Responses of oak species to single and combined abiotic stresses in the global climate change era

Responses of oak species to single and combined abiotic

stresses in the global climate change era

by

Lorenzo Cotrozzi

Ph. D. Thesis

Agriculture, Food and Environment

Department of Agriculture, Food and Environment

University of Pisa

U

U

P

P

Responses of oak species to single and combined

abiotic stresses in the global climate change era

by

Lorenzo Cotrozzi

A thesis submitted in conformity with the requirements

for the degree of Doctor of Philosophy in

Agriculture, Food and Environment

Candidate: Lorenzo Cotrozzi

Supervisor:

Prof. Cristina Nali

Accepted by the Ph.D School

The Coordinator

This thesis is a presentation of my original research work. Wherever contributions of others are involved, every effort is made to indicate this clearly with due reference to the literature and acknowledgement of collaborative research and discussions. This thesis contains no material that has been submitted previously, in whole or in part, for the award of any other academic degree or diploma.

Signature Date

This dissertation is submitted for the degree of Doctor of Philosophy at the University of Pisa. The research described herein was conducted under the supervision of Professor Cristina Nali in the Department of Agriculture, Food and Environment, between November 2013 and February 2017.

Part of this work has been presented in papers published or under review in peer-reviewed journals as follows:

CHAPTER 1

Cotrozzi, L., Couture, J.J., Pellegrini, E., Kingdon C.C., Singh A., Nali C., Lorenzini

G., Townsend P.A. Reflectance spectroscopy: an efficacious approach for better understanding and monitoring the impact of air pollution on Mediterranean plants. Environmental Science and Pollution Research, under review.

Cotrozzi, L., Tonelli, M., Pellegrini, E., 2016. Assessing photosynthetic efficiency

in ornamental urban species. Annali di Botanica 6, 21-37.

Pellegrini, E., Trivellini A., Cotrozzi, L., Vernieri, P., Nali, C., 2016. Involvement of phytohormones in plant reponses to ozone. In: Ahammed, G.J., Yu, J.-Q., (Eds.), Plant hormones under challenging environmental factors. Springer, Dordrecht, Netherland, pp 215-245.

CHAPTER 2

Cotrozzi, L., Remorini, D., Pellegrini, E., Landi, M., Massai, R., Nali, C., Guidi, L.,

Lorenzini, G., 2016. Variations in physiological and biochemical traits of oak seedlings grown under drought and ozone stress. Physiologia Plantarum 157, 69-84.

CHAPTER 3

Cotrozzi, L., Remorini, D., Pellegrini, E., Guidi, L., Lorenzini, G., Massai, R., Nali,

C., Landi, M. Cross-talk between physiological and metabolic adjustments adopted by Quercus cerris to mitigate the effects of severe drought and ozone. Forests, under

Cotrozzi, L., Remorini, D., Pellegrini, E., Guidi, L., Nali, C., Lorenzini, G., Massai,

R., Landi, M. Living in a Mediterranean city in 2050: broadleaf or evergreen “citizens”? Environmental Science and Pollution Research, under review.

CHAPTER 5

Cotrozzi, L., Pellegrini, E., Guidi, L., Landi, M., Lorenzini, G., Massai, R.,

Remorini, D., Tonelli, M., Trivellini, A., Vernieri, P., Nali, C. Losing the warning signal: drought compromises the cross-talk of signalling molecules in Quercus ilex exposed to ozone. Frontiers in Plant Science, under review.

CHAPTER 6

Guidi, L., Remorini, D., Cotrozzi, L., Giordani, T., Lorenzini, G., Massai, R., Nali, C., Natali, L., Pellegrini, E., Trivellini, A., Vangelisti, A., Vernieri, P., Landi, M., 2016. The harsh life of an urban tree: the effect of a single pulse of ozone in salt-stressed Quercus ilex saplings. Tree Physiology, doi:10.1093/treephys/tpw103.

CHAPTER 7

Cotrozzi, L., Couture, J.J., Cavender-Bares, J., Kingdon, C.C., Fallon, E., Pilz, G.,

Pellegrini, E., Nali, C., Townsend, P.A. Using foliar spectral properties to assess the effects of drought on plant water potential. Tree Physiology, under review.

In this era of global warming it is necessary to understand the mechanisms that allow native plant species to tolerate these environmental constraints and the way such mechanisms interact. The main scope of this research work was to investigate the responses of oak species to single and combined abiotic stresses that are common in the Mediterranean urban environments [drought, ozone (O3) and salinity].

Firstly, three Mediterranean oak species (Querucs ilex, Q. pubescens and Q. cerris) with different features (drought tolerance, evergreen or deciduous species) were selected in order to assess their responses under a long period of moderate drought (30% of the effective daily evapotranspiration) and/or O3 stress (80 ppb of O3 for 5 h

d-1 for 77 consecutive days). The chronic O3 treatment had a minor impact compared

to drought, highlighting that the plasticity of the species is dependent on the environment in which they live. Species that inhabit environments characterized by long periods of water deficiency (i.e. Q. ilex and Q. pubescens) are usually more plastic under the same stress compared with those that rarely face the same environmental constraint (Q. cerris). Furthermore, this dataset shows that biochemical and physiological adjustments may reduce the impact of O3 when

combined with the effect of drought.

Following the results came out from this first experiment, and since the mitigative effect of drought against O3 seems more consistent when plant is exposed to short

and harsh period of severe drought rather than to longer O3 exposure but under

moderate drought, other two sets of experiments were performed. Firstly, the interactions of severe drought (20% of the effective daily evapotranspiration) and O3

(80 ppb, 5 h d-1, for 28 consecutive days) were investigated in Q. cerris focusing on its hydric relations, synthesis/production of compatible solutes and lipophilic antioxidant compounds. Although, leaf-intrinsic adjustments occurred (stomatal limitations) and the synthesis of stress-associated metabolites was altered, plants were not able to delay or prevent the negative impact of drought. Furthermore, it was evident that drought alone induced fairly higher effects in comparison to O3, whereas

when O3 was applied together with drought it showed some “mitigating effects”

This response could be interpreted as a photosynthetic acclimation leading to a premature senescence of fully-developed leaves as a strategy to respond to multiple stress conditions, likely addressed to alert younger leaves.

Later, physiological and biochemical responses of Q. ilex (evergreen, isohydric species) were compared to those of the sympatric Q. pubescens (deciduous, anisohydric species) under the same environmental constrains of the previous experiment. This study confirms the higher ability of evergreen species to counteract the effect of drought and O3 when compared to deciduous species in Mediterranean

environment. This ability of evergreens seems to be correlated with the stronger necessity of these species, which inhabits usually unfavorable environments, to protect their long-lived leaves from several negative environmental factors. This peculiarity seems less relevant for highly-demanding, fast-growing deciduous species characterized by shorter leaf lifespan, which have superior fitness than evergreens in non-limiting environment.

Then, the response of Q. ilex (the most studied oak species, as well as the species that has been shown the higher tolerance and plasticity in the first experiment) to drought (20% of the effective daily evapotranspiration) and/or an acute O3-exposure (200

ppb, 5 h) was also investigated. This study, which focused on the interaction between reactive oxygen species (ROS), phytohormones and signalling molecules to evaluate if the response of Q. ilex resembles the biotic defense reactions, shows that: (i) in well-watered conditions, O3 induced a signalling pathway similar to that triggered by

a pathogen only in terms of ROS pattern (showing an O3-sensitive behavior); (ii)

different trends and consequently different roles of phytohormones and signalling molecules were observed in relation to the leaf hydric status and O3 (applied both

singularly and consequently), and (iii) these differences were ascribable to the fact that in drought conditions most defense processes induced by O3 were

compromised/altered.

Furthermore, the response of Q. ilex was also investigated also under salinity and O3. Q. ilex saplings were firstly exposed to salinity (150 mM NaCl, 15 days), and the

effect on photosynthesis, hydric relations and ion partitioning were evaluated (Experiment I). Then, salt-treated plants were exposed to 80 ppb of O3 for 5 h

efficiency on a mass basis because these species invest preferentially in vascular and cell wall formation. This induces these species to decrease intercellular spaces and increase cell wall thickness, increasing CO2 drawdown but also maintaining high

foliar RWC and osmotic stress tolerance. These mechanisms are consistent with a conservative strategy adopted by Q. ilex to preserve its long-lived leaves against different abiotic stresses. Furthermore, the dataset of the second experiment shows that O3 did not exacerbate the oxidative stress observed in salt-treated plants

although a further relevant enhancement of the Halliwell-Asada cycle was necessary to counteract the O3-induced damage when the leaf status was already negatively

affected by a previous salt exposure. This harmonic response is an extra burden for plants, and growth can suffer as a result in the long-term, if these single O3 episodes

take place repeatedly.

Assessing the impact of climate change and air pollution on ecosystems is still a challenging task and the development of adequate monitoring techniques is necessary for assessing vegetation status. Thus, a final study was performed showing that reflectance spectroscopy can be an alternative method for monitoring leaf water potential and also making a posteriori measurements of pre-dawn leaf water potential (PDΨW) on tropical live oak Q. oleoides sampled in four Central American

populations (Belize, Costa Rica, Honduras and Mexico) and grown under differential water availability. This study confirms that spectroscopic approaches are quick and non-destructive, providing the possibility to screen more samples in the field and over multiple time periods. In addition, this dataset demonstrates that spectroscopic retrievals of PDΨW in response to environmental variation (e.g. water availability)

Firstly, I would like to express my sincere gratitude to my advisors Prof. Giacomo Lorenzini and Prof. Cristina Nali for their continuous support of my PhD work and related research, and their patience, motivation and immense knowledge. They enabled me to mature and grow. In particular, I would like to thank Prof. Cristina Nali for being more than a tutor, standing by me during all of the ups and downs. I could not have imagined a better mentor for my PhD study.

A special thanks also goes to Dr. Elisa Pellegrini, who extraordinarily carried me during this long journey. Without her precious and constant support it would not have been possible to conduct this research.

I would like to thank my labmates Dr. Mariagrazia Tonelli, Dr. Alessandra Campanella, and Dr. Romina Papini, as well as the technicians Andrea Parrini, Simona Ciangherotti, and Giuseppe Spinelli for helping me in the research activities. A special thanks goes to Prof. Lucia Guidi, Dr. Marco Landi and Prof. Damiano Remorini for their patience, availability, and for discussion which definitely improved my present work. My sincere gratitude goes also to Prof. Rossano Massai and Prof. Paolo Vernieri.

I am grateful to Prof. Philip A. Townsend who gave me the chance to work in his laboratory at the University of Wisconsin – Madison. I would especially like to thank Prof. John J. Couture, Clayton C. Kingdon, Aidan Mazur, Andrew Ciurro, Robert Phetteplace and Ben Spaier for teaching and helping me during my stay there.

I would also like to thank Dr. Paolo Cherubini, Dr. Massimo Muganu and Prof. Lu Zhang for serving as my committee members.

Above all, I would thank my sister, who during this journey always believed in and supported me with patience and love. Last but not least, I would like to thank my father, my brother-in-law and all my friends and others whose help was fundamental in achieving this result.

Chapter 1:

GENERAL INTRODUCTION AND THESIS OUTLINE

1.1. Climate change and air pollution in the Mediterranean region 1

1.1.1. Ozone 3

1.2. Urban environment 5

1.3. Oak species 7

1.4. Assessing the impact of climate change and air pollution on Mediterranean

plants: vegetation spectroscopy 9

1.4.1. Vegetation spectroscopy 10

1.5. Thesis outline 18

1.6. References 21

Chapter 2:

RESPONSES OF OAK SPECIES TO A LONG PERIOD OF

MODERATE DROUGHT AND/OR OZONE

Abstract 32

2.1. Introduction 33

2.2. Materials and Methods 36

2.2.1. Plant material and experimental design 36

2.2.2. Leaf water status 37

2.2.3. Gas exchange and chlorophyll a fluorescence measurements 37

2.2.4. Proline determination 39

2.2.5. Lipid peroxidation 39

2.2.6. Photosynthetic and accessories pigment analyses 40

2.3. Results 41 2.3.1. Visible symptoms and growth response 41 2.3.2. Water status, proline and MDA-by product content 43 2.3.3. Gas exchanges and chlorophyll a fluorescence 45

2.3.4. Leaf pigments 51

2.3.5. Phenotypic plasticity 53

2.4. Discussion 55

2.5. References 62

Chapter 3:

CROSS-TALK BETWEEN PHYSIOLOGICAL AND METABOLIC

ADJUSTMENT ADOPTED BY QUERCUS CERRIS TO MITIGATE

THE EFFECTS OF SEVERE DROUGHT AND OZONE

Abstract 71

3.1. Introduction 72

3.2. Materials and Methods 72

3.2.1. Plant material and experimental design 72

3.2.2. Leaf water status 73

3.2.3. Gas exchange and chlorophyll a fluorescence measurements 74

3.2.4. Lipid peroxidation 74

3.2.5. Proline determination 75

3.2.6. Abscisic acid determination 75 3.2.7. Photosynthtetic and accessories pigment analyses 75

3.2.8. Hexoses determination 75

3.2.9. Statistical analysis 76

3.3. Results 76

3.3.3. Diurnal profiles of gas exchanges and chlorophyll a fluorescence 80 3.3.4. Leaf water status and osmolyte contents 83

3.3.5. Leaf pigments 85

3.4. Discussion 86 3.5. References 92

Chapter 4:

RESPONSES OF QUERCUS ILEX AND QUERCUS PUBESCENS TO

SEVERE DROUGHT AND OZONE: BROADLEAF OR EVERGREEN

“CITIZENS” IN MEDITERRANEAN CITY?

4.2.1. Plant material and experimental design 98 4.2.2. Leaf water status 99 4.2.3. Gas exchange and chlorophyll a fluorescence measurements 99

4.2.4. Lipid peroxidation 99 4.2.5. Proline, abscisic acid and hexoses determination 99 4.2.6. Photosynthetic and accessories pigment analyses 100

4.2.7. Statistical analysis 100

4.3. Results 100

4.3.1. Visible injury 100

4.3.2. Leaf water status and MDA content 101

4.3.3. Osmolytes and ABA 101

4.3.4. Gas exchange and chlorophyll a fluorescence parameters 102

4.3.5. Chlorophyll and carotenoid content 109

Chapter 5:

LOSING THE WARNING SIGNAL: DROUGHT COMPROMISES

THE CROSS-TALK OF SIGNALLING MOLECULES IN QUERCUS

ILEX EXPOSED TO ACUTE OZONE

Abstract 122

5.1. Introduction 123

5.2. Materials and Methods 124

5.2.1. Plant material and experimental design 124

5.2.2. Leaf water status 125

5.2.3. Gas exchange and chlorophyll a fluorescence measurements 125

5.2.4. Staining and microscope assays 126

5.2.5. ROS determination 126

5.2.6. Phytohormone and signalling molecule bioassays 127

5.2.7. Proline determination 127

5.2.8. Statistical analysis 128

5.3. Results 128

5.3.1. Effects of drought stress 128

5.3.2. Influence of drought stress on the response of plant to acute O3 -exposure 129

5.4. Discussion 138

5.5. References 147

Chapter 6:

THE HARSH LIFE OF AN URBAN TREE: THE EFFECT OF A

SINGLE PULSE OF OZONE IN SALT-STRESSED QUERCUS ILEX

SAPLINGS

6.2. Materials and Methods 156

6.2.1. Plant material and experimental design 156

6.2.2. Leaf water status 158

6.2.3. Osomolality, osmotic potential and relative contribution of each ion to ionic adjustment 158

6.2.4. Gas exchange and chlorphyll a fluorescence measurements 159

6.2.5. Mesophyll conductance and values of Vcmax and Jflu 162

6.2.6. Quantitative limitation analysis 162

6.2.7. Determination of inorganic solutes 163

6.2.8. Photosynthtetic and accessories pigment analyses 163

6.2.9. Proline content and metabolism 163

6.2.10. Abscisic acid determination 164

6.2.11. Lipid peroxidation 164

6.2.12. Antioxidant enzymes 164

6.2.13. Antioxidant molecules: ascorbate and glutathione 165

6.2.14. Statistical analysis 166

6.3. Results 166

6.3.1. Experiment I (Salinty) 166

6.3.1.1. Water status, proline metabolism and abscisic acid 166

6.3.1.2. Photosynthetic parameters 168

6.3.1.3. Photosynthetic and accessories pigments 172

6.3.2. Experiment II (Ozone treatment after salinity) 173

6.3.2.1. Visible symptoms and photosynthetic parameters 173

6.3.2.2. Antioxidant enzyme activities, ascorbic acid and glutathione content and MDA-by product content 174

6.4. Discussion 178

USING FOLIAR OPTICAL PROPERTIES TO ASSESS THE

EFFECTS OF DROUGHT ON PLANT WATER POTENTIAL

Abstract 191

7.1. Introduction 192

7.2. Materials and Methods 194 7.2.1 Experimental design 194 7.2.2. Estimation of leaf water potential 195 7.2.3. Collection of leaf spectra 195 7.2.4. Collection of osmolyte standard spectra 196 7.2.5. Statistical modelling of leaf water potential and pre-dawn leaf water

potential 197

7.3. Results 199

7.3.1. Estimating leaf water potential from leaf water spectra 199 7.3.2. Estimating pre-dawn leaf water potential from dark-acclimated leaves using leaf reflectance spectra 204

7.4. Discussion 207 7.5. References 211

Chapter 8:

GENERAL DISCUSSION

CHAPTER 2

Table 2.1. Biometric parameters estimated in Quercus ilex, Q. pubescens and Q.

cerris plants exposed to drought (daily irrigation with 30% of effective

evapotranspiration), ozone (80 ppb for 77 consecutive days, 5 h day-1) and drought × ozone. Controls were kept in charcoal-filtered air and were well watered. Data are shown as mean ± standard deviation (n = 5). Measurements are made at the end of exposure. For each parameter, different letters indicate significant differences (P ≤ 0.05). Asterisks show the significance of factors/interaction in the two-way ANOVA for: *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05, ns P > 0.05. Abbreviations: TDW, total dry weight; LDW, leaf dry weight.

Table 2.2. Significance of F ratio following two-way ANOVA for pre-dawn water

potential (PDw), leaf proline content and malondialdehyde (MDA) by-products

estimated in Quercus ilex, Q. pubescens and Q. cerris plants exposed to drought (daily irrigation with 30% of effective evapotranspiration), ozone (80 ppb for 77 consecutive days, 5 h day−1) and drought × ozone. *** P ≤ 0.001, ** P ≤ 0.01, *

P≤0.05, ns P > 0.05.

Table 2.3. F values of three-way repeated measures ANOVA of the effects of

drought (daily irrigation with 30% of effective evapotranspiration), ozone (80 ppb for 77 consecutive days, 5 h day−1) and time (6.00, 8.00, 10.00, 12.00, 14.00, 16.00 and 18.00) on daily leaf photosynthesis (A) and stomatal conductance (gs) in Quercus ilex, Q. pubescens and Q. cerris plants. Asterisks show the significance of

factors/interaction: *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05, ns P > 0.05. d.f. represents the degrees of freedom.

Table 2.4. Significance of the F ratio following two-way ANOVA for foliar gas

exchange parameters in Quercus ilex, Q. pubescens and Q. cerris plants exposed to drought (daily irrigation with 30% of effective evapotranspiration), ozone (80 ppb for 77 consecutive days, 5 h day−1) and drought × ozone. P = 0.05. *** P ≤ 0.001, **

P ≤ 0.01, * P ≤ 0.05, ns P > 0.05. Abbreviations: A380, CO2 assimilation rate at light

saturation; gs, stomatal conductance to water vapour; WUEi, intrinsic water use

efficiency, Vcmax, maximum rate of carboxylation.

Table 2.5. Significance of the F ratio following two-way ANOVA for leaf

chlorophyll a fluorescence parameters in Quercus ilex, Q. pubescens and Q. cerris plants exposed to drought (daily irrigation with 30% of effective evapotranspiration), ozone (80 ppb for 77 consecutive days, 5 h day−1) and drought × ozone. *** P ≤ 0.001, ** P ≤ 0.01, * P≤0.05, ns P > 0.05. Abbreviations: Fv/Fm, potential PSII

photochemical activity; PSII, actual PSII photochemical activity; NPQ,

assimilation and non-assimilative processes.

Table 2.6. Leaf pigments content in Quercus ilex, Q. pubescens and Q. cerris plants

exposed to drought (daily irrigation with 30% of effective evapotranspiration), ozone (80 ppb for 77 consecutive days, 5 h day−1) and drought × ozone. Controls were kept in charcoal-filtered air and were well-watered. Data are shown as mean ± standard deviation (n = 3). Measurements are made at the end of exposure. For each parameter, different letters indicate significant differences (P ≤ 0.05). Asterisks show the significance of factors/interaction in the two-way ANOVA for: *** P ≤ 0.001, **

P ≤ 0.01, * P ≤ 0.05, ns P > 0.05. Abbreviations: Chl a/b, chlorophyll a/chlorophyll b; ChlTOT, chlorophyll a+chlorophyll b; VAZ,

Violaxanthin+Anteraxanthin+Zeaxanthin; DW, dry weight.

Table 2.7. Statistical analysis of plasticity index (PI) and deviation of PI groups of

variables from the means values of the three oaks species (mean) of biometric, biochemical and physiological variables as grouped in Table S7. Asterisks show the significance of factors/interaction in the two-way ANOVA with PI (or mean) and species as variability factors (** P ≤ 0.01, * P ≤ 0.05, ns P > 0.05).

Table 2.8. Plasticity index (PI) of Quercus ilex, Q. pubescens and Q. cerris

calculated as in Valladares et al (2000) for all determined variables that are arranged in categories. mean indicates the deviation from the mean values of the three species.

CHAPTER 3

Table 3.1. F values of two-way repeated measures ANOVA of the effects of drought

(daily irrigation with 20% of effective evapotranspiration) and ozone (80 ppb for 28 consecutive days, 5 h day−1, in form of a square wave between 10:00 and 15:00) throughout time (7, 14, 21 and 28 days from the beginning of exposure) on CO2

assimilation rate (A), stomatal conductance to water vapor (gs), intercellular CO2

concentration (Ci), potential PSII photochemical activity (Fv/Fm), actual PSII

photochemical activity (ΦPSII), photochemical (qP), and non photochemical

quenching (qNP) in Quercus cerris plants. Asterisks show the significance of factors/interaction: *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05, ns P > 0.05. d.f. represents the degrees of freedom.

Table 3.2. F values of two-way repeated measures ANOVA of the effects of drought

(daily irrigation with 20% of effective evapotranspiration) and ozone (80 ppb for 28 consecutive days, 5 h day-1, in form of a square wave between 10:00 and 15:00) throughout time (06:00, 08:00, 10:00, 12:00, 14:00, 16:00 and 18:00 hours) on CO2

assimilation rate (A), stomatal conductance to water vapor (gs), intercellular CO2

concentration (Ci), potential PSII photochemical activity (Fv/Fm), actual PSII

photochemical activity (ΦPSII), photochemical (qP), and non-photochemical

represents the degrees of freedom.

Table 3.3. Predawn leaf water potential (PDΨw), leaf osmotic potential (Ψπ),

malondialdehyde (MDA)-by products, relative water content (RWC), and electrolytic leakage (EL) estimated in Quercus cerris plants (i) regularly irrigated to maximum soil water holding capacity and exposed to charcoal filtered air (controls, WW/O3-);

(ii) water stressed and exposed to charcoal filtered air (drought, WS/O3-); (iii)

regularly irrigated and O3 fumigated (ozone, WW/O3+); and (iv) water stressed and

O3 fumigated (drought × ozone, WS/O3+). WS/O3- and WS/O3+ plants daily

received 20% of effective evapotranspiration. WW/O3+ and WS/O3+ plants were

exposed to 80 ppb of O3 for 28 consecutive days (5 h d-1, in form of a square wave

between 10:00 and 15:00). Data are shown as mean ± standard deviation (n = 3). Following two-way ANOVA, for each parameter different letters indicate significant differences (P ≤ 0.05). *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05, ns P > 0.05.

Table 3.4. Proline, abscisic acid (ABA) and hexoses (glucose + fructose) (µmol g-1

DW) estimated in Quercus cerris plants (i) regularly irrigated to maximum soil water holding capacity and exposed to charcoal filtered air (controls, WW/O3-); (ii) water

stressed and exposed to charcoal filtered air (drought, WS/O3-); (iii) regularly

irrigated and O3 fumigated (ozone, WW/O3+); and (iv) water stressed and O3

fumigated (drought × ozone, WS/O3+). WS/O3- and WS/O3+ plants daily received

20% of effective evapotranspiration. WW/O3+ and WS/O3+ plants were exposed to

80±13 ppb of O3 for 28 consecutive days (5 h d-1, in form of a square wave between

10:00 and 15:00). Data are shown as mean (n = 3). Following two-way ANOVA, for each parameter different letters indicate significant differences (P ≤ 0.05). *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05, ns P > 0.05.

Table 3.5. Leaf pigments content in Quercus cerris plants (i) regularly irrigated to

maximum soil water holding capacity and exposed to charcoal filtered air (controls, WW/O3-); (ii) water stressed and exposed to charcoal filtered air (drought, WS/O3-);

(iii) regularly irrigated and O3 fumigated (ozone, WW/O3+); (iv) water stressed and

O3 fumigated (drought × ozone, WS/O3+). WS/O3- and WS/O3+ plants daily

received 20% of effective evapotranspiration. WW/O3+ and WS/O3+ plants were

exposed to 80 ppb of O3 for 28 consecutive days (5 h d-1, in form of a square wave

between 10:00 and 15:00). Data are shown as mean ± standard deviation (n = 3). Following two-way ANOVA, for each parameter different letters indicate significant differences (P ≤ 0.05). *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05, ns P > 0.05. Abbreviations: Car, total carotenoids; Chl, chlorophyll a + chlorophyll b; DW, dry weight; VAZ Violaxanthin + Antheraxanthin + Zeaxanthin.

CHAPTER 4

Table 4.1. Predawn leaf water potential (PDΨw), leaf osmotic potential (Ψπ), relative

water content (RWC), and malondialdehyde (MDA)-by products estimated in

stressed and exposed to charcoal filtered air (WS/O3-); (iii) regularly irrigated and O3

fumigated (WW/O3+); and (iv) water stressed and O3 fumigated (WS/O3+) for 28

consecutive days. WS/O3- and WS/O3+ plants daily received 20% of effective

evapotranspiration. WW/O3+ and WS/O3+ plants were exposed to 80±13 ppb of O3

for 5 h d-1, in form of a square wave between 10:00 and 15:00. Data are shown as mean ± standard deviation (n = 3). Following two-way ANOVA, for each parameter different letters indicate significant differences (P = 0.05). *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05, ns P > 0.05.

Table 4.2. Proline (Pro), abscisic acid (ABA), glucose and fructose estimated in

Quercus pubescens and Quercus ilex plants (i) regularly irrigated to maximum soil

water holding capacity and exposed to charcoal filtered air (WW/O3-); (ii) water

stressed and exposed to charcoal filtered air (WS/O3-); (iii) regularly irrigated and O3

fumigated (WW/O3+); and (iv) water stressed and O3 fumigated (WS/O3+) for 28

consecutive days. WS/O3- and WS/O3+ plants daily received 30% of effective

evapotranspiration. WW/O3+ and WS/O3+ plants were exposed to 80±13 ppb of O3

for 5 h d-1, in form of a square wave between 10:00 and 15:00. Data are shown as mean ± standard deviation (n = 3). Following two-way ANOVA, for each parameter different letters indicate significant differences (P = 0.05). *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05, ns P > 0.05. Abbreviation: DW, dry weight.

Table 4.3. F values of two-way repeated measures ANOVA of the effects of drought

(daily irrigation with 30% of effective evapotranspiration) and ozone (80 ppb for 28 consecutive days, 5 h day−1, in form of a square wave between 10:00 and 15:00) in time (06:00, 08:00, 10:00, 12:00, 14:00, 16:00 and 18:00 hours) on CO2 assimilation

rate (A), stomatal conductance to water vapor (gs), water use efficiency (WUE),

intercellular CO2 concentration (Ci), potential PSII photochemical activity (Fv/Fm),

actual PSII photochemical activity (ΦPSII), and no photochemical quenching (qNP) in Quercus pubescens and Quercus ilex plants. Asterisks show the significance of

factors/interaction: *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05, ns P > 0.05. d.f. represents the degrees of freedom.

Table 4.4. Leaf pigments content in Quercus pubescens and Quercus ilex plants (i)

regularly irrigated to maximum soil water holding capacity and exposed to charcoal filtered air (WW/O3-); (ii) water stressed and exposed to charcoal filtered air

(WS/O3-); (iii) regularly irrigated and O3 fumigated (WW/O3+); (iv) water stressed

and O3 fumigated (WS/O3+) for 28 consecutive days. WS/O3- and WS/O3+ plants

daily received 30% of effective evapotranspiration. WW/O3+ and WS/O3+ plants

were exposed to 80±13 ppb of O3 for 5 h d-1, in form of a square wave between 10:00

and 15:00. Data are shown as mean ± standard deviation (n = 3). Following two-way ANOVA, for each parameter different letters indicate significant differences (P = 0.05). *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05, ns P > 0.05. Abbreviations: car, β-carotene; ChlTOT, chlorophyll a + chlorophyll b; DEPS, depoxidation state; DW, dry

mol-1 ChlTOT).

CHAPTER 5

Table 5.1. Water status and ecophysiological parameters in Quercus ilex plants

well-watered (WW) or water stressed (20% of the effective evapotranspiration daily for 15 days, WS). Data are shown as mean ± standard deviation (n = 3). Asterisks show the significance of Student’s t-test: *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05, ns P > 0.05. Abbreviations: ΦPSII, photochemical efficiency in light conditions; A, CO2

assimilation rate; Ci, intercellular CO2; Fv/Fm, potential PSII photochemical activity;

gs, stomatal conductance to water vapour; PDΨW, pre-dawn leaf water potential; qP,

photochemical quenching; qNP, non-photochemical quenching; RWC, relative water content.

Table 5.2. Biochemical parameters in Quercus ilex plants well-watered (WW) or

water stressed (20% of the effective evapotranspiration daily for 15 days, WS). Data are shown as mean ± standard deviation (n = 3). Asterisks show the significance of Student’s t-test: *** P ≤ 0.001, ** P ≤ 0.01, ns P > 0.05. Abbreviations: ABA, abscisic acid; DW, dry weight; ET, ethylene; FW, fresh weight; H2O2, hydrogen

peroxide; JA, jasmonic acid; O2-, superoxide anion; Pro, proline; SA, salycilic acid.

Table 5.3. F values of one-way repeated measures ANOVA of the effects of acute

ozone exposure (200 ppb for 5 h) in time (0, 5, 24 and 48 h from the beginning of the exposure) on CO2 assimilation rate (A), stomatal conductance to water vapor (gs),

intercellular CO2 concentration (Ci), potential PSII photochemical activity (Fv/Fm),

photochemical efficiency in light conditions (ΦPSII), photochemical quenching (qP)

and non-photochemical quenching (qNP) in Quercus ilex plants well-watered or water stressed (20% of the effective evapotranspiration daily for 15 days). Asterisks show the significance of factors/interaction: *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05, ns P > 0.05. d.f. represents the degrees of freedom.

Table 5.4. F values of one-way repeated measures ANOVA of the effects of acute

ozone exposure (200 ppb for 5 h) in time (0, 5, 24 and 48 h from the beginning of the exposure) on hydrogen peroxide (H2O2), superoxide anion (O2-), ethylene (ET),

salicylic (SA), jasmonic (JA) and abscisic (ABA) acid and proline (Pro) in Quercus

ilex plants well-watered or water stressed (20% of the effective evapotranspiration

daily for 15 days). Asterisks show the significance of factors/interaction: *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05, ns P > 0.05. d.f. represents the degrees of freedom.

CHAPTER 6

Table 6.1. Values of pre-dawn leaf water potential (PDW), osmotic potential (π),

relative water content (RWC), leaf mass per area (LMA) and leaf succulence in control (-Salt) and salt-treated (150 mM NaCl for 15 days; +Salt) plants of Quercus

ilex. Data are shown as mean ± standard deviation (n = 6). In the last row, the

significance of the differences following the Student’s t test is reported. ns P>0.05; *

P≤0.05; ** P≤0.01; *** P≤0.001.

Table 6.2. Values of Proline, Δ1-pyrroline-5-carboxylate synthetase (P5CS), proline dehydrogenase (PHD) activity, and abscisic acid (ABA) content in control (-Salt)

shown as mean ± standard deviation (n = 3). In the last row, the significance of the differences following the Student’s t test is reported. ns P > 0.05; ** P ≤ 0.01; ***: P ≤ 0.001.

Table 6.3. Relative contribution (%) of inorganic solutes to the osmolality in control

(-Salt) and salt-treated (150 mM NaCl for 15 days; +Salt) leaves of Q. ilex. Data are shown as mean ± standard deviation (n = 3). In the last row, the significance of the differences following the Student’s t test is reported. ** P ≤ 0.01; *** P ≤ 0.001.

Table 6.4. Values of parameters derived from light response curves in control (-Salt)

and salt-treated (150 mM NaCl for 15 days; +Salt) leaves of Q. ilex. Data are shown as mean ± standard deviation (n = 3). In the last column, the significance of the differences following the Student’s t test is reported. ns P > 0.05; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. Abbreviations: CO2, apparent maximum quantum yield; A380,

light-saturated rate of photosynthesis; A/gs, intrinsic water use efficiency; Ci,

intercellular CO2 concentration; E, transpiration rate; gs, stomatal conductance to

water vapour; Rdark, rate of mitochondrial respiration rate at zero irradiance.

Table 6.5. Values of different parameters derived from A/Ci response curves in

control (-Salt) and salt-treated (150 mM NaCl for 15 days; +Salt) leaves of Q. ilex. Data are shown as mean ± standard deviation (n = 3). In the last column, the significance of the differences following the Student’s t test is reported. ns: P>0.05; *: P≤0.05; **: P≤0.01; ***: P≤0.001. Abbreviations: Amax, light- and CO2- saturated

photosynthetic rate; Cc, chloroplastic CO2 concentration; gm, mesophyllic

conductance to CO2; gm/gsCO2, mesophyllic/stomatal conductance to CO2, gm/Vcmax

(Cc): mesophyllic conductance/maximal rate of Rubisco carboxylation; gsCO2,

stomatal conductance to CO2; Jflu/Vcmax (Cc), ratio between maximal electron

transport rate and maximal rate of Rubisco carboxylation efficiency at Cc; Jflu/Vcmax

(Ci), ratio between maximal electron transport rate and maximal rate of Rubisco carboxylation efficiency at Ci; Lb, biochemical limitation; Lm, mesophyll limitation;

Ls, stomatal limitation; Vcmax (Cc), maximal rate of Rubisco carboxylation at Cc;

Vcmax (Ci), maximal rate of Rubisco carboxylation at Ci.

Table 6.6. Values of chlorophyll fluorescence parameters determined in control

(-Salt) and salt-treated (150 mM NaCl for 15 days; +(-Salt) leaves of Q.s ilex. Data are shown as mean ± standard deviation (n = 3). In the last column, the significance of the differences following the Student’s t test is reported. *: P≤0.05; **: P≤0.01; ***:

P≤0.001. Abbreviations: PSII: actual efficiency of PSII photochemistry; Fv/Fm:

maximum efficiency of PSII photochemistry in dark-adapted state; Fv’/Fm’:

efficiency of excitation capture by open PSII reaction centres; NPQ: non-photochemical quenching; qP: photochemical quenching coefficient.

Table 6.7. Concentrations of photosynthetic pigments in control (-Salt) and

salt-treated (150 mM NaCl for 15 days; +Salt) leaves of Quercus ilex. Data are shown as mean ± standard deviation (n = 3). In the last column, the significance of the differences following the Student’s t test is reported; ns: P>0.05; *: P≤0.05; **:

P≤0.01; ***: P≤0.001. Abbreviations: Ant, antheraxanthin; Chltot, total chlorophyll;

DES, de-epoxidation state of violaxanthin cycle pigments; Lut, lutein; -Car,  -Carotene; Neo, neoxanthin; VAZ, Violaxanthin+Antheraxanthin+Zeaxanthin; Vio, violaxanthin; Zea, zeaxanthin.

Experiment II following two-way ANOVA with salt and O3 as factors. ns: P>0.05; *:

P≤0.05; **: P≤0.01; ***: P≤0.001. Abbreviations: ΦPSII, actual photochemical

efficiency; A380, light-saturated photosynthesis; ASA, ascorbic acid peroxidase

activity; ASAT; total ascorbate; CAT, catalase activity; Fv/Fm, maximal

photochemical efficiency; gs, stomatal conductance to water vapour; GSH,

glutathione; GSHT, total glutathione; GR, glutathione reductase activity; MDA,

malondialdehyde by-products level; qP, photochemical quenching; SOD, superoxide

dismutase activity.

Table 6.9. Values of total ascorbate (ASAT),total glutathione (GSHT) and the ratio

between their reduced form (ASA and GSH, respectively) and their total contents (ASA/ASAT and GSH/GSHT, respectively) in Q. ilex plants (i) regularly irrigated

and exposed to charcoal filtered air (-Salt/-O3), (ii) salt-treated and exposed to

charcoal filtered air (+Salt/-O3), (iii) regularly irrigated and O3 fumigated

(-Salt/+O3); and (iv) salt-treated and O3 fumigated (+Salt/+O3). +Salt/-O3 and

+Salt/+O3 plants were treated with 150 mM NaCl for 15 days; -Salt/+O3 and

+Salt/+O3 plants were exposed to 80 ppb of O3 for 5 h. Data are shown as mean ±

standard deviation (n = 5). Data with different letters are significant different following two-way ANOVA (P = 0.05). The lack of letters indicates not significant differences of the F ratio.

CHAPTER 7

Table 7.1. Wavelengths range, number of components, R2, root-mean-square error (RMSE) and bias for the preliminary, non-jackknifed models used for the estimation of ΨLW by spectral data collected immediately after the Scholander pressure chamber

measurements of live oak leaves. Final model selected in bold.

Table 7.2. Correlation matrix of foliar traits predicted from spectra with immediate

predictions of leaf water potential (LW) and predictions of pre-dawn leaf water

potential (PDLW) on dark-acclimated leaves. % dm, % dry mass; LMA, leaf mass

per area; NDWI, normalized differential water index; PRI, photochemical reflectance index. Significant relationships (P ≤ 0.05) are in bold.

Table 7.3. Wavelengths range, number of components, R2, root-mean-square error (RMSE) and bias for the preliminary, non-jackknifed models used for the estimation of PDΨLW by spectral data collected approximately 5 hrs after the Scholander

pressure chamber measurements of live oak leaves. Final model selected in bold.

Table 7.4. F and P values of the one-way ANOVA examining the effects of the

different water availability conditions on PDΨLW in live oak in both reference (i.e.,

pressure chamber) measurements and spectroscopy predicted values using dark-acclimated leaves measured stored to preserve PDΨLW and measured later in the day.

CHAPTER 1

Figure 1.1. Solar spectrum with indicated the main atmospheric absorption bands

(modified from Kumar et al. 2001).

Figure 1.2. Leaf reflectance spectra (n=59) of Dwight and Pana genotypes of

soybean collected with a high-spectral-resolution ASD FieldSpec Full-Range spectroradiometer (Analytical Spectral Devices, Boulder, CO, USA) (from Ainsworth et al. 2014).

Figure 1.3. Proposed concept of ‘optical type’ based on the assessment of vegetation

structure, physiology and biochemistry, and phenology – three variables historically contributing to ecological definition of ‘plant functional type’- as well as of stress conditions (e.g. due to air pollution) (modified from Ustin and Gamon 2010).

CHAPTER 2

Figure 2.1. Symptoms in leaves of Quercus ilex, Q. pubescens and Q. cerris exposed

to drought (daily irrigation with 30% of effective evapotranspiration; WS/O3-), ozone

(80 ppb for 77 consecutive days, 5 h day−1; WW/O3+) and drought × ozone

(WS/O3+). Controls were kept in charcoal-filtered air and were well watered

(WW/O3-).

Figure 2.2. Pre-dawn water potential (PDw; a), leaf proline content (b) and

malondialdehyde (MDA) by-products (c) estimated in Quercus ilex, Q. pubescens

and Q. cerris plants exposed to drought (daily irrigation with 30% of effective

evapotranspiration; grey bars), ozone (80 ppb for 77 consecutive days, 5 h day−1; dark grey bars) and drought × ozone (black bars). Controls were kept in charcoal-filtered air and were well watered (white bars). Data are shown as mean ± standard deviation (n = 3). Abbreviations: DW, dry weight; FW, fresh weight.

Figure 2.3. Daily leaf photosynthesis (A) and stomatal conductance (gs) in Quercus ilex, Q. pubescens and Q. cerris plants exposed to drought (daily irrigation with 30%

effective evapotranspiration; closed circle), ozone (80 ppb for 77 consecutive days, 5 h day−1; open square) and drought × ozone (closed square). Controls were kept in charcoal-filtered air and were well watered (open circle). Data are shown as mean ± standard deviation (n = 3).

Figure 2.4. Foliar gas exchange parameters in Quercus ilex, Q. pubescens and Q.

cerris plants exposed to drought (daily irrigation with 30% of effective

evapotranspiration; grey bars), ozone (80 ppb for 77 consecutive days, 5 h day−1; dark grey bars) and drought × ozone (black bars). Controls were kept in charcoal-filtered air and were well-watered (white bars). Data are shown as mean ± standard

gs, stomatal conductance to water vapour (b); WUEi, intrinsic water use efficiency

(c); Vcmax, maximum rate of carboxylation (d).

Figure 2.5. Leaf chlorophyll a fluorescence parameters in Quercus ilex, Q.

pubescens and Q. cerris plants exposed to (daily irrigation with 30% effective

evapotranspiration; grey bars), ozone (80 ppb for 77 consecutive days, 5 h day−1; dark grey bars) and drought × ozone (black bars). Controls were kept in charcoal-filtered air and were well watered (white bars). Data are shown as mean ± standard deviation (n = 6). Quenching analysis parameters are determined at a light intensity of about 600 µmol photon m-2 s-1. Abbreviations: Fv/Fm, potential PSII

photochemical activity (a); PSII, actual PSII photochemical activity (b); NPQ,

non-photochemical quenching (c); Fs/F0, steady-state fluorescence value normalized to

minimal fluorescence (d); PSII/CO2 ratio (e).

Figure 2.6. Quercus ilex, Q. pubescens and Q. cerris ordered by mean phenotypic

plasticity. For each species the percentage contributions of biometric (PB),

biochemical (PBC) and physiological (PP) plasticity to the total phenotypic plasticity

are reported.

CHAPTER 3

Figure 3.1. Profiles of foliar leaf gas exchange and chlorophyll fluorescence

parameters in Q. cerris plants (i) regularly irrigated to maximum soil water holding capacity and exposed to charcoal filtered air (controls, WW/O3-, open circle); (ii)

water stressed and exposed to charcoal filtered air (drought, WS/O3-, open square);

(iii) regularly irrigated and O3 fumigated (ozone, WW/O3+, closed circle); (iv) water

stressed and O3 fumigated (drought × ozone, WS/O3+, closed square). WS/O3- and

WS/O3+ plants daily received 20% of effective evapotranspiration. WW/O3+ and

WS/O3+ plants were exposed to 80 ppb of O3 for 28 consecutive days (5 h d-1, in

form of a square wave between 10:00 and 15:00). Data are shown as mean ± standard error (n = 3). Abbreviations: A, CO2 assimilation rate; gs, stomatal

conductance to water vapor; Ci, intercellular CO2 concentration; ΦPSII, actual PSII

photochemical activity; qP, photochemical quenching; qNP, no photochemical quenching.

Figure 3.2. Daily profiles of foliar leaf gas exchange and chlorophyll fluorescence

parameters in Q. cerris plants (i) regularly irrigated to maximum soil water holding capacity and exposed to charcoal filtered air (controls, WW/O3-, open circle); (ii)

water stressed and exposed to charcoal filtered air (drought, WS/O3-, open square);

(iii) regularly irrigated and O3 fumigated (ozone, WW/O3+, closed circle); (iv) water

stressed and O3 fumigated (drought × ozone, WS/O3+, closed square). WS/O3- and

WS/O3+ plants daily received 20% of effective evapotranspiration. WW/O3+ and

WS/O3+ plants were exposed to 80 of O3 for 28 consecutive days (5 h d-1, in form of

a square wave between 10:00 and 15:00). Data are shown as mean ± standard error (n = 3). Abbreviations: A, CO2 assimilation rate; gs, stomatal conductance to water

qP, photochemical quenching coefficient; qNP, non-photochemical quenching coefficient.

CHAPTER 4

Figure 4.1. CO2 assimilation rate (A) in relation to stomatal conductance to water

vapor (gs) and evapotranspiration (E) in Quercus pubescens and Quercus ilex plants

(i) regularly irrigated to maximum soil water holding capacity and exposed to charcoal filtered air (WW/O3-, open circle); (ii) water stressed and exposed to

charcoal filtered air (WS/O3-, close triangle); (iii) regularly irrigated and O3

fumigated (WW/O3+, open triangle); (iv) water stressed and O3 fumigated (WS/O3+,

closed circle) for 28 consecutive days. WS/O3- and WS/O3+ plants daily received

20% of effective evapotranspiration. WW/O3+ and WS/O3+ plants were exposed to

80±13 ppb of O3 for 5 h d-1, in form of a square wave between 10:00 and 15:00. Data

here reported were collected weekly during the experiment (28 days). Linear regression lines with the coefficients of determination (R2) are shown.

Figure 4.2. Daily of foliar gas exchange parameters in Q. pubescens and Quercus ilex plants (i) regularly irrigated to maximum soil water holding capacity and

exposed to charcoal filtered air (WW/O3-, open circle); (ii) water stressed and

exposed to charcoal filtered air (WS/O3-, open square); (iii) regularly irrigated and

O3 fumigated (WW/O3+, closed circle); (iv) water stressed and O3 fumigated

(WS/O3+, closed square) for 28 consecutive days. WS/O3- and WS/O3+ plants daily

received 20% of effective evapotranspiration. WW/O3+ and WS/O3+ plants were

exposed to 80±13 ppb of O3 for 5 h d-1, in form of a square wave between 10:00 and

15:00. Data are shown as mean ± standard deviation (n = 3). Abbreviations: A, leaf photosynthesis (a, e); gs, stomatal conductance (b, f); WUE, water use efficiency (c,

g); Ci, and intercellular carbon dioxide (d, h).

Figure 4.3. Daily of leaf chlorophyll a fluorescence parameters in Q. pubescens and

Quercus ilex plants (i) regularly irrigated to maximum soil water holding capacity

and exposed to charcoal filtered air (WW/O3-, open circle); (ii) water stressed and

exposed to charcoal filtered air (WS/O3-, open square); (iii) regularly irrigated and

O3 fumigated (WW/O3+, closed circle); (iv) water stressed and O3 fumigated

(WS/O3+, closed square) for 28 consecutive days. WS/O3- and WS/O3+ plants daily

received 20% of effective evapotranspiration. WW/O3+ and WS/O3+ plants were

exposed to 80±13 ppb of O3 for 5 h d-1, in form of a square wave between 10:00 and

15:00. Data are shown as mean ± standard deviation (n = 3). Abbreviations: Fv/Fm,

potential PSII photochemical activity (a, d); PSII, actual PSII photochemical activity

(b, e); qNP, non-photochemical quenching (c, f).

CHAPTER 5

Figure 5.1. Localization of dead cells visualized with Evans blue staining (A-D) and

of hydrogen peroxide (H2O2) visualized the 3,3’-diaminobenzidine (DAB) uptake

15 days) and exposed to charcoal filtered air (WS); (iii) well-watered and exposed to acute ozone (200 pbb for 5 h) (WW+O3); (iv) water stressed and O3 fumigated

(WS+O3). The assays were performed 96 h FBE. Bars 50 µm.

Figure 5.2. Time course of leaf gas exchange parameters in Quercus ilex

well-watered (open square) or water stressed (20% of the effective evapotranspiration daily for 15 days, closed square) and exposed to acute ozone (200 ppb for 5 h). Data are shown as mean ± standard deviation (n = 3). The measurements were carried out at 0, 5, 24 and 48 h from the beginning of exposure. According to the one-way repeated measures ANOVA with treatment as variability factor, different letters indicate significant differences (P ≤ 0.05). Abbreviations: ΦPSII, photochemical

efficiency in light conditions; A, CO2 assimilation rate; gs, stomatal conductance to

water vapour; Ci, intercellular CO2 concentration; qP, photochemical quenching;

qNP, non-photochemical quenching. The thick line indicates the time (5 h) of ozone exposure.

Figure 5.3. Time course of Reactive Oxygen Species in Quercus ilex well-watered

(open square) or water stressed (20% of the effective evapotranspiration daily for 15 days, closed square) and exposed to acute ozone (200 ppb for 5 h). Data are shown as mean ± standard deviation (n = 3). The measurements were carried out at 0, 1, 2, 5, 8 and 24 h from the beginning of exposure. According to the two-way ANOVA with treatment and time as variability factors, different letters indicate significant differences (P ≤ 0.05). Abbreviations: DW, dry weight; H2O2, hydrohen peroxide;

O2-, superoxide anion. The thick line indicates the time (5 h) of ozone exposure.

Figure 5.4. Time course of phytohormones in Quercus ilex well-watered (open

square) or water stressed (20% of the effective evapotranspiration daily for 15 days, closed square) and exposed to acute ozone (200 ppb for 5 h). Data are shown as mean ± standard deviation (n = 3). The measurements were carried out at 0, 1, 2, 5, 8 and 24 h from the beginning of exposure. According to the two-way ANOVA with treatment and time as variability factors, different letters indicate significant differences (P ≤ 0.05). Abbreviations: DW, dry weight; ET, ethylene; FW, fresh weight; JA, jasmonic acid; SA, salycilic acid. The thick line indicates the time (5 h) of ozone exposure.

Figure 5.5. Time course of abscisic acid (ABA) and proline (Pro) in Quercus ilex

well-watered (open square) or water stressed (20% of the effective evapotranspiration daily for 15 days, closed square) and exposed to acute ozone (200 ppb for 5 h). Data are shown as mean ± standard deviation (n = 3). The measurements were carried out at 0, 1, 2, 5, 8 and 24 h from the beginning of exposure. According to the two-way ANOVA with treatment and time as variability factors, different letters indicate significant differences (P ≤ 0.05). Abbreviation: DW, dry weight. The thick line indicates the time (5 h) of ozone exposure.

CHAPTER 6

Figure 6.1. Response of CO2 assimilation rate (A) to intercellular CO2 concentration

(Ci) at three irradiances levels (50, 200 and 400 mol m-2 s-1, 400-700 nm) determined in control (-Salt, above) and salt-treated (150 mM NaCl for 15 days; +Salt, below) leaves of Quercus ilex.

treated (150 mM NaCl for 15 days; +Salt) tissues of Q. ilex (leaf, stem and root). Bars respresent means ± standard deviation (n = 3). –Salt vs +Salt plants were compared with the Student’s t-test. *** P ≤ 0.001.

Figure 6.3. Response of net CO2 assimilation to photosynthetically active radiation

determined in control (-Salt, open circles) and salt-treated (150 mM NaCl for 15 days; +Salt, closed circles) leaves of Q. ilex.

Figure 6.4. The response of net CO2 assimilation to internal CO2 concentration (Ci)

determined on young leaves of Q. ilex grown in control conditions (open circle) and at 150 mM NaCl for 15 days (closed circle).

Figure 6.5. Relationship between the DES of xantophylls and the efficiency of

excitation capture by open PSII reaction centres (Fv’/Fm’, closed circle), the actual

PSII efficiency (PSII, closed square) and NPQ coefficient (open square) measured at

midday in control or salt-treated (150 mM NaCl for 15 days) leaves of Q. ilex.

Figure 6.6. Light-saturated rates of photosynthesis (A380), stomatal conductance to

water vapour (gs), maximal and optimal photochemical efficiency of PSII (Fv/Fm and

ΦPSII, respectively), proportion of open reaction centres (qP) and non-photochemical

quenching (NPQ) measured in Q. ilex plants (i) regularly irrigated and exposed to charcoal filtered air (-Salt/-O3), (ii) salt-treated and exposed to charcoal filtered air

(+Salt/-O3), (iii) regularly irrigated and O3 fumigated (-Salt/+O3); and (iv)

salt-treated and O3 fumigated (+Salt/+O3). +Salt/-O3 and +Salt/+O3 plants were treated

with 150 mM NaCl for 15 days; -Salt/+O3 and +Salt/+O3 plants were exposed to 80

ppb of O3 for 5 h. Data are shown as mean ± standard deviation (n = 5). For each

parameter, the lack of letters above bars indicates the absence of significant interaction between salt and O3 factors (P = 0.05), following two-way ANOVA.

Figure 6.7. Activity of superoxide dismutase (SOD), catalase (CAT), ascorbate

peroxidase (APX), and glutathione reductase (GR) in Q. ilex plants (i) regularly irrigated and exposed to charcoal filtered air (-Salt/-O3), (ii) salt-treated and exposed

to charcoal filtered air (+Salt/–O3), (iii) regularly irrigated and O3 fumigated

(-Salt/+O3); and (iv) salt-treated and O3 fumigated (+Salt/+O3). +Salt/-O3 and

+Salt/+O3 plants were treated with 150 mM NaCl for 15 days; -Salt/+O3 and

+Salt/+O3 plants were exposed to 80 ppb of O3 for 5 h. Data are shown as mean ±

standard deviation (n = 5). For each parameter, bars keyed with different letters indicate significant differences among treatments (P = 0.05), following two-way ANOVA. The lack of letters above bars indicates the absence of significant interaction between salt and O3 factors.

Figure 6.8. Values of malondialdehyde (MDA) by-products measured in Q. ilex

plants (i) regularly irrigated and exposed to charcoal filtered air (-Salt/-O3), (ii)

salt-treated and exposed to charcoal filtered air (+Salt/-O3), (iii) regularly irrigated and O3

fumigated (-Salt/+O3); and (iv) salt-treated and O3 fumigated (+Salt/+O3). +Salt/-O3

and +Salt/+O3 plants were treated with 150 mM NaCl for 15 days; -Salt/+O3 and

+Salt/+O3 plants were exposed to 80 ppb of O3 for 5 h. Data are shown as mean ±

standard deviation (n = 5). Bars keyed with different letters indicate significant differences among treatments (P = 0.05), following two-way ANOVA.

Figure 7.1. Error distributions of (a) R2, (b) root-mean-square error (RMSE) and (c) bias for validation data generated using 500 random permutations of the data with 80% used for calibration and 20% used for validation to predict LW values from

foliar spectra from live oak. (d) Predicted vs observed values of LW of live oak.

Dotted line is 1:1 relationship.

Figure 7.2. Mean (solid), 5th, and 95th percentile (dotted) of LW (a, black) and

PDLW (b, blue) standardized model coefficients and (c) variable important to the

projection (VIP) selection values by wavelengths. Grey vertical bars represent the main absorption water features of the spectrum. Red vertical dotted lines represent prominent absorption features of common foliar osmolyes (see Figure 7.3).

Figure 7.3. Reflectance profiles of purified osmolyte standards. Red vertical dotted

lines represent prominent absorption features.

Figure 7.4. Error distributions of (a) R2, (b) root-mean-square error (RMSE) and (c) bias for validation data generated via cross-validation using 500 random permutations of the data with 80% used for calibration and 20% used for validation for models predicting PDLW values from foliar spectra collected ~5 hrs after the

reference measurement from live oak. (d) Instantaneous predicted vs a posteriori predicted values of PDLW of live oak. Dotted line is 1:1 relationship.

Figure 7.5. PDLW predicted values using the model built with the data collected

instantaneously after the Scholander pressure chamber measurements vs PDLW

predicted values using the model built from a posteriori measurements on dark-acclimated leaves in live oak.

Chapter 1

General introduction and thesis outline

1.1. Climate change and air pollution in the Mediterranean region

The Working Group I contribution to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change (IPCC 2013) has highlighted three main conclusions: (i) the warming of the climate system is unequivocal (i.e. the last three decades have been successively warmer at the Earth’s surface than any preceding decade since 1850), (ii) the human influence has been the dominant cause of this phenomenon since the mid-20th century, and (iii) the continued emissions of greenhouse gases will cause further increasing of temperatures and changes in all components of the climate system. Climate change is a scientific certainty as the effects of biotic and abiotic stresses on ecosystems are already detectable and will likely be more evident in the next years.

The European Mediterranean region is considered an aesthetically appealing area, containing a rich amount of biodiversity, exclusive combinations of landscapes due to the climate, relief and soil conditions and as had human occupation for centuries (de Jong et al. 2012). However, in this area the impact of climate change is particularly severe in comparison to the other areas worldwide as the Mediterranean basin, characterized by a unique climate regime with wet and mild winters, hot and dry summers and an inconsistent inter-annual and -seasonal distribution of precipitation (Luterbacher et al. 2006), is greatly sensitive to even minor changes in global atmospheric dynamics. Here, climate change manifests itself in two fundamentally different ways: increasing the yearly temperature (aridity) and/or modifying the frequency and the intensity of extreme meteorological events, such as heat waves, rain pulses and dust events (Rumukainen 2012; Gaetani and Pasqui 2014). In the last 100 years a significant annual warming trend of +0.75 °C has been found (Joel and Kaniewski 2015), and the heat waves are by now a familiar feature of the Mediterranean area as several anomalous warm summers have occurred in southern Europe over the last 50-60 years (e.g., the dramatic summers of 2003 and 2012, Lorenzini et al. 2014a). Moreover, climatic models indicate that the

Mediterranean basin will be one of the areas subjected to the most drastic reductions in precipitation and increases in warming globally, with a predicted reduction in precipitation of 25 to 30% by the end of the 21st century and an increase in air temperature of between 2-3 °C by 2050 (Christensen et al. 2007). Both variations are expected to be larger in summer, as meteorological models also predict an increasing frequency of heat waves in the next 50 years, with consequences on the total length of this season and the number of heat days (temperature higher than 30 °C, Simolo et al. 2014). Furthermore, the exacerbation of human population growth, anthropogenic activities and environmental alterations (Matesanz and Valladares 2014) strongly contributes the substantial vulnerability of ecosystems in the Mediterranean under future predicted patterns of climate change.

Atmospheric pollution is one of the most difficult environmental tasks society currently face. Airborne particles induce several negative impacts on human health (WHO 2013). Similarly, exposure to tropospheric ozone (O3), nitrogen oxides (NOx),

sulphur dioxide (SO2) or heavy metals can cause cardiovascular and lung disorders,

premature deaths and have carcinogenic effects. In addition, these air pollutants may also affect ecosystem functioning and agricultural productivity, having pronounced adverse effects on growth, development and longevity of plants (Cerro et al. 2015). Finally, air pollution will have a major effect on climate (IPCC 2013).

The Mediterranean placed between the tropical and mid-latitudes, is an intersection of air masses coming from Europe, Asia and Africa, where anthropogenic emissions, mostly from Europe, Balkans and Black Sea, meet with natural emissions from Saharan dust, vegetation and the sea, as well as from biomass burning. Furthermore, atmospheric pollution is favored by the South European climate and is likely to grow in the future due to rapid urbanization (Kanakidou et al. 2011). For these reasons, the Mediterranean basin can be considered a hot-spot not only in terms of climate change (Giorgi and Lionello 2008), but also for air-quality (Cristofanelli et al. 2016).

All levels of biota may be affected by climate change and atmospheric pollution including aboveground (e.g. crops, trees, animals etc.) and belowground (e.g. microbiota, invertebrates) species that are intimately related. The sensitivity of Mediterranean plant species to abiotic stresses (such as warming, drought, O3,

field and controlled experiments (Bussotti et al. 2014). However, understanding how the climate change and atmospheric pollution will modulate plant responses and how they will affect plant growth and distribution is still largely unknown.

1.1.1. Ozone

The most widespread pollutant in the Mediterranean region is O3 (Cristofanelli and

Bonasoni 2009), which frequently exceeds the limit values established for the protection of natural vegetation and crops (EEA 2016). Despite efforts to control this pollutant over recent decades, the background mean concentrations in the Northern Hemisphere have more than doubled to 35-40 ppb since the industrial revolution, and daily peak concentrations continue to exceed the WHO guideline values of 50 ppb in many regions (Mills et al. 2016). Although the impacts of surface O3 on human

health on vegetation have prompted O3 precursor emission reductions in the

European Union and United States (whereas emissions have increased in East Asia) (Lefohn et al. 2017), ground levels of O3 are expected to increase in Mediterranean

regions as a consequence of climate change because its formation and accumulation in the atmosphere is connected to elevated solar radiation and high temperature and is favored by high pressure conditions (Lorenzini et al. 2014b). Elevated O3 may

contribute to increased rates of climate change directly, due to its status as an important greenhouse gas, and indirectly, due to its effect on rates of carbon dioxide (CO2) uptake by terrestrial ecosystems.

Ozone is a secondary product formed by the pollution of the atmosphere, as it is generated when primary polluting substances [such as carbon monoxide (CO), hydrocarbons, nitrogen oxides (NOx) and halogens] of automobile exhaust and

industrial wastes are exposed to the solar light. If in the stratosphere O3 is decreasing,

its concentration in the troposphere is increasing with negative impacts on the (i) environment and (ii) health of living organisms. O3 is a greenhouse gas and

especially is one of the most powerful oxidants known (with a redox potential of 2.07 V). It is recognized that O3 sources in the continental boundary layer of the

atmosphere include both transport from the stratosphere and in situ photochemical production. In the troposphere, O3 is produced by O2/O3 equilibrium reactions