or salinity

Scientia Horticulturae 195 (2015) 56–66

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Growth and nutritional quality improvement in two differently pigmented lettuce cultivars grown under elevated CO2 and/or salinity Usue Pérez-López a,∗ , Jon Miranda-Apodaca a , Maite Lacuesta b , Amaia Mena-Petite a , a ˜ Alberto Munoz-Rueda a

Departamento de Biología Vegetal y Ecología, Facultad de Ciencia y Tecnología, Universidad del País Vasco, UPV/EHU, Apdo. 644, E-48080 Bilbao, Spain Departamento de Biología Vegetal y Ecología, Facultad de Farmacia, Universidad del País Vasco, UPV/EHU, P◦ de la Universidad 7, 01006 Vitoria-Gasteiz, Spain b

a r t i c l e

i n f o

Article history: Received 5 June 2015 Received in revised form 28 July 2015 Accepted 20 August 2015 Keywords: Antioxidants Elevated CO2 Lettuce Minerals Nutraceutical value Salt stress

a b s t r a c t The interest of improving yield and quality of vegetables has increased in the recent years due to their benefits on human health. The aim of this study was to investigate if salt stress and elevated CO2 applied alone or in combination can improve the growth and nutritional quality of two differently pigmented (green and red) Lactuca sativa (L.) cultivars. Seedlings grown under ambient (400 ± 20 ␮mol mol−1 ) or elevated (700 ± 20 ␮mol mol−1 ) CO2 concentration for 35 days were subsequently supplied with 0 or 200 mM NaCl for 4 days. Then, biomass production, antioxidant capacity and minerals, nitrates, carbohydrates, proteins and hydrophilic and lipophilic antioxidant concentrations were measured. Red-pigmented lettuce showed higher nutritional quality than green-pigmented lettuce due to higher concentrations of Ca, P, Zn, and higher concentrations of lipophilic (Chl-a, Chl-b, and carotenoids) and hydrophilic (reduced ascorbate, total phenolics, and anthocyanins) antioxidants. Under elevated CO2 , both lettuce cultivars increased the uptake of almost all minerals to adjust to the higher growth rates, reaching similar concentrations to the ones detected under ambient CO2 ; only Mg and Fe were reduced. Furthermore, the antioxidant capacity, Chl-b and glutathione concentration increased in both cultivars. Under salt stress, the N and K concentrations decreased in both cultivars, while Ca, Mg, and P concentrations were also reduced in the red cultivar, probably due to a blockage in the uptake of these nutrients. Both lipophilic and hydrophilic antioxidant compounds increased in order to defend against the oxidative stress caused by an imbalance in ATP and NADPH production and consumption. The magnitude of the response was dependent on the cultivar. When salt stress was imposed under elevated CO2 , each cultivar responded differently. The red cultivar seemed to gain a greater advantage from elevated CO2 than the green cultivar because it better adjusted both mineral uptake and antioxidant metabolism. We conclude that elevated CO2 alone or in combination with short environmental salt stress permits us to increase the nutritional quality (increasing the concentration of some minerals and antioxidants) of lettuce without yield losses or even increasing production; however, the choice of the best growing conditions is dependent on the attributes we wish to improve. © 2015 Elsevier B.V. All rights reserved.

1. Introduction

Abbreviations: ASA, reduced ascorbate; DAS, days after sowing; DHA, oxidized ascorbate; DMSO, dimethyl sulphoxide; DW, dry weight; EDTA, ethylenediaminetetraacetic acid; FW, fresh weight; GSH, reduced glutathione; GSSG, oxidized glutathione; OL, red-pigmented lettuce cultivar; PB, green-pigmented lettuce cultivar. ∗ Corresponding author. Fax: +34 94 601 3500. E-mail addresses: [email protected] (U. Pérez-López), [email protected] (J. Miranda-Apodaca), [email protected] (M. Lacuesta), [email protected] ˜ (A. Mena-Petite), [email protected] (A. Munoz-Rueda). http://dx.doi.org/10.1016/j.scienta.2015.08.034 0304-4238/© 2015 Elsevier B.V. All rights reserved.

Diets containing a high proportion of fruits and vegetables have been shown to reduce the incidence of chronic diseases thanks to their effective antioxidant and other health-promoting properties (Wu et al., 2004; Llorach et al., 2008). Thus, there is an increasing demand from consumers for safe and nutritious foods that improve physical performance, reduce the risk of diseases, and increase life span. Lettuce (Lactuca sativa L.) is one of the most important consumed fresh vegetables (22.5 g FW day−1 per capita in Europe; WHO, 2003), containing a number of nutritive and health-promoting compounds. Indicators of lettuce nutritional

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quality include attributes such as antioxidant capacity (a measure of hydrophilic [ascorbate, glutathione, phenols] and lipophilic [chlorophylls and carotenoids] antioxidant compounds), mineral, carbohydrate and protein concentrations (Mou, 2005). Some authors have demonstrated that the genotype is an important factor affecting the concentration of phenols, ascorbate, carbohydrates and proteins, as well as the total antioxidant capacity (Llorach et al., 2008; Mou, 2009; Pérez-López et al., 2013a; Neocleous et al., 2014; Pérez-López et al., 2014b; Pérez-López et al., 2015). In addition to genotype differences, growing plants under non-optimal conditions for a short period of time may influence nutritional quality of lettuce. Under non-optimal conditions, the plant usually increases its antioxidant metabolism to counteract oxidative stress, thus, improving nutritional quality from a human point of view. In order to prevent oxidative damage caused by different salt concentrations, some authors have obtained increases and/or decreases in certain antioxidant compounds, although the response is cultivar- and species-specific (Kim et al., 2008; Younis et al., 2009; Leyva et al., 2011; Tarchoune et al., 2013; Neocleous et al., 2014). Conversely, the changes caused by Na and Cl in some ion activities in the root may trigger a nutritional imbalance due to the uptake blockage of other cations such as K, Ca, and Mg or anions such as nitrate and phosphate, provoking changes in mineral, carbohydrate, and protein concentrations, finally provoking a reduction in growth (Martínez-Ballesta et al., 2010). Some authors have detected changes in the aforementioned compounds in lettuce although the response was genotype- and mineral-dependent (Eraslan et al., 2007; Leyva et al., 2011; Neocleous et al., 2014). Elevated CO2 is also an environmental factor that could alter the nutritional quality of lettuce. The increased CO2 /O2 ratio at the photoreduction sites usually increases the photosynthetic rate and finally growth and production (Champigny and Mosseau, 1999). This increase in growth is generally accompanied by a dilution in mineral and protein content, decreasing the nutritional quality of leaves (Sicher and Bunce, 1997; Loladze, 2014). Other authors have not detected changes in minerals or they have detected even increases (Baslam et al., 2012; Pérez-López et al., 2014a). Baslam et al. (2012) detected that the green-leaf cultivar accumulated higher levels of the most part of macro and micronutrients when cultivated under elevated CO2 , while in the red-leaf cultivar only the Fe concentration was increased by elevated CO2 . On the other hand, the higher photosynthetic rates may increase ATP and NADPH utilization and consequently reduce the rate of oxygen activation and oxyradical formation (Halliwell and Gutteridge, 1999) detecting a relaxation of antioxidant metabolism (Goufo et al., 2014). Conversely, the higher carbohydrate availability could permit a greater allocation to secondary metabolites such as phenolics or other antioxidant compounds (Jaafar et al., 2012). It would therefore be expected that environmental enrichment with elevated CO2 could enhance the bioactive compounds in lettuce and consequently enhance the nutraceutical value of this vegetable. However, the responses detected thus far are species-specific as both decreases and increases in antioxidants have been detected in response to elevated CO2 (Wang et al., 2003; Jaafar et al., 2012). Besides, cultivar specific responses have also been detected. Baslam et al. (2012) detected that elevated CO2 increased the concentration of anthocyanins in outer leaves of green-leaf cultivar and increased the concentration of phenolic compounds in red-leaf cultivar, while reduced ascorbate was not modified in neither of them. To the best of our knowlegde, few studies have utilized salt irrigation combined with CO2 -enriched atmospheres to enchance a crop’s nutraceutical value. Thus, the objective of the present work was to evaluate and understand how elevated CO2 and salinity alone or in combination change the biomass production

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and the nutritional quality of lettuce and to determine if differently pigmented lettuce cultivars respond similarly. The achievement of these two objectives will permit the implementation of a better cultivation practice to obtain high yielding cultivars of higher nutritional quality.

2. Materials and methods 2.1. Plant material, growth conditions and experimental design Two differently pigmented lettuce (L. sativa L.) cultivars, Blonde of Paris Batavia (green-leaf) and Oak Leaf (red-leaf), were used as plant material. The plants grew in a controlled environment growth chamber under a daily light regimen of 14 h of light and 10 h of darkness, with an average day/night temperature of 25/18 ◦ C and a relative humidity of 60/80% day/night. The photosynthetic photon flux density in the chamber was 400 ␮mol photons m−2 s−1 throughout the entire light period. To minimize the effects of intra-chamber environmental gradients, plants were randomly repositioned within the chamber each week. The plants were allowed to grow from seeds in ambient (400 ± 20 ␮mol mol−1 ) or elevated (700 ± 20 ␮mol mol−1 ) CO2 concentrations, 24 h per day. The atmospheric CO2 concentration was continuously recorded by a CO2 sensor, the signal being received by a computer that activated, when necessary, CO2 injection into the chamber so as to reach the desired CO2 concentration. Six seeds per pot were grown in a mixture of perlite/vermiculite (3:1), and the most uniformly sized lettuce was selected seven days after sowing (DAS), leaving one lettuce per pot. The seedlings were watered with Hoagland’s solution (Arnon and Hoagland, 1940) every 2 days until 35 DAS. Subsequently, salt treatment was administered by adding 0 or 200 mM NaCl to Hoagland’s solution, which was supplied every day for 4 days. It has been indicated that the lower nutritional value of some lettuce varieties is due to the high enclosure of their leaves in the head structure because most of the edible portion of head structure includes leaves that are not exposed to light. Hence, using the external and mature leaves permits to obtain more homogenized results since all the leaves have receive light in the same extent. Moreover, it has been stated that in some lettuce cultivars, the potentially beneficial compounds appeared in higher levels in the outer than in the inner leaves (Baslam et al., 2013 and references there in). Accordingly, at the end of the experimental period (39 DAS), the fully matured leaves were randomly selected and harvested for all measurements. Six independent plants (one plant per pot) were measured for each treatment and cultivar.

2.2. Biomass production, water content, and concentration of minerals, nitrates, carbohydrates and proteins Shoot biomass production was measured at the end of the growing period (39 DAS). The plants were harvested and weighed to determine the fresh weight (FW). The samples were then dried at 70 ◦ C for 48 h, and their dry weight (DW) was determined. The leaf water content (WC) was determined using the equation WC = (FW–DW)/DW. To determine the C and N concentrations, the plant material was oven-dried, weighed, and milled. C and N were determined in leaf samples (2 mg DW) using an elemental analyser (FlashEA 1112; ThermoFinnigan, Germany). For mineral analysis, samples (0.5 g DW) from six plants per treatment were dissolved in 10 mL nitric acid (1%). K, Ca, Mg, Na, and P were determined using inductively coupled plasma atomic emission spectroscopy (ICP-AES, Horiba Yobin Yvon Activa). Cu, Zn, Mn, B and Fe were determined by inductively coupled plasma mass spectrometry (ICPMS, Agilent 7700).

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The soluble sugars (fructose, glucose, and sucrose) and starch were extracted and measured with an enzymatic kit (Boehringer Mannheim/R-Biofarm, Germany) as described by Pérez-López et al. (2012). The total soluble proteins and nitrates were extracted with 50 mM Hepes-KOH (pH 7.4). The concentration of soluble proteins in the leaf was measured using the protein dye-binding method described by Bradford (1976) with bovine serum albumin as a standard. The level of nitrates in the leaf was determined as described by Cataldo et al. (1974) using KNO3 as standard.

the means were compared by Duncan’s test at a probability of 95%. Statistical analyses were performed using SPSS 21.0 (SPSS, Inc., Chicago, IL, USA). Prior to analyses, we tested whether the assumptions of an ANOVA, homogeneity of variances and normally distributed errors were achieved. The homogeneity of variances for all the studied parameters was evaluated by Levene’s test and the distribution of the residuals was assessed by Kolmogorov–Smirnov test. 3. Results

2.3. Chlorophylls, carotenoids, and anthocyanins 3.1. Shoot biomass production and antioxidant capacity The chlorophylls and carotenoids were extracted with DMSO, and their concentrations were measured according to Wellburn (1994); the results are expressed on a DW basis. The anthocyanins were extracted with 3 M HCl/H2 O/methanol (1/3/16 in v/v/v), and their concentration was measured as described by Gould et al. (2000). The absorbance of anthocyanins at 524 nm was corrected by subtracting the interference by phaeophytin as A524 -0.24A653 (Murray and Hackett, 1991). 2.4. Reduced ascorbate (ASA), oxidized ascorbate (DHA), reduced glutathione (GSH), and oxidized glutathione (GSSG) Previously frozen fresh leaf samples (0.15 g) were homogenized in 2.25 mL of 1% HCl and 1 mM EDTA using a cold mortar and pestle, and the resultant mixture was centrifuged at 16,100 g for 10 min. Subsequently, the concentration of metabolites in the neutralized supernatant was measured as described by Pérez-López et al. (2010). 2.5. Total phenolics Total phenolics were extracted and measured according to Oh et al. (2009) with some modifications. The total concentration of phenolic compounds (0.2 g FW) was measured in 2.5 mL of ethanolic extract (85% ethanol/1% HCl), which was obtained using a cold mortar and pestle, followed by centrifuging the mixture at 16,100 g for 10 min. The ethanol was evaporated at 80 ◦ C, and 1 mL of milliQ water was subsequently added. Folin-Ciocalteu reagent was used to determine the total phenol concentration as described by Nguyen and Niemeyer (2008) with some modifications. Fifty microlitres of the sample extract was mixed with 1150 ␮L of milliQ water and 200 ␮L of Folin–Ciocalteu reagent and allowed to stand at 25 ◦ C for 3 min before Na2 CO3 solution (400 ␮L, 35%) was added.

We observed no differences in WC between the greenpigmented lettuce cultivar (PB) and the red-pigmented lettuce cultivar (OL) under control (plants grown at 0 mM NaCl under ambient CO2 ) conditions; at elevated CO2 the WC was higher in both cultivars (Fig. 1A). Salt treatment reduced WC in both cultivars. This reduction was greater at ambient CO2 than at elevated CO2 in OL, while no differences between CO2 treatments were detected in PB. Under control conditions, the shoot biomass production, estimated as DW per plant (Fig. 1B), was greater in PB than in OL, while the antioxidant capacity was greater in OL than in PB (Fig. 1C). The positive effect of elevated CO2 alone caused an increase of approximately 42% in PB and 62% in OL for biomass production and of 126% in PB and 293% in OL for antioxidant capacity. At ambient CO2 , salt stress did not affect plant production in either of the cultivars (Fig. 1B); however, the antioxidant capacity of OL lettuce increased by 179% (Fig. 1C). Under combined conditions of salt stress and elevated CO2 , the growth increased by 56% in PB and by 61% in OL and total antioxidant capacity increased by 61% in PB and by 109% in OL compared to salt stress and ambient CO2 . 3.2. Mineral analysis

The antioxidant capacity (defined as DDPH free radical scavenging activity) was measured by using the 1,1-diphenyl-2picrylhydrazyl (DPPH) method (Boo et al., 2011). Thirty microlitres of lettuce leaf extract (acetic acid/H2 O/methanol in 7/70/23, v/v/v) was added to 270 ␮L of DPPH (0.27 mM) working solution, and the absorbance was measured at 515 nm on a spectrophotometer. The DPPH scavenging activity (reported as ␮moles of Trolox equivalent) of the lettuce leaves was determined based on the antioxidant capacity on a per-gram DW basis.

The concentrations of different minerals varied among different lettuce cultivars (Figs. 2 and 3). The concentrations of Ca, P, and Zn were higher (17%, 12%, and 34%, respectively) in OL than in PB, while the concentrations of the rest of minerals were similar in both cultivars. Compared to control conditions, under elevated CO2 alone, the concentration of B and Mn increased by 16% and 28% in PB; the concentration of K, Cu, and Mn increased by 6%, 17%, and 22% in OL. On the other hand, both cultivars decreased the concentration of Mg and Fe by 19% and 55% in PB, and by 16% and 40% in OL, while in PB we also detected decreases in K. The concentrations of the other minerals remained constant. Under salt stress alone, in PB lettuce, Na increased 8 times and N, K, and B decreased by 10%, 12%, and 17%, respectively, while the concentrations of the other minerals remained constant. In OL lettuce, Na increased 7 times, and N decreased by 5%, K by 8%, Ca by 17%, Mg by 12%, and P by 8%. Finally, under combined conditions of salt stress and elevated CO2 and compared to salt stress and ambient CO2 , in PB, Na decreased by 33%, N by 6%, K by 5%, Ca by 14%, Mg by 23%, P by 8%, and Fe by 36%. Cu increased by 21%, B by 24%, and Mn by 33%. In OL, Na decreased by 14% and Zn increased by 23%. The concentrations of the other minerals remained constant compared to salt stress and ambient CO2 conditions.

2.7. Statistical analysis

3.3. Nitrates, soluble proteins, soluble sugars and starch analysis

The experiment was set up in a completely randomized design. Each treatment contained six independent pots and each pot contained one lettuce plant. The results are reported as the mean ± standard error (SE) values of six biological replicates (n = 6). The SE was calculated directly from crude data. Data were evaluated by analysis of variance (ANOVA) and differences between

Under control conditions, the levels of nitrates were similar between the cultivars, but lower in OL compared to PB under elevated CO2 (Fig. 4A). Analysing the effects of the different treatments, nitrates decreased under salt stress alone in both cultivars and in OL subjected to elevated CO2 and salt stress. Conversely, in PB subjected to elevated CO2 , nitrates increased compared to

2.6. Determination of DDPH free radical scavenging activity

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Fig. 1. Effects of salt treatment and CO2 concentration on water content (A), biomass production (B) and antioxidant capacity (C) on PB and OL. White bars (control) represent plants grown at 0 mM NaCl and ambient (400 ␮mol mol−1 ) CO2 , light grey bars represent plants grown at 200 mM NaCl and ambient (400 ␮mol mol−1 ) CO2 , black bars represent plants grown at 0 mM NaCl and elevated (700 ␮mol mol−1 ) CO2 , and dark grey bars represent plants grown at 200 mM NaCl and elevated (700 ␮mol mol−1 ) CO2 . Each value represents mean ± standard error (n = 6). Within each cultivar, significant differences (at P < 0.05) are indicated by different letters. * indicates differences between cultivars.

control conditions. The total soluble proteins were similar in both lettuce cultivars and remained almost constant independent of the treatment (Fig. 4B). Regarding carbohydrates, we detected higher concentration of total soluble sugars in OL than in PB under control conditions (Fig. 4C), while the starch concentration was similar between cultivars (Fig. 4D). Compared to control conditions, under elevated CO2 the concentration of total soluble sugars increased only in PB (59%) (Fig. 4C). Under salt stress and ambient CO2 , soluble sugars increased by 59% in both cultivars, while starch concentration decreased by 31% in PB and by 59% in OL. When salt stress was submitted under elevated CO2 , the soluble sugars and the starch concentration did not change compared to salt stress alone, in either of the cultivars.

3.4. Lipophilic antioxidants: concentration of chlorophylls and carotenoids The concentrations of Chl-a and Chl-b were around 122% higher in OL than in PB under control conditions (Fig. 5A–B). Similarly, the carotenoids were also higher (130%) in OL than in PB (Fig. 5C). Under elevated CO2 alone, the concentration of Chl-b increased by 52% and 39% in PB and OL, respectively. The concentration of Chl-a and carotenoids remained constant in both cultivars. Under salt stress and compared to control conditions, in PB the concentration of Chl-a and carotenoids increased (32% and 18%, respectively), while the concentration of Chl-b remained constant. In OL, the concentration of Chl-a, Chl-b and carotenoids

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Fig. 2. Effects of salt treatment and CO2 concentration on the concentrations of C (A), N (B), K (C), Ca (D), Mg (E), and P (F) on PB and OL. White bars (control) represent plants grown at 0 mM NaCl and ambient (400 ␮mol mol−1 ) CO2 , light grey bars represent plants grown at 200 mM NaCl and ambient (400 ␮mol mol−1 ) CO2 , black bars represent plants grown at 0 mM NaCl and elevated (700 ␮mol mol−1 ) CO2 , and dark grey bars represent plants grown at 200 mM NaCl and elevated (700 ␮mol mol−1 ) CO2 . Each value represents mean ± standard error (n = 6). Within each cultivar, significant differences (at P < 0.05) are indicated by different letters. * indicates differences between cultivars.

increased (21%, 14%, and 14%, respectively). Under combined conditions of salt stress and elevated CO2 , compared to salt stress and ambient CO2 conditions, the concentration of Chl-b increased by 77% in PB and by 29% in OL, while the concentration of carotenoids remained similar; the concentration of Chl-a decreased by 24% and 12% in PB and OL, respectively. 3.5. Hydrophilic antioxidants: concentration of ascorbate, glutathione, total phenolics, and anthocyanins The concentration of ASA, total phenolics and anthocyanins was 64%, 194% and 10 times, respectively, higher in OL than in PB (Fig. 6B, E–F). The elevated CO2 increased GSH + GSSG by 52% in PB and by 43% in OL (Fig. 6C) and increased GSH by 37% in both cultivars (Fig. 6D), while it reduced the anthocyanin concentration

compared to control conditions by 25% and 40% in PB and OL, respectively (Fig. 6F). Under salt stress, GSH, GSH + GSSG, and anthocyanins increased by 30%, 31%, and 71%, respectively, while ASA, ASA + DHA, and total phenolics remained constant. On the other hand, red lettuce increased its GSH, GSH + GSSG, and anthocyanins concentration by 85%, 82%, and 53%, respectively; reduced its ASA concentration (34%), its ASA + DHA concentration (30%), while the total phenolics remained constant. Under combined conditions of salt stress and elevated CO2 , and compared to salt stress and ambient CO2 conditions, in PB ASA + DHA, GSH + GSSG, and total phenolics increased (26%, 84%, and 36%, respectively); in OL these components remained constant. Regarding anthocyanins, under combined conditions of salt stress and elevated CO2 , they decreased by 45% and 53% in PB and OL, respectively, compared to salt stress and ambient CO2 conditions.

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Fig. 3. Effects of salt treatment and CO2 concentration on the concentrations of Na (A), B (B), Fe (C), Mn (D), Cu (E), and Zn (F) on PB and OL. White bars (control) represent plants grown at 0 mM NaCl and ambient (400 ␮mol mol−1 ) CO2 , light grey bars represent plants grown at 200 mM NaCl and ambient (400 ␮mol mol−1 ) CO2 , black bars represent plants grown at 0 mM NaCl and elevated (700 ␮mol mol−1 ) CO2 , and dark grey bars represent plants grown at 200 mM NaCl and elevated (700 ␮mol mol−1 ) CO2 . Each value represents mean ± standard error (n = 6). Within each cultivar, significant differences (at P < 0.05) are indicated by different letters. * indicates differences between cultivars.

4. Discussion 4.1. Differences between lettuce cultivars in biomass production and mineral, nitrate, protein, carbohydrate and antioxidant composition under control or elevated CO2 conditions The OL cultivar possessed a higher antioxidant capacity than PB (Fig. 1C). The present study confirms the existence of differences between cultivars in some specific antioxidant compounds. Indeed, higher constitutive amounts of lipophilic (Fig. 5) and hydrophilic (Fig. 6) antioxidants were detected in OL than in PB. Additionally, differences in some mineral and carbohydrate concentrations were also detected (Figs. 2–4). The OL cultivar showed higher chlorophylls and carotenoid concentrations than PB (Fig. 5A–C). Different concentrations were also observed in different lettuce cultivars by Mou (2005). Our results suggest that a higher proportion of resources were allocated to

the photosynthetic machinery in OL than in PB, probably because the absorbance attributed to anthocyanins in OL (Fig. 6F) may reduce the light absorbed by photosynthetic pigments (Pietrini and Massacci, 1998); thus, there is a need to increase the quantity of light absorbed by these pigments in order to maintain an adequate photosynthetic rate. The higher concentration of carotenoids has been correlated with the lower risk of cardiovascular diseases and certain cancers (Sies and Krinsky, 1995; Johnson et al., 2000). Likewise, according to Sgherri et al. (2011), chlorophylls show antioxidant activity and have hypothetical anti-inflammatory activity (Mulabagal et al., 2010). Concerning the hydrophilic antioxidant compounds, we detected higher ASA in OL than in PB (Fig. 6A). Differences in ASA have also been detected between cultivars of lettuce (Mou, 2009), although other authors have not detected differences (Baslam et al., 2011). We also detected higher total phenolics (Fig. 6E) and anthocyanins (Fig. 6F) in OL than in PB. Llorach et al. (2008)

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Fig. 4. Effects of salt treatment and CO2 concentration on the concentrations of NO3 − (A), soluble proteins (B), soluble sugars (C), and starch on PB and OL. White bars (control) represent plants grown at 0 mM NaCl and ambient (400 ␮mol mol−1 ) CO2 , light grey bars represent plants grown at 200 mM NaCl and ambient (400 ␮mol mol−1 ) CO2 , black bars represent plants grown at 0 mM NaCl and elevated (700 ␮mol mol−1 ) CO2 , and dark grey bars represent plants grown at 200 mM NaCl and elevated (700 ␮mol mol−1 ) CO2 . Each value represents mean ± standard error (n = 6). Within each cultivar, significant differences (at P < 0.05) are indicated by different letters. * indicates differences between cultivars.

and Pérez-López et al. (2014b) found higher caffeic acid derivatives, flavones, flavonols and anthocyanins in red-leafed cultivars of lettuce compared to the green ones explaining these differences. Phenolic compounds have a great interest for their health properties due their antioxidant capacity (de Pascual-Teresa and Sanchez-Ballesta, 2008). Thus, the higher concentration of chlorophylls, carotenoids, ascorbate, phenols and anthocyanins in OL than in PB makes OL nutritionally more valuable cultivar than the green cultivar. Soluble proteins and nitrates concentrations were similar between cultivars. Several studies have demonstrated toxic effects of nitrates on human health (Addiscott and Benjamin, 2004). Accordingly, in many countries the maximum level of nitrates allowed for the consumption of lettuce is 4000 ppm. Our values in all treatments and cultivars were below this value (the highest was 1200 ppm for PB lettuce under elevated CO2 ). Therefore, nitrate levels in PB and OL would be far below those of the maximum. Finally, we detected higher concentration of minerals (Ca, P, and Zn) in OL than in PB (Figs. 2 and 3). Neocleous et al. (2014) also detected differences in the values of minerals among different lettuce cultivars. Since we detected similar transpiration rates in both cultivars (data not shown), we hypothesize that the higher concentrations in OL may be attributed to a more selective uptake of these minerals in the red cultivar. Consequently, this cultivar is more nutritive than the green one since Ca, P, and Zn are essential for human health, participating in the biological functions of several tissues (musculoskeletal, nervous and cardiac system, bones and teeth, and parathyroid gland) and acting in a high number of enzyme reactions (Martínez-Ballesta et al., 2010). Regarding Na concentration, there were 10.79 mg/100 g FW in PB

and 19.04 mg/100 g FW in OL. These values would contribute with 0.11% and 0.20% of the recommended daily sodium uptake (WHO, 2012), assuming a consumption of 22.5 g FW of lettuce per day and per capita in Europe (WHO, 2003). Under elevated CO2 , the higher photosynthetic rates (PérezLópez et al., 2013a) would increase the sugar availability to synthesize more biomass (Fig. 1B) and antioxidant compounds (Fig. 1C). We also observed a higher Chl-b and GSH+GSSG concentration, indicating an active synthesis, which could contribute to the higher antioxidant capacity (Sgherri et al., 2011) detected. On the contrary, under elevated CO2 the concentration of anthocyanins decreased compared to ambient CO2 conditions. Anthocyanins absorb light between 400 and 600 nm (Pietrini and Massacci, 1998) reducing the visible light availability for photosynthesis. Under elevated CO2 , the need for ATP and NADPH generated by the light reactions to fix the extra CO2 (dark reactions) is higher, so plants would decrease their anthocyanin concentration, in order to increase the light arriving to the photosynthetic pigments to carry on light reactions. Ren et al. (2014) also detected decreases in anthocyanins due to elevated CO2 in Sorghum bicolor. On the other hand, the higher biomass production achieved at elevated CO2 (Fig. 1B) would imply a dilution effect for some compounds, reducing the ratio between the nutritional and caloric values of crops and increasing the micronutrient malnutrition problem in the human diet (Loladze, 2014). This fact occurred for Mg and Fe in both cultivars (Figs. 2E and 3C). Fe is a key element of haemoglobin formation and together with Mg is also required for energy production (Martínez-Ballesta et al., 2010). However, the response was mineral- and genotype-dependent since the concentration of the other minerals determined in our study increased (Mn

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Fig. 5. Effects of salt treatment and CO2 concentration on the concentrations of Chl-a (A), Chl-b (B), and carotenoids (C) on PB and OL. White bars (control) represent plants grown at 0 mM NaCl and ambient (400 ␮mol mol−1 ) CO2 , light grey bars represent plants grown at 200 mM NaCl and ambient (400 ␮mol mol−1 ) CO2 , black bars represent plants grown at 0 mM NaCl and elevated (700 ␮mol mol−1 ) CO2 , and dark grey bars represent plants grown at 200 mM NaCl and elevated (700 ␮mol mol−1 ) CO2 . Each value represents mean ± standard error (n = 6). Within each cultivar, significant differences (at P < 0.05) are indicated by different letters. * indicates differences between cultivars.

and B in PB; Mn, K and Cu in OL) or remained constant. This fact indicates that the uptake of these minerals was increased at comparable pace to the growth rate. Similar differences in the mineral response between cultivars were detected by Baslam et al. (2012). 4.2. Effects of salt treatment alone and in combination with elevated CO2 on biomass production and mineral, nitrate, protein, carbohydrate and antioxidant composition The salt treatment imposed on lettuce cultivars for 4 days did not affect production under either ambient or elevated CO2 (Fig. 1B). Both mineral and antioxidant compositions were altered by salt, however, in a cultivar and CO2 concentration dependent manner (Figs. 2–6).

The salt treatment increased the chlorophylls and carotenoids concentration in both cultivars under ambient CO2 . Other authors also detected increases in chlorophylls (Wang and Nii, 2000), lutein and ␤-carotene (Kim et al., 2008) in response to salt stress. The increase in carotenoids under salt stress has been associated with protective functions in plants because they can function in thermal energy dissipation (Demmig-Adams and Adams, 1992). They are also able to detoxify various forms of ROS such as 1 O2 (Young, 1991). Thus, their accumulation due to active synthesis (as growth was not reduced under salt stress) could be a defence response. Regarding hydrophilic compounds, salt stress increased GSH + GSSG in both cultivars (Fig. 6D). Glutathione has been seen to increase under salinity in other species due to its protective function (Mittova et al., 2003). In addition, under salt stress, the

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Fig. 6. Effects of salt treatment and CO2 concentration on the concentration of total ascorbate (A), reduced ascorbate (B), total glutathione (C), reduced glutathione (D), total phenolics (E), and anthocyanins (F) on PB and OL. White bars (control) represent plants grown at 0 mM NaCl and ambient (400 ␮mol mol−1 ) CO2 , light grey bars represent plants grown at 200 mM NaCl and ambient (400 ␮mol mol−1 ) CO2 , black bars represent plants grown at 0 mM NaCl and elevated (700 ␮mol mol−1 ) CO2 , and dark grey bars represent plants grown at 200 mM NaCl and elevated (700 ␮mol mol−1 ) CO2 . Each value represents mean ± standard error (n = 6). Within each cultivar, significant differences (at P < 0.05) are indicated by different letters. * indicates differences between cultivars.

photosynthetic rate decreased (Pérez-López et al., 2013a), which usually implies an increase of photorespiration rate (Pérez-López et al., 2012). The higher photorespiration rates could produce extra glycine, which could be used to synthesize de novo GSH, as appears to be the case in our cultivars (Fig. 6D). Moreover, salt stress increased anthocyanins in both cultivars (Fig. 6F), but to a higher extent in OL than in PB. Anthocyanins have been seen to increase under salinity in other species due to their protective function (Kennedy and De Filippis, 1999). The increase of these molecules with salt stress, together with chlorophylls and carotenoids, would indicate that the short treatment with saline irrigation increases the nutritional and health qualities of both cultivars without decreasing biomass production. Regarding mineral concentration, under saline conditions and ambient CO2 , PB lettuce showed decreases in N, K (Fig. 2B–C), and B (Fig. 3B) concentrations, while OL lettuce showed decreases in N, K, Ca, Mg, and P concentrations (Fig. 2B–F). These decreases were probably due to a competition between Na and Cl with

the other minerals (Pérez-López et al., 2013b, 2014a and literature therein). These findings would suppose a reduction of the nutritional quality of lettuce under the perspective of the mineral contribution. Regarding the concentration of Na under salt treatment, the increase of 7–8 times could be hazardous for health because it could provoke the increase in blood pressure and cause cardiovascular disease. The World Health Organization (WHO, 2012) recommends the intake of 2 g day−1 of Na (5 g day−1 of salt) in adults. Assuming an estimated mean daily consumption of 22.5 g of lettuce per person in Europe (WHO, 2003), the concentrations obtained in our study represent only the 1.24% in PB and 1.15% in OL of the daily intake recommend by the World Health Organization. Nevertheless, it could be necessary to recommend not adding salt in these salt-treated lettuces when are raw consumed in salads. Under combined conditions of salt stress and elevated CO2 , compared to salt stress and ambient CO2 conditions, Chl-a decreased in both cultivars, while the carotenoids concentration was similar and Chl-b concentration increased. These results indicate that

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besides showing higher biomass production capacity than under salt conditions alone, both lettuce cultivars grown under combined conditions of salt stress and elevated CO2 were able to maintain (for carotenoids) and even increase (for Chl-b) the synthesis of these antioxidant compounds, probably because the carbohydrate supply was higher under elevated CO2 . On the other hand, as reported in the previous section, under elevated CO2 the risk of oxidative damage is usually lower. In our study both cultivars showed lower anthocyanin concentration under combined conditions than under salt stress alone. The lower concentration of anthocyanins under elevated CO2 could respond to an adequate adjustment between light and dark reactions as mentioned in the previous section. However, regarding other metabolites each cultivar responded differently. In PB, we detected higher ASA + DHA (Fig. 6A), GSH + GSSG (Fig. 6C) and total phenolics (Fig. 6E) under combined conditions than under salt stress alone which could indicate that under elevated CO2 , plants could accumulate antioxidants more rapidly than biomass, resulting in a stronger antioxidant metabolism (Schwanz and Polle, 2001). However, in OL, the values of GSH, ASA and total phenolics remained similar under combined conditions than under salt stress alone, indicating that under elevated CO2 and salt stress, plants accumulated antioxidants and biomass at comparable rates. Hence, in this cultivar, the antioxidant metabolism was not enhanced compared to PB, probably because OL is endowed constitutively with higher antioxidant compounds. The response is therefore cultivar specific and would depend on the constitutive antioxidant compounds concentrations. However, we detected a higher increase in antioxidant capacity under combined conditions in OL than in PB, despite the fact that the antioxidants compounds increase in PB but not in OL. Kim et al. (2007) have reported that phenolic compounds were highly correlated with antioxidant activity in lettuce; however, we did not detect such a correlation. Złotek et al. (2014) also failed to detect a correlation between total phenolics and antioxidant activity in lettuce; in fact, they detected that the DPPH scavenging activity of lettuce was significantly and positively correlated with chlorogenic acid and quercetin concentrations, but significantly and negatively correlated with caffeic acid and luteolin concentrations. In a previous study performed using the same lettuce cultivars under non-stressful conditions (Pérez-López et al., 2014b), we detected differential contributions of the different phenolics to their total amount, so we hypothesize that elevated CO2 , under salt stress, could cause an increase in specific phenolics such as quercetin and chlorogenic acid and a decrease in the amount of caffeic acid and luteolin. This hypothesis should be demonstrated in further analysis, but it is beyond the scope of this paper. Regarding minerals, in PB, when salt stress was submitted under elevated CO2 , we detected higher decreases in N and K (lower concentrations of N and K) than the decreases detected under salt stress and ambient CO2 (Fig. 2B and C). We also detected decreases in Ca, Mg, and P, which we did not detect under ambient CO2 (Fig. 2D–F). This indicates that the elevated CO2 may have provoked, in concert with the lower uptake caused by salinity, a dilution of all of the macronutrients, as the biomass production was higher (Fig. 1B) and the transpiration rate lower. In OL lettuce, however, we detected a different behaviour. Under combined conditions of salt stress and elevated CO2 , the decrease in the K concentration disappeared compared to salt stress conditions alone (Fig. 2C). Further, the N, Ca, Mg, and P (Fig. 2B, D–F) concentrations were similar to those detected under salt stress conditions alone, not detecting a dilution effect despite the fact that we observed higher biomass production under combined conditions than under salt stress alone (Fig. 1B). This indicates that OL lettuce was able to maintain the growth and the uptake and translocation of N, K, Ca, Mg, and P at comparable rates under combined conditions of salt stress and elevated CO2 . These results indicate that not all cultivars of lettuce will

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respond to elevated CO2 in the same way; it seems that for the same species, the cultivar that under ambient CO2 is more limited by its growth (in our case the red cultivar) takes more advantage of CO2 enrichment. This fact is important because under future environmental conditions, the most successful cultivars will be the ones that can best adapt their metabolism to new environmental conditions. Another interesting implication from our results is that under combined conditions of salt stress and elevated CO2 , the reduction of Fe detected under elevated CO2 alone was reverted in both cultivars (Fig. 3C). Additionally, the micronutrients tended to increase, improving the nutraceutical value.

5. Conclusions Our results confirm the higher antioxidant power in red versus green lettuce, attributing this higher nutraceutical quality to higher levels of chlorophylls and carotenoids and phenolic compounds in the former, standing out the higher concentration of anthocyanins. Conversely, salt stress increased Chl-a, carotenoids, GSH and anthocyanin concentrations (in order to face possible oxidative stress) in both cultivars without yield losses. Finally, elevated CO2 alone or in combination with brief environmental salt stress allows the concentration of some minerals and antioxidants to increase, indicating that there is a real possibility to improve the nutritional quality of lettuce; in addition, it increased biomass production. However, the choice of the best growing conditions is dependent on the attributes we wish to improve.

Acknowledgements This research was financially supported by the following grants: Training and Research Units from the University of the Basque Country (UFI11/24), EHUA14/19, and GRUPO Gobierno VascoIT577-13. Technical and human support by Ph.D. Azucena González, Phytotron Service, SGIker (UPV/EHU) is gratefully acknowledged. The authors thank the technicians of SGIker’s General Analysis Service, financed by the National Program for the Promotion of Human Resources within the National Plan of Scientific Research, Development and Innovation, “Ministerio de Ciencia e Innovación”, “Fondo Social Europeo (FSE)” and “Gobierno Vasco/Eusko Jaurlaritza, Dirección de Política Científica” for the mineral measurements.

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