Effect of High Salinity on Atriplex Portulacoides: Growth, Leaf Water Relations and Solute Accumulation in Relation with Osmotic Adjustment

South African Journal of Botany 95 (2014) 70–77

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South African Journal of Botany

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Effect of high salinity on Atriplex portulacoides: Growth, leaf water relations and solute accumulation in relation with osmotic adjustment

Maali Benzarti a,1, Kilani Ben Rejeb a,b,1, Dorsaf Messedi a, Amira Ben Mna c, Kamel Hessini a, Mustapha Ksontini c, Chedly Abdelly a, Ahmed Debez a,⁎ a Laboratoire des Plantes Extrêmophiles, Centre de Biotechnologie de Borj-Cedria (CBBC), BP 901, Hammam-Lif 2050, Tunisia b Adaptation des plantes aux contraintes environnementales, UR5, Université Pierre et Marie Curie (UPMC), Case 156, 4 Place Jussieu, 75252 Paris cedex 05, France c Unité d'agrosylvopastoralisme, Institut National de Recherches en génie Rural, Eau et Forêts (INRGREF), Ariana 2080, Tunisia article info abstract

Article history: Atriplex (Halimione) portulacoides is a halophyte with potential interest for saline soil reclamation and Received 10 December 2013 phytoremediation. Here, we assess the impact of salinity reaching up to two-fold seawater concentration Received in revised form 20 August 2014 (0–1000 mM NaCl) on the plant growth, leaf water status and ion uptake and we evaluate the contribution Accepted 24 August 2014 of inorganic and organic solutes to the osmotic adjustment process. A. portulacoides growth was optimal at Available online xxxx 200 mM NaCl but higher salinities (especially 800 and 1000 mM NaCl) significantly reduced plant growth. + − Edited by JM Farrant Na and Cl contents increased upon salt exposure especially in the leaves compared to the roots. Interestingly, no salt-induced toxicity symptoms were observed and leaf water content was maintained even at the highest sa- fi Keywords: linity level. Furthermore, leaf succulence and high instantaneous water use ef ciency (WUEi) under high salinity Halophyte significantly contributed to maintain leaf water status of this species. Leaf pressure–volume curves showed that 100 Salinity salt-challenged plants adjusted osmotically by lowering osmotic potential at full turgor (Ψπ )alongwithade- Pressure–volume curves crease in leaf cell elasticity (values of volumetric modulus elasticity (ε) increased). As a whole, our findings indi- Water relations cate that A. portulacoides is characterized by a high plasticity in terms of salt-response. Preserving leaf hydration Osmotic adjustment and efficiently using Na+ for the osmotic adjustment especially at high salinities (800–1000 mM NaCl), likely through its compartmentalization in leaf vacuoles, are key determinants of such a performance. The selective absorption of K+ over Na+ in concomitance with an increase in the K+ use efficiency also accounted for the overall plant salt tolerance. © 2014 SAAB. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

1. Introduction terrastat). In general salinity can reduce plant growth or damage the plant through: (i) osmotic effect (caused by low water potential + − Salinity is among the most adverse environmental issues for agricul- (Ψw)), (ii) toxic effect (mainly due to Na and Cl ) and (iii) alteration ture, since it affects about 5% of the cultivated areas throughout the of the nutritional balance. To survive with the detrimental effects of salt world. The United Nations Food and Agriculture Organization estimates stress, plants have evolved various combating mechanisms. Among that there are currently 4 million km2 of salinized land and a similar these, ion exclusion, along with other strategies such as adjustment of + area that is affected by sodicity, a condition in which Na ions represent the cell osmotic potential (Ψπ) is of special importance (Türkan and more than 15% of the exchangeable cations (www.fao.org/agl/agl1/ Demiral, 2009). The latter response termed as osmotic adjustment (OA) involves transport, active accumulation, and compartmentaliza-

Abbreviations: A, net CO2 assimilation rate; AWC, apoplastic water content; DW, dry tion of inorganic ions and organic compounds (Flowers and Colmer, weight; FAA, free amino acids; FW, fresh weight; GB, glycine betaine; KAE, potassium ab- 2008). During osmotic adjustment cells tend to compartmentalize fi fi – sorption ef ciency; KUE, potassium use ef ciency; OA, osmotic adjustment; P V curves, most of the absorbed ions in vacuoles at the same time as they synthe- pressure–volume curves; RWC, relative water content; RWC0, relative water content at + + size and accumulate compatible organic solutes in the cytoplasm, in theturgor loss point; SK/Na, selectivity of K over Na ;T,transpirationrate;TSS,totalsoluble sugar;WC,watercontent; WUEi, instantaneouswater use efficiency; ε, volumetric modulus order to maintain the osmotic equilibrium between these two compart- Ψ Ψ Ψ elasticity; s, osmotic potential of each measured solute; w, water potential; π,osmotic ments (Parida and Das, 2005). Clearly, tolerance in the form of OA plays Ψ0 Ψ100 potential; π, osmotic potential at the turgor loss point; π , osmotic potential at full an important role in salt-tolerant plants thriving in saline environments turgor. (Flowers and Colmer, 2008). ⁎ Corresponding author. Tel.: +216 79 325 848; fax: +216 79 325 638. fi E-mail address: [email protected] (A. Debez). Halophytes are de ned as plants that live in naturally saline habitats 1 Both authors contributed equally. or that complete their life cycle at a salt concentration of at least

http://dx.doi.org/10.1016/j.sajb.2014.08.009 0254-6299/© 2014 SAAB. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/). M. Benzarti et al. / South African Journal of Botany 95 (2014) 70–77 71

200 mM NaCl (Flowers et al., 2010). Facing the increasing salinization with 0.5% (w/v) toluidine blue, were examined using a Zeiss light mi- throughout the world, domestication of these plants as cash crop spe- croscope. Axio software was used to measured leaf cross-section cies, better known as biosaline agriculture, is a currently emerging ap- thickness. proach (Debez et al., 2011). Better understanding of the physiological and molecular mechanisms enabling halophytes to survive and main- 2.3. Water content, succulence and instantaneous water use efﬁciency tain productivity in saline environments is also a critical issue for re- (WUEi) searchers. A large group of species of Atriplex genus belonging to halophytes having ecological and agronomic importance are frequent Leaf water content was calculated as the difference between fresh in many arid and semi-arid regions of the world, particularly in habitats weight (FW) and dry weight (DW) and is expressed on a dry mass that combine relatively high soil salinity with aridity (Benzarti et al., basis WC = (FW − DW) / DW. Leaf succulence was estimated by divid-

2013) and therefore constitute a useful material for the identification ing leaf water content by leaf surface area (Debez et al., 2004). WUEi of physiological mechanisms involved in salt stress resistance. Atriplex was calculated as the ratio: net CO2 assimilation rate (A) / transpiration (Halimione) portulacoides (Sea purslane) is a widespread C3-type peren- rate (T). Total leaf surface area was measured by a leaf area meter (por- nial halophyte common on salt marshes of Europe, North Africa and table area meter LI/3000A, LI-COR). The gas exchange measurements South-West Asia. Its crunchy and salt tasting leaves are edible as a nat- were made with a portable photosynthesis system (LCA4). ural condiment (Wright, 2009). Interestingly, recent data indicate that this plant could be valorized as a source of valuable phytochemicals, in- 2.4. Inorganic cation contents cluding phenolic compounds (Rodrigues et al., 2014; Vilela et al., 2014). High productivity and capacity to grow in heavy metal contaminated Dried samples of roots, stems and leaves were finely ground. Ion ex- + + soils (Sousa et al., 2008) also make this species potentially useful for sa- traction was achieved in 0.5% HNO3.Na and K were assayed by flame line soil reclamation and phytoremediation purposes and even as fod- emission photometry (Corning, UK) and Cl− by coulometry (Büchler der plants. Recently, we reported that A. portulacoides displayed an chloridometer), while Ca2+ was determined using atomic absorption effective antioxidant defense protecting the photosynthetic machinery spectrometry (VARIAN, Spectra AA 220 FS). + + from salt-induced photodamage under extreme (up to 1000 mM The selectivity of K over Na (SK/Na) was estimated from ion con- NaCl) salinity (Benzarti et al., 2012). Yet, the involvement of the osmotic tents as (Debez et al., 2004): adjustment in the salt stress tolerance of this species especially when hihi ¼ þ= þ þ þ = þ= þ þ þ : challenged with extreme salinity is still unknown. It has to be pointed SK=Na K K Na K K Na leaves medium out that in soils in Mediterranean areas like Tunisia, salinity level may dramatically rise during the long summer season as a result of both The potassium absorption efficiency (KAE) and potassium use effi- high evaporation rate and reduced water availability, resulting in salin- ciency (KUE) were calculated as: ity levels that my exceed seawater salinity. Therefore, we evaluated here the responses of Tunisian population of A. portulacoides when ex- þ KAE μmol K =mg DW posed up to twofold seawater NaCl salinity (1000 mM) in terms of roots ¼ þ = growth, water relations and organic and inorganic solute accumulation Total plant K amount average root DW in relation to osmotic adjustment. where the average root DW is the logarithmic average of the root dry 2. Material and methods weight, =μ þ ¼ = þ : 2.1. Plant material and culture conditions KUE mg DW mol K Whole plant DW total K amount

Young plants of A. portulacoides were obtained using cuttings col- lected from mother plants. Uniformly rooted plantlets were trans- 2.5. Organic solute content ferred to pots containing inert sand. They were irrigated for 40 days with a complete nutrient solution (Hewitt, 1960)added Total soluble sugar (TSS) and free amino acids (FAA) were extracted with different salinities (0, 200, 400, 800, and 1000 mM NaCl). To re- by boiling 80% ethanol with 100 mg of leaf fresh tissue. The ethanol frac- duce osmotic shock on plants, salt treatments were daily increased tion was evaporated under a vacuum to dryness and soluble compounds by 50 mM NaCl up to 200 mM NaCl. For the subsequent treatments, were redissolved with 4 mL of distilled water. the daily increment was 200 mM NaCl. After reaching the final con- Total FAA was determined as described by Chen et al. (2007). One centration (1000 mM NaCl), pots were irrigated every 2 days with milliliter of 0.1 M sodium acetate acetic acid (pH = 4.3) and 1 mL of nin- a volume of 250 mL per pot. The culture was carried out under green- hydrin (5% ninhydrin in ethanol) were added to 1 mL of the sample. The house conditions (400 μmol m−2 s−1 photosynthetic active radia- samples were vortexed, then immersed in a hot water bath (95 °C) for tion (PAR), 25 ± 5 °C temperature, and 60 ± 10% relative humidity). 15 min, and finally cooled to room temperature. Samples were measured at 570 nm using a spectrophotometer. A calibration curve with 2.2. Plant growth and leaf anatomy glycine was used as a standard. TSS was determined by the classical anthrone method using a spectrophotometer. A standard curve was At harvest, leaf, stem and roots' fresh weight (FW) was immediately established using glucose. estimated and dry weight (DW) was determined after their drying at Free proline content was measured according to the Bates et al. 60 °C until constant weight. (1973) method. 50 mg of leaf sample was grounded, homogenized in For anatomical studies, at the end of the experiment samples were 1.5 mL 3% sulfosalicylic acid and centrifuged at 14,000 ×g for 10 min cut from the middle section of the fully matured leaf at the second at 4 °C. To the 1 mL extract, 1 mL acid-ninhydrin and 1 mL of glacial node of control and 200–800 mM NaCl treated plants and were subse- acetic acid were added and the reaction mixture incubated at 100 °C quently fixed in a solution containing 1.25% glutaraldehyde and 1.25% for 1 h. The reaction mixture was stopped by placing the tubes on ice. paraformaldehyde for 1 h. They were then post-fixed for 45 min in 1% The red color that has developed was extracted with 2 mL toluene. osmium tetroxide and embedded in Eponaraldite resin after dehydra- Upper phase was taken out to read the absorbance at 520 nm. The pro- tion in an ethanol series. Semi-thin sections (0.7 μm), cut from plastic line content was calculated by using a standard curve drawn with the embedded tissue on a Reichert ultracut-E ultramicrotome and stained known concentrations of proline. 72 M. Benzarti et al. / South African Journal of Botany 95 (2014) 70–77

GB estimation was done according to Grieve and Grattan (1983).The 3. Results absorbance was measured at 365 nm with spectrophotometer. A calibration curve with GB was used as a standard. 3.1. Plant growth and leaf hydration

2.6. Pressure–volume (P–V) curves Plant exposure to 200 mM NaCl resulted in a 30% increase of the whole plant dry weight as compared to plants grown in salt-free medi- P–V curves were determined using the Scholander pressure cham- um (control treatment), whereas this parameter was significantly ber technique (Scholander et al., 1965). The P–V curves of each leaf inhibited at 800 and 1000 mM NaCl (Fig. 1). The anatomical study were obtained by expressing the relationship between relative water showed that high salt stress (800 mM NaCl) resulted in a significant in- content (RWC) values and the reciprocals of the measured water poten- crease of the leaf thickness and of almost all histological components, as 100 tials (−1/Ψw). Osmotic potential at full turgor (Ψπ ) was estimated well as of the average area of the spongy mesophyll cells (Table 1, via linear regression of data in the straight-line region of the P–V Fig. 2). No significant impact of salinity was observed on leaf water con- curve (Mguis et al., 2012). Osmotic potential at the turgor loss point tent (Fig. 3A). Yet succulence and WUEi (Fig. 3B and C, respectively) in- 0 (Ψπ)wasderivedfromtheRWCand−1/Ψw coordinates respectively creased significantly in response to salt application, especially at 800 of the first point in the straight-line region of the P–V curves (Patakas and 1000 mM NaCl. and Noitsakis, 1999). The osmotic adjustment (OA) was defined as the 100 difference in Ψπ between stressed and control plants: 3.2. Inorganic cation accumulation

+ 100 100 Leaf Na contents increased signiﬁcantly with increasing NaCl con- OA ¼ Ψπ − Ψπ : stressed control centrations, the highest value being registered in plants submitted to 1000 mM NaCl (Fig. 4A). Stem and root Na+ contents showed the same tendency up to 200 mM NaCl, but remained relatively stable at The apoplastic water content (AWC) or intercellular water is esti- the higher salt levels. At 1000 mM NaCl, leaf Na+ content was about 2 mated from the extension of the linear part to the axis (OX). The relative and 3.5 fold higher than that of stems and roots, respectively. Cl− con- water content at the turgor loss point (RWC ) was obtained from the 0 tent in roots and leaves was characterized by a similar accumulation extension of the intersection of curve and linear portions. The volumet- pattern to that observed for Na+ but the values were much lower for ric modulus elasticity (ε) was calculated as the slope of the relationship Cl− (Fig. 4B). between turgor pressure and RWC: High accumulation of Na+ and Cl− in leaves was concomitant with a + signiﬁcant decrease in K content of these organs (about 60% reduction ε ¼ Ψ100−Ψ0 ðÞ− =ðÞ− : at 1000 mM NaCl as compared to the control) (Fig. 4C). Salt-induced max π π 1 AWC 1 RWC0 changes in Ca2+ content were similar since values in 1000 mM NaCl- treated plants were about 33% of the control values (Fig. 4D). To assess the ability of A. portulacoides to achieve selective uptake of K+ in the + 2.7. Contribution of solutes to osmotic adjustment presence of Na excess, the selectivity ratio SK/Na was calculated from leaf ion contents and ion concentrations in the medium. Results indicat- + + Concentrations of organic solutes as well as inorganic ions were cal- ed a marked selectivity for K over Na with SK/Na values increasing in culated for the symplastic water volume at full turgor, according to the leaves with increasing NaCl concentration in the culture medium different fractions in control and stressed leaves estimated by pressure– (Table 2). volume technique (Patakas et al., 2002). These concentrations were used to estimate the contribution of each solute to osmotic adjustment, 3.3. Organic solute accumulation assuming that 40 μmol·g−1 of symplastic water corresponds to 0.1 MPa (Hessini et al., 2008). The contribution of each solute (s)tothetotalos- A marked increase in proline concentration was observed in leaves motic adjustment (OAt) was calculated using the formula: of plants challenged with 400 mM or higher NaCl concentrations, values reaching up to 6 μmol g FW−1 at 1000 mM NaCl, which represents a 25- ½ −½ : fold increase as compared with the non-treated plants (Fig. 5A). Leaf s stressed s control 0 1 100 OAðÞin % ¼ =OA : FAA content was constant up to 400 mM NaCl before decreasing at s 40 t higher salinity levels (Fig. 5B). The leaf TSS content showed a transient increase (2-fold) at 200 mM NaCl before decreasing to values close to Ψ The estimated osmotic contribution of each measured solute s to 5 100 the leaf osmotic potential at full turgor Ψπ was obtained using the Leaves van't Hoff equation according to Meloni et al. (2001). c 4 Stems

-1 b 2.8. Statistical analysis Roots 3 b All statistical analyses were done using the SPSS software (SPSS 16.0 d 2 for Windows, IBM Company, Inc., Chicago, USA). The parameters ana- c g DW plant lyzed in the experiment were tested for assumptions of normality and b a a homogeneity of variance with the Kolmogorov–Smirnov and Levene's 1 c a c b a tests, respectively, and indicated a normal distribution and variance a a that were homogeneous in all cases. To evaluate whether the plant 0 0 200 400 800 1000 physiological parameters analyzed were affected by the treatments, a NaCl (mM) one way analysis of variance (ANOVA) at P b 0.05 significance level, followed by Duncan post-hoc test for mean comparisons was applied Fig. 1. Effect of NaCl on plant growth of different organs (leaves, stems and roots) of fi with salinity treatment as xed factors and the parameters analyzed Atriplex portulacoides exposed to different NaCl concentrations for 40 days. Means (n = as dependant variables. 4 ± SE) with different letters are significantly different at P b 0.05. M. Benzarti et al. / South African Journal of Botany 95 (2014) 70–77 73

12 Table 1 b Anatomical changes in cross-sections of fully expanded leaves of Atriplex portulacoides ex- A posed for 40 days to 0, 200 and 800 mM NaCl. Means (n = 12 ± SE) with different letters 10 were significantly different at P b 0.05. DW) -1 a a a 8 NaCl (mM) a 0 200 800 6 Adaxial palisade thickness (μm) 50.8 ± 2.1a 60.3 ± 3.5a 74.4 ± 3.9b μ a a b Abaxial palisade thickness ( m) 55.1 ± 3.9 53.2 ± 3.3 72.8 ± 1.2 4 Spongy mesophyll layer thickness (μm) 199 ± 2.8a 188 ± 15a 231 ± 15b Spongy mesophyll cell area (mm2) 2.10 ± 0.3a 3.61 ± 0.5a 6.4 ± 0.6b Total thickness (μm) 304 ± 4.2a 312 ± 12a 391 ± 5.6b 2 Water content (mL g 0 that of the control under the other treatments (Fig. 5C). Leaf glycine be- 0,1 taine (GB) content also increased significantly in response to salt stress, especially at 800 and 1000 mM NaCl (Fig. 5D). ) B c -2 0,08 b 3.4. P–Vcurveanalysis 0,06 100 Both osmotic potentials at full turgor Ψπ and at turgor loss point 0 Ψπ decreased in stressed plants (Table 3). Yet, the percentage of a a 0,04 a apoplastic water content (AWC) and RWC0 increased significantly as compared with control plants, especially at the highest salinities. The values of bulk modulus of elasticity increased with increasing salinity. 0,02 100 OA estimated as the difference between the Ψπ in the control and sa- Succulence ratio (mL cm linized plants was 1.6 and 2.99 MPa for 200 and 1000 mM NaCl treat- 0 ments respectively. 6 O)

2 C 4. Discussion b b H 5 -1 a a a The genus Atriplex contains many species which are able to complete 4

their life cycles under extreme environmental conditions such as mmol drought, high temperature and high salinity (Silveira et al., 2009). In 2 3 the present study A. portulacoides growth was significantly stimulated at 200 mM NaCl, but were reduced at higher salinities (800 and 2 1000 mM NaCl). Yet it is noteworthy that even at these salt concentrations, plants remain alive and did not exhibit any symptom of toxicity 1 such as chlorosis and leaf necrosis. Salt-induced growth stimulation under moderate salinities has been previously documented for WUEi (µmol CO 0 0 200 400 800 1000 A. portulacoides (Redondo-Gómez et al., 2007; Benzarti et al., 2012) and many other salt-requiring halophytic species such as Cakile NaCl (mM) maritima and Batis maritima (Debez et al., 2008, 2010). In A. portulacoides, the stimulation of the plant growth at mild salinities Fig. 3. Water content (A), succulence (B) and instantaneous water use efficiency (C) in was correlated with similar changes in the photosynthetic gas exchange leaves of Atriplex portulacoides exposed to different NaCl concentrations for 40 days. Means (n = 4 ± SE) with different letters are significantly different at P b 0.05. rate and the carboxylation capacity, whereas high salinity showed to hardly affect the photochemical (PSII) apparatus as well as the carboxylation capacity (Redondo-Gómez et al., 2007). The greatest impact of toxicity of this ion. For example, Atriplex amnicola growninahighlysa- salinity on photosynthesis appears to be via the regulation of stomatal line soil was reported to tolerate over 900 mM Na+ in mesophyll tissue conductance and its consequences for intercellular leaf CO2 concentra- without drying (Aslam et al., 1986). X-ray microanalysis of Salicornia tion. Some halophytes accumulate high amounts of sodium (up to 50% europea cells growing in 200 mM NaCl demonstrated that Na+ was of dry weight) in their above ground tissues despite the potential compartmentalized predominantly into the cell vacuoles of shoots

Fig. 2. Semi-thin cross sections of Atriplex portulacoides leaves grown at 0 (A), 200 (B) and 800 mM NaCl (C). Each section shows, from the top to the bottom: adaxial palisade parenchyme, spongy mesophyll cells and abaxial palisade parenchyme. Scale bar: 200 μm. 74 M. Benzarti et al. / South African Journal of Botany 95 (2014) 70–77

7 6 Leaves AB 6 5 Stems 5

Roots 4 DW) DW) -1 -1 4 3 3 (mmol g (mmol g 2 - 2 + Cl

Na 1 1

0 0 1,2 0,5 CD 1 0,4 DW) DW) 0,8 -1 1 - 0,3 0,6

0,2 (mmol g (mmol g 0,4 2+ + Ca K 0,2 0,1

0 0 0 200 400 800 1000 0 200 400 800 1000 NaCl (mM) NaCl (mM)

Fig. 4. Na+ (A), Cl− (B), K+ (C) and Ca2+ (D) content in different organs (leaves, stems and roots) of Atriplex portulacoides exposed to different NaCl concentrations for 40 days. Means of four replicates ± SE. endodermis tissues (Lv et al., 2012). In our study, salt uptake by control compartmentalization. The protection of cytosolic structures and bio- plants was found to be surprisingly considerable, a phenomenon rather molecules in some Atriplex species is also ensured by sequestering saline common in the Atriplex species as documented by several authors ions into vacuoles of specialized cells, termed trichomes and vesicles, (Debez et al., 2003; Araújo et al., 2006; Silveira et al., 2009). However, which are located in the leaf epidermis (Smaoui et al., 2010). These spe- it is noteworthy that despite the large accumulation of salt ions in cialized cells are believed to be very efficient to exclude toxic ions from leaves, neither leaf dehydration nor leaf injury symptoms occurred, cytosol as well as contribute to the overall osmotic adjustment of leaf which provides an indirect evidence for the effective vacuolar compart- cells (Silveira et al., 2009). mentalization of Na+ and its use for the osmotic adjustment needs. This The restriction of A. portulacoides growth observed at high salinities hypothesis is further strengthened by the fact that both leaf succulence could result from salt-induced nutritional imbalances. The increased and leaf thickness were significantly higher in salt-treated plants. Debez Na+ accumulation in A. portulacoides leaves was associated with a sig- et al. (2004) showed that succulence (to dilute accumulated Na+)isone nificant reduction in K+ uptake which was however offset by an in- of the mechanisms evolved by C. maritima to deal with the high internal crease in the K+ and use efficiency (i.e. the quantity of biomass ion concentrations. Increases in leaf thickness and succulence may be produced per unit of absorbed ion) (Table 2). Hence, A. portulacoides is achieved by an increase in size of the mesophyll cells and the relative able to use economically K+ for specific functions when challenged size of their vacuoles or an increase in the number of spongy cell layers with salinity, and K+ could be replaced by Na+ for osmotic adjustment (Shabala and Mackay, 2011). by accumulating it in the vacuoles. In addition, A. portulacoides showed + + Based on the anatomical study, our findings suggest that the increase to be strongly selective for K over Na since the selectivity ratio SK/Na, in the spongy mesophyll cell size rather than the increase in cell number calculated from leaf ion contents and ion concentrations in the medium accounted for the higher leaf thickness observed at high salinity remained significantly higher in A. portulacoides leaves than in the cul- (800 mM NaCl). According to Katschnig et al. (2013), higher succulence ture medium, even at the high external NaCl concentration (1000 mM and thickening of the leaf tissues in response to salinity are likely direct NaCl). This means that efficient transport systems selective for K+ are responses in order to increase storage area needed for salt also at work in cells of A. portulacoides to maintain mineral nutrition and also to protect and preserve the metabolic activity under saline conditions. Table 2 + + OA is considered to be an important component of salt tolerance Selectivity of K over Na (SK/Na), potassium absorption efficiency (KAE) and potassium use efficiency (KUE) in Atriplex portulacoides exposed to different NaCl levels for 40 days. mechanisms in plants; it is usually defined as a decrease in the cell sap Means (n = 4 ± SE) with different letters were significantly different at P b 0.05. osmotic potential resulting from a net increase in intracellular osmolytes to prevent the loss of cell water. The intracellular osmolytes include in- NaCl (mM) SK/Na KAE KUE + −1 −1 + + − + (μmol K mg DWroots) (mg DW μmol K ) organic ions (mainly absorbed from medium such as Na ,Cl ,K , 2+ 0 0.36 ± 0.01a 7.8 ± 1.1c 0.98 ± 1.8a Ca ) and/or compatible organic solutes (mainly include soluble 200 5.55 ± 0.4b 5.1 ± 0.6b 1.81 ± 0.1b sugar, free amino acids and proline) (Zhou and Yu, 2009). The occur- 400 8.13 ± 0.89c 3.2 ± 0.4a 2.10 ± 0.06c rence of the osmotic adjustment mechanism in A. portulacoides leaves e a c 800 13.6 ± 1.63 2.9 ± 0.6 1.90 ± 0.1b in this study is also reinforced by the analysis of P–V curve data which 1000 11.92 ± 0.4d 2.6 ± 0.5a 2.10 ± 0.1c showed that the plant growing in presence of NaCl adjusted osmotically M. Benzarti et al. / South African Journal of Botany 95 (2014) 70–77 75

7 20 ABe b

6 FW) b b

16 -1 d FW) 5 -1 4 12

3 8 c a 2 a

Proline (µmol g 4 1 b a 0 0 Free amino acids (µmol g d

FW) CD d FW) -1 100 200 c -1 80 150 c 60 b b b 100 40 a 50 a a 20 Glycine betaine (µmol g 0 Total soluble sugars (µmol g 0 0 200 400 800 1000 0 200 400 800 1000 NaCl (mM) NaCl (mM)

Fig. 5. Proline (A), free amino acids (B), total soluble sugars (C) and glycine betaine (D) concentration in leaves of Atriplex portulacoides exposed to different NaCl concentrations for 40 days. Means (n = 4 ± SE) with different letters are signiﬁcantly different at P b 0.05. The concentration of free amino acids (FAA) reported here is the difference between total FAA and proline concentrations.

100 0 as reﬂected by the decrease in Ψπ and Ψπ with increasing salinity as the maintenance of lower water potential at any given volume better compared to the control (Table 3). Succeeding in lowering Ψπ likely than elastic walls (Clifford et al., 1998; Martinez et al., 2004). These maintained a potential gradient for water uptake and made possible changes are important in plants as a mechanism for maintaining osmot- positive turgor potential at all salinity treatments. Total OA estimates ic and water homeostasis in saline environments. 100 as the differences between the Ψπ in the control and salinized Plants dealing with salt respond by decreasing osmotic potential and

A. portulacoides plants were 1.6 and 2.99 MPa for 200 and 1000 mM increasing ε in a manner that would lower Ψleaf and thus enhance water NaCl treatments, respectively. These results agree with other reports flow through the plant without major loss of relative water content showing OA to salinity and maintenance of turgor in higher plants de- (Touchette et al., 2009a). We found that salt stress led to a significant in- spite low water potentials associated with high rhizosphere salt crease in AWC compared with the control (Table 3). Increasing water (Suárez and Sobrado, 2000; Hajlaoui et al., 2010; Kachout et al., 2011). reserve of apoplast compartment could be due to thicker cell walls. Turgor regulation during changes in plant water status may preserve For instance, water apoplast protects tissues against water loss from the metabolic processes of the plant and contribute to growth mainte- the symplast. The increase of AWC played a role in maintaining or in- nance. Turgor regulation can result from (i) the decrease in the cell vol- creasing the turgidity. This behavior would have a buffer role during ume, (ii) the decrease in the symplast with respect to the apoplast the installation of water deficit to preserve symplast water (Mguis volume, and (iii) the changes induced at the elastic modulus (Larher et al., 2012). Further, A. portulacoides was able to increase water use ef- et al., 2009). In the present study, salt stress decreased significantly ficiency under high salinities (Fig. 3C) in concomitance with a signifi- A. portulacoides leaf cell elasticity (ε values increased). Higher ε follow- cant reduction in transpiration rate. Chen et al. (2003) reported that ing salt exposure has been also observed in other plant species lowering the transpiration rate under salt stress contributes to improv- (Paliyavuth et al., 2004; Touchette et al., 2009a,b). In species that ing their ability to control Na+ influx into roots and subsequently to the show OA and accumulate significantly high solute concentrations, a shoots. However, A. portulacoides actively accumulated Na+ to high con- rigid cell wall may be necessary to maintain cell/tissues integrity or re- centration in its leaves at high salt concentration; therefore, the dehydration following a period of stress, and rigid cell walls may facilitate crease in the transpiration rate does not seem to be essentially

Table 3 Water relation parameters as derived from pressure–volume curve analysis in leaf of Atriplex portulacoides exposed to different NaCl levels for 40 days. Means (n = 4 ± SE) with different letters were signiﬁcantly different at P b 0.05.

100 0 NaCl (mM) Ψπ (MPa) Ψπ (MPa) RWC0 (%) AWC (%) ε (MPa) OAt (MPa) 0 2.52 ± 0.12a 3.13 ± 0.1a 64.2 ± 3.1a 33.17 ± 2.7a 0.31 ± 0.04a 200 3.68 ± 0.01b 4.10 ± 0.05b 72.83 ± 0.6b 36.5 ± 2a 0.21 ± 0.06a 1.16 ± 0.11a 400 4.54 ± 0.03c 5.26 ± 0.01c 66.9 ± 2.1a 31.5 ± 4.9a 0.33 ± 0.11a 2.02 ± 0.11b 800 5.14 ± 0.04d 6.03 ± 0.01d 80.97 ± 3.7c 60.73 ± 8.1b 0.67 ± 0.13b 2.62 ± 0.15c 1000 5.51 ± 0.03e 6.30 ± 0.1e 84.93 ± 2.2c 67.13 ± 3.2b 0.63 ± 0.09b 2.99 ± 0.09d

100 0 Osmotic potential at full turgor (Ψπ ), osmotic potential at turgor loss point (Ψπ), relative water content at turgor loss point (RWC0), apoplastic water content (AWC), bulk modulus of elasticity (ε) and total osmotic adjustment (OA). 76 M. Benzarti et al. / South African Journal of Botany 95 (2014) 70–77

Table 4 100 Relative contribution (%) of inorganic and organic solutes to the leaf osmotic potential at full turgor (Ψπ )ofAtriplex portulacoides exposed to different NaCl levels for 40 days. Means (n = 4 ± SE) with different letters were signiﬁcantly different at P b 0.05.

NaCl (mM)

0 200 400 800 1000

Na+ 22.1 ± 2.8a 23.9 ± 0.8ab 26.8 ± 1.6b 31.1 ± 1.9c 42.2 ± 0.6d Cl− 11.2 ± 1.1a 15.2 ± 1.1b 17.4 ± 1.8b 29.1 ± 1.6c 37.4 ± 0.9d K+ 12.2 ± 3.9b 3.91 ± 0.4a 3.11 ± 0.2a 3.11 ± 0.4a 2.71 ± 0.1a Ca2+ 7.21 ± 0.7b 1.64 ± 0.1a 1.64 ± 0.12a 1.39 ± 0.1a 1.62 ± 0.08a a a b c ∑inorganic ions⁎ 49.9 ± 5.7a 44.7 ± 2.1 48.6 ± 2.4 64.6 ± 2.4 84.1 ± 1.4 Proline 0.02 ± 0.001a 0.04 ± 0.006b 0.11 ± 0.005c 0.25 ± 0.003d 0.31 ± 0.003e TSS 10.3 ± 1.7d 15.2 ± 1.3e 7.91 ± 0.4c 4.91 ± 0.3b 2.51 ± 0.1a FAA 1.75 ± 0.08d 1.14 ± 0.01c 0.93 ± 0.06b 0.19 ± 0.07a 0.15 ± 0.06a GB 1.75 ± 0.02a 1.26 ± 0.07a 3.10 ± 0.12b 4.72 ± 0.27c 5.05 ± 0.5d c d c b a ∑organic solutes⁎⁎ 13.9 ± 1.6 17.7 ± 1.2 12.2 ± 0.4 10.1 ± 0.3 8.09 ± 0.18 ⁎ ∑, sum of inorganic ions (Na+,Cl−,K+ and Ca2+). ⁎⁎ ∑, sum of organic solutes (proline, TSS, FAA and GB). achieved as a mechanism to avoid excessive Na+ influx (Nemat Alla is one of the most organic solutes that accumulate in the et al., 2011). The reduction in the transpiration rate in A. portulacoides Chenopodiaceae. In our study, GB was significantly increased at the could be considered as one of the tolerance mechanisms to prevent high salt stress (800 and 1000 mM NaCl) with a relative contribution leaf dehydration under salt stress. of ca. 5% to the osmotic potential. In addition, the relative contributions Plant species differ in terms of OA capacity under salt stress condi- of FAA (0.35%–1.75% of the total osmotic potential) and proline (0.02%– tions and the nature of the major solutes contributing to osmotic poten- 0.3% of the total osmotic potential) to OA were negligible whether in the tial. In A. portulacoides, the main inorganic ions involved in OA under control conditions or under low and high salinity (Table 4). In contrast salt-free conditions were Na+ followed by K+,Cl− and Ca2+.Asthe to inorganic ions, organic solutes formed the lowest component contrib- salt concentration increased, the contribution of K+,Ca2+ was replaced uting to OA in A. portulacoides (Table 5). The production of organic sol- + − + − 100 by that of Na and Cl .Na plus Cl contributed by up to 80% to Ψπ at utes is metabolically expensive and potentially limits plant growth by the highest salt concentration (1000 mM NaCl) (Table 4) whereas K+ consuming significant quantities of carbon that could otherwise be and Ca2+ each accounted for only 2.7% and 1.6% respectively. Thus, in used for growth (Patakas et al., 2002). The energetic cost of osmotic ad- salt-treated A. portulacoides, OA is ensured mainly by inorganic ions justment using inorganic ions is much lower than that of using organic (up to 95% at 1000 mM NaCl) (Table 5). This is consistent with the re- molecules synthesized in the cells (Yeo, 1983). Thus, by using this alter- cent findings of Silveira et al. (2009) who observed an effective partici- native mechanism of inorganic ion accumulation to adjust their osmotic pation of Na+ and Cl− ions in the OA in salt-treated Atriplex nummularia potential, A. portulacoides plants seem to save energy, which enables leaves. The overall contribution of inorganic ions to the cell osmotic ad- them to grow in less favorable conditions. This advantage may also be justment was estimated to be 73% for Avicennia species (Suárez et al., related to their ability to withstand higher internal saline ion concentra- 1998). The importance of these inorganic solutes (Na+ and Cl−)in tions. Other roles proposed for proline and GB besides OA in stressed achieving OA in A. portulacoides plants exposed to salt stress is plants include acting as reactive oxygen species (ROS) scavenger, stabi- highlighted by their relative low cost (less energy and carbon demand). lization of proteins and macromolecular complexes, regulator of cellular The accumulation of inorganic ions during OA requires an accumula- redox potential under stress and as a sink for carbon and nitrogen for tion of organic solutes. Glycine betaine, soluble sugars, free amino acids stress recovery (Rhodes and Hanson, 1993; Szabados and Savouré, in general and proline in particular, have been suggested as important 2010). organic solutes contributing to OA in some plant species (Hare et al., 1998). Accumulation of these compatible solutes reduces osmotic po- 5. Conclusion tential in the cytoplasm and contributes to maintaining water homeostasis among several cellular compartments (Sairam and Tyagi, 2004). In conclusion, our results indicate that A. portulacoides is able to At 200 mM NaCl, TSS content increased strongly in leaves (Fig. 5C) as grow optimally in the presence of 200 mM NaCl and remains alive compared to the control. According to the van't Hoff equation, the mea- when challenged with 1000 mM NaCl, which is twice the salinity of sea- 100 + − sured TSS concentrations represented about 15% of the Ψπ (Table 4). water. The higher Na and Cl levels in leaves than in the stem or roots However, under high salt stress, their contribution was reduced to of salt-treated plants indicated that in A. portulacoides an ion inclusion 2.5%. This result agrees with those of other authors (Ben Hassine and mechanism operated. With this respect, the fact that leaves in salt- Lutts, 2010) showing that TSS is a major organic solute contributor to treated plants showed neither dehydration nor leaf injury symptoms the Ψπ of cell sap in the Atriplex species under moderate salt stress. GB could be related to an efficient salt ion compartmentalization. The analysis of P–V curve led also to the suggestion that A. portulacoides responded to salt stress with an active osmotic adjustment. The quanti- tative contribution of Na+ and Cl− was higher in the osmotic adjust- Table 5 ment thereby maintaining a positive water balance and protecting the + − + 2+ Percent contribution of total inorganic ions (Na ,Cl ,K and Ca ) and organic solutes photosynthetic machinery. These mechanisms together with the (proline, TSS, FAA and GB) to total osmotic adjustment in leaves of Atriplex portulacoides sustained antioxidant activity (Benzarti et al., 2012) would be determi- exposed to different NaCl levels for 40 days. Means (n = 4 ± SE) with different letters were significantly different at P b 0.05. nant for the high survival aptitude of A. portulacoides under extreme salinity conditions. Contribution to total osmotic adjustment (%) NaCl (mM) Inorganic ions Organic solutes Acknowledgments 200 31.4 ± 5.7a 25.2 ± 4.3b 400 48.8 ± 9.1b 10.1 ± 2.5a We thank Dr. Zouhaier Barhoumi and Dr. Isabel Le Disquet for their c a 800 81.3 ± 6.2 7.41 ± 1.1 technical assistance during the anatomical studies. This work was sup- 1000 95.39 ± 6.8c 3.87 ± 1.5a ported by the Tunisian Ministry of Higher Education and Scientific M. Benzarti et al. / South African Journal of Botany 95 (2014) 70–77 77

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