Potassium deficiency alters growth, photosynthetic performance, secondary metabolites content, and related antioxidant capacity in Sulla carnosa grown under moderate salinity

Accepted Manuscript Potassium deficiency alters growth, photosynthetic performance, secondary metabolites content, and related antioxidant capacity in Sulla carnosa grown under moderate salinity Chokri Hafsi, Hanen Falleh, Mariem Saada, Riadh Ksouri, Chedly Abdelly PII:

S0981-9428(17)30253-X

DOI:

10.1016/j.plaphy.2017.08.002

Reference:

PLAPHY 4956

To appear in:

Plant Physiology and Biochemistry

Received Date: 28 April 2017 Revised Date:

19 July 2017

Accepted Date: 2 August 2017

Please cite this article as: C. Hafsi, H. Falleh, M. Saada, R. Ksouri, C. Abdelly, Potassium deficiency alters growth, photosynthetic performance, secondary metabolites content, and related antioxidant capacity in Sulla carnosa grown under moderate salinity, Plant Physiology et Biochemistry (2017), doi: 10.1016/j.plaphy.2017.08.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1

Running title: Response of Sulla carnosa to salinity and K+ deficiency

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Corresponding author: Chokri Hafsi*.

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Laboratoire des plantes Extrêmophiles, Centre de Biotechnologie de Borj-Cédria, BP 901,

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2050 Hammam-Lif, Tunisia Tel: (+216) 71 430 855

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Fax: (+216) 71 430 934

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E-mail: [email protected]

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ACCEPTED MANUSCRIPT 15 Potassium deficiency alters growth, photosynthetic performance, secondary metabolites

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content, and related antioxidant capacity in Sulla carnosa grown under moderate

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salinity

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Chokri Hafsi*a, Hanen Fallehb, Mariem Saadab, Riadh Ksourib, Chedly Abdellya

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2050 Hammam-Lif, Tunisia

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Laboratoire des plantes Extrêmophiles, Centre de Biotechnologie de Borj-Cédria, BP 901,

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Cédria, BP 901, 2050 Hammam-Lif, Tunisia

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Laboratoire des Plantes Aromatiques et Médicinales, Centre de Biotechnologie de Borj-

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Abstract

Salinity and K+ deficiency are two environmental constraints that generally occur

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simultaneously under field conditions, resulting in severe limitation of plant growth and

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productivity. The present study aimed at investigating the effects of salinity, either separately

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applied or in combination with K+ deficiency, on growth, photosynthetic performance,

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secondary metabolites content, and related antioxidant capacity in Sulla carnosa. Seedlings

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were grown hydroponically under sufficient (6000 µM) or low (60 µM) K+ supply with 100

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mM NaCl (C+S and D+S treatments, respectively). Either alone or combined with K+

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deficiency, salinity significantly restricted the plant growth. K+ deficiency further increased

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salt impact on the photosynthetic activity of S. carnosa, but this species displayed

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mechanisms that play a role in protecting photosynthetic machinery (including non

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photochemical quenching and antioxidant activity). In contrast to plants subjected to salt

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stress alone, higher accumulation of phenolic compounds was likely related to antioxidative

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defence mechanism in plants grown under combined effects of two stresses. As a whole, these

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data suggest that K+ deficiency increases the deleterious effects of salt stress. The quantitative

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and qualitative alteration of phenolic composition and the enhancement of related antioxidant

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capacity may be of crucial significance for S. carnosa plants growing under salinity and K+

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deficient conditions.

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Keywords: Antioxidants; Photosynthesis; Potassium deficiency; Salinity; Sulla carnosa

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1. Introduction

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Salt stress is one of the biggest environmental factors impacting plant growth and productivity

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in many parts of the world, particularly in irrigated lands of arid and semi-arid regions (Hafsi

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et al., 2007; Mostek et al., 2015). This constraint affects more than 800 million hectares of

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land, representing more than 6% of the total global area of the Earth (Munns and Tester, 3

ACCEPTED MANUSCRIPT 2008). In Tunisia, about 1.5 million ha are salt affected which represent 10% of the whole

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territory (Hachicha et al., 1994).

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The competitive restriction of K+ uptake by Na+ into plant cells, due to physicochemical

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similarities between these two cations, is among the major effects caused by salinity (Rubio et

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al., 1995; Hafsi et al., 2007). In fact, NaCl excess in the medium can cause a down-regulation

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of genes involved in K+ transport (Zhu, 2003). In addition, Shabala et al. (2003) demonstrated

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that the depolarization of the plasma membrane with a consequent higher K+ efflux may also

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occur because of NaCl-exposure. Lipid peroxidation and K+ loss from cells by activating K+

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efflux channels is another consequence of over-production of reactive oxygen species (ROS)

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induced by salinity (Demidchik et al., 2003; Cuin and Shabala, 2007). Therefore, to survive

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under salinity conditions, it is pivotal for plants to maintain K+ homeostasis. In this context, a

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strong relationship between K+ status in roots and salinity tolerance has been found in barley

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(Chen et al., 2005, 2007a, 2007b) and wheat (Cuin et al., 2012). Hafsi et al. (2007) reported

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that the tolerance of Hordeum maritimum to salinity stress may be related to the improvement

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of K+ transport towards shoots and an increase of its use efficiency for biomass production.

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More recently, a correlation between salinity tolerance and the capacity of photosynthetically

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active mesophyll cells to retain K+ was found in a screening experiment conducted on wheat

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and barley genotypes (Wu et al., 2013, 2014a, 2014b).

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At the cellular level, K+ plays important roles including activation of enzymes, stabilization of

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protein synthesis, neutralization of negatively charged proteins, and maintenance of cytosolic

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pH homeostasis (Shabala, 2003; Dreyer and Uozum, 2011). Therefore, several authors

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suggest that the regulation of Na+ over K+ selectivity is pivotal for maintaining an adequate

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K+/Na+ ratio in the cytoplasm, and hence preserving biochemical and biophysical-K+

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dependent processes (Munns and Tester, 2008; Shabala and Cuin, 2008). It was shown that

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selection of plants with higher K+/Na+ ratios in their tissues may be sufficient to select salt-

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ACCEPTED MANUSCRIPT tolerant genotypes (Chen et al., 2007a,b). Evidence has shown that the addition of K+ can

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mitigate the deleterious effects of salt stress abiotic (Cakmak, 2005; Degl’Innocenti et al.,

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2009; Abbasi et al., 2015).

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It was observed in several studies that photosynthesis, one of the most important metabolic

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processes in plants, was sensitive to salt stress (Koyro, 2006; Degl’Innocenti et al., 2009;

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Huang et al., 2014). A reduction in net CO2 assimilation rate, transpiration rate, and stomatal

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conductance was frequently observed (Ouerghi et al., 2000; Liao and Guizhu, 2007;

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Degl’Innocenti et al., 2009). Salt-induced effects on photosynthesis were mostly due to a low

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osmotic potential of the soil solution, specific ion toxicities, nutritional imbalances or a

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combination of these factors (Ashraf, 1994; Zhu, 2003).

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Salinity leads to increased ROS production in plant cells such as singlet oxygen, hydrogen

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peroxide molecules, superoxide, and hydroxyl radicals. These reactive oxygen species are

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produced in different sites including chloroplasts, mitochondria, peroxisomes, the

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endoplasmic reticulum, and plasma membranes (del Río et al., 2006; Foyer and Noctor,

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2009). ROS overproduction can lead to oxidative damage to lipids, proteins, nucleic acids,

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and photosynthetic components (Meloni et al., 2003; Implay, 2003) or even to cell death

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(Jones, 2000). In order to reduce the deleterious effects of ROS, plant cells contain different

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protective mechanisms either through anti-oxidative enzymes or anti-oxidant secondary

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metabolites (Sharma and Dubey, 2005). Numerous non-enzymatic antioxidants such as α-

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tocopherol, β-carotene, ascorbate, and glutathione (Kim et al., 2001), and polyphenols (Ksouri

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et al., 2007; Valifard et al., 2014) are implicated in ROS-scavenging. Polyphenol synthesis

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and accumulation are stimulated in plants under salt stress, which might play an important

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role in ROS scavenging (Hernández, 2007; Taârit et al., 2012). These are attributable to (i) the

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high reactivity of polyphenols as hydrogen or electron donors (ii) the capacity of these

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ACCEPTED MANUSCRIPT compounds in chelating transition metal ions, and (iii) the ability of radicals derived from

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polyphenols to stabilize and delocalize the unpaired electron (Huang et al., 2005).

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Literature related to the interaction between salt stress and K+ availability is still limited. In a

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previous work (Hafsi et al., 2010) we showed that adding 100 mM NaCl salinity was

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beneficial for the facultative halophyte H. maritimum when exposed to K+ deficiency, due to

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the enhancement of the plant antioxidant response. More recently, we demonstrated in Sulla

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carnosa that K+ deficiency modulates the composition of secondary metabolites and their

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antioxidant properties (Hafsi et al., 2016). Nevertheless, data related to polyphenol

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accumulation and related antioxidative activities of plants subjected to salt-K+ deficiency

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mixed stresses are still scarce. In this study, we investigated the effects of salinity in

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combination with low or sufficient K+ supply on growth, photosynthesis, polyphenolic

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compound contents, and related antioxidative capacities in the legume S. carnosa. Sulla

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constitutes an important genetic resource and contributes to pastoral production particularly in

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semi-arid regions because of its drought tolerance and enrichment of soil due to its nitrogen

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fixing capacity (Trifi-Farah et al., 2002). Furthermore, the genus Sulla contains various

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chemical constituents. To date, 155 compounds have been isolated through different

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chromatography methods , including flavonoids, triterpenes, coumarins, lignanoids, nitrogen

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compounds, sterols, carbohydrates, fatty compounds, and benzofuran which contribute to the

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antioxidant, anti-tumor, anti-aging, anti-diabetic, and anti-hypertensive properties of these

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plants (Dong et al., 2013).

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2. Material and methods

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2.1. Plant material and growth conditions

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Seeds of S. carnosa were collected from Kalbia sabkha (a saline area in the center of Tunisia

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bioclimatic climate: semiarid, altitude: 34 m, location: 10°8’29” N, 35°48’26” E). Seeds were

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ACCEPTED MANUSCRIPT scarified, disinfected for 2 min with 1% NaClO and washed several times with distilled water.

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After, they were germinated in Petri dishes on filter paper moistened with distilled water.

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Three-day-old seedlings were transferred into plastic pots (14 plants pot-1) and irrigated with

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5 L of modified Hewitt’s nutrient solution (Hewitt, 1966). The composition of nutrient

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solution was: 1.5 mM MgSO4, 7H2O, 3.5 mM Ca (NO3)2, 4H2O, 5.4 mM NaNO3, 2 mM

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NH4H2PO4, and 6 mM KCl for macronutrients. The micronutrients (µM) were: Mn (0.5), Cu

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(0.04), Zn (0.05), B (0.5), Mo (0.02) (Arnon and Hoagland, 1940) and Fe (3) as Na2-Fe-

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EDTA complex.

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After 28 days pretreatment period, plants were divided into three lots (three replicates for

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each lot). The following treatments: C = control (complete medium (CM) containing 6 mM

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K+), C+S = salt treatment (CM containing 6 mM K+ with 100 mM NaCl), and D+S =

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interactive treatment (CM containing 60 µM K+ and 100 mM NaCl) were used. The culture

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was performed in a greenhouse with day/night temperatures of 25°C/18°C, a 16 h

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photoperiod, a photon flux density of 400 µmol m-2 s-1, and a relative humidity of 70-75%. At

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the end of treatment period (30 days), plants were harvested and divided into roots, stems, and

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leaves. The fresh weights (FW) were immediately determined. Samples were then oven-dried

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for 48 h at 60oC and dry weight (DW) was measured.

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2.2. Pigment determination

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After extraction in 80% acetone, chlorophyll and carotenoid concentrations (mg g-1 FW) were

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quantified spectrophotometrically from fresh leaves following the method of Arnon (1949).

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2.3. Gas exchange and chlorophyll fluorescence measurements

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Photosynthetic gas exchange was determined with a portable photosynthesis system (LCA4)

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(Bio-Scientific, Great Amwell, Herts, UK) at the end of the treatment period. Measurements

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ACCEPTED MANUSCRIPT were carried out between 10:00 and 13:00 h on the youngest fully emerged leaves at

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photosynthetically active radiation 1350 µmol m-2 s-1 ± 249, 27 ± 2oC leaf temperature, 65 ±

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5% relative humidity, and 380 µmol mol-1 ambient CO2 concentration. Parameters of

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chlorophyll fluorescence were measured using a modulated chlorophyll fluorimeter (OS1-FL)

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following the method described by Genty et al. (1989). The minimal (F0) and maximal (Fm)

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Chl a fluorescence were assessed in leaves after 20 min of dark adaptation. The following

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ratios were calculated according to Maxwell and Johnson (2000). The maximum quantum

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yield of PSII was calculated as Fv/Fm = (Fm - F0)/Fm. The relative quantum yield of PSII at

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steady-state was determined as ΦPSII= (F’m - Fs)/F’m, where Fs and F’m are fluorescence at

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steady-state and maximum fluorescence in the light, respectively. Non-photochemical

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quenching of fluorescence (NPQ) was calculated as NPQ = (Fm – F’m)/F’m.

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2.4. Colorimetric quantification of antioxidants

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2.4.1. Total phenolic content

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Total polyphenol contents were quantified as described by Dewanto et al. (2002). Powdered

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leaves of S. carnosa were extracted with methanol 80%. An aliquot (0.125 ml) of sample

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extract was mixed with 0.5 ml distilled water and 0.125 ml of Folin–Ciocalteu reagent. After

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an incubation of 3 min, 1.25 ml of 7% Na2CO3 solution was added to the mixture and the final

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volume was adjusted to 3 ml using distilled water. Finally and after 90 min of incubation in

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the dark, the absorbance of each mixture was measured at 760 nm. The phenol contents were

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expressed as milligram gallic acid equivalent per gram of dry weight (mg GAE g-1 DW) using

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a calibration curve of gallic acid (0–400 µg ml−1).

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ACCEPTED MANUSCRIPT 2.4.2. Total flavonoid content

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The measurement of flavonoid concentrations was based on the method described by

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Dewanto et al. (2002). An aliquot of the samples was added to test tubes containing 75 µl of a

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5% NaNO2 solution, and mixed for 6 min. Then, 0.15 ml of a freshly prepared 10% AlCl3

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solution was added. After 5 min at room temperature, 0.5 ml of 1 N NaOH was also added.

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The final volume was adjusted to 2.5 ml with distilled water and thoroughly mixed.

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Absorbance of the mixture was determined at 510 nm and the concentrations of flavonoid

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compounds were calculated according to the equation obtained from the standard (+)-catechin

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graph, and were expressed as mg catechin equivalents g-1 DW (mg CE g-1 DW).

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2.4.3. Total condensed tannins assay

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Contents of condensed tannin were assessed using Sun et al. (1998) procedure. Fifty

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microliters of extracts were mixed with 3 ml of 4% vanillin and 1.5 ml 1 N hydrochloric acid.

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The mixture was allowed to stand for 15 min and the absorbance was measured at 500 nm.

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The final result was expressed as mg catechin equivalents per gram dry weight (mg CE g-1

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DW).

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2.5. Assessment of antioxidant activities

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2.5.1. Evaluation of total antioxidant capacity

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Total antioxidant capacity of alcoholic extracts was assessed using the green phosphate/Mo5+

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complex following the method described by Prieto et al. (1999). Firstly, 0.1 ml of suitably

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diluted samples were combined with 1 ml of reagent solution (0.3 N sulfuric acid, 28 mM

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sodium phosphate and 4 mM ammonium molybdate). Secondly, the mixtures were incubated

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in a boiling water bath for 90 min. Then, the obtained solutions were cooled to room

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temperature and the absorbance was read at 695 nm in UV-Visible spectrophotometer

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(Anthelie Advanced 2, Secoman). As for total polyphenol content, the total antioxidant

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capacity was expressed as mg gallic acid equivalent per gram dry weight (mg GAE g−1 DW).

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The capacity of the corresponding extracts to donate hydrogen atoms or to accept electrons

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was monitored according to the method described by Hanato et al. (1988). One ml of various

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concentrations of plant extracts was added to 250 µl of 0.2 mM DPPH radical solution. The

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mixture was shaken vigorously than allowed to stand for 30 min in the dark. The absorbance

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of the resulting solution was monitored at 517 nm. Inhibition of DPPH radical was calculated

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as follows:

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DPPH° scavenging effect (%) = [(Ac-As)/Ac]*100

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where Ac and As are the absorbance at 30 min of the control and the sample, respectively.

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The antiradical activity was expressed as IC50 (µg.ml-1), the extract dose required to cause a

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50% decrease of the absorbance at 517 nm. A lower IC50 value corresponds to a higher

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antioxidant activity. All samples were analyzed in triplicate.

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2.5.3. Chelating effect on ferrous ions

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The chelating activity of S. carnosa leaf extracts was tested according to Zhao et al. (2006).

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Several concentrations of sample extracts were mixed with 0.05 ml of FeCl2 × 4H2O solution

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(2 mM) and left at room temperature for 5 min. After that, 0.1 ml of ferrozine (5 mM) and

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deionized water were added and the obtained solutions were shaken vigorously, and left at

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room temperature for 10 min. Absorbance was finally measured at 562 nm. The percentage of

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inhibition of ferrozine-Fe2+ complex formation was calculated using formula (1) and results

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were expressed as EC50: efficient concentration corresponding to 50% ferrous iron chelating.

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ACCEPTED MANUSCRIPT 2.5.4. Determination of reducing power

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The reducing power of plant extracts was determined through the transformation of Fe3+ to

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Fe2+ according to the method of Oyaizu (1986). One ml of extracts was mixed with 2.5 ml of

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phosphate buffer (0.2 mol l-1, pH 6.6) and 2.5 ml of K3Fe(CN)6. After incubation at 50 °C for

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25 min, 2.5 ml of trichloroacetic acid (10%) was added and the mixture was centrifuged at

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650 × g for 10 min. Finally, 2.5 ml of the upper layer was mixed with 2.5 ml of distilled water

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and 0.5 ml of FeCl3 (0.1%). A higher absorbance indicates a higher reducing power. EC50

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value (mg ml-1) is the effective concentration giving an absorbance of 0.5 for reducing power

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and was obtained from the linear regression analysis.

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2.5.5. β-Carotene bleaching test

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Firstly, β-Carotene (2 mg) was dissolved in 20 ml chloroform and to 4 ml of this solution,

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linoleic acid (40 mg) and Tween 40 (400 mg) were added (Koleva et al., 2002). Chloroform

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was evaporated using a vacuum at 40 °C than 100 ml of oxygenated water was added and the

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obtained emulsion was vigorously shaken. From this emulsion, 150 µl were distributed in

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each of the wells of 96-well microtiter plates and fraction solutions of the test samples (10 µl)

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were added. These microtiter plates were incubated at 50 °C for 120 min, and the absorbance

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was read using a model EAR 400 microtiter reader (Labsystems Multiskan MS) at 470 nm.

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Readings of all samples were performed immediately and after 120 min of incubation. The

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antioxidant activity (AA) of the extracts was evaluated in terms of β-carotene bleaching using

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the following formula:

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AA% = [(S−C120) / (C0-C120)]*100:

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where C0 and C120 are the absorbance values of the control at 0 and 120 min, respectively, and

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S is the sample absorbance at 120 min. The results were expressed as IC50 values (µg ml−1).

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ACCEPTED MANUSCRIPT 2.5.6. Analysis of individual phenolic compounds by RP-HPLC

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The identification of individual phenolics was performed with an Agilent 1100 series HPLC

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system equipped with an online degasser (G 1322A), a quaternary pump (G 1311A), a

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thermostatic autosampler (G 1313A), a column heater (G 1316A), and a diode array detector

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(G 1315A). Instrument control and data analysis was carried out using Agilent HPLC

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Chemstation 10.1 editon through Windows 2000. The separation was carried out on a reverse

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phase ODS C18 column (5 µm, 250 × 4.6 mm, Hypersil) used as a stationary phase at

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ambient temperature. Extracts were filtered through a polytetrafluoroethylene (PTFE)

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membrane (0.45 µm) prior to HPLC analysis. Then, 10 µl of each filtrate were injected into

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the HPLC system. The column was maintained at 30°C and the flow rate was 1 mL.min-1. The

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mobile phase used was a gradient of solvent B (acetonitrile) and solvent A (2.5% acetic acid).

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The following linear gradient was applied: 3% B; 0-5 min, 9% B; 5-15 min, 16% B; 15-45

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min, 50% B; and finally 45-51 min, 90% B to wash the column before initial condition

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recovery. Peaks were monitored at 280 nm and the identification was obtained by comparing

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the retention time and the UV spectra with those of pure standards purchased from Sigma (St.

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Louis, MO, USA).

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2.6. Statistical analysis

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All data were analyzed by one-way ANOVA test, and means were compared using Duncan’s

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multiple-range test at p ≤ 5 % level of significance by means of IBM SPSS 20 for Windows.

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3. Results

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3.1. Plant growth parameters

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Salinity alone or in combination with K+ deficiency significantly reduced growth-related

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parameters (Figure 1). The depressive effects were more pronounced in plants subjected to the

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ACCEPTED MANUSCRIPT interactive effects of the two constraints. C+S treatment decreased leaf DW, stem DW, root

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DW, and whole plant DW by 48.3%, 76.5%, 50%, and 55.1%, respectively, as compared to

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the control. More marked decrease was observed under D+S treatment for leaf DW, stem

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DW, root DW, and whole plant DW, reaching 71.3%, 91.2%, 73%, and 76.2%, respectively,

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as compared to the control. Stem growth was the most affected.

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3.2. Pigment contents

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Leaf pigment (Chl a, Chl b, and carotenoids) concentrations (Table 1) were significantly

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decreased following all treatments applied. Salinity alone (C+S treatment) led to a significant

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reduction in Chl a, Chl b, and carotenoids concentrations by 70.2%, 63.9%, and 68.2%,

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respectively, as compared to the control. The interactive effects of the two stresses (D+S

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treatment) decreased Chl a, Chl b, and carotenoids concentrations, by 76.6%, 75%, and 72.7%

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respectively, as compared to the control. Total Chl contents decreased by 68.5% and 75.4% at

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100 mM NaCl, either alone (C+S) or in combination with K+ deficiency (D+S), respectively

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(Table 2).

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3.3. Leaf gas exchange

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Control plants showed the highest CO2 assimilation rate (A), transpiration rate (E), and

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stomatal conductance (gs) (Fig. 2a-d). C+S treatment significantly decreased A, E, and gs by

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59.5%, 22.5%, and 54.7%, respectively, compared to control plants. D+S treatment impact

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was more marked on these parameters (-74.6%, -60.1%, and -83% for A, E, and gs,

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respectively). Water use efficiency (WUE) was decreased significantly by 47.2% and 36% in

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plants subjected to salt stress alone or in combination with K+ deficiency, respectively (Fig.

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2e). Intrinsic water use efficiency (IWUE) was not affected by salinity alone while increased

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by 50.3% in D+S treated-plants compared to control (Fig. 2f).

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ACCEPTED MANUSCRIPT 3.4. Chlorophyll fluorescence parameters

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Fv/Fm, which measures the maximum quantum yield of photochemistry, was not affected by

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all treatments applied (Table 2). While NPQ was not significantly affected in C+S treated-

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plants, the interactive effects of the two constraints (D+S treatment) increased this parameter

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by 29.5%. Electron transport rate (ETR) was decreased by 44.5% and 54% in C+S and D+S

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treated-plants, respectively. Nevertheless, salinity alone or in combination with K+ deficiency

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significantly increased ETR/A by 34.3% and 79.3%, respectively, as compared to control

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plants (Table 2).

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3.5. Quantification and identification of phenolic compounds

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3.5.1. Total polyphenol, flavonoid, and condensed tannins contents

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Total polyphenol content increased by 47.7% and 46.8% in C+S and D+S treated-plants,

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respectively (Fig. 3). Flavonoid content was only increased by the combined effects of the

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two stresses (+17.7% of the control), whereas salinity alone had no significant effect on this

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parameter (Fig. 3). Salinity alone or in combination with K+ deficiency decreased condensed

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tannins content (Fig. 2) by 33.3% and 30.2%, respectively, as compared to the control.

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3.5.2. Antioxidant activities

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Salinity (C+S treatment) decreased total antioxidant activity by 31.60% whereas the

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interactive effects of salt stress and K+ deficiency (D+S treatment) increased this parameter by

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12.6% as compared to the control (Fig. 4a). S+C treatment had no significant effect on DPPH

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radical-scavenging capacity and iron reducing power (Fig. 4b and 4c), whereas D+S treatment

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decreased these two parameters by 70% and 29.3%, respectively. Polyphenolic extracts were

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not able to chelate ferrous ions (Fig. 4d). In addition, β-carotene bleaching test was increased

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by 17.6% and 75.9% in C+S and D+S treated-plants, respectively (Fig. 4e).

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ACCEPTED MANUSCRIPT 3.5.3. Phenolics identification by RP-HPLC

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Phenolic compounds were identified by matching their retention times with those of known

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standards (Table 3). The assays performed on extracts from control S. carnosa leaves

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demonstrated that they contained fourteen phenolics compounds among which six were

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identified. In C+S treated-plants, ten phenolic compouds were detected, among which six

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were identified. Approximately 1.7-fold increase in total polyphenol contents was observed in

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these plants as compared to control ones. In D+S treated-plants, of the thirteen phenolic

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compounds that were detected, six were identified. A 2.1-fold increase in total phenolic

346

compound contents was registered in D+S-plants in comparison to control ones.

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4. Discussion

349

Salinity is one of the major environmental stresses affecting more than 800 million hectares of

350

land, equivalent to more than 6% of the total global area of the Earth (Munns and Tester,

351

2008). This constraint is a serious problem for crop production in many regions of the world

352

(Yamaguchi and Blumwald, 2005). In plants grown under salinity stressful conditions, an

353

impairment of K+ nutrition frequently occurs (Rubio et al., 1995; Hafsi et al., 2007). So,

354

maintaining K+ supply is crucial for plant survival under saline conditions (Akram et al.,

355

2009b). The present research work investigated the effect of salinity either alone or in

356

combination with K+ deficiency on S. carnosa plants. Our data show that plant growth was

357

negatively affected by all the treatments applied with a more pronounced reduction was

358

observed when the two constraints are applied simultaneously (Figure 1). Therefore,

359

improving K+ nutrition of plants may promote growth and alleviated the salt-induced growth

360

reduction as suggested by Cakmak (2005). Similar results were observed in strawberry (Kaya

361

et al., 2001), cucumber (Kaya et al., 2003), and barley (Degl’Innocenti et al., 2009). Leaf

362

injury symptoms (chlorosis and necrosis) were observed in aged leaves of D+S treated-plants

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ACCEPTED MANUSCRIPT after three weeks of treatment. These symptoms may be explained by increased chlorophyll

364

oxidative damage as a consequence of enhanced ROS generation during photosynthesis as

365

suggested by Cakmak (2005).

366

It has been demonstrated that photosynthetic pigments contents were reduced in plants

367

following exposure to salt stress (Huang et al., 2014; Gengmao et al., 2015). This depressive

368

impact was more pronounced when plants are cultivated under combined effects of salinity

369

and K+ deficiency (Qu et al., 2012; Abbasi et al., 2015). Our results are in accordance with

370

these findings. In fact, we demonstrated that pigments contents (chlorophyll and carotenoids)

371

were reduced by salinity and especially by the interactive effects of the two stresses (Table 1).

372

This suggested a more pronounced inhibition of photosynthetic pigments biosynthesis and/or

373

an acceleration of their biodegradation (Rabhi et al., 2007). However, the decrease in

374

photosynthetic pigments could be considered as an adaptive mechanism, since it may lead to

375

decrease the over reduction of the photosynthetic electron transport and consequently the

376

generation of free oxygen radicals (Wang et al., 2003; Christian, 2005). In contrast,

377

Degl’Innocenti et al. (2009) showed that exposure of barley plants to salinity alone or in

378

combination with K+ deprivation did not result in any significant impact in leaf photosynthetic

379

pigments contents.

380

It is well documented that photosynthesis was sensitive to numerous constraints, including

381

salinity (Garcia-Sanchez et al., 2002; Munns et al., 2006). Stomatal (closure of stomata) and

382

non-stomatal (including damage to photosynthetic apparatus) factors may be involved in the

383

reduction of the photosynthetic activity (Kao et al., 2003). This negative impact on

384

photosynthetic process was more pronounced under combined effects of salinity and K+

385

deficiency (Degl’Innocenti et al., 2009; Qu et al., 2012). Our data (Fig. 2) showed a marked

386

decrease in leaf photosynthetic parameters [net photosynthesis rate (A), transpiration rate (E),

387

and stomatal conductance (gs)], the strongest impact being registered in plants subjected to the

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ACCEPTED MANUSCRIPT combined effects of salinity and K+ deficiency. This is indicative of the significance of K+

389

nutrition in mitigating the deleterious effects of salinity stress on plant photosynthesis. In case

390

of stomatal-regulated photosynthesis, the role of K+ in stomatal regulation is of prime

391

importance (Shabala et al., 2002). In fact, decreased gs was a consequence of the loss of K+ by

392

guard cells (Peaslee and Moss, 1968). The reduction in gs is the initial and most important

393

cause of a decrease in CO2 assimilation rate (James et al., 2002). Our data demonstrated that

394

gs was strongly reduced either by salt alone (54.7% of reduction) or in combination with K+

395

deficiency (approximately 83% of reduction) suggesting that the decline of A could be

396

resulted from the stomatal closure (Fig. 2). However, reduced stomatal conductance

397

minimized loss of water through transpiration, resulting in lower transpiration rate (Fig. 2) at

398

the expense of CO2 fixation.

399

The reduction in gas exchange parameters was not accompanied by a decrease in the

400

intercellular CO2 concentration values (Ci) which remained unchanged (Fig. 2). This may

401

indicate that photosynthetic process decline under our experimental conditions might be also

402

caused by non-stomatal factors such as decreasing Rubisco and chlorophyll contents as

403

suggested by Koyro et al. (2013). Similar results were observed by Degl’Innocenti et al.

404

(2009) on barley species. In salt-stressed sunflower plants, Akram et al. (2009a) demonstrated

405

that the foliar application of different sources of K+ had no significant effects on Ci

406

suggesting that stomatal limitation was not the sole factor controlling photosynthetic process.

407

Furthermore, decreased photosynthetic capacity may be also attributed to increased leaf

408

K+/Na+ ratio. In fact, it was observed in several glycophytes species that the combination of

409

high Na+ and low K+ may affect K+ nutrition and cause Na+ toxicity (Munns and Tester, 2008;

410

Schubert et al., 2009). In addition, Rodrigues et al. (2013) demonstrated on Jatropha curcas

411

plants that a favorable ionic balance K+/Na+ ratio induced by the progressive increase in the

412

external K+ levels was capable of improving both photosynthesis and plant growth even under

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ACCEPTED MANUSCRIPT toxic NaCl concentrations in the culture medium. The salt-induced decrease in photosynthesis

414

capacity is considered as one of the most important factors restricting plant growth and

415

productivity (Zhao et al., 2007). In our case, growth parameters varied in the same manner

416

with gas exchange properties which could indicate a relationship.

417

WUE was calculated by rationing the A to E values. The increase in WUE is considered an

418

important adaptive trait for plant survival under high salinity (Debez et al., 2008). Hence, in

419

the present study, the decrease in WUE (Fig. 2) appears to be due to a more reduction in

420

photosynthetic rate as compared to transpiration rate. Similar results were observed by Rivelli

421

et al. (2002) on sunflower plants grown under salinity. Contrarily, IWUE (A/gs) was

422

significantly increased in plants subjected to the combined effects of salinity and K+

423

deficiency (D+S treatment) which may be owing to the larger decrease in stomatal

424

conductance (gs) compared to CO2 assimilation rate (A) (Fig. 2). Fernández-García et al.

425

(2014) observed an increase in IWUE suggesting that efficient water and carbon use, controls

426

henna plant performance grown under high salinity conditions.

427

Fv/Fm, the maximal quantum efficiency of PSII, is widely used as an indicator of the

428

photoinhibition or of the stress damage to the PSII (Maxwell and Johnson, 2000). No

429

significant effects of different treatments applied on Fv/Fm were observed in our study (Table

430

3) indicating no impact on PSII photochemistry. ETR/A was significantly increased in plants

431

subjected to salt stress alone (+34.3%) and especially by the combined effects of the two

432

constraints (+79.3%) (Table 2). This suggests that more electrons were driven to other sinks

433

including oxidation of molecular O2 at the expense of reduced CO2 assimilation (Wang et al.,

434

2012). NPQ is one of several mechanisms that have been developed by plants to cope with

435

excessive excitation energy during photosynthesis (Demmig-Adams and Adams, 2002). This

436

parameter increased significantly only in S. carnosa subjected to the interactive effects of

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ACCEPTED MANUSCRIPT salinity and K+ deficiency (Table 2) which may serve to dissipate excess excitation energy

438

and this determines the observed decrease in ΦPSII

439

As discussed above, salinity stress reduced S. carnosa growth as a consequence of decreased

440

photosynthetic capacity (reductions in pigments contents, CO2 net assimilation rate, stomatal

441

conductance, transpiration rate, and electron transport rate). This negative impact was

442

aggravated by K+ deficiency. Such unfavourable conditions could led to an overproduction of

443

ROS such as singlet oxygen (1O2), superoxide radical (O2.-), hydroxyl radical (OH.), and

444

hydrogen peroxide (H2O2) (Hochida et al., 2000), leading to oxidative damage to the

445

biomolecules such as lipids, proteins, and nucleic acids (Meloni et al., 2003; Implay, 2003).

446

Antioxidant activity either through anti-oxidative enzymes or anti-oxidant secondary

447

metabolites plays a crucial role in ROS scavenging (Sharma and Dubey, 2005). Increased

448

total polyphenol content (Fig. 3) is consistent with previous research works on different plant

449

species grown under salt stress (Ksouri et al., 2007; Taârit et al., 2012) and could have an

450

efficient scavenging capacity against harmful radicals. Flavonoid content was only increased

451

by salt stress alone while condensed tannins content was decreased by salinity alone or in

452

combination with K+ deficiency (Fig. 3). These results suggest the ability of S. carnosa to

453

alter and modulate the accumulation of phenolic compounds in response to different applied

454

stresses. The results of present work revealed that salt stress alone or in combination with K+

455

deficiency had an impact on the phenolic composition. In fact, some phenolic compounds

456

tended to disappear while others tended to appear according to applied treatment (Table 3).

457

This redistribution in phenolic composition is crucial to maintain antioxidant capacity in order

458

to limit oxidative damage. Similar results were observed on Mesembryanthemum edule

459

subjected to salt stress (Falleh et al., 2012).

460

Leaf methanolic extracts of S. carnosa were evaluated for their capacity to scavenge ROS.

461

Total antioxidant capacity was decreased by salinity when applied alone. Similar results were

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ACCEPTED MANUSCRIPT observed by Falleh et al. (2012) on M. edule subjected to increasing salinity. The decrease of

463

total antioxidant activity under salt was not accompanied by a decline in total polyphenol

464

content (Fig. 3). Interestingly, the increased antioxidant capacity in D+S-treated plants could

465

help to protect these plants from damaging effects by scavenging free radicals. Similarly, in

466

H. maritimum subjected to the interactive effects of salinity and K+ deprivation, Hafsi et al.

467

(2010) observed an increase in antioxidative response both enzymatic and non-enzymatic as

468

compared to plants grown only under salt stress. In the present study, four different assays

469

(DPPH.radical scavenging ability, iron reducing power, ferrous ion chelating ability, and β-

470

carotene bleaching test) were considered to evaluate antioxidant capacity of S. carnosa leaf

471

extracts. Plant secondary metabolites such as polyphenols (Sgherri et al., 2004) and

472

flavonoids (Hernandez et al., 2004) are low molecular antioxidants that can effectively

473

scavenge harmful free radicals. Leaf polyphenolic extracts from C+S-treated plants had no

474

significant effects on DPPH scavenging and on iron reducing power capacities (Fig. 4). These

475

two activities were enhanced by K+ deficiency. In fact, D+S-treated plants presented high iron

476

reducing power (EC50 decreased by 29.3% as compared to control) as well as a strong DPPH

477

scavenging activity (IC50 decreased by 70% in comparison to control). These activities may

478

be positively correlated with phenol and flavonoid contents and consequently to their free

479

radical scavenging capacities (Fig. 3). The β-carotene bleaching test increased by 17.6% and

480

75.9% in plants subjected to salt stress alone or in combination to K+ deficiency, respectively.

481

This indicates that the capacity to prevent the bleaching of β-carotene was altered. Besides,

482

polyphenolic extracts were not able to chelate ferrous ions (Fig. 4).

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483 484

5. Conclusion

485

This study showed that salinity alone or in combination with K+ deficiency results in a

486

significant reduction of S. caranosa growth with a more pronounced decline was observed

20

ACCEPTED MANUSCRIPT when the two constraints are applied simultaneously. In addition, photosynthetic process was

488

impacted. Thus, improving K+ nutrition is crucial to promote growth and mitigates the

489

unfavorable effects of salt stress. Contrarily to plants subjected to salt stress alone, increased

490

polyphenol accumulation and the parallel increase in related antioxidant activity registered in

491

plants exposed to D+S treatment may have contributed to scavenge free oxygen radicals

492

generated under these conditions and hence preventing oxidative damage. Other experiments

493

will be needed to investigate the regulation of polyphenol synthesis in response to salinity

494

alone or in combination with K+ deficiency.

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Author contributions Chokri HAFSI conceived, designed research, conducted experiments,

497

and wrote the manuscript. Hanen FALLEH analyzed the data and corrected the manuscript.

498

Mariem SAADA helped for the analysis of polyphenols and antioxidant activity. Riadh

499

KSOURI and Chedly ABDELLY provided scientific advices and supported the work. All

500

authors read and approved the manuscript.

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Conflicts of interest The authors declare that they have no conflicts of interest.

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Acknowledgements

505

This work was supported by Tunisian Ministry of Higher Education, and Scientific Research

506

(LR15CBBC02). We wish to thank Mrs. Fethia ZRIBI and Khaoula MKADMINI for

507

technical assistance. We thank also Dr. Ahmed DEBEZ for English revision of the

508

manuscript.

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ACCEPTED MANUSCRIPT Table 1. Changes in pigment contents in leaves of S. carnosa grown for 30 days in a nutrient

2

solution containing different concentrations (mM) of K+ and NaCl:. C: Control (6 mM K+ and

3

0 mM NaCl), C+S: Salt treatment (6 mM K+ and 100 mM NaCl), and D+S: interactive

4

treatment between K+ deficiency and salt (60 µM K+ and 100 mM NaCl). Data are the mean

5

of three replicates ± SE. Treatment means followed by the same letters are not significantly

6

different at p ≤ 5% according to the Duncan’s Multiple Range Test.

7 Parameters

C 0.94±0.11a

0.28±0.03b

Chl b (mg g-1 FW)

0.36±0.05a

0.13±0.02b

Chl a+b (mg g-1 FW)

1.30±0.15a

0.41±0.05b

0.22±0.03a

0.07±0.004b

Carotenoids (mg g FW)

10 11

17 18 19 20

0.32±0.03c

0.06±0.004c

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0.09±0.01c

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0.22±0.03c

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C+S

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21 22 23 24

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ACCEPTED MANUSCRIPT Table 2. Changes in chlorophyll fluorescence parameters recorded in leaves of S. carnosa

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grown for 30 days in a nutrient solution containing different concentrations of K+ and NaCl:.

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C: Control (6 mM K+ and 0 mM NaCl), C+S: Salt treatment (6 mM K+ and 100 mM NaCl),

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and D+S: interactive treatment between K+ deficiency and salt (60 µM K+ and 100 mM

29

NaCl). Data are the mean of three replicates ± SE. Treatment means followed by the same

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letters are not significantly different at p ≤ 5% according to the Duncan’s Multiple Range

31

Test.

32 Parameters

C+S

D+S

0.8±0.5a

0.8±0.04a

NPQ

2.8±0.5b

1.9±0.8bc

ΦPSII

0.6±0.03a

0.4±0.1b

0.3±0.06c

ETR

94.4±5.5a

52.4±8.5b

43.4±7.6c

ETR/A

3.8±0.2c

5.2±0.6b

6.8±0.4a

0.8±0.03a 3.7±0.7a

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33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59

C

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ACCEPTED MANUSCRIPT Table 3. HPLC identification of leaf phenolic compounds in methanolic extract of control and

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K+-deficient Sulla carnosa plants. Concentrations are given in milligrams per gram of dry

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weight (mg g-1 DW). Signal was collected at 280 nm. Unk: Unknown compounds, nd: not

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detected, RT: Retention Time.

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C+S nd 9.14 8.83 45.07 23.98 nd 2.50 10.66 nd nd nd 2.42 1.85 1.50 1.69 nd nd nd nd 107.64

D+S nd 14.41 12.42 56.11 17.29 6.55 4.71 10.52 nd 2.03 1.16 nd nd 1.84 nd nd 1.61 3.73 1.31 133.69

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RT (min) Compounds C 2.54 Unk 1 1.46 2.81 Unk2 nd 2.85 Unk 3 7.68 2.99 Unk 4 23.11 3.23 Unk 5 6.63 3.34 Unk 6 3.87 3.56 gallic acid 3.48 3.70 tannic acid 6.46 4.55 3,4-dihydroxybenzoic acid 2.14 17.09 ferrulic acid nd 17.34 naringin nd 17.85 naringenin-7-o-glucoside 2.27 18.07 coumarin nd 18.34 3,4-methoxyphenyl propionic acid 1.80 19.91 genistein 1.58 21.15 Unk 7 1.56 21.37 Unk 8 0.79 22.07 Unk 9 1.42 23.09 5,7 dihydroxyflavone nd Total 64.25

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ACCEPTED MANUSCRIPT Figure captions Figure 1. Changes in dry weights (DW) in S. carnosa grown in a nutrient solution containing different concentrations of K+ and NaCl:. C: Control (6 mM K+ and 0 mM NaCl), C+S: Salt treatment (6 mM K+ and 100 mM NaCl), and D+S: interactive treatment between K+

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deficiency and salt (60 µM K+ and 100 mM NaCl). Data are the mean of three replicates ± SE. Treatment means followed by the same letters are not significantly different at p ≤ 5%

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according to the Duncan’s Multiple Range Test.

Figure 2: Changes in gas exchange parameters (A: net photosynthesis rate, E: transpiration

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rate, gs: stomatal conductance to water vapor, Ci: intercellular CO2 concentration, water use efficiency (WUE), and intrinsic water use efficiency (IWUE) in leaves of S. carnosa grown for 30 days in a nutrient solution containing different concentrations of K+ and NaCl:. C: Control (6 mM K+ and 0 mM NaCl), C+S: Salt treatment (6 mM K+ and 100 mM NaCl), and D+S: interactive treatment between K+ deficiency and salt (60 µM K+ and 100 mM NaCl).

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Data are the mean of three replicates ± SE. Treatment means followed by the same letters are not significantly different at p ≤ 5% according to the Duncan’s Multiple Range Test.

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Figure 3: Changes in total polyphenols (mg GAE g-1 DW), flavonoids (mg CE g-1 DW), and condensed tannins contents (mg CE g-1 DW) in leaves of S. carnosa grown for 30 days in a

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nutrient solution containing different concentrations of K+ and NaCl:. C: Control (6 mM K+ and 0 mM NaCl), C+S: Salt treatment (6 mM K+ and 100 mM NaCl), and D+S: interactive treatment between K+ deficiency and salt (60 µM K+ and 100 mM NaCl). Data are the mean of three replicates ± SE. Treatment means followed by the same letters are not significantly different at p ≤ 5% according to the Duncan’s Multiple Range Test. Figure 4. Changes in total antioxidant activity, DPPH scavenging, and reducing power capacities, chelating ability, and β-carotene bleaching test in leaves of S. carnosa grown for

ACCEPTED MANUSCRIPT 30 days in a nutrient solution containing different concentrations of K+ and NaCl:. C: Control (6 mM K+ and 0 mM NaCl), C+S: Salt treatment (6 mM K+ and 100 mM NaCl), and D+S: interactive treatment between K+ deficiency and salt (60 µM K+ and 100 mM NaCl). Data are the mean of three replicates ± SE. Treatment means followed by the same letters are not

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significantly different at p ≤ 5% according to the Duncan’s Multiple Range Test.

ACCEPTED MANUSCRIPT

2 C

a b

0.8 0.4

a

c

a

c b c

b c

0 Stem

Root

Whole plant

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DW, g plant-1

1.6

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C+S D+S

c

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D+S

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6

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c

2 1 C

C+S

(c)

0.6 b

0.2

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Figure 2

C+S Treatments

2

D+S

C+S

D+S

(e)

c

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D+S

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C

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200

a

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bc

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WUE (A/E)

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Ci (µmol CO2 mol-1)

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IWUE (A/gs)

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Total polyphenols Flavonoids

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DPPH scavenging capacity (IC50 mg ml-1)

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16

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4

12

Chelating ability (EC50 mg ml-1) 0

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β-carotene bleaching test (IC 50 mg ml-1)

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C

a

C+S

a

a

C

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C C+S Treatments

C+S

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C+S

Treatments

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Total antioxidant activity (mg GAE g-1 DW) 0

2,5

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1,5

1

0,5

0

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Figure 4

Reducing power (EC 50 mg ml-1 )

(d)

D+S

a (e)

D+S

ACCEPTED MANUSCRIPT •

The effects of salinity alone or in combination with K+ deficiency were investigated in Sulla carnosa plants.

•

K+ supply influences growth and photosynthetic activity

•

Phenolic composition and their antioxidant capacity were modulated by salinity and

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K+ deficiency.

ACCEPTED MANUSCRIPT Author contributions Chokri HAFSI conceived, designed research, conducted experiments, and wrote the manuscript. Hanen FALLEH analyzed the data and corrected the manuscript. Mariem SAADA helped for the analysis of polyphenols and antioxidant activity. Riadh

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authors read and approved the manuscript.

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KSOURI and Chedly ABDELLY provided scientific advices and supported the work. All