Effects of potassium supply on growth, gas exchange, phenolic composition, and related antioxidant properties in the forage legume Sulla carnosa

Accepted Manuscript Title: Effects of potassium supply on growth, gas exchange, phenolic composition, and related antioxidant properties in the forage legume Sulla carnosa Author: Chokri Hafsi Hanen Falleh Mariem Saada Mokded Rabhi Khaoula Mkadmini Riadh Ksouri Chedly Abdelly Abderrazek Smaoui PII: DOI: Reference:

S0367-2530(16)30061-5 http://dx.doi.org/doi:10.1016/j.flora.2016.04.012 FLORA 50968

To appear in: Received date: Revised date: Accepted date:

9-11-2015 24-4-2016 27-4-2016

Please cite this article as: Hafsi, Chokri, Falleh, Hanen, Saada, Mariem, Rabhi, Mokded, Mkadmini, Khaoula, Ksouri, Riadh, Abdelly, Chedly, Smaoui, Abderrazek, Effects of potassium supply on growth, gas exchange, phenolic composition, and related antioxidant properties in the forage legume Sulla carnosa.Flora http://dx.doi.org/10.1016/j.flora.2016.04.012 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.

Running title:Responses of Sulla carnosa Desf. to potassium deficiency

Effects of potassium supply on growth, gas exchange, phenolic composition, and related antioxidant properties in the forage legume Sulla carnosa

Chokri Hafsi*, Hanen Falleh, Mariem Saada, Mokded Rabhi, Khaoula Mkadmini, Riadh Ksouri, Chedly Abdelly, Abderrazek Smaoui

Laboratory of Extremophile Plants, Biotechnology Center of Borj-Cedria, P. O. Box 901, 2050 Hammam-Lif, Tunisia Tel: (+216) 71 430 855 Fax: (+216) 71 430 934 Corresponding author: Chokri Hafsi*, E-mail: [email protected]

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Highlights 

The effects of K+ deficiency were investigated on Sulla carnosa plants.

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Growth of vegetative organs was decreased by K+ deficiency.

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Photosynthetic gas exchange and pigment contents were affected by low K+ conditions.

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S. carnosa was able to modulate the metabolism of secondary metabolites and their antioxidant activity.

Abstract The effects of potassium (K+) deficiency were investigated in Sulla carnosa plants. Plants were grown hydroponically for one month in K+-sufficient (6 mM K+, Control) and K+deficient (60 µm K+) solutions inside the greenhouse in Biotechnology Center of Borj Cedria, Tunisia. Growth, water status, pigment contents, photosynthetic gas exchange, photosystem II (PSII) photochemistry and leaf principal secondary metabolites (polyphenols, flavonoids, and condensed tannins), and their antioxidant properties (DPPH (1.1-diphenyl-2-picrylhydrazyl) scavenging capacity, ferric reducing power, chelating effect on ferrous ions, and β-carotene bleaching test) were determined. Growth of vegetative organs was decreased by some 50% by K+ deficiency with stems more affected (-68%) than roots (-42%) and leaves (-45%). Water content decreased in the three vegetative organs. Photosynthetic gas exchange and pigment contents were affected by low K+ conditions. In contrast to condensed tannins which remained constant, total polyphenols and flavonoids contents increased under K+ deficiency (by 62.7 and 14.5%, respectively). Furthermore, total antioxidant activity increased by 33.5% compared to control plants. Except for β-carotene bleaching test that increased, DPPH scavenging capacity, ferric reducing antioxidant power, and chelating effect on ferrous ions decreased owing to K+ deficiency. An increased and/or de novo synthesis of individual polyphenols was also observed by RP-HPLC analysis. As a whole, these data suggest that S.

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carnosa was able to modulate the metabolism of secondary metabolites and their antioxidant activity under conditions favouring reactive oxygen species (ROS) production in order to minimize the deleterious effects of these oxygen species. Key words: Chlorophyll fluorescence; K+ deficiency; Leaf pigments; Photosynthesis; Polyphenols.

1. Introduction Potassium (K+) is one of the essential mineral elements and is the second most abundant nutrient in plants comprising between 2 and 10% of the plant dry weight (Leigh and Wyn Jones, 1984). K+ is involved in many physiological and biochemical functions such as photosynthesis, activation of numerous enzymes, protein synthesis, osmoregulation, and the maintenance of cation:anion balance (Marschner, 1995; Zhao et al., 2001). For this reason, it is widely included in fertilization management strategies to increase crop production (Hafsi et al., 2014). K+ deficiency often leads to plant growth restriction as a consequence of decreased photosynthetic activity (Zhao et al., 2001; Wang et al., 2012). K+ influences the photosynthetic process at many levels, mainly through limitation of mesophyll and stomatal conductance to CO2, restriction of photoassimilates transport in the phloem, ATP synthesis, the activation of the enzymes involved in photosynthesis, the balance of the electric charges required for photophosphorylation in chloroplasts, low chlorophyll content, poor chloroplast ultrastructure, and acts as the counter ion to light-induced H+ flux across the thylakoid membranes (Marschner, 1995; Zhao et al., 2001; Gerardeaux et al., 2010). Like for most of other biotic and abiotic environmental stresses, it has been reported in several studies that K+ deficiency induces oxidative damage to proteins, lipids, carbohydrates,

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and DNA catalysed by reactive oxygen species (ROS) like superoxide radical (O 2.-), hydrogen peroxide (H2O2), hydroxyl radical (OH.), and singlet oxygen (1O2) (Cakmak, 2005; Tewari et al., 2007; Hafsi et al., 2011). This situation is due to impairment in (i) stomata regulation, (ii) conversion of light energy into chemical energy, (iii) phloem export of photosynthates from source leaves into sink organs, and (iv) an enhancement in NADPH-dependent O2.- generation (Cakmak, 2005). Oxidative stress occurs when there is a serious imbalance between ROS production and their scavenging by antioxidant defense systems (Ahmad et al., 2010). Plant cells have evolved very efficient defense mechanisms including enzymatic (superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase, monodehydroascorbate reductase, dehydroascorbate reductase, glutathione peroxidase, guaicol peroxidase, and glutathione-S-transferase) and non-enzymatic (ascorbic acid, glutathione, alkaloids, nonprotein amino acids, α-tocopherols, and phenolic compounds) antioxidants which work in concert to protect cells from oxidative damage by scavenging of ROS (Noctor and Foyer, 1998; Apel and Hirt, 2004). Polyphenol production is known to be induced by a wide range of environmental stresses, including salinity (Ksouri et al., 2007), drought (Bettaieb et al., 2011), and mineral deficiencies such as N (Kováčik et al., 2007; Galieni et al., 2015), P (Kandlbinder et al., 2004), Ca (Ruiz et al., 2003), and Fe (Msilini et al., 2013). The beneficial effects of those molecules are related to their antioxidant activity (Heim et al., 2002), including their ability to scavenge free radicals, to donate hydrogen atoms or electrons, or to chelate metal cations (Balasundram et al., 2006). Several studies have been focusing on the physiological responses and primary metabolism of plants to K+ deficiency (Zhao et al., 2001; Gerardeaux et al., 2010), but data relative to the secondary metabolism of plants under K+-deficient conditions are scarce. For this reason, the present study was carried out to assess the effect of K+ deficiency on growth, photosynthesis, production of phenolic compounds and the corresponding antioxidant capacity in the forage

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legume Sulla carnosa Desf. The impact of K+ availability on physiological parameters, biosynthesis of polyphenols and related antioxidant properties of S. carnosa has not been previously determined. Sulla carnosa constitues an important genetic resource contributing to pastoral production particularly in semi-arid regions because of its drought tolerance and enrichment of soil due to its nitrogen fixing capacity (Trifi-Farah et al., 2002).

2. Materials and methods 2.1. Plant material and culture conditions Seeds of Sulla carnosa Desf. were collected from Kalbia Sebkha (a saline region in the center of Tunisia, semi-arid bioclimatic climate). Seeds were disinfected for 3 min with NaClO (1%), abundantly rinsed in distilled water and germinated in darkness at 20 °C in Petri dishes on filter paper moistened with distilled water. Four days after sowing, seedlings were transferred into plastic pots (twenty plants per pot) and irrigated with 5 L of modified Hewitt nutrient solution (Hewitt, 1966). The nutrient solution contained the following macronutrients (mM): 1.5 MgSO4 × 7H2O, 3.5 Ca(NO3)2 × 4H2O, 5.4 NaNO3, 2 NH4H2PO4, and 6 KCl. The micronutrients (µM) were: MnCl2 × 4H2O (0.5), CuSO4 × 5H2O (0.04), ZnSO4 × 7H2O (0.05), H3BO3 (0.5), Mo7O24(NH4)6(4H2O) (0.02) (Arnon and Hoagland, 1940) and Fe (III) as Na2-Fe-EDTA complex. Nutrient solutions were continuously aerated and renewed weekly to prevent nutrient depletion. After a pretreatment period of 28 days, plants were divided into two lots (three replicates for each lot): one lot was cultivated on the standard nutrient solution containing 6 mM K+ (Control), the second one was grown in the same nutrient solution but with low K+ concentration (60 µM K+) (K+-deficient treatment). K+ deficiency was created by replacing KCl by an equivalent amount of NaCl. The pH of the nutrient solution was 6.2. Culture was

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carried out in a growth chamber with day/night temperatures of 25 °C/18 °C, a 16 h photoperiod, a photon flux density of 400 µmol m-2 s-1 and 70–75% relative humidity. At the end of the experiment (30 d of treatment) plants were divided into roots, stems, and leaves and fresh weights (FW) were immediately measured. Samples were then oven-dried for 48 h at 60 oC for dry weight (DW) determination. 2.2. Relative growth rate (RGR) determination The mean relative growth rate (RGR, d-1), i.e., the rate of increase in dry weight per unit of plant dry weight (DW), was calculated according the following equation (Hunt, 1990): RGR = (ln DW2-ln DW1) / (t2-t1), with DW, leaf, stem or root dry weight (g), t = time (d), and the subscripts 1 and 2 = initial and final harvest.

2.3. Pigment determination Chlorophyll and carotenoids were extracted from fresh leaf samples cut into discs in five milliliters of 80% acetone. The extraction took place in the darkness at 4 oC for 72 h. The extract absorbances were measured at 645, 663, and 460 nm. Pigment concentrations (mg g -1 FW) were calculated according to the equations reported by Arnon (1949).

2.4. Gas exchange and chlorophyll fluorescence measurements Net photosynthetic rate (A), stomatal conductance (g s), intracellular CO2 concentration (Ci), and intrinsic water use efficiency (IWUE = A/gs) were determined with a portable photosynthesis system (LCA4) (Bio-Scientific, Great Amwell, Herts, UK) at the end of the treatment period. Measurements were carried out between 10:00 and 13:00 on the youngest fully emerged leaves (n = 5 per treatment). The photosynthetically active radiation during measurement under sunlight conditions was about 1350 ± 249 µmol m-2 s-1. During gas

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exchange measurements, the leaf temperature was 27 ± 2 oC, relative humidity was 65 ± 5%, and the ambient CO2 concentration was 380 µmol mol-1. Data were collected every minute after the plant reached a steady photosynthesis rate. Chlorophyll fluorescence was measured using a modulated chlorophyll fluorimeter (OS1-FL). Leaves previously selected for the measurement of photosynthetic gas exchange were used for fluorescence measurements following the procedure described by Genty et al. (1989). The minimal (F0) and maximal (Fm) Chl a fluorescence were assessed in leaves after 20 min of dark adaptation. The maximum quantum efficiency of PSII photochemistry was calculated as Fv/Fm = (Fm - F0)/Fm. The relative quantum yield of PSII at steady-state was calculated as PSII= (F’m - Fs)/F’m, where Fs and F’m are fluorescence at steady-state and maximum fluorescence in the light, respectively. Non-photochemical quenching of fluorescence (NPQ) was calculated as NPQ = (Fm – F’m)/F’m according to Maxwell and Johnson (2000).

2.5. Water content determination (WC %) Water content was calculated utilizing the following equation according to Kováčik et al. (2014): WC = (100-(DW*100 / FW)) with DW, dry weight and FW, fresh weight.

2.6. Colorimetric quantification of antioxidants 2.6.1. Total phenolic content Colorimetric quantification of total polyphenols was determined as described by Dewanto et al. (2002). An aliquot (0.125 ml) of appropriately diluted sample extract was mixed with 0.5 ml distilled water and 0.125 ml of Folin–Ciocalteu reagent. After 3 min, 1.25 ml of Na2CO3 solution (7%) was added and the final volume was made up to 3 ml with distilled water. The absorbance of the resulting solution was measured at 760 nm, after incubation for 90 min. The

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phenol contents were expressed in terms of milligram gallic acid equivalent per gram of dry weight (mg GAE g-1 DW) using a calibration curve with gallic acid (0–400 µg mL−1).

2.6.2. Total flavonoid content Total flavonoids were measured colorimetrically according to Dewanto et al. (2002). An aliquot of suitable diluted samples was added to 0.075 ml of NaNO 2 and mixed for 6 min, before adding 0.15 ml of a freshly prepared AlCl3 (10%). After 5 min, 0.5 ml of NaOH (1 M) solution was added. The final volume was adjusted to 2.5 ml with distilled water and thoroughly mixed. Absorbance of the mixture was determined at 510 nm. Total flavonoid content was expressed as mg catechin per gram of dry weight (mg CE g-1 DW).

2.6.3. Total condensed tannins assay The analysis of condensed tannins (Proanthocyanidins) was carried out according to the method of Sun et al. (1998). Three milliliters of 4% methanolic vanillin solution and 1.5 ml of concentrated H2SO4 were added to 0.05 ml of suitably diluted sample. The mixture was allowed to stand for 15 min, and the absorbance was measured at 500 nm. The amount of total condensed tannins was expressed as mg (+)-catechin equivalent g-1 DW.

2.7. Assessment of antioxidant activities 2.7.1. Evaluation of total antioxidant capacity Total antioxidant capacity of methanolic extracts was evaluated through the assay of a green phosphate/Mo5+ complex according to the method described by Prieto et al. (1999). An aliquot (0.1 mL) of diluted samples was combined with 1 mL of reagent solution (0.3 N sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). Methanol was used instead of sample for the blank. The tubes were incubated in a boiling water bath for 90

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min. Then, the samples were cooled to room temperature and the absorbance was measured at 695 nm against blank in a UV-Visible spectrophotometer (Anthelie Advanced 2, Secoman). Antioxidant capacity was expressed as mg gallic acid equivalent per gram dry weight (mg GAE g−1 DW).

2.7.2. DPPH radical-scavenging activity The free radical scavenging activity (RSA) of the fraction extracts was measured using the DPPH (1.1-diphenyl-2-picrylhydrazyl) method based on measurement of hydrogen donating or radical-scavenging ability using the stable DPPH method (Hanato et al., 1988). One ml of fraction extracts was mixed with 0.25 ml of methanolic solution of DPPH (0.2 mmol/l) and allowed to react in the dark for 30 min. The absorbance was then measured at 517 nm. The antiradical activity was expressed as IC50 (μg/ml), the extract dose required to cause a 50% inhibition. A lower IC50 value corresponds to a higher antioxidant activity of plant extract. Radical-scavenging activity (RSA) was estimated as RSA% = [(A0 − A1) / A0] × 100, where A0 is the absorbance of the control reaction and A1 is the absorbance of the test extract.

2.7.3. Chelating effect on ferrous ions The ferrous ion chelating activity of leaf S. carnosa extracts was assessed as described by Zhao et al. (2006). Different concentrations of organ extracts were added to 0.05 ml of FeCl2 × 4H2O solution (2 mM) and left for incubation at room temperature for 5 min. Then, the reaction was initiated by adding 0.1 ml of ferrozine (5 mM), and the mixture was adjusted to 3 ml with deionized water, shaken vigorously, and left standing at room temperature for 10 min. Absorbance of the solution was then measured spectrophotometrically at 562 nm. The percentage of inhibition of ferrozine-Fe2+ complex formation was calculated using the following formula:

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Metal chelating effect % = [(A0 − A1) / A0] × 100 where A0 is the absorbance of the control reaction and A1 is the absorbance of the test extract. Results were expressed as EC50: efficient concentration corresponding to 50% ferrous iron chelating.

2.7.4. Determination of reducing power The ability of the extracts to reduce Fe3+ was assayed by the method of Oyaizu (1986). Briefly, 1 ml of fraction extracts was mixed with 2.5 ml of phosphate buffer (0.2 mol/l, pH 6.6) and 2.5 ml of K3Fe(CN)6. After incubation at 50 °C for 25 min, 2.5 ml of trichloroacetic acid (10%) was added and the mixture was centrifuged at 650 × g for 10 min. Finally, 2.5 ml of the upper layer was mixed with 2.5 ml of distilled water and 0.5 ml of aqueous FeCl 3 (0.1%). The absorbance was measured at 700 nm. A higher absorbance indicates a higher reducing power. EC50 value (mg/ml) is the effective concentration giving an absorbance of 0.5 for reducing power and was obtained from the linear regression analysis.

2.7.5. β-Carotene bleaching test (BCBT) A slightly modified method of Koleva et al. (2002) was employed. β-Carotene (2 mg) was dissolved in 20 ml chloroform and to 4 ml of this solution, linoleic acid (40 mg) and Tween 40 (400 mg) were added. Chloroform was evaporated under vacuum at 40 °C and 100 ml of oxygenated pure water was added, then the emulsion was vigorously shaken. An aliquot (150 μl) of the β-carotene:linoleic acid emulsion was distributed in each of the wells of 96-well microtiter plates and fraction solutions of the test samples (10 μl) were added. Three replicates were prepared for each of the samples. The microtiter plates were incubated at 50 °C for 120 min, and the absorbance was measured at 470 nm using a model EAR 400 microtiter reader (Labsystems Multiskan MS). Readings of all samples were performed

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immediately (t = 0 min) and after 120 min of incubation. The antioxidant activity (AA) of the extracts was evaluated in terms of β-carotene bleaching using the following formula: AA% = [(S−C120) / (C0-C120)] × 100 where C0 and C120 are the absorbance values of the control at 0 and 120 min, respectively, and S is the sample absorbance at 120 min. The results were expressed as IC 50 values (µg mL−1).

2.8. Analysis of individual phenolic compounds by RP-HPLC The separation of phenolics was performed with an Agilent 1100 series HPLC system equipped with an online degasser (G 1322A), a quaternary pump (G 1311A), a thermostatic autosampler (G 1313A), a column heater (G 1316A), and a diode array detector (G 1315A). Instrument control and data analysis was carried out using Agilent HPLC Chemstation 10.1 editon through Windows 2000. The separation was carried out on a reverse phase ODS C18 column (5 µm, 250 mm × 4.6 mm, Hypersil) used as a stationary phase at ambient temperature. Methanolic extracts were filtered through a polytetrafluoroethylene (PTFE) membrane (0.45 µm) prior to HPLC analysis. Then, ten µl of each filtrate were injected into the HPLC system. The column was maintained at 30 °C and the flow rate was 1 mL min-1. The mobile phase used was a gradient of solvent A (acetonitrile) and solvent B (2.5% acetic acid). The following linear gradient was applied: 3% A; 0–5 min, 9% A; 5–15 min, 16% A; 15–45 min, 50% A; and finally 45–51 min, 90% A to wash the column before initial condition recovery. Peaks were monitored at 280 nm and the identification was obtained by comparing the retention time and the UV spectra with those of pure standards which were purchased from Sigma (St. Louis, MO, USA).

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2.9. Statistical analysis All data were analyzed by one-way ANOVA test, and means were compared using Duncan’s multiple-range test at 5 % level of significance by means of IBM SPSS 20 for Windows.

3. Results 3.1. Plant growth parameters Excepting root length that was increased by K+ deficiency, shoot height, leaf number, leaf, stem, and root dry weights were all significantly decreased by this constraint (Table 1). Stems were more affected than leaves and roots. The ratio root to shoot (i.e. stem + leaves) dry weight was unaltered. Concerning RGR, a significant decrease was observed in leaves, stems, roots, and whole plants. Furthermore, a significant decrease in water content occurred in all vegetative organs in response to K+ deficiency (Table 1). >> insert Table 1 here 3.2. Pigment concentrations The concentrations of total chlorophyll, Chl a, Chl b, and carotenoids (Table 2) were decreased significantly in K+-deficient plants in comparison to the control. Chl b was the most affected pigment by a diminution of 33% as compared to the control. While Chl a/b ratio was increased in K+-deficient leaves, the chlorophyll/carotenoids ratio was diminished compared to the control. >> insert Table 2 here

3.3. Gas exchange K+ deficiency lead to a decrease in net photosynthesis (A), stomatal conductance to water vapor (gs), and intercellular CO2 concentration (Ci) but increased intrinsic water use

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efficiency (IWUE). The most affected parameter was g s, which was diminished by about 48% compared to the control (Fig. 1). >> insert Fig. 1 here

3.4. Chlorophyll fluorescence While no significant changes in the ratio Fv/Fm were recorded in S. carnosa subjected to K+ deficiency, there were significant decreases in non-photochemical quenching (NPQ), the quantum yield of PSII (PSII), and the electron transport rate (ETR) parameters compared to the control (Table 3). >> insert Table 3 here

3.5. Quantification and identification of phenolics 3.5.1. Total polyphenol, flavonoid, and condensed tannin contents Total phenolics extracted from S. carnosa leaves increased by about 63% by K+ deficiency. Total flavonoids increased by 14.5%, whereas contents of condensed tannins were not changed significantly (Table 4). >> insert Table 4 here

3.5.2. Antioxidant activities Global antioxidant activity of leaf extracts as expressed as the number of gallic acid equivalents was increased by 33.5% in response to K+ deficiency in comparison to the control (Fig. 2). DPPH scavenging capacity, iron reducing power, and ferrous ion chelating ability were significantly decreased by 70.0%, 17.7%, and 12.8%, respectively. In contrast, βcarotene bleaching was increased by 33.2%.

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>> insert Fig. 2 here 3.5.3. Phenolics identification by RP-HPLC Phenolic compounds were identified by matching their retention times with those of known standards. The assays performed on extracts from control S. carnosa leaves (Table 5) demonstrated that they contained fourteen phenolics compounds; among them six were identified viz. gallic acid, tannic acid, 3,4- dihydroxybenzoic acid, naringinin -7- o- glucoside, 3,4-methoxyphenyl propionic acid, and genistein while nineteen compounds were detected in leaves of K+-deficient plants with ten identified components viz. gallic acid, tannic acid, 3,4dihydroxybenzoic acid, rosmarinic acid, naringin, naringinin -7-o-glucoside, coumarin, 3,4methoxyphenyl propionic acid, genistein, and 5,7 dihydroxyflavone. >> insert Table 5 here The most abundant phenolic compounds were unfortunately not identified (Unk 2, Unk 3, and Unk 4). Gallic and tannic acids constituted the major components among the identified phenolics. K+ deficiency increased the contents of the majority of these compounds (Table 5). Thus phenolic compounds not found in control plants were identified under K+-deficient conditions.

4. Discussion The present study was conducted to evaluate the effects of K+ deficiency on Sulla carnosa plants. K+ deficiency led to a reduction in S. carnosa biomass production which might be due to combined effect of decreased shoot height, leaf number (Table 1), and leaf surface area (visual observations). Growth inhibition following exposure of plants to K+ deficiency is also reported in other studies (Zhao et al., 2001; Hafsi et al., 2011; Wang et al., 2012). Several investigations demonstrated that K+ deficiency affects the partitioning of dry matter between shoot and roots (Tewari et al., 2004, 2007; Hafsi et al., 2011). The variation of the root/shoot

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DW ratio under K+ deficiency depends on the species and conditions of culture (Andrews et al., 1999). In the present study, K+ deficiency had no significant effect on biomass distribution between shoots and roots (Table 1) which is in agreement with results obtained by Tewari et al. (2004) and Gruber et al. (2013) on maize and Arabidopsis thaliana plants, respectively. A decrease in root/shoot ratio is reported by Cakmak (1994) in K+-deficient plants, whereas an increase in this parameter was observed in mulberry (Tewari et al., 2007) and H. maritimum (Hafsi et al., 2011). Sulla carnosa plants grown on K+-deficient media displayed classical symptoms of deficiency of this macronutrient after three weeks of treatment. In fact, old leaves of those plants were reduced in size and pale yellow in colour, indicating decreased chlorophyll contents, which may represent a survival strategy adopted by plants exposed to K+ deficiency consisting in the mobilization of K+ from mature and senescing organs to make it available for the youngest ones (Hewitt, 1963; Cochrane and Cochrane, 2009). Consistent with previous studies (Zhao et al., 2001; Wang et al., 2012; Chen et al., 2013), we found a significant decrease in the contents of photosynthetic pigments (Table 2). This could be explained either by a decrease of synthesis and/or an increase of degradation of chlorophyll, and by a stimulation of chlorophyllase activity (Santos, 2004). The reduction of pigment contents can be also attributed to K+ deficiency inducing an enhancement of ROS production which can cause damage to the organelle membranes as suggested by Cakmak (2005). In addition, the depressive effects of K+ deficiency on leaf chlorophyll contents might be considered an adaptative mechanism which may lead to decrease the over-reduction of the photosynthetic electron transport and consequently the generation of ROS as suggested by Wang et al. (2003). Furthermore, leaf Chl a/b ratio increased indicating that Chl b was more affected than Chl a, likely owing to the fact that the first step in Chl b degradation involves its conversion to Chl a (Fang et al., 1998). In contrast, in cotton plants subjected to K+ deficiency, Zhao et al.

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(2001) observed no significant difference in the Chl a/b ratio suggesting that contents of both photosynthetic pigments were reduced synchronously. K+ is an important macronutrient required in high concentrations for photosynthetic metabolism owing to multiple functions accomplished by this element (Marschner and Marschner, 2012). In the present work, gas exchange analysis (Fig. 1) indicates that photosynthesis was reduced by K+ deficiency. However, the decrease in CO2 assimilation (reduction by 28%) was not as severe as the decrease in stomatal conductance (reduction by 47%), which resulted in a higher intrinsic water use efficiency (38% increase). Pervez et al. (2004) suggested that K+ plays an important role in stomatal function by maintaining turgor pressure. The reduction in gas exchange parameters (Fig. 1) observed in our work and the corresponding decrease in intercellular CO2 concentration (Fig. 1d) may reflect that K+ deficiency effects on photosynthesis of S. carnosa were mainly due to increased stomatal closure which leads to a substantial reduction of CO2 diffusion to the carboxylation sites. However, the decrease in stomatal conductance can be considered as one of the main responses of plants grown under K+ deficiency to minimize water loss at the expense of CO 2 fixation. The principal way by which PSII reaction centers dissipate excess energy is non-radiative dissipation via non-photochemical quenching (Degl’Innocenti et al., 2009). In the present study, a decrease in NPQ was observed (Table 3) indicating that thermal dissipation of excitation energy was not the main protecting process from over-reduction of the photosynthetic reaction centers. This can be accomplished by an oxygen-dependent alternative electron sink including photorespiratory metabolism, photoreduction of O2 in the water–water cycle, and a chlororespiratory alternative oxidase (Niyogi, 2000). The photosynthetic apparatus and particularly PSII are known to be sensitive to different environmental stress conditions (Yıldıztugay et al., 2011). Chlorophyll fluorescence is a non-

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invasive analysis which is widely used to examine photosynthetic performance in vivo (Baker, 2008). The chlorophyll fluorescence parameter Fv/Fm reflects the maximum quantum efficiency of photosystem II (PSII) photochemistry and has been widely used for early stress detection in plants (Sharma et al., 2015). In the present study, no significant changes in Fv/Fm ratio were recorded (Table 3) indicating optimal functioning of PSII and that photosynthetic efficiency of PSII is well protected under K+-deficient conditions. Compared with Fv/Fm, actual quantum yield of photosynthesis (PSII) was more sensitive to K+ deficiency. The reduction of PSII indicates lower electron transport to carbon fixation, and thus decreases the CO2 assimilation rate (Maxwell and Johnson, 2000). As discussed above, the reduction in photosynthetic activity can lead to an over-reduction of the photosynthetic electron transport chain that enhances the production of ROS and may cause oxidative damage to biomolecules such as lipids, proteins, and nucleic acids as suggested by Kanazawa et al. (2000). In this context, it has been reported in several studies that K+ deficiency induces oxidative damage in plant tissues (Hafsi et al., 2011). To protect themselves against the deleterious effects of ROS, plant cells have developed a complex antioxidant system which includes enzymatic antioxidative enzymes as well as non-enzymatic antioxidants (Noctor and Foyer, 1998; Hafsi et al., 2011). The accumulation of secondary metabolites is known to be a defense mechanism that can help plants to respond and adapt to oxidative stress by altering cellular metabolism to face various challenges (Ksouri et al., 2007). In the present study, except condensed tannins contents which remained constant, K+ deficiency significantly increased total leaf polyphenols and flavonoids contents (Table 4). This suggests that S. carnosa responds to K+ deficiency by activating the biosynthesis of antioxidants as an adaptive mechanism. The increase in polyphenols compounds might be due to increased soluble carbohydrates as suggested by Ibrahim and Jaafar (2011). In this context,

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it is well established that K+ deficiency results in a restriction of sucrose export from the source to the sink organs which leads to a strong accumulation of carbohydrates in the source leaves (Cakmak, 2005; Gerardeaux et al., 2010) and hence there is more carbon to be allocated to secondary metabolites. Furthermore, the enhancement of phenolics accumulation can be due to a stimulation of a phenylalanine ammonia-lyase (PAL, E.C.4.3.1.5) activity, which is the main enzyme in the synthesis pathway of phenolic compounds in plants (Ritter and Schulz, 2004). In contrast, Mudau et al. (2007) and Nguyen et al. (2010) reported that total polyphenols significantly increased with K+ application. Similar results were obtained in some other studies. Bettaieb et al. (2011) observed that in general water deficit increased the level of total and individual polyphenols in Salvia officinalis plants. These results were confirmed in Cakile maritima (Ksouri et al., 2007), and Nigella sativa (Bourgou et al., 2012) subjected to salt stress. On the other hand, some other works have observed quite the opposite effect. Król et al. (2014) found that drought stress causes a decrease in the total polyphenols contents in grapevine leaves and roots. Such large variation in accumulation of polyphenols can be attributed to differences in abiotic stresses (e.g. type of stress, intensity, duration, stages of plant development) and the biological material (e.g. whole seedlings or different organs of plants) as suggested by Weidner et al. (2009). As shown in Figure 2, K+ deficiency increased total antioxidant activity by 34% (Fig. 2a). This is probably a consequence of the enhancement of the total polyphenol accumulation (Table 4). Antioxidant and antiradical activity of leaf extracts of S. carnosa was investigated using several methods such as DPPH. radical scavenging ability, iron reducing power, ferrous ion chelating ability, and β-carotene bleaching test. The above results suggest that, except βcarotene bleaching which was increased by K+ deficiency (Fig. 2e), all other leaf extracts had antioxidative properties. The extracts are more efficient in scavenging DPPH. free radicals which decreased by 70% in comparison to the control (Fig. 2c). In accordance with our

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results, a decrease on DPPH free-radical scavenging capacity and ferric reducing antioxidant power by low K+ application was observed by Nguyen et al. (2010) on basil plants. The identification of phenolic compounds using high-performance liquid chromatography revealed that K+ deficiency provokes a 1.5-fold increase in total polyphenol compounds (Table 5). Unfortunately, the most prevalent phenolic detected peaks could not be identified, and one of the unknown compouds was the most abundant phenolic acid. K+ deficiency induced appreciable qualitative and quantitative modification of the phenolic profiles when compared with the control treatment. In fact, the production of the majority of these components increased by K+ deficiency. Interestingly, K+ deficieny induced the accumulation of five new components, four of them were identified viz., naringin, coumarin, 5,7 dihydroxyflavone, and especially rosmarinic acid. Such a result may be explained by regulation of individual enzymes involved in the biosynthesis of phenolic compounds as a response to oxidative stress. This may explain the enhancement of antioxidant capacity (Fig. 2). Nguyen et al. (2010) demonstrated for basil plants that K+ application rates (1, 2, 4, and 5 mM K+) had a significant effect on rosmarinic and chicoric acid concentrations which are much higher at the highest K+ concentration.

5. Conclusion The present study demonstrated that K+ deficiency led to a reduction of Sulla carnosa growth and photosynthetic gas exchange parameters, and an increase in total polyphenols and flavonoids contents. A modulation of secondary metabolites biosynthesis and an enhancement of antioxidant activity evaluated by four different test systems (scavenging ability on DPPH radical, β-carotene bleaching test, ferrous ion chelating and iron reducing power assays) suggest an adequate protection against oxidative damage which confers a certain tolerance to

19

K+ deficiency to this species. As a result, K+ deficiency appears to be a suitable manipulation for the production of secondary metabolites in S. carnosa grown in hydroponics.

Conflicts of interest The authors declare that there are no conflicts of interest.

Acknowledgments This work was supported by Tunisian Ministry of Higher Education, and Scientific Research (LR15CBBC02). We wish to thank Mrs. Fethia Zribi and Rebey Ben Amar for technical assistance.

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Figures captions Figure 1: Changes in gas exchange parameters (A: net photosynthesis rate, g s: stomatal conductance to water vapor, Ci: intercellular CO2 concentration), and intrinsic water use efficiency (A/g s) in leaves of Sulla carnosa grown for 30 days in a nutrient solution containing high (Control) or low K+ concentration (K+-deficient). Data are the mean of three replicates ± SE. Asterisks represent values that are significantly different between control and K+-deficient treatment at 5% according to the Duncan’s Multiple Range Test.

Figure 2: Changes in total antioxidant, iron reducing power capacity, DPPH scavenging capacity, ferrous ion chelating ability, and β-carotene bleaching test in leaves of Sulla carnosa grown for 30 days in a nutrient solution containing high (Control) or low K+ concentration (K+-deficient). Data are the mean of three replicates ± SE. Asterisks represent values that are significantly different between control and K+-deficient treatment at 5% according to the Duncan’s Multiple Range Test.

28

Figure 1

29

*

Reducing power (EC 50 mg ml-1 )

12 10

8 6

4 2

DPPH scavenging capacity (IC50 mg ml-1 )

(b) *

8 6 4 2 0

0 Control

K+-deficient

2.5

Control

2 1.5 1

*

0.5

K+-deficient

25

(c)

(d)

20

*

15 10

5 0

0 Control

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

10

(a)

Chelating ability (EC50 mg ml-1 )

Total antioxidant activity (mg GAE g-1 DW)

14

K+-deficient

35 *

30 25

Control (e)

K+-deficient

Treatments

20 15 10 5

0 Control

K+-deficient Treatments

Figure 2

30

Table 1. Changes in shoot height, root length, leaf number, dry weight, water content, RGR, and root/shoot DW ratio in Sulla carnosa grown for 30 days in a nutrient solution containing high (Control) or low K+ concentration (K+-deficient). Data are the mean of three replicates ± SE. Asterisks represent values that are significantly different between control and K+deficient treatment at 5% according to the Duncan’s Multiple Range Test.

K+-deficient

Parameters

Control

Change %

Shoot height (cm plant-1)

28.33 ± 1.69

19.42 ± 0.67*

-31

Root length (cm plant-1)

48.67 ± 1.75

60.67 ± 1.95*

+25

Leaf number plant-1

47.83 ± 1.99

21.83 ± 1.19*

-54

Leaf DW (g plant-1)

0.87 ± 0.12

0.48 ± 0.03*

-45

Stem DW (g plant-1)

0.34 ± 0.12

0.11 ± 0.01*

-68

Root DW (g plant-1)

0.26 ± 0.06

0.15 ± 0.02*

-42

Whole plant DW (g plant-1)

1.47 ± 0.25

0.74 ± 0.05*

-50

Leaf WC (%)

91.50 ± 0.45

87.38 ± 0.67*

-4

Stem WC (%)

88.59 ± 0.19

85.26 ± 0.51*

-3

Root WC (%)

93.87 ± 0.31

91.72 ± 0.65*

-2

Leaf RGR (day-1)

0.122 ± 0.005

0.103 ± 0.002*

-16

Stem RGR (day-1)

0.168 ± 0.011

0.133 ± 0.004*

-21

Root RGR (day-1)

0.138 ± 0.008

0.119 ± 0.004*

-14

Whole plant RGR (day-1)

0.131 ± 0.006

0.109 ± 0.002*

-17

Root/Shoot DW ratio

0.22 ± 0.05

0.24 ± 0.03

+9

31

Table 2. Changes in pigment contents in leaves of Sulla carnosa grown for 30 days in a nutrient solution containing high (Control) or low K+ concentration (K+-deficient). Data are the mean of three replicates ± SE. Asterisks represent values that are significantly different between control and K+-deficient treatment at 5% according to the Duncan’s Multiple Range Test.

Parameters

Control

K+-deficient

Change %

Chl a (mg g-1 FW)

0.94 ± 0.11

0.69 ± 0.10*

-27

Chl b (mg g-1 FW)

0.36 ± 0.05

0.24 ± 0.03*

-33

Chl a+b (mg g-1 FW)

1.30 ± 0.15

0.93 ± 0.12*

-28

Chl a/b

2.66 ± 0.21

2.89 ± 0.20*

+9

Carotenoids (mg g-1 FW)

0.22 ± 0.03

0.18 ± 0.02*

-18

Chl/Carotenoids

5.86 ± 0.39

5.23 ± 0.34*

-11

32

Table 3. Changes in chlorophyll fluorescence parameters recorded in leaves of Sulla carnosa grown for 30 days in a nutrient solution containing high (Control) or low K+ concentration (K+-deficient). Data are the mean of three replicates ± SE. Asterisks represent values that are significantly different between control and K+-deficient treatment at 5% according to the Duncan’s Multiple Range Test.

K+-deficient

Change %

0.76 ± 0.52

0.77 ± 0.07

-

NPQ

2.85 ± 0.52

1.63 ± 0.45*

-42.8

PSII

0.59 ± 0.03

0.36 ± 0.10*

-39.0

ETR

94.40 ± 5.57

57.12 ± 15.53*

-39.5

Parameters

Control

Fv/Fm

33

Table 4. Changes in total polyphenols, flavonoids, and condensed tannins contents in leaves of Sulla carnosa grown for 30 days in a nutrient solution containing high (Control) or low K + concentration (K+-deficient). Data are the mean of three replicates ± SE. Asterisks represent values that are significantly different between control and K+-deficient treatment at 5% according to the Duncan’s Multiple Range Test. Parameters

Control

K+-deficient

Change %

Total polyphenols (mg

2.20 ± 0.26

3.58 ± 0.18*

+62.7

0.62 ± 0.01

0.71 ± 0.01*

+14.5

0.63 ± 0.06

0.58 ± 0.04

-7.9

GAE g-1 DW) Flavonoids (mg CE g-1 DW) Condensed tannins (mg CE g-1 DW)

34

Table 5. HPLC identification of leaf phenolic compounds in methanolic extract of control and K+-deficient Sulla carnosa plants. Concentrations are given in milligrams per gram of dry weight (mg g-1 DW). Signal was collected at 280 nm. Unk: Unknown compounds, nd: not detected, RT: Retention Time (min).

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

RT Compounds Control 2.54 Unk 1 1.46 2.85 Unk 2 7.68 2.99 Unk 3 23.11 3.23 Unk 4 6.63 3.34 Unk 5 3.87 3.56 gallic acid 3.48 3.70 tannic acid 6.46 4.55 3,4-dihydroxybenzoic acid 2.14 7.12 Unk 6 nd 13.42 rosmarinic 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 20.02 Unk 7 nd 21.15 Unk 8 1.56 21.37 Unk 9 0.79 22.07 Unk 10 1.42 23.09 5,7 dihydroxyflavone nd Total 64.26

K+-deficient 2.15 10.67 32.95 10.69 nd 7.98 6.89 2.71 6.40 4.56 0.67 0.52 0.69 1.23 1.56 0.95 2.37 2.02 2.06 0.45 97.52

35