotenoid to chlorophyll ratio and rates of violaxanthin de-epoxidation. This allowed in an increase in thermal dissipation of the excess radiant energy, but led to rather small values of light utilization in photochemistry (it averaged from 31% in the morning and 29% in the evening). In contrast, in Eucalyptus globulus photosynthetic rates were highest, thermal dissipation of absorbed radiation lowest and, values of light utilization in photochemistry were approximately 60%. These findings show from one hand, highly coordinated response mechanisms involving stomatal control photosynthetic capacity, and biochemical adjustments and from the other hand largely different physiological-biochemical 14
Page 16 of 53
responses to the same environmental constrain adopted by Mediterranean plants. This is in some relation with the high level of biodiversity usually observed in Mediterranean ecosystems.
ip t
Photochemical efficiency of PSII attributable to an impairment of LHC (Oliveira and Peñuelas, 2000) can also occur in forest vegetation in continental Mediterranean areas, as
cr
usually observed during winter. Photo-inhibition is particularly severe in sunny days following
us
overnight frosts, as frequently occurs during Mediterranean winter. Garcia-Plazaola et al. (1999) reported three different strategies to cope with the winter stress in Mediterranean
an
evergreens. The first showed by some Mediterranean sclerophyllus in which reversible reductions of PSII efficiency (i.e. reduction in Fv/Fm ratio) related to the activity of the
M
xanthophyll cycle allowed to an adaptation of the photosynthetic apparatus to winter stress.
d
Among these species, Q. ilex showed the higher ability to adapt its photochemical efficiency
te
and this can be explained by its wide altitudinal distribution. A second type of photo-protection is showed by malacophyllous semideciduous Cistus species which maintained a higher
Ac ce p
photosynthetic rate dissipating a large amount of excitation energy by carbon assimilation and very low down-regulation of PSII efficiency under moderate stress conditions. Under more stressful conditions, these species lost their leaves as reported also by Oliveira and Peñuelas (2004). Finally, the third way of adaptation is showed by Boxus sempervirens that contains high amount of ascorbate and tocopherol and a high carotenoid/Chl and Zea/Chl ratios. In Boxus photochemical efficiency of PSII was highly reduced because of the loss of D1 protein and/or the retention of de-epoxidized xanthophylls. In Mediterranean plants, response mechanisms to low or high temperatures do not substantially differ. Silva-Cancino et al. (2012) used CFI to analyze species-specific 15
Page 17 of 53
responses to cope with winter stress in evergreens: Buxus semprevirens, Cistus albidus and Arctostaphylus uva-ursi. In Buxus semprevirens a decrease in Fv/Fm over winter, was accompanied by a massive accumulation of red pigments in sub epidermal cells. Recovery
ip t
from winter photo-inhibition was characterized by leaf re-greening and decrease in VAZ pool pigments (Silva-Cancino et al., 2012). Photosynthetic decreases, as well as reductions in
cr
photochemical PSII activity at extreme temperatures, are often regulatory mechanisms
us
associated with photo-protective strategies that avoid irreversible damage to photosystem by so-called ‘dynamic’ photo-inhibition. These strategies may be reversed when environmental
an
conditions become favorable for growth (Larcher, 2000).
M
3.3 Water stress
d
The scarcity of soil water is likely the major environmental constrain for plants inhabiting
te
Mediterranean areas. Photosynthetic CO2 assimilation in Mediterranean species is impaired by drought in different ways, but stomatal closure represents is primarily involved in drought-
Ac ce p
induced depression of carbon assimilation (Chaves et al., 2002). Mesophyll conductance to CO2 (gm) has been reported that in drought-adapted species diffusional limitations account for most of the observed water stress-induced depression in photosynthesis. gm has long been reported to depend on leaf and cell anatomy (Laisk et al., 1970; Nobel et al., 1975), but recently aquaporins have been reported to greatly affect gm. Aquaporins effectively regulate CO2, not only water diffusion through membranes (Heckwolf et al., 2011). Details about the key role of gm in photosynthesis have been recently reviewed (Flexas et al., 2012; Griffiths and Helliker, 2013).
16
Page 18 of 53
Overall, Mediterranean evergreens display low responsiveness to environmental changes: they have phenotypic stability and conservative resource-use strategy (Valladares et al., 2000). Conversely, Quercus coccifera and Pinus halepensis show a high plastic
ip t
response of gas exchange to drought, in terms of stomatal control, and hence in net CO2 assimilation and transpiration rates. However, these species have low plasticity of leaf
cr
photochemistry. Photochemical efficiency was little affected during drought stress, as
us
physiological and biochemical features, including photo-respiration, pigments and antioxidant defenses mostly varied during stress episodes (Baquedano et al., 2008).
an
As already mentioned, drought stress occurs in concomitance with high sunlight irradiance during summer in Mediterranean areas. Balancing light capture and energy use is
M
therefore a great challenge for Mediterranean plants under drought stress, with dramatic
d
consequences on the performance of the photosynthetic (Chaves et al., 2009). Drought-
te
induced restriction of CO2 assimilation ultimately results in a massive accumulation of ROS and, hence, in oxidative damage. However, Mediterranean plants have evolved strategies
Ac ce p
and are equipped with photoprotective mechanisms such as xanthophyll cycle pigments (Munné-Bosch and Peñuelas, 2003; Galmes et al., 2007) and antioxidants (enzymatic and non-enzymatic) (Munné-Bosch and Peñuelas, 2003; Melgar et al., 2009). Sunlight irradiance strongly modulates plant responses to water deficit (Fini et al., 2012). Guidi et al. (2008) have shown that in Ligustrum vulgare under mild drought, net photosynthesis decreased more in plants growing under full sun than in plants growing under partial shading, as consequence of greater reductions in Fv/Fm, PSII and gs. Nonetheless, oxidative stress and damage were lower in sun than in shade plants, as in sun plants antioxidant defenses were more active than in shade plants. 17
Page 19 of 53
A drought-avoidance strategy has been identified as a general feature of evergreen oaks inhabiting xeric sites and is characterized by low leaf area per shoot volume ratio, strong stomatal control, moderate leaf osmotic potential, and high relative water content. Deciduous
ip t
species usually show a drought-tolerant behavior mediated by phenol-morphological features such as short leaf lifetimes, or leaf pubescence (Oliveira and Peñuelas, 2004). A comparative
cr
study in Quercus ilex (evergreen) vs Q. robur (deciduous) revealed different photosynthetic
us
strategies to cope with severe water stress (Koller et al., 2013). Both oak taxa decreased CO 2 assimilation rate at saturating light due to stomatal closure and Fv/Fm ratio decreased
an
significantly only in Q. ilex while remained unaffected in Q. robur. Chl fluorescence measurements shown that in the former species there were a high amount of non-functional
M
PSII reaction centers not involved in electron transport but that dissipated excitation energy
d
independently from the xanthophyll cycle (no changes in NPQ). Conversely, in Q. robur the
te
unaffected photoactive reaction centers under water stress together with the strongly decreased in photosynthetic rate enforced the strong decrease in qP and the increase in NPQ
Ac ce p
under actinic light. In this species additionally and/or alternatively, dissociation of PSII antennae from the reaction centers and emission of absorbed light through heat dissipation by the antenna carotenoids could represent an option to release electron pressure in the electron transport chain.
In conclusion, the alterations that occur in response to drought in Mediterranean plants include the restriction of CO2 diffusion to the chloroplast, as well as metabolic changes, and the modulation of expression of photosynthesis-related genes. However, the involvement of the different mechanisms is strictly related to the experimental conditions, the different species and the severity of imposed stress. 18
Page 20 of 53
3.4. Salinity Soil and water salinity are major environmental challenges for Mediterranean plants. Salinity detrimentally affects the performance of plants through potential toxicity driven by
ip t
specific ions and a general constrain due to osmotic unbalance (Munns and Tester, 2008). High concentration of salts in the soil reduces a plant’s ability to extract water from the soil
cr
thus resulting in water stress.
us
Physiological and biochemical processes fundamental for plant metabolism, such as photosynthesis and respiration, are highly sensitive to excess of salts (Munns, 1993; Stepien
an
and Johnson, 2009; Duarte et al., 2013).Early responses to salinity do not markedly differ from responses to drought stress (Munns, 2002), particularly in Mediterranean plants (Chaves
M
et al., 2009).
d
Biomass reduction (Degl’Innocenti et al., 2009; Jaoudé et al., 2012; Cocozza et al.,
te
2013), the first visible effect induced by salt stress, is caused by the increased energy consumption for various tolerance mechanisms, e.g. for synthesizing compatible organic
Ac ce p
solutes and proteins (Gucci and Tattini, 1997; Ben Hassine et al., 2009; Chen and Jiang, 2010; Rajavindran and Natarajan, 2012; Gill et al., 2013). Notably, salt tolerance greatly differs in co-occurring Mediterranean plants. Three Mediterranean evergreens differ greatly in their strategies for salt allocation: the ‘saltexcluders’ Olea europaea and Phyllirea latifolia (both Oleaceae); and Pistacia lentiscus, which, instead, mostly uses Na+ and Cl- for osmotic adjustment. Both Oleaceae spp. undergo severe leaf dehydration, and reduce net photosynthesis and whole-plant growth to a significantly greater degree than Pistacia lentiscus (Tattini et al., 2009). These contrasting strategies to manage the allocation of potentially toxic ions in sensitive leaf organs were 19
Page 21 of 53
reflected in markedly different biochemical adjustments. These included the activation of violaxanthin cycle pigments and antioxidant defences, aimed at avoiding and countering saltinduced reductions in the use of radiant energy to photosynthesis. In salt-excluding O.
ip t
europaea and P. latifolia leaf biochemistry was greatly altered as compared with P. lentiscus to minimize oxidative load (Tattini et al., 2009).
cr
Olea europea, is tolerant to salinity stress (Therios and Misopolinos, 1988; Gucci and
us
Tattini, 1997). Survival and not growth performance follows response mechanisms operating in olive plants in response to salinity stress, as occurs in most Mediterranean (Munns and
an
Tester, 2008; Cimato et al., 2010). These mechanisms are based mostly in reducing transpiration and hence water mass flow-transport of potentially toxic ions to sensitive shoot
M
organs. This imposes severe leaf dehydration and steep reduction in gas exchange
d
performance during salt stress, but preserves young leaves from massive accumulation of
te
toxic ions. Interestingly, reduction in transpiration rate and CO2 photo-assimilation are adaptive mechanisms to salinity stress in olive and other salt-excluding evergreens (Tattini et
Ac ce p
al., 2002; Cimato et al., 2010).
During drought in the Mediterranean area, salinity stress occurs in concomitance with high sunlight irradiance (Chaves et al., 2003). In olive, Melgar et al. (2009) found that sunexposed leaves had higher Na+, Cl− and mannitol content than shaded leaves. The high concentration of VAZ-cycle pigments in sun-exposed leaves suggests that Zea may protect the chloroplast from photo-oxidative damage through ROS scavenging rather than dissipating excess excitation energy via NPQ. In fact recently it has been reported that Zea plays a significant role as chloroplastic antioxidant, specifically protecting highly unsaturated chloroplast membranes from photo-oxidation (Havaux and Niyogi, 1999; Havaux et al., 2000). 20
Page 22 of 53
Given that most of the light-related increase in the VAZ pool resulted in increases in the ‘free’ VAZ, membrane protection from peroxidation may be the primary role of light-related enhancements of VAZ pool size. Beside zeaxanthin playing a role in NPQ and acting as an
ip t
antioxidant, the xanthophyll cycle may also be involved in the protection of plants from stress (Havaux and Tardy, 1996). In addition, the results obtained from Melgar et al. (2009) suggest
cr
that when olive was subjected to salinity under partial shading, the antioxidant defense
us
system may be ineffective to counter salt-induced oxidative damage (Remorini et al., 2009). Interestingly, Melgar et al. (2009) found a steep decline in midday Fv/Fm because of high
an
sunlight in control plants (from 0.826 in shade to 0.794 in sun-exposed leaves), whereas the midday Fv/Fm value was greater in salt-treated sun-exposed leaves (0.822) than in
M
corresponding shaded leaves (0.808) (Figure 2). The authors found also a light-induced
d
increase in the carotenoid to Chltot ratio, which mostly regarded the relative concentration of
te
violaxanthin-cycle pigments and concluded that xanthophyll in addition to their contribute to protect PSII photochemistry from irreversible photo-damage via NP quenching, likely served
Ac ce p
important antioxidant functions. Zeaxanthin is unlikely to participate in the thermal dissipation of excess excitation energy in the chloroplast, when the concentration of violaxanthin-cycle pigment exceeds 50 mmol mol−1 Chltot, as suggested by Havaux and Niyogi (1999). To conclude salinity represent a serious constrain in Mediterranean species that show integrated anatomical, physiological and biochemical adjustment to survive in this environment. Photosynthesis is certainly the principal target of salinity and Mediterranean species showed different mechanisms aimed to contrast the salt-induced oxidative load through photo-protective mechanisms.
21
Page 23 of 53
3.5. Ozone Ozone (O3) is one of the most important pollutants in the Mediterranean area, the concentration of which increases because of high sunlight irradiance high temperatures, and
ip t
recirculation processes of polluted air masses (Sanz et al., 2007). As a consequence, the Mediterranean area suffers from critical level of this photochemical oxidant. In fact, levels of
us
protection (Gimeno et al., 1999; Calatayud and Bareno, 2001).
cr
O3 during spring and summer exceed UN-ECE critical level guidelines for vegetation
Ozone affects growth, photosynthetic performance and induces oxidative damage in
an
Mediterranean plants (Ribas et al., 2005; Velikova et al., 2005; Bussotti, 2008) even though evergreen broadleaves seems to be quite O3-tolerant (Manes et al., 1998; Calatayud et al.,
M
2010). Indeed, Pinus halepensis and Pinus pinea are sensitive to O3 (Nali et al., 2004).
d
Furthermore, intra-specific variability in O3-sensitivity has been frequently observed.
te
Differential response of three cabbage varieties to O3 has been reported (Table 1; Calatayud et al., 2002). However, Bussotti et al. (2011) analyzing the effect of O3 on Fv/Fm in woody
Ac ce p
plants, found that O3 did not affect Fv/Fm in 48% of examined experiments. Furtheremore, Calatayud et al. (2007) observed no significant effects of ozone on Fv/Fm in four maple species (Acer campestre, A. opalus, A. monspessulanum and A. pseudoplatanus), which are important components in humid Mediterranean regions, A. pseudoplatanus and A. opalus were most sensitive as revealed by O3-induced reductions in PSII and in the quantum efficiency of excitation capture by oxidized reaction centers in PSII (exc) and in qP.. These findings were consistent with a decrease in CO2 assimilation because of ozone treatment. (Calatayud et al., 2007). Studies in Pinus uncinata, the dominant specie in subalpine Pyrenees forest (Díaz-de-Quijano et al., 2012), did not detect significant changes in Fv/Fm 22
Page 24 of 53
ratio and Chl content under 1.8 x O3 ambient. Changes conversely were observed on photosynthetic rate and stomatal conductance, higher in O 3-treated plants as compared to the controls. Thus, the surplus of CO2 photoassimilation is supposed to represent an investment
ip t
of the assimilate carbon towards the pathways for the synthesis of antioxidants (Diaz-deQujano et al., 2012).
cr
It is known as a large part of diffusive resistance to O3 relies in the stomata and that
us
the different sensitivity to this pollutant could be explained by differences in leaf conductance (Reich, 1987). Stomata are also the way by which O3 entries into plants and so usually an O3-
an
induced reduction in stomata is significantly coupled with a reduction in CO2 (Weber et al., 1993; Clark et al., 1996).
M
It is difficult to compare the results from various research studies; in fact, plant
d
response depends on experimental conditions, age of the leaves and heterogeneity inside the
te
leaf, evergreen vs deciduous species and sensitivity to air pollutants. For example, Quercus ilex is tolerant to on both short-term and long-term basis. (Velikova et al., 2005; Calatayud et
Ac ce p
al., 2011; Mereu et al., 2011). All authors did found negligible effects of ozone on gas exchange and Chl fluorescence parameters. Nonetheless, aged leaves were more sensitive than one-year-old leaves, as the result of lower stomatal conductance and hence reduced O3 uptake (Velikova et al., 2005). Significant reduction in photosynthetic rate, stomatal conductance, Rubisco carboxylation efficiency, ribulose-1.5-bisphosphate regeneration capacity, Fv/Fm, PSII and qP were instead observed in deciduous Mediterranean species, such Pistacia terebinthus and Viburnum lantana. In contrast evergreen relatives, i.e. Pistacia lentiscus and Viburnum tinus were tolerant to ozone (Calatayud et al., 2010). Ozonetolerance in evergreen species was mostly related with the activation of antioxidant defenses, 23
Page 25 of 53
possibly as a consequence of minor reduction in gas exchange performance as compared with their deciduous relatives (Fini et al., 2012). Plant species and cultivars exhibit a wide range of O3 sensitivity evident a s heritable
ip t
characteristics related to identifiable metabolic and molecular processes that affect sensitivity to O3 (Fiscus et al., 2005). Overall, plant response to O3 seems mostly mediated by stomatal
cr
conductance (see Fiscus et al., 2005) and the mechanisms that determine plants sensitivity or
us
tolerance are not clearly understood. However, plants with similar gs largely differ in O3tolerances (Calatayud et al., 2010). It has been suggested that an integrated network of
an
antioxidant defenses, which includes antioxidant enzymes and low molecular-weight antioxidants (ascorbate, glutathione or -tocopherol), not only stomatal aperture contribute to
M
ozone tolerance in a wide range of species (Nali et al., 2004).
d
Tolerance to ozone in Mediterranean evergreens has been suggested to depend on
te
their sclerophylly, which translates into low gas exchange capacity, but also on a ‘constitutively’ effective pool of antioxidant defenses (Manes et al., 1998; Nali et al., 2004). It
Ac ce p
has been reported that more than one mechanism is involved in the tolerance to O 3 (Guidi et al., 2010). Such tolerance might overlap with their level of tolerance to other environmental constraints in Mediterranean area such as for example water stress and is related to the high constitutive levels, and/or O3-induced increases in antioxidants.
3.6 Climate change Models of global climate change predict a further 1.8-4°C warming by 2100 (Mc Arthy et al., 2001) and changing patterns of rainfall as a result of rising atmospheric CO 2 concentration and other greenhouse gases. Climate change could be particularly important in 24
Page 26 of 53
the Mediterranean basin (Christensen et al., 2007), affecting vegetation structure and plant productivity. Elevated CO2 concentration, increases in air temperature, and drought represent the main environmental constraints on crop and forest ecosystems. A prediction for the
ip t
effects of climate change on plants takes into account of these variables as well as their interactions. However, such a comprehensive picture for the effects of multiple stresses on
cr
plants, including those inhabiting the Mediterranean area has not been rarely drawn. In Pinus
us
taeda (Wertin et al., 2010) the combined effect of elevated CO2 (700 nL L-1), air temperature (+2ºC above air temperature), and water stress (25% respect to well watered conditions ) did
an
not affect photosynthesis and Fv/Fm while steeply decreasing the gs (-60%). WUE increased markedly, conforming to current model in which CO2 decreases stomatal conductance with
M
little effect on CO2 assimilation rate.
d
In general terms, combined drought and elevated temperature had more detrimental
te
effects than either stress alone under ambient CO2 concentration. Elevated CO2 mitigated the degree of change in photosynthesis parameters under heat stress and drought, at least for
Ac ce p
short-term exposures where photosynthetic activity can be stimulated or maintained. Photosynthetic down-regulation has been proposed to explain the acclimation to climate change (see in Aranjuelo et al., 2005; Erice et al., 2006) under middle and long-term exposure. Down-regulation can be caused by non-stomatal and/or stomatal limitations. Nonstomatal limitations are proposed to explain the decrease in PSII efficiency as a result of the accumulation of inactive PSII reaction centres, and the decrease in LHCs (Kalina et al., 1997) and/or a reduced carboxylation efficiency (Long et al, 2004), or a reduced amount/activity of Rubisco. Moreover, safe energy dissipation mediated by non-photochemical processes modulated by xanthophylls cycle has occurred and this is combined with an effective 25
Page 27 of 53
antioxidant system. Stomatal limitations explain the decrease in photosynthesis because the depletion of the intercellular CO2 concentration. The protecting effect of elevated CO2 against water stress under high temperature is associated with mechanisms for maintaining higher
ip t
relative water content and turgor potential with a higher WUE and osmotic adjustment (Tuba and Lichtenthaler 2007). Many other studies suggest that photosynthetic down-regulation
cr
results from an insufficient sink capacity (Ainsworth et al., 2004). Plants grown under elevated
us
CO2 concentration have a greater carbohydrate content in leaves than leaves of plants grown under ambient CO2 (Geiger et al., 1999). The carbohydrates accumulate and the gene
an
expression for photosynthetic apparatus is suppressed through the possible increase in hexose cycling within the leaves – resulting in decreased photosynthetic capacity and a
M
notable decrease in the amount of Rubisco (Erice et al., 2006).
d
The down-regulated photosynthetic response to climate change depends on genetic,
te
relative availability of the other co-limiting environmental factors, and on the degree of pressure of each stress (CO2, water, and temperature) on the ecosystem (Aranjuelo et al.,
Ac ce p
2005). Plant adaptation to climate change will depend on the buffer capacity to maintain metabolic functions and the interaction between the soil-plant-atmosphere system and this constitutes a complex network.
4. Conclusions
The Mediterranean ecosystem is complex and many abiotic environmental factors can affect plant species simultaneously. The large number of rare species that have survived for tens to hundreds of thousands of years enables us to accumulate knowledge on the physiological, cellular, and molecular responses of plants to these environmental constraints 26
Page 28 of 53
(drought, salinity, high light, high temperature, etc.) including the signaling of events occurring under these stresses. To further understand the complexity of plant response to stresses in the Mediterranean area, including the effects on photosynthesis, it is necessary to strengthen
ip t
multilevel genomic and physiological studies, and cover the differing intensities and timing of the imposition of stresses in genotypes with differing sensitivities to stress.
cr
Clearly, many difficulties occur in these studies. In addition to the difficulties posed by
us
the experimental conditions (length of time exposure, field as compared to greenhouse conditions, species utilized, etc.) there is the added difficulty of studying the interaction among
an
different stresses. Drought and salinity represent the most important stresses in the Mediterranean area but plant response is also linked to the intensity and quality of light.
M
Photosynthesis is limited in Mediterranean plant species by environmental constraints
d
either direct (as the diffusion limitations through the stomata and the mesophyll and the
te
alterations in photosynthetic metabolism) or secondary, such as the oxidative stress arising from the superimposition of multiple stresses. Gas exchange and chlorophyll fluorescence
Ac ce p
permit to collect a large pool of data regarding photosynthetic process in Mediterranean species subjected to many environmental stresses. These non-invasive methodologies give important information on the functionality of photosynthetic apparatus that combines with other physiological, biochemical and molecular studies. Furthermore, they sometimes permit, to understand the mechanisms on the basis of the plant response. In spite of the reliability of Chl fluorescence and gas exchange techniques for early stress diagnostic, they have some weakness: there is a variety of equipment and differing measuring protocols, various stress-dose applications and parameters make comparison among experiments difficult. These discordances should be overcome by standardizing and 27
Page 29 of 53
defining common protocols so that the experiments can be reproducible and compared. In addition, in field experiments it is difficult to discover the type of stress that is affecting the plant through Chl fluorescence and gas exchange. In our opinion, the effects induced by
ip t
abiotic stresses on Mediterranean plants are similar to those induced in plants from other biomes.
cr
In view of above, Chl fluorescence and gas exchange are useful approaches but until
us
now they have not suitable for understanding the mechanisms on the basis of plant
an
responses in Mediterranean area.
Acknowledgments
M
The authors thank Dr. Marco Landi and phD-Student Consuelo Penella for their contribution
d
in this review. This work has been financed by INIA (Spain) through project RTA2010-00038-
Ac ce p
(Fondi di Ateneo 2007-2012).
te
C03-01 and the European Regional Development Fund (ERDF) and by University of Pisa
References
Adams, W.W.III, Demmig-Adams, B., 2004. Chlorophyll fluorescence as a tool to monitor plant response to the environment. In: Papageorgiou, G.C., Govindjee (Eds.), Chlorophyll a Fluorescence: A Probe of Photosynthesis. Springer, Dordrecht, pp. 583604. Agati, G., Biricolti, S., Guidi, L., Ferrini, F., Fini, A., Tattini, M., 2011. The biosynthesis of flavonoids is enhanced similarly by UV radiation and root zone salinity in L. vulgare leaves. Journal of Plant Physiology 168, 204-212. 28
Page 30 of 53
Agati, G., Tattini, M., 2010. Multiple functional roles of flavonoids in photoprotection. New Phytologist 186, 786-793. Agrawal, S.B., Rathore, D., 2007. Changes in oxidative stress defense system in wheat
ip t
(Triticum aestivum L.) and mung bean (Vigna radiata L.) cultivars grown with and without mineral nutrients and irradiated with supplemental ultraviolet-B. Environmental and
cr
Experimental Botany 59, 21-33.
us
Ainsworth, E.A., Rogers, A., Nelson, R., Long, S.P., 2004. Testing the ‘source-sink’ hypothesis of down-regulation of photosynthesis in elevated [CO2] in the field with a
an
single gene substitution in Glycine max. Agricultural and Forest Meteorology 122, 85-94. Aranjuelo, I., Pérez, P., Hernández, L., Irigoyen, J.J., Zita, C., Martínez-Carrasco, R.,
M
Sánchez Díaz, M., 2005. The response of nodulated alfalfa to water supply,
te
123, 348-358
d
temperature, and elevated CO2: photosynthetic down regulation. Physiologia Plantarum
Avenson, T.J., Ahn, T.K., Zigmantas, D., Niyogi, K.K., Li, Z., Ballottari, M., Bassi, R., Fleming,
Ac ce p
G.R., 2008. Zeaxanthin radical cation formation in minor light-harvesting complexes of higher plant antenna. Journal of Biological Chemistry 283, 3550-3558. Ballaré, C.L., Caldwell, M.M., Flint, S.D., Robinson, S.A., Bornman, J.F., 2011. Effects of solar ultraviolet radiation on terrestrial ecosystems. Patterns, mechanisms, and interactions with climate change. Photochemical and Photobiological Science 10, 226241. Ballottari, M., Girardon, J., Dall'Osto, L., Bassi, R., 2012. Evolution and functional properties of Photosystem II light harvesting complexes in eukaryotes. Biochimica et Biophysica Acta (BBA) - Bioenergetic 1817, 143-157. 29
Page 31 of 53
Baquedano, F.J., Valladares, F., Castillo, F.J., 2008. Phenotypic plasticity blurs ecotypic divergence in the response of Quercus coccifera and Pinus halepensis to water stress. European Journal of Forest Research 127, 495-506.
ip t
Barnes, P.W., Kersting, A.R. Flint, S.D., Beyschlag, W., Ryel, R.J., 2013. Adjustments in epidermal UV-transmittance of leaves in sun-shade transitions. Physiologia Plantarum
cr
149, 200-213.
us
Bauwe, H., Hagemann, M., Fernie, A.R., 2010. Photorespiration: players, partners and origin. Trends in Plant Science 15, 330-336.
an
Ben Hassine, A., Ghanem, M.E., Bouzid, S., Lutts, S., 2009. Abscisic acid has contrasting effects on salt excretion and polyamine concentrations of an inland and a coastal
M
population of the Mediterranean xero-halophyte species Atriplex halimus. Annals of
d
Botany 104, 925-936.
te
Berry, J., Bjorkman, O., 1980. Photosynthetic response and adaptation to temperature in higher plants. Annual Review of Plant Physiology 31, 491-543,
Ac ce p
Bilger, W., Björkman, O., 1991. Temperature dependence of violaxanthin de-epoxidation and non-photochemical fluorescence quenching in intact leaves of Gossypium hirsutum L. and Malva parviflora L. Planta 184, 226-234. Bongi, G., Loreto, F., 1989. Gas-exchange properties of salted stressed olive (Olea europea L.) leaves. Plant Physiology 90, 1408-1416. Bussotti, F. 2008. Functional leaf traits, plant communities and acclimation processes in relation to oxidative stress in trees: a critical overview. Global Change Biology 14, 27272739.
30
Page 32 of 53
Bussotti, F., Desotgiu, R., Cascio, C., Pollastrini, M., Gravano, E., Gerosa, G., Marzuoli, R., Nali, C., Lorenzini, G., Salvatori, E., Manes, F., Schaub, M., Strasser, R.J., 2011. Ozone stress in woody plants assessed with chlorophyll a fluorescence. A critical reassessment
ip t
of existing data. Environmental and Experimental Botany 73, 19-30.
Calatayud, A., Alvarado, J.W., Barreno, E., 2002. Differences in ozone sensitivity in three
cr
varieties of cabbage (Brassica oleracea L.) in the rural Mediterranean area. Journal
us
Plant Physiology 159, 863-868.
Calatayud, A., Barreno, E., 2001. Chlorophyll a fluorescence, antioxidant enzymes and lipid
an
peroxidation in tomato in response to ozone and benomyl. Environmental Pollution 115, 283-289.
M
Calatayud, V., Cerveró, J., Calvo, E., García-Breijo, F.-J., Reig-Armiñana, J., Sanz, M.J.,
d
2011. Responses of evergreen and deciduous Quercus species to enhanced ozone
te
levels. Environmental Pollution 159, 55–63. Calatayud, V., Cerveró, J., Sanz, M.J., 2007. Foliar, physiological and growth responses of
Ac ce p
four maple species exposed to ozone. Water, Air and Soil Pollution 185, 239-254. Calatayud, V., Marco, F., Cerveró, J., Sánchez-Peña, G., Sanz, M.J., 2010. Contrasting ozone sensitivity in related evergreen and deciduous shrubs. Environmental Pollution 158, 3580–3587.
Caldwell, M.M., Bornman, J.F., Ballaré, C.L., Flint, S.D., Kulandaivelu, G., 2007. Terrestrial ecosystems, increased solar ultraviolet radiation, and interactions with other climate change factors. Photochemical and Photobiological Science 6, 252-266.
31
Page 33 of 53
Chaves, M M., Pereira, J.S., Maroco, J., Rodrigues, M.L., Ricardo, C.P.P.,. Osório, M.L., Carvalho, I., Faria, T., Pinheiro, C., 2002. How plants cope with water stress in the field? Photosynthesis and growth. Annals of Botany 89, 907-916.
ip t
Chaves, M.M., Flexas, J., Pinheiro, C., 2009. Photosynthesis under drought and salt stress: regulation mechanisms from whole plant to cell. Annals of Botany 103, 551-560.
cr
Chaves, M.M., Maroco, J., Pereira, J.S., 2003. Understanding plant responses to drought –
us
from genes to whole plant. Functional Plant Biology 30, 239-264.
Chen, H., Jiang, J.-G., 2010. Osmotic adjustment and plant adaptation to environmental
an
changes related to drought and salinity. Environmental Reviews 18(NA), 309-319. Christensen, J., Carter, T., Rummukainen, M., Amanatidis, G., 2007. Evaluating the
M
performance and utility of regional climate models: the PRUDENCE project. Climatic
d
Change 81, 1–6.
te
Cimato, A., Castelli, S., Tattini, M., Traversi, M.L., 2010. An ecophysiological analysis of salinity tolerance in olive. Environmental and Experimental Botany 68, 214-221.
Ac ce p
Clark, C.S., Weber, J.A., Lee, E.H., Hogsett, W.E. 1996. Reductions in gas exchange of Populus tremuloides caused by leaf ageing and ozone exposure. Canadian Journal of Forest Research 26 1384-1391. Cocozza, C., Pulvento, C., Lavini, A., Riccardi, M., d'Andria, R., Tognetti, R., 2013. Effects of increasing salinity stress and decreasing water availability on ecophysiological traits of quinoa (Chenopodium quinoa Willd.) grown in a Mediterranean-type agroecosystem. Journal of Agronomy and Crop Science (in press). DOI: 10.1111/jac.12012
32
Page 34 of 53
Davis, P.A., Caylor, S., Whippo, C.W., Hangarter R.P., 2011. Changes in leaf optical properties associated with light-dependent chloroplast movements. Plant, Cell & Environment 34, 2047-2059.
ip t
de Bianchi, S., Ballottari, M., Dall’Osto, L., Bassi, R., 2010. Regulation of plant light harvesting by thermal dissipation of excess energy. Biochemical Society Transaction 38, 651-660.
cr
De Lillis, M., 1991. An ecomorphological study of the evergreen leaf. Braun-Blanquetia 7: 1-
us
127.
Degl’Innocenti, E., Hafsi, C., Guidi, L., Navari-Izzo, F., 2009. The effect of salinity on
an
photosynthetic activity in potassium-deficient barley species. Journal of Plant Physiology 166, 1968-1981.
M
Adams, W.W. , Zarter, C.R., Mueh, K.E., Amiard, V., Demmig-Adams, B., 2006. Energy
d
dissipation and photoinhibition: a continuum of photoprotection. In: Demmig-Adams, B.,
te
Adams, W.W., Mattoo, A.K. (Ed.), Photoprotection, Photoinhibition, Gene Regulation, and Environment. Springer, Dordrecht, pp. 49-64.
Ac ce p
Díaz-de-Quijano, M., Schaub, M., Bassin, S., Volk, M., Peñuelas, J., 2012. Ozone visible symptoms and reduced root biomass in the subalpine species Pinus uncinata after two years of free-air ozone fumigation. Environmental Pollution 169, 250-257. Dias, A.S., Semedo, J., Ramalho, J.C., Lidon, F.C., 2011. Bread and durum wheat under heat stress: a comparative study on the photosynthetic performance. Journal of Agronomy and Crop Science 197, 50-56. Duarte, B. Santos, D., Marques, J.C., Caçador, I. 2013. Ecophysiological adaptations of two halophytes to salt stress: Photosynthesis, PS II photochemistry and anti-oxidant
33
Page 35 of 53
feedback – Implications for resilience in climate change. Plant Physiology and Biochemistry 67, 178-188. Erice, G., Irigoyen, J.J., Pérez, P., Martínez-Carrasco, R., Sánchez-Díaz, M. 2006. Effect of
a cutting regrowth cycle. Physiologia Plantarum 126, 458-468.
ip t
elevated CO2, temperature and drought on photosynthesis and nodulated alfalfa during
cr
Evans, J.R., von Caemmerer, S., 1996. Carbon dioxide diffusion inside leaves. Plant
us
Physiology 110, 339-346.
Faria, T., Silvério, D., Breia, E., Cabral, R., Abadia, A., Abadia, J., Pereira, J.S., Chaves, M.M.
an
1998. Differences in the response of carbon assimilation to summer stress (water deficits, high light and temperature) in four Mediterranean tree species. Physiologia
M
Plantarum 102, 419-428.
d
Fini, A., Guidi, L., Ferrini, F., Brunetti, C., Di Ferdinando, M., Biricolti, S., Pollastri, S.,
te
Calamai, L., Tattini, M., 2012. Drought stress has contrasting effects on antioxidant enzymes activity and phenylpropanoid biosynthesis in Fraxinus ornus leaves: An excess
Ac ce p
light stress affair? Journal of Plant Physiology 169, 929-939. Fiscus, E.L., Booker, F.L., Burkey, K.O., 2005. Crop responses to ozone: uptake, modes of action, carbon assimilation and partitioning. Plant, Cell & Environment 28, 997-1011. Flexas, J., Barbour, M.M., Brendel, O., Cabrera, H.M., Carriquí, M., Díaz-Espejo, A., Douthe, C., Dreyer, E., Ferrio, J.P., Gago, J., Gallé, A., Galmés, J., Kodama, N., Medrano, H., Niinemets, U., Peguero-Pina, J.J., Pou, A., Ribas-Carbó, M., Tomás, M., Tosens, T., Warren, C.R., 2012. Mesophyll diffusion conductance to CO2: An unappreciated central player in photosynthesis. Plant Science 193–194, 70-84.
34
Page 36 of 53
Flexas, J., Diaz-Espejo, A., Gago, J., Gallé, A., Galmés, J., Gulías, J., Medrano, H., 2013. Photosynthetic limitations in Mediterranean plants: A review. Environmental and Experimental Botany in press (http://dx.doi.org/10.1016/j.envexpbot.2013.09.002).
ip t
Galmés, J., Medrano, H., Flexas J., 2007. Photosynthetic limitations in response to water stress and recovery in Mediterranean plants with different growth forms. New Phytologist
cr
175, 81-93.
us
García-Plazaola, J.I., Artetxe, U., Duñabeitia, M.K., Becerril, J.M., 1999. Role of photoprotective systems of Holm-Oak (Quercus ilex) in the adaptation to winter
an
conditions. Journal of Plant Physiology 155, 625–630.
Geiger, M., Haake, V., Ludewig, F., Sonnewald, U., Stitt, M., 1999. The nitrate and
M
ammonium nitrate supply have a major influence on the response of photosynthesis,
d
carbon metabolism, nitrogen metabolism and growth to elevated carbon dioxide in
te
tobacco. Plant Cell & Environment 22, 1177-1199. Genty, B., Briantais, J.M., Baker, N.R., 1989. The relationship between the quantum yield of
Ac ce p
photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochimica et Biophysica Acta 990, 87–92.
Gill, R., Boscaiu, M., Lull, C., Bautista, I., Lidan, A., Vicente, O., 2013. Are soluble carbohydrates ecologically relevant for salt tolerance in halophytes? Functional Plant Biology (in press) DOI: 10.1007/s11103-013-0031-6. Gimeno, B.S., Bermejo, V., Reinert, R.A., Zheng, Barnes, J.D., 1999. Adverse effects of ambient ozone on watermelon yield and physiology at a rural site in Eastern Spain. New Phytologist 144, 245–260.
35
Page 37 of 53
Gorbe, E., Calatayud, A., 2012. Applications of chlorophyll fluorescence imaging technique in horticultural research: A review. Scientia Horticulturae 138, 24–35. Govindjee, 1995. Sixty-three years since Kautsky: chlorophyll a fluorescence. Australian
ip t
Journal of Plant Physiology 22, 131-160.
Griffiths, H., Helliker, B.R., 2013. Mesophyll conductance: internal insights of leaf carbon
cr
exchange. Plant, Cell & Environment 36, 733-735.
us
Gucci, R., Tattini, M., 1997. Salinity tolerance in olive. Horticultural Review 17, 177-204. Guidi, L., Degl’Innocenti, E., Remorini, D., Biricolti, S., Fini, A., Ferrini, F., Nicese, F.P.,
an
Tattini, M., 2011. The impact of UV-radiation on the physiology and biochemistry of
Experimental Botany 70, 88-95.
M
Ligustrum vulgare exposed to different visible-light irradiance. Environmental and
d
Guidi, L., Degl’innocenti, E., Remorini, D., Massai, R., Tattini, M., 2008. Interactions of water
te
stress and solar irradiance on the physiology and biochemistry of Ligustrum vulgare. Tree Physiology 28, 873–883.
Ac ce p
Guidi, L., Degl’Innocenti, E., Giordano, C., Biricolti, S., Tattini, M. 2010. Ozone tolerance in Phaseolus vulgaris depends on more than one mechanism. Environmental Pollution 158, 3164-3171.
Havaux, M., Niyogi, K.K., 1999. The violaxanthin cycle protects plants from photooxidative damage by more than one mechanism. PNAS 96, 8762-8767. Havaux, M., Bonfils J.-P., Lütz C., Niyogi K.K., 2000. Photodamage of the photosynthetic apparatus and its dependence on the leaf developmental stage in the npq1 Arabidopsis mutant deficient in the xanthophyll-cycle enzyme violaxanthin deepoxidase. Plant Physiology 124, 273-284. 36
Page 38 of 53
Havaux, M., Tardy, F. 1996. Temperature-dependent adjustment of the thermal stability of photosystem II in vivo: possible involvement of xanthophyll-cycle pigments. Planta 198, 324-333.
ip t
Heckwolf, M., Pater, D., Hanson, D.T., Kaldenhoff, R., 2011. The Arabidopsis thaliana aquaporin AtPIP1;2 is a physiologically relevant CO2 transport facilitator. Plant Journal
cr
67, 795-804.
us
Hernández, I. Alegre, L., Munné-Bosch, S., 2011. Plant aging and excess light enhance flavan-3-ol content in Cistus clusii. Journal of Plant Physiology 168, 96-102.
an
Hernández, I., Van Breusegem, F., 2010. Opinion on the possible role of flavonoids as energy escape valves: Novel tools for nature's Swiss army knife? Plant Science 179, 297-301.
M
Horton, P., Wentworth, M., Ruban, A., 2005. Control of the light harvesting function of
te
FEBS Letters 579, 4201-4206.
d
chloroplast membranes: the LHCII-aggregation model for non-photochemical quenching.
IPCC. 2007. Summary for policymakers In: Solomon, S., Qin, D., Manning, M., Chen, Z.,
Ac ce p
Marquis, M., Averyt, K.B., Tignor, M., Miller, H.L. (Eds.), Climate change 2007: the physical science basis. Contribution of working group I to the fourth assessment report of the intergovernmental panel on climate change. Cambridge University Press, Cambridge, pp. 747-846.
Jahns, P., Holzwarth, A.R., 2012. The role of the xanthophyll cycle and of lutein in photoprotection of photosystem II. Biochimica et Biophysica Acta 1817, 182-193. Jansen, M.A.K., Gaba, V., Greenberg, B.M., 1998. Higher plants and UV-B radiation: balancing damage, repair and acclimation. Trends in Plant Science 3, 131-135.
37
Page 39 of 53
Jaoudé, R.A., de Dato, G., De Angelis, P., 2012. Photosynthetic and wood anatomical responses of Tamarix africana Poiret to water level reduction after short-term fresh- and saline-water flooding. Ecological Research 27, 857-866.
ip t
Johnson, M.P., Goral, T.K., Duffy, C.D.P., Brain, A.P.R. Mullineaux, C.W., Ruban, A.V., 2011. Photoprotective energy dissipation involves the reorganization of Photosystem II light-
cr
harvesting complexes in the grana membranes of spinach chloroplasts. The Plant Cell
us
23, 1468-1479.
Johnson, M.P., Ruban, A.V., 2011. Restoration of rapidly reversible photoprotective energy
an
dissipation in the absence of PsbS protein by enhanced ΔpH. The Journal of Biological Chemistry 286, 19973-19981.
M
Kalina, J., Cajanek, M., Spunda, V., Marck, M.V., 1997. Changes of the primary
d
photosynthetic reaction of Norway sprunce under elevated CO 2. In: Mohren, G.M.J.,
te
Sabaté, S., (Eds.), Impacts of Global Change on Tree Physiology and Forest Ecosystems. Kluwer Academic Publishers, Dordrecht, pp. 59-69.
Ac ce p
Kangasjärvi, S., Neukermans, J., Li, S., Aro, E.-M., Noctor, G., 2012. Photosynthesis, photorespiration, and light signalling in defence responses. Journal of Experimental Botany 63, 1619-1636.
Kitajima, M., Butler, W.L., 1975. Quenching of chlorophyll fluorescence and primary photochemistry in chloroplasts by dibromothymoquinone. Biochimica et Biophysica Acta 376,105 -115. Koller, S., Holland, V., Brűggermann, W., 2013. Effects of drought stress on the evergreen Quercus ilex L., the deciduous Q. robur and their hybrid Q. x turneri Wild. Photosynthetica 51, 574-582 . 38
Page 40 of 53
Kramer, D.M. Johnson, G. Kiirats, O. Edwards G.E., 2004. New fluorescence parameters for the determination of QA redox state and excitation energy fluxes. Photosynthesis Research 79, 209-218.
ip t
Krupa S.V., 2000. Ultraviolet-B radiation, ozone and plant biology. Environmental Pollution 110, 193-194.
chlorophyll
fluorescence
(Ribulose-1,5-Bisphosphate
carboxylase/oxygenase
us
and
cr
Laisk, A., Loreto, F., 1996. Determining photosynthetic parameters from leaf CO2 exchange
specificity factor, dark respiration in the light, excitation distribution between
an
photosystems, alternative electron transport rate, and mesophyll diffusion resistance. Plant Physiology 110, 903-912.
M
Laisk, A., Oja, V., Rahi, M., 1970. Diffusion resistances as related to the leaf anatomy.
d
Fiziologiya Rastenii 17, 40-48.
te
Laisk, A., Oja, V., Rasulov, B., Rämma, H., Eichelmann, H., Kasparova, I., Pettai, H., Padu, E., Vapaavuori, E., 2002. A computer-operated routine of gas exchange and optical
Ac ce p
measurements to diagnose photosynthetic apparatus in leaves. Plant, Cell & Environment 25, 923-943.
Larcher, W., 2000. Temperature stress and survival ability of Mediterranean sclerophyllous plants. Plant Biosystems 134, 279–295. Leshem, Y.Y., Kuiper, P.J.C. 1996. Is there a gas (general adaptation syndrome) response to various types of environmental stress? Biologia Plantarum 38, 1-18. Long. S.P., Ainsworth, E.A., Rogers, A., Ort, D.R., 2004. Rising atmospheric carbon dioxide: Plant face the future. Annual Review of Plant Biology 55, 591-628.
39
Page 41 of 53
Makino, A.,Miyake, C., Yokota, A., 2002. Physiological functions of the water–water cycle (Mehler reactions) and the cyclic electron flow around PSI in rice leaves. Plant and Cell Physiology 43, 1017-1026.
ip t
Manes, F., Vitale, M., Donato, E., Paoletti, E. 1998. O3 and O3+CO2 effects on a Mediterranean evergreen broadleaf tree, holm oak (Quercus ilex L.). Chemosphere 36,
cr
801-806.
us
Maxwell, K., Johnson, G.N., 2000. Chlorophyll fluorescence-a practical guide. Journal of Experimental Botany 51, 659–68.
an
McCarthy, J.J., Canziani, O., Leary, N.A., Dokken, D.J., White, K.S., 2001. Climate Change 2001: Impacts, Adaptation and Vulnerability. IPCC Working Group II, Cambridge
M
University Press, Cambridge.
d
McKenzie, R.L., Aucamp, P.J., Bais, A.F., Björn, L.O., Ilyas, M., 2007. Changes in
te
biologically-active ultraviolet radiation reaching the Earth's surface. Photochemistry and Photobiology Science 6, 218-231.
Ac ce p
Melgar, J.C., Guidi, L., Remorini, D., Agati, G., Degl’innocenti, E., Castelli, S., Baratto, M.C., Faraloni, C., Tattini, M., 2009. Antioxidant defences and oxidative damage in salt-treated olive plants under contrasting sunlight irradiance. Tree Physiology 29, 1187-1198. Mereu, S., Gerosa, G., Marzuoli, R., Fusaro, L., Salvatori, E., Finco, A., Spano, D., Manes, F. (2011). Gas exchange and JIP-test parameters of two Mediterranean maquis species are affected by sea spray and ozone interaction. Environmental and Experimental Botany 73, 80-88.
40
Page 42 of 53
Mooney, H.A., 1981. Primary production in Mediterranean-climate regions. In: Di Castri, F., Goodall, D.W., Specht, R.L. (Ed.), Mediterranean-type Shrublands. Ecosystems of the World 11. Elsevier Scientific Publishing, Amsterdam, pp. 249-256.
ip t
Morales, L.O., Tegelberg, R., Brosché, M., Keinänen, M., Lindfors, A., Aphalo, P.J., 2010.
in Betula pendula leaves. Tree Physiology 30, 923-934.
cr
Effects of solar UV-A and UV-B radiation on gene expression and phenolic accumulation
us
Munné-Bosch, S., Peñuelas, J. 2013. Photo- and antioxidative protection during summer leaf senescence in Pistacia lentiscus L. grown under Mediterranean field conditions. Annals
an
of Botany 92, 385-391.
Munns, R., 1993. Physiological processes limiting plant growth in saline soils: some dogmas
M
and hypotheses. Plant, Cell & Environment 16, 15-24.
te
25, 239-250.
d
Munns, R., 2002. Comparative physiology of salt and water stress. Plant, Cell & Environment
Munns, R., Tester, M., 2008. Mechanisms of salinity tolerance. Annual Review of Plant
Ac ce p
Biology 59, 651-681.
Murata, N., Allakhverdiev, S.I., Nishiyama, Y., 2012. The mechanism of photoinhibition in vivo: Re-evaluation of the roles of catalase, α-tocopherol, non-photochemical quenching, and electron transport. Biochimica et Biophysica Acta (BBA) - Bioenergetics 1817, 11271133.
Nali, C., Paoletti, E., Marabottini, R., Della Rocca, G, Lorenzini, G., Paolacci, A.R., Ciaffi, M., Badiani, M., 2004. Ecophysiological and biochemical strategies of response to ozone in Mediterranean evergreen broadleaf species. Atmospheric Environment 38, 2247-2257.
41
Page 43 of 53
Nedbal, L., Whitmarsh, J., 2004. Chlorophyll fluorescence imaging of leaves and fruits. In: Govindjee,
Papageorgiou,
G.,
Ed.,
Chlorophyll
fluorescence:
A
signature
of
photosynthesis. Kluwer Academic Publishers., Dordrecht, The Netherlands, pp. 389–
ip t
407.
Niinemets, U., 2010. A review of light interception in plant strands from leaf to canopy in
cr
different plant functional types and in species with varying shade tolerance. Ecological
us
Research 25, 693-714.
Nobel, P.S., Zaragoza, L.T., Smith, W.K., 1975. Relation between mesophyll surface area,
an
photosynthetic rate, and illumination level during development for leaves of Plectranthus parviflorus Henckel. Plant Physiology 55, 1067-1070.
M
Nogués, S., Baker, N.R., 1995. Evaluation of the role of damage to photosystem II in the
te
Environment 18, 781-787.
d
inhibition of CO2 assimilation in pea leaves on exposure to UV-B. Plant, Cell &
Ogaya, R., Peñuelas, J., Asensio, D., Llusià, J., 2011. Chlorophyll fluorescence responses to
Ac ce p
temperature and water availability in two co-dominant Mediterranean shrub and tree species in a long-term field experiment simulating climate change. Environmental and Experimental Botany 73, 89–93. Oliveira, G., Peñuelas, J., 2000. Comparative photochemical and phenomorphological responses to winter stress of an evergreen (Quercus ilex L.) and a semi-deciduous (Cistus albidus). Acta Oecologica 21, 97–107. Oliveira, G., Peñuelas, J., 2004. The effect of winter cold stress on photosynthesis and photochemical efficiency of PSII of two Mediterranean woody species - Cistus albidus and Quercus ilex. Plant Ecology 175, 179-191. 42
Page 44 of 53
Osório, M.L., Osório, J., Vieira, A.C., Gonçalves, S., Romano, A., 2011. Influence of enhanced temperature on photosynthesis, photooxidative damage, and antioxidant strategies in Ceratonia siliqua L. seedlings subjected to water deficit and rewatering.
ip t
Photosynthetica 49, 3–12.
Paul, N.D., Moore, J.P., McPherson, M., Lambourne, C., Croft, P., Heaton, J.C., Wargent,
cr
J.J., 2012. Ecological responses to UV radiation: interactions between the biological
us
effects of UV on plants and on associated organisms. Physiologia Plantarum 145, 565581.
an
Peguero-Pina, J.J., Gil-Pelegrín, E., Morales, F., 2013. Three pools of zeaxanthin in Quercus coccifera leaves during light transitions with different roles in rapidly reversible
M
photoprotective energy dissipation and photoprotection. Journal of Experimental Botany
d
64, 1649-1661.
te
Peterson, R.B., 1989. Partitioning of noncyclic photosynthetic electron transport to O2dependent dissipative processes as probed by fluorescence and CO 2 exchange. Plant
Ac ce p
Physiology 90, 1322-1328.
Prado, F.E., Rosa, M., Prado, C., Podazza, G., Interdonato, R., González, J.A., Hilal, M., 2012. UV-B radiation, its effects and defense mechanisms in terrestrial plants. In: Ahmad, P., Prasad, M.N.V. (Eds.), Environmental Adaptations and Stress Tolerance of Plants in the Era of Climate Change. Springer, Dordrecht, pp. 57-83. Prieto, P., Peñuelas, J., Llusià, J., Asensio, D., Estiarte, M., 2009. Effects of long-term experimental night-time warming and drought on photosynthesis, Fv/Fm and stomatal conductance in the dominant species of a Mediterranean shrubland. Acta Physiologiae Plantarum 31, 729–739. 43
Page 45 of 53
Rajaravindran, M., Natarajan, S., 2012. Effect of NaCl stress on biochemical and enzymes changes of the halophyte Suaeda maritime Dum. International Journal of Research in Plant Science 2, 1-7.
ip t
Reich, P.B., 1987. Quantifying plant response to ozone: a unifying theory. Tree Physiology 3, 63-91.
cr
Remorini, D., Melgar, J.C., Guidi, L., Degl’Innocenti, E., Castelli, S., Traversi, M.L., Massai,
us
R., Tattini, M., 2009. Interaction effects of root-zone salinity and solar irradiance on the physiology and biochemistry of Olea europaea. Environmental and Experimental Botany
an
65, 210-219.
Ribas, A., Peñuelas, J., Elvira, S., Gimeno, B.S. 2005. Ozone exposure induces the activation
M
of leaf senescence-related processes and morphological and growth changes in
d
seedlings of Mediterranean tree species. Environmental Pollution 134, 291-300.
te
Rochaix, J.-D., 2011. Regulation of photosynthetic electron transport. Biochimica et Biophysica Acta - Bioenergetics 1807, 375-383.
Ac ce p
Sánchez-Gómez, D., Valladares, F., Zavala, M.A., 2006. Performance of seedlings of Mediterranean woody species under experimental gradients of irradiance and water availability: trade-offs and evidence for niche differentiation. New Phytologist 170, 795806.
Sanz, M.J., Calatayud, V., Sánchez-Peña, G., 2007. Measures of ozone concentrations using passive sampling in forests of South Western. Environmental Pollution 145, 620–628. Schreiber, U., Schliwa, U., Bilger, W., 1986. Continuous recording of photochemical and nonphotochemical chlorophyll fluorescence quenching with a new type of modulation fluorometer. Photosynthesis Research 10, 51–62. 44
Page 46 of 53
Silva-Cancino, M.C., Esteban, R., Artetxe, U., Plazaola, J.I.G., 2012. Patterns of spatiotemporal distribution of winter chronic photoinhibition in leaves of three evergreen Mediterranean species with contrasting acclimation responses. Physiologia Plantarum
ip t
144, 289–301.
Stepien, P., Johnson, G.N., 2009. Contrasting responses of photosynthesis to salt stress in
cr
the glycophyte Arabidopsis and the halophyte Thellungiella: role of the plastid terminal
us
oxidase as an alternative electron sink. Plant Physiology 149, 1154-1165. Stirbet, A., Govindjee, 2012. Chlorophyll a fluorescence induction: a personal perspective of
an
the thermal phase, the J-I-P rise. Photosynthesis Research 113, 15-61. Strasser, R.J., Srivastava, A., Tsimilli-Michael, M., 2004. Analysis of fluorescence transient.
M
In: Papageorgiou, G.C., Govindjee, (Eds.) Chlorophyll a Fluorescence: A signature of
te
321-362.
d
photosynthesis. Advances in Photosynthesis and Respiration. Springer, Dordrecht, pp.
Takahashi, S., Badger, M.R. 2011. Photoprotection in plants: a new light on photosystem II
Ac ce p
damage. Trends in Plant Science 16, 53-60. Takahashi, S., Milward, S.E., Fan, D.-Y., Chow, W.S., Badger, M.R., 2009. How does cyclic electron flow Alleviate photoinhibition in Arabidopsis? Plant Physiology 149, 1560-1567. Tattini, M., Montagni, G., Traversi M.L., 2002. Gas exchange, water relations and osmotic adjustment in Phillyrea latifolia grown at various salinity concentrations. Tree Physiology 22, 403-412. Tattini, M., Traversi, M.L., Castelli, S. Biricolti, S., Guidi, L. Massai, R., 2009. Contrasting response mechanisms to root-zone salinity in three co-occurring Mediterranean woody
45
Page 47 of 53
evergreens: a physiological and biochemical study. Functional Plant Biology 36, 551563. Terashima, I., Hanba, I.T., Tholen, D., Niinemets, U., 2011. Leaf functional anatomy in
ip t
relation to photosynthesis. Plant Physiology 155, 108-116.
Therios, I.N. Misopolinos, N.D., 1988. Genotypic response to sodium chloride salinity of four
cr
major olive cultivars (Olea europaea L.). Plant and Soil 106, 105-111.
us
Tuba Z, Lichtenthaler H. K., 2007. Long-term acclimation of plants to elevated CO2 and its interaction with stresses. Annals of the New York Academy of Sciences 1113, 135-46.
an
Valladares, F., Chico, J., Aranda, I., Balaguer, L., Dizengremel, P., Manrique, E., Dreyer, E. 2002. The greater seedling high-light tolerance of Quercus robur over Fagus sylvatica is
M
linked to a greater physiological plasticity. Trees 16, 395-403.
d
Valladares, F., Martinez-Ferri, E., Balaguer, L., Perez-Corona, E., Manrique E., 2000. Low
te
leaf-level response to light and nutrients in Mediterranean evergreen oaks: a conservative resource-use strategy? New Phytologist 148, 79-91.
Ac ce p
Vaz, M., Maroco, J., Ribeiro, N., Gazarini, L.C., Pereira, J.S., Chaves, M.M., 2011. Leaf-level responses to light in two co-occurring Quercus (Quercus ilex and Quercus suber): leaf structure, chemical composition and photosynthesis. Agroforestry Systems 82, 173-181. Velikova, V., Tsonev, T., Pinelli, P., Alessio, G.A., Loreto, F., 2005. Localized ozone fumigation system for studying ozone effects on photosynthesis, respiration, electron transport rate and isoprene emission in field-grown Mediterranean oak species. Tree Physiology 12, 1523-1532. Verdaguer, D., Llorens, L., Bernal, M., Badosa, J., 2012. Photomorphogenic effects of UVB and UVA radiation on leaves of six Mediterranean sclerophyllous woody species 46
Page 48 of 53
subjected to two different watering regimes at the seedling stage. Environmental and Experimental Botany 79, 66-75. Weber, J.A., Scott Clark, C., Hogsett, W.E. 1993. Analysis of the relationships among O3
ip t
uptake, conductance, and photosynthesis in needles of Pinus ponderosa. Tree Physiology 13, 157-172.
cr
Werner, C., Correia, O., Beyschlag, W. 2002. Characteristic patterns of chronic and dynamic
us
photoinhibition of different functional groups in a Mediterranean ecosystem. Functional Plant Biology 29, 999-1011-
an
Wertin, T.M., Mcguire, M.A., Teskey, R.O., 2010. The influence of elevated temperature, elevated atmospheric CO2 concentration and water stress on net photosynthesis of
M
loblolly pine (Pinus taeda L.) at northern, central and southern sites in its native range.
d
Global Change Biology 16, 2089–2103.
te
Yin, X., Sun, Z., Struik, P.C., Gu, J. 2011. Evaluating a new method to estimate the rate of leaf respiration in the light by analysis of combined gas exchange and chlorophyll
Ac ce p
fluorescence measurements. Journal of Experimental Botany 62, 3489-3499.
47
Page 49 of 53
Table 1. Changes in dark-adapted F0, Fm and Fv/Fm in cabbage leaves at the end of the growing station (30 days in the field) with charcoal-filtered air (CFA), non-filtered air (NFA) and NFA+O3 (63 nL L-1) treatments. Values are the means of ten samples. For comparison of means, variance analysis (ANOVA) followed by the least significance
ip t
differences (LSD) test, calculated at 95% confidence level, were performed. Values followed by the same letter indicate no significant differences (from Calatayud et al.,
Caramba F0
Fm
Sentinel Fv/Fm
F0
Fm
Othelo
us
Treatment
cr
2002).
Fv/Fm
F0
Fm
Fv/Fm
0.236a 1.116a 0.789a 0.236a
1.127a 0.790a 0.248a 1.203a 0.794a
NFA
0.242ab 0.955b 0.743b 0.241ab 1.052ab 0.770a 0.256a 1.220a 0.789a
NFA+O3
0.257b 0.793c 0.675c 0.255b
M
an
CFA
Ac ce p
te
d
0.870b 0.705b 0.226b 1.045b 0.780a
48
Page 50 of 53
Figure legend Figure 1. Maximum (Fv/Fm, A) and actual (ΦPSII, B) quantum efficiency of PSII photochemistry, non-photochemical quenching (qNP, C), and excitation pressure on PSII
ip t
(1−qP, D) in leaves of Ligustrum vulgare exposed to 35% (grey bars) or 100% sunlight irradiance (open bars) in the absence or in the presence (hutched bars) of UV-radiation. Data
cr
is mean±standard deviation (n=5), and at each sampling date, when not accompanied by the
us
same letter, significantly differ for P<0.05 using a least significant difference (LSD) test (from Guidi et al., 2011).
an
Figure 2. Time course of maximum quantum efficiency of PSII photochemistry (Fv/Fm) in leaves of O. europaea plants exposed to 15% (circles, shade) or 100% sunlight (circles, sun)
M
and supplied with 0 (open symbols) or 125 mM NaCl (closed symbols). Measurements were conducted at midday, after 7, 14 and 35 days of treatment. Summary of the results of a three-
d
way ANOVA (total error df = 59) for variation in Fv/Fm, with light (L), salt (S) and treatment
te
period (t) as fixed factors, with their interaction factors (L×S; L×t; S×t, L×S×t) is shown.
Ac ce p
**P<0.001 and data are mean±SD, n=4 (from Melgar et al., 2009).
49
Page 51 of 53
M
an
us
cr
ip t
Figure 1
Ac
ce pt
ed
Figure 1.
Page 52 of 53
Ac
ce pt
ed
M
an
us
cr
ip t
Figure 2
Page 53 of 53