Regulation of hormonal responses of sweet pepper as affected by salinity and elevated CO2 concentration

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M. Carmen Piñeroa, Fabrice Houdusseb, Jose M. Garcia-Minac,d, María Garnicac and Francisco M. del Amora,*

a

Equipo de Calidad Alimentaria, Instituto Murciano de Investigación y Desarrollo Agrario y

Alimentario (IMIDA), C/Mayor s/n, 30150 Murcia, Spain b

Centre de Recherche International en Agroscience, CRIA-TAI, Groupe Roullier, 55 boulevard Jules

Verger, 35800 Dinard, France

c

R&D Department (CIPAV-Timac Agro Roullier Group), Polígono Arazuri-Orcoyen, c/C No. 32,

31160 Orcoyen (Navarra), Spain d

Department of Chemistry and Soil Chemistry, Faculty of Sciences, University of Navarra, P.O. Box

273, 31080 Pamplona (Navarra), Spain

*Corresponding author, e-mail: [email protected] Received 26 July 2013; revised 3 October 2013

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/ppl.12119

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This study examines the extent to which the predicted CO2-protective effects on the inhibition of growth, impairment of photosynthesis and nutrient imbalance caused by saline stress are mediated by an effective adaptation of the endogenous plant hormonal balance. Therefore, sweet pepper plants (Capsicum annuum, cv. Ciclón) were grown at ambient or elevated [CO2] (400 or 800 µmol mol–1)

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with a nutrient solution containing 0 or 80 mM NaCl. The results show that, under saline conditions, elevated [CO2] increased plant dry weight, leaf area, leaf relative water content and net photosynthesis

compared with ambient [CO2], whilst the maximum potential quantum efficiency of photosystem II was not modified. In salt-stressed plants, elevated [CO2] increased leaf NO3– concentration and

reduced Cl– concentration. Salinity stress induced ABA accumulation in the leaves but it was reduced in the roots at high [CO2], being correlated with the stomatal response. Under non-stressed conditions, IAA was dramatically reduced in the roots when high [CO2] was applied, which resulted in greater

root DW and root respiration. Additionally, the observed high CK concentration in the roots (especially tZR) could prevent downregulation of photosynthesis at high [CO2], as the N level in the leaves was increased compared with the ambient [CO2], under salt-stress conditions. These results demonstrate that the hormonal balance was altered by the [CO2], which resulted in significant changes

at the growth, gas exchange and nutritional levels. Abbreviations – ABA, abscisic acid; ACO2, net CO2 assimilation; Ci, internal CO2 concentration; ci/ca, ratio intercellular/external CO2 concentration; CKs, cytokinins; cZR, cis-zeatin riboside; DHZ, dehydrozeatine; DHZR, dehydrozeatine riboside; DW, dry weight; GAs, gibberellins; gs, stomatal conductance; IAA, indole-3-acetic acid; iP, N6-isopentenyladenine; iPR, N6-isopentenyladenosine; SLA, specific leaf area; tZR, trans-zeatin-riboside; Z, zeatin.

Introduction The predicted change in average air temperatures is likely to be accompanied by changes in the precipitation and storm patterns and alterations in drought intensity and frequency (Batjes 1999). Additionally, it is thought that, by 2050, 15–37% of species and taxa will be ‘committed to extinction’ (Thomas et al. 2004). Under such a scenario, the net primary production could be limited by reductions in both water supply and water quality and, especially in the Mediterranean region, global warming could aggravate salinity stress as the projections of climate change impacts in this area include pronounced increases in water shortage (Metzger et al. 2006, Giorgi and Lionello 2008). Therefore, agriculture requires new tools to identify plant resilience and adaptation mechanisms under the new climate scenario. Plant responses to elevated [CO2] vary as a result of complex responses of

underlying growth and development to changes in [CO2] and other environmental conditions (Kimball et al. 2002). An elevated CO2 concentration usually increases the rate of photosynthesis, as it directly increases CO2 intake, and induces partial closure of stomata, reducing water loss by transpiration

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(Korner 2000). However, nutrient restrictions (i.e. low N availability) could limit the enhancement of biomass accumulation (Duval et al. 2012). In semi-arid climates, secondary salinization (irrigation without appropriate drainage) could be counterbalanced by the reported positive effects of elevated CO2 concentration on both yield and water use efficiency (Yeo 1999). The osmotic effects, restriction of gas exchange, ion toxicity and nutrient

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imbalance are the main negative effects of salinity regarding plant growth (Greenway and Munns 1980). Thus, salinity affects the plant at morphological, physiological and molecular levels (Munns 2002, Dajic 2006) and interferes with growth and development (Zhu 2002). High salinity causes ion toxicity in plants (Zhu 2002, Munns and Tester 2008), as it leads to the increase of Na+ and Cl– in the

cytosol of cells. In addition, salinity can reduce the concentration of NO3– in plants whilst Cl– is augmented (Pessarakli 1991). However, in comparison with salinity, the application of a high CO2 concentration has contrasting effects on C3 plants. It is often stated that CO2 improves photosynthesis

but decreases photorespiration and oxidative stress (Rogers et al. 2004). Elevated CO2 affects cell

division, elongation and differentiation within apical meristems and, therefore, can accelerate plant growth and development (Masle 2000, Li et al. 2002). Plant hormones regulate these cellular processes (Yong et al. 2000), and modification of these hormones, including auxins, gibberellins (GAs), cytokinins (Cks), and abscisic acid (ABA), could have an important role in regulating the ontogeny of plants under high CO2 concentrations (Teng et al. 2006). Moreover, these hormonal regulations not only can affect the adaptive response but also modify the normal development of the fruits and thus impact economic productivity (Albacete et al. 2008). Several studies reported that elevated CO2 concentrations could ameliorate the redox homeostasis, photosynthesis and damaged cell structure of plants exposed to salinity (Pérez-López et al. 2013, Ratnakumar et al. 2013); however, this response is altered by sex-specific morphological, physiological and biochemical characteristics of the plant (Li et al. 2013). Pepper (Capsicum annuum) is an important crop species for greenhouse cultivation in Europe,

and is generally considered to be salt sensitive (del Amor and Cuadra-Crespo 2012). The effect of the CO2 concentration on the hormonal response has not been thoroughly studied in sweet pepper; nevertheless, this aspect is essential for a broad and integrative understanding of plant responses to a

rise in CO2 concentration under the predicted climate scenario, linked with a reduction in water

quality. Therefore, to gain insight into the physiological mechanisms of the response to salinity in sweet pepper, in relation to climate change predictions, the main objectives of this study were (i) to identify the extent to which – under high CO2 concentration – plant growth, photosynthesis, stomatal conductance, maximum potential quantum efficiency of photosystem II, chlorophyll content and leaf NO3– and Cl– concentrations are affected in pepper plants under salinity stress, and (ii) to determine the extent to which, under these conditions, the endogenous hormonal response of ABA, indole-3acetic acid (IAA), zeatin (Z), trans-zeatin-riboside (tZR), Cis-zeatin riboside (cZR), dehydrozeatin (DHZ), dehydrozeatin riboside (DHZR), N6-isopentenyladenine (iP) and N6-isopentenyladenosine

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(iPR) is altered. Such information about the role of endogenous hormones, in an important horticultural crop such as pepper, could establish new ways for managing plant resilience to climate change.

Material and methods

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Plant material, growth conditions and treatments Sweet pepper plants (Capsicum annuum), cv. Ciclón (Zeta Seeds S.A.) Lamuyo type, were grown in 5l black containers filled with coconut coir fiber (Pelemix, Alhama de Murcia, Murcia, Spain). The plants were irrigated with a modified Hoagland solution with the following composition in mM; NO3–:

12.0; H2PO4–:1.0; SO42–:3.5; K+: 7.0; Ca2+: 4.5; Mg2+: 2.0. Each container was rinsed with 3 l of demineralized water before transplanting. Irrigation was supplied by self-compensating drippers (2 l h–

1

) and fresh nutrient solution was applied to avoid salt accumulation, with a minimum of 35% drainage

(del Amor and Cuadra-Crespo 2012). The amount of water used to irrigate plants was modified over time, depending on the demand of the plants, but always maintaining a drainage of 35% to avoid concentration of salts in the substrate. Plants were grown in a climate chamber designed by our department specifically for plant research proposes (del Amor et al. 2010), with fully-controlled environmental conditions: 70% relative humidity, 16/8 h day/night photoperiod, 26/18°C and a photosynthetically-active radiation (PAR) of 250 µmol m–2 s–1 provided by a combination of fluorescent lamps (TL-D Master reflex 830 and 840, Koninklijke Philips Electronics N.V., the Netherlands) and high-pressure sodium lamps (Son-T Agro, Philips). Two irrigation solutions were applied: the control solution (0 mM NaCl) and 80 mM NaCl. The experiment was performed at [CO2]

of 400 or 800 µmol mol–1 with a duration of 30 days each. The CO2 concentration was regulated by injection of external compressed air or CO2 (bottle [CO2] ≥ 99.9%), controlled by a Dräger Polytron IR CO2. Thus, four treatments were studied, corresponding to two nutrient solutions and two ambient CO2 concentrations.

Growth, leaf area and specific leaf area At the end of each experiment (30 days after transplanting), the leaf area and fresh and dry weights of each plant, individually, and of the roots and stem (including petioles) were measured. Dry weight (DW) was determined after a minimum of 72 h at 70°C. DW partitioning into roots was calculated as plant DW/Root DW. Leaf area was measured in a Li-Cor LI-3100 (LI-COR, Inc. Lincoln, NE, USA). Specific leaf area

(SLA) was calculated as the ratio between the area and the DW (obtained after 48 h at 80°C) of leaf discs of 6.91 cm2. Disc fresh weight was also determined at the time of collection. Leaf relative water content (LRWC) was calculated as:

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where FW is the fresh mass of the leaves, TW is the turgid mass after re-hydrating the leaves in distilled water for 24 h, and DW is the dry mass after oven-drying at 80°C for 48 h (Bota et al. 2004).

Gas exchange At the end of each experimental period, the net CO2 assimilation (ACO2), internal CO2 concentration

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(Ci) and stomatal conductance to CO2 (gs) were measured in the youngest fully-expanded leaf of each

plant, using a CIRAS-2 (PP system, Amesbury, MA, USA) with a PLC6 (U) Automatic Universal Leaf Cuvette, measuring both sides of the leaves. The cuvette provided light (LED) with a photon flux of 1300 µmol m−2 s−1, 400 or 800 µmol mol–1 CO2 and a leaf temperature of 25°C. Root respiration was measured for each plant with the CIRAS-2 and the SRC-1 Soil Respiration Chamber (PP system, Amesbury, MA, USA). The chamber (volume: 1171 cm3 and area: 78.5 cm2) was placed on the substrate at the top of each container, after holding the chamber in air to flush it out with the ambient CO2 concentration (15 s). Calibration was performed before each measurement, and readings were taken after CO2 stabilization (maximum 60 s).

Maximum potential quantum efficiency of PSII The ratio between the variable fluorescence from a dark-adapted leaf (Fv) and the maximal

fluorescence from a dark-adapted, youngest fully-expanded leaf (Fm), which is called the maximum potential quantum efficiency of PSII (Fv/Fm), was determined with an ADC Fim 1500 (ADC BioScientific, Herts., England). This ratio is the one used most-widely in research employing the fluorescence technique and is considered a good indicator of the in vivo functionality of the photosynthetic apparatus in plants under stress (Maxwell and Johnson 2000). A special leaf clip holder was allocated to each leaf to maintain dark conditions for at least 30 min before reading.

Chlorophyll content Chlorophylls a and b were extracted from youngest-leaf samples with N, N-dimethylformamide, for 72 h, in darkness at 4°C. Subsequently, the absorbance was measured with a spectrophotometer at 750, 664 and 647 nm, and the quantities were calculated according to the method of Porra et al. (1989). The relative chlorophyll content (SPAD) of the youngest leaf was determined with a SPAD-502 (KonicaMinolta Sensing, Osaka, Japan). This optical device takes instant readings without destroying the plant tissue and the readings are performed in a very short time; it determines the greenness and the interaction of thylakoid chlorophyll with incident light (Jifon et al. 2005). Three measurements (nondimensional) were made on each leaf. One leaf per plant and six plants per treatment were evaluated.

Leaf ion concentrations The leaf Cl– and NO3– concentrations were determined in dry matter. These ions were extracted from ground material (0.4 g) with 20 ml of deionized water and were analyzed in an ion chromatograph

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(METROHM 861 Advanced Compact IC; METROHM 838 Advanced Sampler); the column used was a METROHM Metrosep A Supp7 250/4.0 mm.

IAA and ABA analyses The extraction and purification of plant material followed the procedure described by Bacaicoa et al.

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(2009): 0.5 g of frozen plant tissue (previously triturated in a mortar to a powder with liquid nitrogen) was homogenized with 5 ml of pre-cooled (–20°C) methanol:water (80:20, v/v) and 2.5 mM sodium diethyldithiocarbamate. Deuterium-labeled internal standards ([2H5] indole-3-acetic acid, (D-IAA); [2H6] (+)-cis, trans-abscisic acid, (D-ABA); from Olchemim, Olomouc, Czech Republic) were added

(100 µl of a stock solution of 400 ng·ml–1 of each standard in methanol) to the extraction medium. After overnight extraction at –20°C, solids were separated by centrifugation, at 12 000 rpm for 10 min, at 4°C, using a Centrikon T-124 centrifuge with an A8.24 rotor (Kontron Instruments, Cumbernauld, United Kingdom), and re-extracted for 1 h with an additional 4 ml of extraction mixture. The supernatants were passed through a Strata C18-E cartridge (3 cm3, 200 mg) Ref. 8B-S001-FBJ

(Phenomenex, Torrance, CA), pre-conditioned with 4 ml of methanol followed by 2 ml of extraction medium. After evaporation at 40°C to leave the aqueous phase, using a Labconco Vortex Evaporator (Labconco Co., Kansas City, MO), 0.5 ml of 1 M formic acid (pH ≈ 3.5) was added. Afterwards, the hormones were extracted successively with two portions of 5 and 4 ml of diethyl ether, and the organic phase was evaporated to dryness. The residue was re-dissolved in 250 µl of methanol:0.5% acetic acid (40:60, v/v). The solution was centrifuged at 8000 rpm for 5 min before its injection in the LC/MS/MS system. Liquid chromatography-mass spectrometry quantification of IAA and ABA: the hormones were quantified by HPLC (2795 Alliance HT; Waters Co., Milford, MA) linked to a 3200 Q TRAP LC/MS/MS System (Applied Biosystems/MDS Sciex, Ontario, Canada), equipped with an electrospray interface, using a reverse-phase column (Synergi 4 µm Hydro-RP 80A, 150 × 2 mm; Phenomenex, Torrance, CA). A linear gradient of methanol (A) and 0.5% acetic acid in water (B) was used: 35% A for 1 min, 35% to 95% A in 9 min, 95% A for 4 min and 95% to 35% A in 1 min, followed by a stabilization time of 5 min. The flow rate was 0.20 ml·min–1, the injection volume was

40 µl and the column and sample temperatures were 30 and 20°C, respectively. The detection and quantification were performed by multiple reaction monitoring (MRM) in the negative-ion mode, employing multilevel calibration curves with deuterated hormones as internal standards. Compounddependent parameters are listed in Appendix Table S1, in Supporting Information. The source parameters are: curtain gas: 172.37 kPa, GS1: 310.26 kPa, GS2: 413.69 kPa, ion spray voltage: –4000 V, and temperature: 600°C. Data samples were processed using Analyst 1.4.2 Software from Applied Biosystems/MDS Sciex (Ontario, Canada).

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Cytokinins analysis The extraction and purification of plant material followed the procedure described by Dobrev et al. (2002) and Garnica et al. (2010), with slight modifications: 0.5 g of frozen plant tissue, (previously triturated in a mortar to a powder with liquid nitrogen), was homogenized with 5 ml of pre-cooled (– 20°C) methanol:water:formic acid (15:4:1, v/v/v). Deuterium-labeled CKs internal standards, ([2H5]

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trans-zeatin, (D-Z); [2H5] trans-zeatin riboside, (D-ZR); [2H6] N6-isopentenyladenine, (D-iP); [2H6]

N6-isopentenyladenosine, (D-iPR); from Olchemim, Olomouc, Czech Republic), were added (40 µl of a stock solution of 50 ng·ml–1 of each standard in methanol) to the extraction medium. After overnight extraction at –20°C, solids were separated by centrifugation, at 12 000 rpm for 10 min, at 4°C, using a Centrikon T-124 centrifuge with an A8.24 rotor (Kontron Instruments, Cumbernauld, United Kingdom), and re-extracted for 1 h with an additional 4 ml of extraction mixture. The supernatants were passed through a Strata C18-E cartridge (3 cm3, 200 mg) Ref. 8B-S001-FBJ (Phenomenex,

Torrance, CA), pre-conditioned with 4 ml of methanol followed by 2 ml of extraction medium. After evaporation at 40°C to leave the aqueous phase, using a Labconco Vortex Evaporator (Labconco Co., Kansas City, MO), 2 ml of 1 M formic acid were added and the extract was applied to an Oasis MCX column (3 cm3, 60 mg) Ref. 186000254 (Waters Co., Milford, MA) pre-conditioned with 4 ml of methanol and 2 ml of 1 M formic acid. The column was washed successively with 2 ml of 1 M formic acid, 2 ml of methanol and 2 ml of 0.35 M NH4OH, and the CKs bases, ribosides and glucosides were

eluted with 2 ml of 0.35 M NH4OH in 60% (v/v) methanol. This eluted fraction was evaporated to

dryness in the vortex evaporator and re-dissolved in 250 µl of methanol:0.05% formic acid (40:60, v/v). The solution was centrifuged at 8000 rpm for 5 min before its injection into the LC/MS/MS system. Liquid chromatography-mass spectrometry quantification of CKs: the CKs were quantified by HPLC (2795 Alliance HT; Waters Co., Milford, MA) linked to a 3200 Q TRAP LC/MS/MS System (Applied Biosystems/MDS Sciex, Ontario, Canada), equipped with an electrospray interface, using a reverse-phase column (Tracer Excel 120 ODSA 3 µm, 100 × 4.6 mm; Teknokroma, Barcelona, Spain). A linear gradient of methanol (A) and 0.05% formic acid in water (B) was used: 35% to 95% A in 11 min, 95% A for 3 min and 95% to 35% A in 1 min, followed by a stabilization time of 5 min. The flow rate was 0.25 ml·min–1, the injection volume was 40 µl and the column and sample temperatures were 30 and 20°C, respectively. Detection and quantification were performed by MRM in the positive-ion mode, employing multilevel calibration curves with deuterated CKs as internal standards. Compounddependent parameters are listed in Appendix Table S2, in Supporting Information. The source parameters are: curtain gas: 172.37 kPa, GS1: 344.74 kPa, GS2: 413.69 kPa, ion spray voltage: 5000 V, and temperature: 600°C. Data samples were processed using Analyst 1.4.2 Software from Applied Biosystems/MDS Sciex (Ontario, Canada).

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Statistical analysis The data were tested first for homogeneity of variance and normality of distribution, and Duncan’s multiple range test was used to determine differences between means (P≤0.05). Four combinations of treatments were used, involving two salinity levels (control and 80 mM) and two ambient [CO2], with

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six plants per combination.

Results Plant growth Under non-stress conditions, increasing [CO2] led to a significant increase in the total plant DW (52%), and a differential response was found when salinity was applied (Fig. 1A). Salinity stress (80 mM NaCl) led to a significant reduction in total plant DW at both [CO2]; however, under such saline

conditions, high [CO2] increased DW production from 2.52 g to 4.79 g, an increase of 89.8%,

compared with ambient [CO2]. A similar pattern was observed for stems and leaves and roots (Fig. 1B, 1C, and 1D). The DW partitioning into the roots was increased by salinity when growth took place at ambient [CO2] but it was reduced at high [CO2] (Fig. 1E). The total leaf area was also increased at

elevated [CO2], from 0.15 m2 to 0.21 m2 with the standard nutrient solution and from 0.05 m2 to 0.09 m2 with 80 mM NaCl (Fig. 1F); thus, under saline conditions [CO2] boosted leaf area by 88.3%. The

leaf relative water content was significantly decreased by both increasing salinity and [CO2] (Fig. 1G), whilst the SLA was reduced by increasing the [CO2] (Fig. 1H).

Leaf gas exchange, maximum potential quantum efficiency of PSII and chlorophyll content The net CO2 assimilation increased from 13.4 µmol m–2 s–1 at ambient [CO2] to 18.8 µmol m–2 s–1 at high [CO2] when plants were grown with the control nutrient solution, and from 10.6 µmol m–2 s–1 to

16.1 µmol m–2 s–1 when plant grew with high NaCl in the nutrient solution (Fig. 2A). The intercellular/external CO2 concentration ratio (ci/ca) was also reduced by salinity but it was increased

by elevated [CO2], by 32.5% in non-salinized plants and by 46.6% in the salinized ones (Fig. 2B). The Gs was significantly reduced by salinity at both ambient and high [CO2] (Fig. 2C), whilst the Fv/Fm

was not affected by [CO2] or salinity (Fig. 2D). The chlorophyll a concentration was only reduced by salinity when plants were grown at ambient

but not at high [CO2] (Fig 3A), whilst the chlorophyll b had the inverse pattern (Fig 3B). Thus, the

effect of salt stress was greater with regard to increasing the chlorophyll b content (50.3%) than for decreasing the chlorophyll a content (3.50%) at ambient [CO2]. The chlorophyll a/b ratio and the relative chlorophyll content (SPAD) were only affected by salinity but not by [CO2] (Fig. 3C, 3D).

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Root respiration Root respiration was affected by both [CO2] and salinity in the nutrient solution. Thus, it was increased by elevated [CO2], from 9.6 to 11.75 µmol CO2 m–2 s–1 (21.4%) in non-salinized plants, and from 5.7 to 8.45 µmol CO2 m–2 s–1 (46%) in salinized plants (Fig. 4).

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Leaf Cl– and NO3– concentrations

When plants were grown in a highly-saline nutrient solution, the leaf NO3– concentration was significantly reduced (by 36.5% or 46.1% for ambient or high [CO2]) (Fig. 5A). Additionally, elevated [CO2] had a significant effect on leaf NO3–, increasing it by 42.7% or 79.9% for salinized or non-

salinized plants, respectively. The concentration of Cl– in the leaves of salinized plants was reduced

significantly by increasing [CO2], from 1046.2 mmol kg–1 to 827.8 mmol kg–1 (20.8%) (Fig. 5B).

Abscisic acid, indole-3-acetic and cytokinins concentrations Salinity produced a significant increase in ABA concentration and this effect was higher in the leaves than in the roots (Fig. 6A, 6B). Thus, in the leaves, the ABA concentration increased notably, from 94 pmol g–1 to 999 pmol g–1 at ambient [CO2] or from 229 pmol g–1 to 978 pmol g–1 at elevated [CO2]. In

the roots a decrease in ABA concentration was observed in salinized plants when grown at elevated [CO2], compared with those plants grown at ambient [CO2]. The concentration of IAA in the leaves was not altered by salinity or [CO2]. In the roots of non-salinized plants, IAA was reduced by increasing [CO2] whilst this effect was not statistically significant for salinized plants (Fig. 6C, 6D). The total CKs concentration was not affected in the leaves of salinized plants compared with the nonsalinized, and in the roots the high [CO2] significantly increased total CKs in salinized plants, but did

not affect their levels under non-saline conditions (Fig. 6E, 6F). The different forms of CKs (Z, tZR, DHZ, cZR, DHZR, iP and iPR) were analyzed in leaves (Fig.

7A) and roots (Fig. 7B) and expressed as percentages of the total CKs. The concentration of Z was

only high enough to be detected in plants (salinized or non-salinized) grown at ambient [CO2] but not at high [CO2]. tZR accounted for ca. 38% of total CKs in the leaves of non-salinized plants but it fell to 33% in the salinized ones. In the roots, CK levels were modified by salinity and [CO2]. Thus, under

non-saline conditions, [CO2] increased tZR from 15% to 54%, and under salinity stress from 17% to 83%. In the leaves, the highest percentage of cZR was found in non-salinized plants grown in ambient [CO2] (26%); this percentage was reduced by increasing salinity (to 15%) or by increasing [CO2] (to 9%). In the root, cZR maintained similar relative values at ambient [CO2] (34%–38%) but they were

significantly reduced by increasing [CO2]: to 22% in non-salinized and to only 5% in salinized plants. In the leaves, iP was increased by increasing [CO2] but was not affected by salinity. In the root, an inverse pattern was observed: those treatments that gave lower values in the leaves produced higher values in the root. iPR showed its lowest value in the leaves when non-stressed plants grew at ambient [CO2] (28%); this was increased significantly by salinity and elevated [CO2], by ca. 45%. DHZ and

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DHZR were not detected in either leaves or roots.

Discussion Growth The defense mechanisms to counterbalance the effects of salinity on plants may differ between species

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(Greenway and Munns 1980). Thus, to reduce the negative effects of salinity, it is necessary to know the specific mechanisms involved in the salt tolerance of crops, which can be affected by both environmental conditions and cultural practices (De Pascale et al. 2003, Lycoskoufis et al. 2005). Sweet pepper (Capsicum annuum) is commonly classified as salt-sensitive (Maas and Hoffman 1977, del Amor and Cuadra-Crespo 2012), which is consistent with our salinity-stress response (reduced growth) and agrees with the conclusions of Lycoskoufis et al. (2005), del Amor and Cuadra-Crespo (2012) and Ben-Gal et al. (2008). Thus, the increase of salt in the leaves, leading to salt toxicity in the plant, can result in a reduction in the total photosynthetic leaf area and therefore in the supply of photosynthate to the plant, modifying the carbon balance required to maintain growth (Munns 2002). Additionally, our data agree with those of Juan et al. (2007), who concluded that high CO2 increased

the nutrient-stress tolerance in tomato seedlings. The SLA (leaf area per unit leaf biomass) was decreased by increasing [CO2]; this reduction could

counteract the increased rate of photosynthesis per unit leaf area and, as a result, the enhancement of growth could be lower than that expected on the basis of the stimulation of the rate of photosynthesis alone (Poorter and Pérez-Soba 2002). This decrease is linked partly to the build-up of starch and other non-structural carbohydrates (Korner et al. 1995). Root DW was significantly increased at high [CO2]

under both control and salinity-stress conditions, but a differential response in the root respiration rate was found. Thus, with control nutrient solution, an increase in root DW of 85% produced an increase of 21% in respiration, but at high NaCl concentration, an increase of 73% in root DW accounted for a 46% rise in respiration, which indicates a greater specific root response under salinity. Regarding growth, our results show that in the control plants under salinity there was a significant

reduction in the dry weight of the aerial parts (leaves plus stem) and root, although the ratio root/shoot remained unchanged. The water content decreased slightly. However, among the pepper genotypes, there is a large variation in salt tolerance (Aktas et al. 2006).

Gas exchange Diminished growth in plants under saline stress is often associated with a reduction in photosynthesis (Munns 2002, Sayed 2003). Salt stress has been reported to affect predominantly the diffusion of CO2 in leaves, due to decreases in the stomatal and mesophyll conductances, but not the biochemical capacity to assimilate CO2 (Flexas et al. 2004). However above certain levels, it has been described that Rubisco activity is impaired (Galmes et al. 2013). The application of high CO2 throughout the day

has been reported to increase the tolerance of plants to salinity, this being attributed mainly to an

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increased rate of carbon assimilation since a higher internal leaf [CO2] allows high rates of carbon assimilation even with the partial stomatal closure induced by salt (Munns 1993), and could more than compensate for any damaging effect of high [CO2] at night, which may result from partial suppression of dark respiration (Reuveni et al. 1997). Our data agree with the finding that a highly-saline nutrient solution led to a long-term reduction of gs in pepper plants, showing that one consequence of salt

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stress in this crop is a reduced ability of the plant to supply CO2 to the photosynthetic apparatus

(Lycoskoufis et al. 2005). In this study, salinity had a negative effect on the gs and ci/ca. Some previous studies have shown

that plants grown under elevated [CO2] had higher ci/ca ratios, compared with the controls (Bryant et al. 1998), but others found that CO2 enrichment did not significantly alter the coordination of gs and

photosynthesis, ci/ca being insensitive to atmospheric CO2 (Xu and Hsiao 2004, Ainsworth and Long 2005). Additionally, our results show that the reduction of gs under salinity stress was large relative to the control, while that of ci/ca was small. Chlorophyll fluorometry can be a useful tool in the evaluation of both plant photosynthetic efficiency and the extent to which it is limited by photochemical and non-photochemical processes (Baker and Oxborough 2004). Our data showed that the Fv/Fm was not affected (photodamage was not induced), indicating that photosynthesis was not limited by non-stomatal factors, in agreement with previous findings (del Amor and Cuadra-Crespo 2011), whilst Azuma et al. (2010) attributed it to both stomatal and non-stomatal factors. Thus, this behavior corroborated the cultivar-specific response of sweet pepper to salinity (Aktas et al. 2006). Additionally, the reduced effect of CO2 on this cultivar indicates that the priority for these salinitystressed plants does not seem to be decreased water loss, but rather the minimization of photorespiration and the maximization of photosynthetic rates and energy gain (Geissler et al. 2009). Thus, the high energy demand of salinity-tolerance mechanisms could be satisfied partly by this strategy. Mavrogianopoulos et al. (1999) indicated that when plants grew under saline conditions, [CO2]

enrichment increased significantly the leaf growth and the chlorophyll content. Our data show that the chlorophyll a concentration was maintained under salinity at high [CO2] whilst that of chlorophyll b increased at ambient [CO2]. The chlorophyll concentration under saline conditions was maintained in barley (Abadía et al. 1999) and increased in salinized Xanthium strumarium (Reuveni et al. 1997), whilst Vu et al. (2001) pointed out that the chlorophyll content in soybean increased in mature leaves after they were exposed to a high CO2 concentration.

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Ion response The nitrogen concentration was lower in the leaves of plants irrigated with salt. This effect has been described previously for maize (Tuna et al. 2008), tomato (Cerdá and Martinez 1998) and pepper (del Amor and Cuadra-Crespo 2012). This inhibition of nitrogen uptake may occur by NO3−/Cl− interaction at the sites of ion transport (Cram 1983), as sodium is reported to cause severe membrane

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depolarization in plants (Suhayda et al. 1990), being linked to non-competitive inhibition of nitrate uptake (Hawkins and Lewis 1993). However, our data show that high [CO2] could counteract this effect, reducing Cl− and increasing NO3− concentrations in the leaves, which agrees with the results of del Amor (2013) for tomato. Thus, the controversial effect of lesser NO3− assimilation at high [CO2] is clarified, since high-frequency irrigation fully satisfied the plant N requirements. In our study, a decrease in the leaf Cl– concentration at high [CO2] was linked with reduced leaf

transpiration (Maggio et al. 2002), while reductions in Cl–, despite reductions in gs, were not statistically significant at high [CO2].

Hormonal response The ABA-mediated adaptive responses are reported to be of paramount importance when plants have to face salinity stress, as such stressors can act as a trigger for the accumulation of ABA, which in turn activates various stress-associated genes that are thought to function in the accumulation of osmoprotectants and proteins, signaling, transcriptional regulation, etc. (Bartels and Sunkar 2005). In our study, the ABA concentration in the leaves influenced stomatal conductance and the two parameters were correlated (R2=0.72) (data not shown), in agreement with Hose et al. (2000).

Additionally, increases in the leaf ABA concentration in salt-stressed plants were correlated with growth inhibition, while the ABA concentration in roots was maintained – although it tended to decrease. Jeschke et al. (1997) found that reduced leaf conductance and inhibition of leaf growth were correlated with an increased ABA concentration in the xylem. High [CO2] reduced the ABA

concentration in the roots grown at high salinity, compared with non-stressed plants at ambient or high [CO2] and, similarly to other reports, we found a much-higher ABA concentration in the shoot relative to the roots (Mulholland et al. 2003). Moreover, Li et al. (2007) reported that the ABA content in ginkgo leaves cultivated at an elevated CO2 concentration was 63% lower compared with the control. IAA plays a major role in the regulation of plant growth, as it controls vascular tissue

development, cell elongation and apical dominance (Wang et al. 2001); higher concentrations of IAA are linked to decreased growth (Ribaut and Pilet 1994). We found a clear effect of CO2 with regard to

increasing the dry weight of both leaves and roots of salinized plants, while partitioning to the roots remained constant. Most striking is the reduction in leaf IAA concentration, compared to the control in the root. The normal pathway of IAA synthesis is aerial, from where it is translocated to the root. The IAA values in the aerial parts were slightly lower than for the controls, but there were no significant differences. In the roots, under non-saline conditions, IAA was dramatically reduced when high [CO2]

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was applied, coinciding with higher root DW and root respiration. What changed dramatically was the IAA/CKs ratio: it was much higher in control plants than in plants under elevated CO2, which promotes the development of aerial parts, making the transport of nutrients easier. Therefore, the ratio ABA/IAA was increased, which promotes stomatal closure. In non-stressed leaves, the high [CO2] caused an important reduction in the total CKs

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concentration, whereas in the roots high salinity and high [CO2] triggered high CKs concentrations. A

pathway to convert cZ into tZ was proposed following partial purification of a putative cis-transisomerase (Bassil et al. 1993), and the relationship between side chain variation and activity has been investigated by classical bioassays and the characterization of cytokinin signaling components (Kudo et al. 2012). Thus, tZ showed the highest activity, followed by iP and cZ, in callus growth assays using tobacco (Nicotiana tabacum) (Mok et al. 1978). However, recent studies also suggested that cZ functions as an active cytokinin and that its metabolism contributes to the control of cytokinin activity (Kudo et al. 2012). Sakakibara et al. (2006) reported that nitrogenous nutrition is associated with the synthesis of CKs, indicating that CKs act as N-sensors because of their role in the regulation of genes involved in the uptake of nutrients. An interesting conclusion is that changes in CKs with salinity seem to be ruled by a different mechanism, the CO2 response being better adjusted to the mechanism

proposed by Sakakibara (NO3––CKs translocation) and resulting in an increase in leaf CKs

concentration due to the conversion of c-Z into t-Z. This effect is also supported by the active CKs level, which increased in non-salinized plants under elevated CO2, due to lower cZ and increased tZ, indicating that transformation of cZ (lower activity) into tZ (more active form) was induced. This transformation has been linked to nitrate translocation to aerial parts (Sakakibara et al. 2006), a result that we also obtained, and this can help to maintain chlorophyll concentration and chlorophyll fluorescence (anti-senescent). In any case, the ratio ABA/CKs (total or active) continues to increase with salinity, which also favors stomatal closure. This is reflected in the decrease of stomatal conductance with salinity. Although the index of chlorophyll fluorescence was not altered by salinity, the chlorophyll content was slightly affected; therefore, the chlorophyll a/b ratio changed significantly. These data suggest that the high rate of growth at elevated CO2 is the result of increased net

photosynthesis and an increase in the distribution of active CKs in the root and leaf, favoring the translocation of solutes and minerals (sink effect of the CKs) resulting from the demand of the plant to increase photosynthesis. With salinity, the same happened in the case of the ratio IAA/CKs, but the mechanism was different since in this case it was the CKs which greatly increased in the root. In leaves it was also observed, although to a lesser extent, the transformation of cZ into tZ. They seem to be alternative mechanisms in the presence or absence of salt stress, as reflected in the IAA/CKs hormonal balance, with the same goal: to promote the growth of the plant. Hence, it seems to be a synergistic effect between increased net photosynthesis and the ratio IAA/CKs under elevated CO2 (mainly active CKs). These changes in active CKs in the leaf at elevated CO2, either with or without salt, are also reflected in the distribution of active CKs in the root, with an increase in tZ to the

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detriment of cZ, when CO2 increased in the presence or absence of salinity. In this way, the plant is able to regulate its CKs levels under non-optimal conditions (salinity) and at high [CO2], and high tissue NO3– concentrations can be achieved. Kuiper (1988) discussed the role of CKs in growth regulation, demonstrating that the reduction in CKs contents in Plantago major plants exposed to nutrient deficiencies was due to diminished endogenous CKs and not a reduction in the nutrient

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concentrations in the plant tissues. Thus, the higher CK concentrations in the roots (especially tZR) observed here could prevent down-regulation of photosynthesis at high [CO2], as the N status in the leaves was maintained and photosynthesis increased in pepper plants, throughout the study. As salinity decreased both the leaf area and the leaf dry weight, so cell division and elongation

were both decreased. Shoot CKs are also most likely involved in these processes since they were strongly decreased by salinity in the shoot tissues: the leaf levels of Z+ZR by about 58% (Ghanem et al. 2008). In addition, the leaf CK (ZR and Z+ZR) concentrations were also correlated positively with the overall shoot growth capacity, which may be explained not only by promotion of cell division but also by delayed leaf senescence under saline conditions. This could be relevant when further analyzing the crop performance under these conditions. In summary, our results present further evidence that the hormonal balance operates as a signal of

stress conditions (salinity), promoting different response mechanisms. Salinity stress induced ABA accumulation in the leaves, which was correlated with the stomatal response. Regarding the ABA/IAA ratio in leaves at ambient and elevated CO2 concentrations, it behaved as in salinized plants, which favors stomatal closure, as reflected in the stomatal conductance results. On the other hand, many studies pointed out that downregulation is the result of an insufficient plant sink capacity, caused by a reduced N supply. Therefore, the high CKs concentrations in the roots (especially tZR) could have prevented the down-regulation of photosynthesis at high [CO2], as the N level in the leaves was

increased compared with the ambient [CO2], N availability being a critical factor in the acclimation

response to elevated CO2. Our results also suggest that the application of high CO2 altered the

hormonal balance, resulting in significant changes at the growth, gas exchange and nutritional levels. Acknowledgements – The authors thank Dr. D.J. Walker for correction of the written English, G. Ortuño for his help regarding measurements of plant biomass, leaf gas exchange and mineral composition and Angel M. Zamarreño regarding hormonal analyses. M.C. Piñero received support from an INIA predoctoral fellowship. This study was supported by the Instituto Nacional de Investigaciones Agrarias (INIA), under the project RTA2011-00026-C02-01, with the collaboration of FEDER Operative Program of the Region of Murcia 2007–2013 (POI 07-021).

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References Abadia J, Morales F, Abadia A (1999) Photosystem II efficiency in low chlorophyll, iron-deficient leaves. Plant Soil 215: 183–192 Ainsworth EA, Long SP (2005) What have we learned from 15 years of free-air CO2 enrichment

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(FACE)? A meta-analytic review of the responses of photosynthesis, canopy. New Phytol 165: 351–371

Aktas H, Abak K, Cakmak I (2006) Genotypic variation in the response of pepper to salinity. Sci Hortic 110: 260–266

Albacete A, Ghanem ME, Martinez-Andujar C, Acosta M, Sanchez-Bravo J, Martinez V, Lutts S, Dodd IC, Perez-Alfocea F (2008) Hormonal changes in relation to biomass partitioning and shoot growth impairment in salinized tomato (Solanum lycopersicum L.) plants. J Exp Bot 59: 4119– 4131

Azuma R, Ito N, Nakayama N, Suwa R, Nguyen Tran N, Larrinaga-Mayoral JA, Esaka M, Fujiyama H, Saneoka H (2010) Fruits are more sensitive to salinity than leaves and stems in pepper plants (Capsicum annuum L.). Sci Hortic 125: 171–178

Bacaicoa E, Zamarreño AM, Leménager D, Baigorri R, García-Mina JM (2009) Relationship between the hormonal balance and the regulation of iron deficiency stress responses in cucumber (Cucumis sativus L.) plants. J Am Soc Hort Sci 134: 589–601

Baker RB, Oxborough K (2004) Chlorophyll fluorescence as a probe of photosynthetic productivity. In: Papageorgiou G, Govindjee (eds) Chlorophyll fluorescence: a signature of photosynthesis, 1st Edn. Kluwer Academic, Dordrecht, pp 65–82

Batjes NH (1999) Management options for reducing CO2-concentrations in the atmosphere by increasing carbon sequestration in the soil. Dutch National Research Programme on Global Air Pollution and Climate Change report 410-200-031 and ISRIC technical paper 30, International Soil Reference and Information Centre, Wageningen, The Netherlands

Bartels D, Sunkar R (2005) Drought and salt tolerance in plants. CRC Crit Rev Plant Sci 24: 23–58 Bassil NV, Mok D, Mok MC (1993) Partial purification of a cis-trans-isomerase of zeatin from immature seed of Phaseolus vulgaris L. Plant Physiol 102: 867–872

Ben-Gal A, Ityel E, Dudley L, Cohen S, Yermiyahu U, Presnov E, Zigmond L, Shani U (2008) Effect of irrigation water salinity on transpiration and on leaching requirements: A case study for bell peppers. Agr Water Manage 95: 587–597

Bota J, Mendrano H, Flexas J (2004) Is photosynthesis limited by decreased Rubisco activity and RuBP content under progressive water stress? New Phytol 162: 671–681 Bryant J, Taylor G, Frehner M (1998) Photosynthetic acclimation to elevated CO2 is modified by source: sink balance in three component species of chalk grassland swards grown in a free air carbon dioxide enrichment (FACE) experiment. Plant Cell Environ 21: 159–168

Cerda A, Martinez V (1988) Nitrogen fertilization under saline conditions in tomato and cucumber

This article is protected by copyright. All rights reserved.

plants. J Hortic Sci 63: 451–458 Cram WJ (1983) Choride accumulation in plant-cells as a homeostatic system – Energy supply as a dependent variable. J Membr Biol 74: 51–58 Dajic Z (2006) Salt stress. In: Madhava KV, Raghavendra AS, Janardhan K (eds) Physiology and molecular biology of stress tolerance in plants. Springer, Dordrecht, pp 41–99

Accepted Article

De Pascale S, Ruggiero C, Barbieri G, Maggio A (2003) Physiological responses of pepper to salinity and drought. J Am Soc Hortic Sci 128: 48–54

del Amor FM, Gomez-Lopez MD (2009) Agronomical response and water use efficiency of sweet pepper plants grown in different greenhouse substrates. HortScience 44: 810–814

del Amor FM, Cuadra-Crespo P, Walker DJ, Camara JM, Madrid R (2010) Effect of foliar application of antitranspirant on photosynthesis and water relations of pepper plants under different levels of CO2 and water stress. J Plant Physiol 167: 1232–1238

del Amor FM, Cuadra-Crespo P (2011) Alleviation of salinity stress in broccoli using foliar urea or methyl-jasmonate: analysis of growth, gas exchange, and isotope composition. Plant Growth Regul 63: 55–62

del Amor FM, Cuadra-Crespo P (2012) Plant growth-promoting bacteria as a tool to improve salinity tolerance in sweet pepper. Functional Plant Biology 39: 82–90

del Amor FM (2013) Variation in the leaf δ13C is correlated with salinity tolerance under elevated CO2 concentration. J Plant Physiol 170: 283–290

Dobrev VK, Doebner HD, Twarock R (2002) Quantum mechanics with difference operators. Rep Math Phys 50: 409–431

Duval BD, Blankinship JC, Dijkstra P, Hungate BA (2012). CO2 effects on plant nutrient concentration depend on plant functional group and available nitrogen: a meta-analysis. Plant Ecol 213: 505– 521

Flexas J, Bota J, Loreto F, Cornic G, Sharkey TD (2004) Diffusive and metabolic limitations to photosynthesis under drought and salinity in C(3) plants. Plant Biol 6: 269–279

Galmés J, Perdomo JA, Flexas J, Whitney SM (2013) Photosynthetic characterization of Rubisco transplantomic lines reveals lterations on photochemistry and mesophyll conductance. Photosynth Res 115: 153–166

Garnica M, Houdusse F, Zamarreno AM, Garcia-Mina JM (2010) The signal effect of nitrate supply enhances active forms of cytokinins and indole acetic content and reduces abscisic acid in wheat plants grown with ammonium. J Plant Physiol 167: 1264–1272

Geissler N, Hussin S, Koyro H-W (2009) Elevated atmospheric CO2 concentration ameliorates effects of NaCl salinity on photosynthesis and leaf structure of Aster tripolium L. J Exp Bot 60: 137–151 Ghanem ME, Albacete A, Martinez-Andujar C, Acosta M, Romero-Aranda R, Dodd IC, Lutts S, Perez-Alfocea F (2008) Hormonal changes during salinity-induced leaf senescence in tomato (Solanum lycopersicum L.). J Exp Bot 59: 3039–3050

This article is protected by copyright. All rights reserved.

Giorgi F, Lionello P (2008) Climate change projections for the Mediterranean region. Glob Planet Change 63: 90–104 Greenway H, Munns R (1980) Mechanisms of salt tolerance in non-halophytes. Annu Rev Plant Physiol Plant Mol Biol 31: 149–190

Accepted Article

Hawkins HJ, Lewis OAM (1993) Effect of NaCl salinity, nitrogen form, calcium and potassium concentration on nitrogen uptake and kinetics in Triticum aestivum L. cv. Gamtoos. New Phytol 124: 171–177

Hose E, Steudle E, Hartung W (2000) Abscisic acid and hydraulic conductivity of maize roots: a study using cell- and root-pressure probes. Planta 211: 874–882

Jeschke WD, Peuke AD, Pate JS, Hartung W (1997) Transport, synthesis and catabolism of abscisic acid (ABA) in intact plants of castor bean (Ricinus communis L.) under phosphate deficiency and moderate salinity. J Exp Bot 48: 1737–1747

Jifon JL, Syvertsen JP, Whaley E (2005) Growth environment and leaf anatomy affect nondestructive estimates of chlorophyll and nitrogen in Citrus sp leaves. J Am Soc Hortic Sci 130: 152–158

Juan L, Jian-Min Z, Zeng-Qiang D, Chang-Wen D, Huo-Ya W (2007) Effect of CO2 enrichement on the growth and nutrient uptake of tomato seedlings. Pedosphere 17: 343–351

Kimball BA, Kobayashi K, Bindi M (2002) Responses of agricultural crops to free-air CO2 enrichment. Adv Agron 77: 293–368

Korner C, Pelaezriedl S, Vanbel AJE (1995) CO2 responsiveness of plants – A possible link to phloem loading. Plant Cell Environ 18: 595–600

Korner C (2000) Biosphere responses to CO2 enrichment. Ecol Appl 10: 1590–1619 Kudo T, Makita N, Kojima M, Tokunaga H, Sakakibara H (2012) Cytokinin activity of cis-Zeatin and phenotypic alterations induced by overexpression of putative cis-Zeatin-O-glucosyltransferase in rice. Plant Physiol 160: 319–331

Kuiper D (1988) Growth-responses of Plantago major L. ssp. Pleiosperma (Pilger) to changes in mineral supply – Evidence for regulation by cytokinins. Plant Physiol 87: 555–557

Li CR, Gan LJ, Xia K, Zhou X, Hew CS (2002) Responses of carboxylating enzymes, sucrose metabolizing enzymes and plant hormones in a tropical epiphytic CAM orchid to CO2 enrichment. Plant Cell Environ 25: 369–377

Li L, Zhanga Y, Luoc J, Korpelainend H, Li C (2013) Sex-specific responses of Populus yunnanensis exposed to elevated CO2 and salinity. Physiol Plant 147: 477–488

Lycoskoufis IH, Savvas D, Mavrogianopoulos G (2005) Growth, gas exchange, and nutrient status in pepper (Capsicum annuum L.) grown in recirculating nutrient solution as affected by salinity imposed to half of the root system. Sci Hortic 106: 147–161

Maggio A, Dalton FN, Piccinni G (2002) The effects of elevated carbon dioxide on static and dynamic indices for tomato salt tolerance. Eur J Agron 16: 197–206 Masle J (2000) The effects of elevated CO2 concentrations on cell division rates, growth patterns, and

This article is protected by copyright. All rights reserved.

blade anatomy in young wheat plants are modulated by factors related to leaf position, vernalization, and genotype. Plant Physiol 122: 1399–1415 Mavrogianopoulos GN, Spanakis J, Tsikalas P (1999) Effect of carbon dioxide enrichment and salinity on photosynthesis and yield in melon. Sci Hortic 79: 51–63

Accepted Article

Maas EV, Hoffman GJ (1977) Crop salt tolerance – Current assessment. J Irrig Drain Div 103: 115– 134

Maxwell K, Johnson G (2000). Chlorophyll fluorescence – a practical guide. J Exp Bot 51: 659–668

Metzger MJ, Rounsevell MDA, Acosta-Michlik L, Leemans R, Schrotere D (2006) The vulnerability of ecosystem services to land use change. Agric Ecosyst Environ 114: 69–85

Mok MC, Mok DW, Armstrong DJ (1978) Differential cytokinin structure activity relationships in Phaseolus. Plant Physiol 61: 72–75

Mulholland BJ, Taylor IB, Jackson AC, Thompson AJ (2003) Can ABA mediate responses of salinity stressed tomato. Environ Exp Bot 50: 17–28

Munns R (1993) Physiological processes limiting plant-growth in saline soils – some dogmas and hypotheses. Plant Cell Environ 16: 15–24

Munns R (2002) Comparative physiology of salt and water stress. Plant Cell Environ 25: 239–250 Munns R, Tester M (2008) Mechanisms of salinity tolerance. Annu Rev Plant Biol 59: 651–681 Pérez-López U, Miranda-Apodaca J, Mena-Petite A, Muñoz-Rueda A (2013) Barley growth and its underlying components are affected by elevated CO2 and salt concentration. J Plant Growth Regul, DOI: 10.1007/s00344-013-9340-x

Pessarakli M (1991) Dry-matter yield, N-15 absorption, and water-uptake by green bean under sodium-chloride stress. Crop Sci 31: 1633–1640

Porra RJ, Thompson WA, Kriedemann PE (1989) Determination of accurate extinction coefficients and simultaneous-equations for assaying chlorophyll a and chlorophyll b extracted with 4 different solvents – verification of the concentration of chlorophyll standards by atomicabsorption spectroscopy. Biochim Biophys Acta 975: 384–394

Poorter M, Pérez-Soba M (2002) Plant growth at elevated CO2. The Earth system: biological and ecological dimensions of global environmental change. In: Mooney HA, Canadell JG (eds) Encyclopedia of Global Environmental Change, Vol 2. John Wiley & Sons Ltd, Chichester, pp 489–496

Ratnakumar P, Rajendrudu G, Swamy PM (2013) Photosynthesis and growth responses of peanut (Arachis hypogaea L.) to salinity at elevated CO2. Plant Soil Environ 59: 410–416

Reuveni J, Gale J, Zeroni M (1997) Differentiating day from night effects of high ambient CO2 on the gas exchange and growth of Xanthium strumarium L. exposed to salinity stress. Ann Bot 79: 191– 196 Ribaut JM, Pilet PE (1994) Water stress and indol-3yl-acetic acid content of maize roots. Planta 193: 502–507

This article is protected by copyright. All rights reserved.

Rogers A, Allen DJ, Davey PA, Morgan PB, Ainsworth EA, Bernacchi CJ, Cornic G, Dermody O, Dohleman FG, Heaton EA, Mahoney J, Zhu XG, Delucia EH, Ort DR, Long SP (2004) Leaf photosynthesis and carbohydrate dynamics of soybeans grown throughout their life-cycle under Free-Air Carbon dioxide Enrichment. Plant Cell Environ 27: 449–458

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Sakakibara H (2006). Cytokinins: Activity, Biosynthesis, and Translocation. Annu Rev Plant Biol 57: 431–449

Sakakibara H, Takei K, Hirose N (2006) Interactions between nitrogen and cytokinin in the regulation of metabolism and development. Trends Plant Sci 11: 440–448

Sayed OH (2003) Chlorophyll fluorescence as a tool in cereal crop research. Photosynthetica 41: 321– 330

Suhayda CG, Giannini JL, Briskin DP, Shannon MC (1990) Electrostatic changes in Lycopersicon esculentum root plasma-membrane resulting from salt stress. Plant Physiol 93: 471–478

Teng N, Wang J, Chen T, Wu X, Wang Y, Lin J (2006) Elevated CO2 induces physiological, biochemical and structural changes in leaves of Arabidopsis thaliana. New Phytol 172: 92–103

Thomas CD, Cameron A, Green RE, Bakkenes M, Beaumont LJ, Collingham YC, Erasmus BFN, de Siqueira MF, Grainger A, Hannah L, Hughes L, Huntley B, van Jaarsveld AS, Midgley GF, Miles L, Ortega-Huerta MA, Peterson AT, Phillips OL, Williams SE (2004) Extinction risk from climate change. Nature 427: 145–148

Tuna AL, Kaya C, Dikilitas M, Higgs D (2008) The combined effects of gibberellic acid and salinity on some antioxidant enzyme activities, plant growth parameters and nutritional status in maize plants. Environ Exp Bot 62: 1–9

Vu JCV, Gesch RW, Pennanen AH, Allen LH, Boote KJ, Bowes G (2001) Soybean photosynthesis, Rubisco and carbohydrate enzymes function at supraoptimal temperatures in elevated CO2. J Plant Physiol 158: 295–307

Wang YY, Mopper S, Hasenstein KH (2001) Effects of salinity on endogenous ABA, IAA, JA, and SA in Iris hexagona. J Chem Ecol 27: 327–342

Xu LK, Hsiao TC (2004) Predicted versus measured photosynthetic water-use efficiency of crop stands under dynamically changing field environments. J Exp Bot 55: 2395–2411

Yeo A (1999) Predicting the interaction between the effects of salinity and climate change on crop plants. Sci Hortic 78: 159–174

Yong JWH, Wong SC, Letham DS, Hocart CH, Farquhar GD (2000) Effects of elevated CO2 and nitrogen nutrition on cytokinins in the xylem sap and leaves of cotton. Plant Physiol 124: 767– 779 Zhu JK (2002) Salt and drought stress signal transduction in plants. Annu Rev Plant Biol 53: 247–273

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Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1. Optimal parameters for the multiple reaction monitoring detection of indole-3-acetic acid and abscisic acid used in the HPLC mass spectrometry analysis.

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Table S2. Optimal parameters for the multiple reaction monitoring detection of cytokinins used in the

HPLC mass spectrometry analysis.

Edited by J. Flexas

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Figure legends Fig. 1. Effect of the elevated CO2 concentration and salinity on sweet pepper plants: (A) total plant dry weight; (B) stem dry weight; (C) leaf dry weight; (D) root dry weight; (E) dry weight partitioning into

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the root; (F) leaf area; (G) leaf relative water content and (H) specific leaf area. Data are means ±SE of 6 plants.

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Fig. 2. Effect of the elevated CO2 concentration and salinity on sweet pepper plants: (A) net photosynthesis rate; (B) internal/ambient CO2 concentration ratio; (C) stomatal conductance; (D)

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chlorophyll fluorescence. Data are means ±SE of 6 plants.

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Fig. 3. Effect of the elevated CO2 concentration and salinity on sweet pepper plants, regarding leaf chlorophyll: (A) chlorophyll a; (B) chlorophyll b; (C) chlorophyll a/b; (D) relative chlorophyll content

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(SPAD). Data are means ±SE of 6 plants.

Fig. 4. Effect of the elevated CO2 concentration and salinity on sweet pepper plants, regarding the root respiration. Data are means ±SE of 6 plants.

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Fig. 5. Effect of the elevated CO2 concentration and salinity on sweet pepper plants: (A) leaf NO3–

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concentration and (B) leaf Cl– concentration. Data are means ±SE of 6 plants.

Fig. 6. Effect of the elevated CO2 concentration and salinity on sweet pepper plants: regarding hormone concentrations: ABA in the leaves (A) or roots (B); IAA in the leaves (C) or roots (D); total cytokinins in the leaves (E) or roots (F). Data are means ±SE of 6 plants.

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Fig. 7. Effect of the elevated CO2 concentration and salinity on sweet pepper plants, regarding

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different cytokinin forms in the leaves (A) or roots (B). Data are means ±SE of 6 plants.

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