Salinity induction of recycling Crassulacean acid metabolism and salt tolerance in plants of Talinum triangulare

Salinity induction of recycling Crassulacean acid metabolism and salt tolerance in plants of... Abstract Background and Aims Crassulacean acid metabolism (CAM) can be induced by salinity, thus conferring the plant higher water-use efficiency. Talinum triangulare does not frequently encounter salt in its natural habitat but is cultivated in soils that may become salinized. Here we examined whether plants of T. triangulare can grow in saline soils and show salt-induced CAM. Methods Leaf gas exchange, carbon isotopic ratio (δ13C), nocturnal acid accumulation (ΔH+), water relations, photosynthetic pigment and mineral contents, leaf anatomy and growth were determined in greenhouse in plants irrigated with 0, 150, 300 and 400 mm NaCl. Key Results Salinity reduced gas exchange and induced CAM, ΔH+ reaching 50.2 μmol H+ g−1 fresh mass under 300 mm NaCl. No nocturnal CO2 uptake, but compensation, was observed. Values of δ13C were lowest under 0 and 400 mm NaCl, and highest under 150 and 300 mm. The difference in osmotic potential (ψs) between control and treated plants averaged 0.45 MPa for the three [NaCl] values, the decrease in ψs being accounted for by up to 63 % by Na+ and K+. Pigment contents were unaffected by treatment, suggesting lack of damage to the photosynthetic machinery. Changes in stomatal index with unchanged stomatal density in newly expanded leaves suggested inhibited differentiation of epidermal cells into stomata. Whole-leaf and parenchymata thickness increased under 150 and 300 mm NaCl. Only plants irrigated with 400 mm NaCl showed reductions in biomass (stems, 41 %; reproductive structures, 78 %). The K/Na molar ratio decreased with [NaCl] from 2.0 to 0.4. Conclusions The operation of CAM in the recycling mode was evidenced by increased ΔH+ with no nocturnal CO2 uptake. Talinum triangulare can be classified as a halo-tolerant species based on its low K/Na molar ratio under salinity and the relatively small reduction in growth only at the highest [NaCl]. CAM, Crassulacean acid metabolism, Talinum triangulare, mineral content, salinity INTRODUCTION Plants can respond to salinity by either tolerating osmotic stress, excluding Na+ or Cl−, or accumulating Na+ or Cl−; these responses can vary in time and work in combination with each other (Munns and Tester, 2008). Osmotic adjustment is a mechanism which some plants employ to reduce osmotic potential (ψs) and increase turgor potential against water deficit conditions, including salinity (Munns and Tester, 2008). Osmotic adjustment has been shown to occur under salinity in many glycophytes, such as Nicotiana glauca, which are not usually subjected to saline soils (González et al., 2012). Induction by drought of Crassulacean acid metabolism (CAM) has been reported in Talinum triangulare by, among others, Herrera et al. (1991, 2015), Taisma and Herrera (1998), and Pieters et al. (2003). The possibility exists that salinity may be an inducer of CAM in T. triangulare, as in Mesembryanthemum crystallinum (Winter and Holtum, 2002a). In plants of M. crystallinum under exposure to 400 mm NaCl, δ13C values were higher (−21.0 ‰) than without salt (between −28.3 and −29.5 ‰; Winter and Holtum, 2002a), evidencing a switch from C3 metabolism to CAM. During a daily cycle of gas exchange in a CAM plant, four phases can be distinguished, phase I taking place during the night, phase II at dawn and phases III and IV during the day; for a description of the four phases, see Niewiadomska et al. (2004). CAM can occur in several modes, which include the constitutive or obligate mode, the cycling mode, with nocturnal acid accumulation but no dark CO2 fixation in watered plants, and inducible/facultative or C3-CAM (Herrera, 2009), with moderate ΔH+ and low dark CO2 fixation. CAM induction is considered an adaptive strategy that increases water use efficiency (Herrera, 2009), which is particularly important for plants living in arid and/or saline environments (Niewiadomska et al., 2004). The main diagnostic characters of CAM are nocturnal acid accumulation (ΔH+) and nocturnal stomatal opening; mesophyll succulence apparently is a requisite for CAM performance (Osmond, 1978; but see Herrera, 2009). Values of δ13C of photosynthetic organs may help in assessing the operation of CAM (Herrera, 2009). The δ13C of plants that acquire one-third or less of their carbon in the dark may be confused with that of C3 plants when the carbon fixation pathway is assigned solely on the basis of δ13C (Winter and Holtum, 2002b). Values of δ13C between −30 and −21 ‰ suggest operation of weak CAM in two of its modes, i.e. cycling or facultative (Herrera, 2009). Plants of T. triangulare and T. paniculatum do not frequently grow in saline soils. Plants of T. triangulare tolerated up to 560 mm NaCl (Bamidele et al., 2007) and those of T. paniculatum, up to 300 mm NaCl (Assaha et al., 2017). In T. triangulare decreases in average fresh mass (FM) of leaves, stems and roots of 87 % and dry mass (DM) of 82 % were noted, leaf succulence remaining unchanged at up to 280 mm NaCl (Bamidele et al., 2007). Thus far, no report of CAM induction by salinity has been published for either species. Leaves of T. triangulare are an unconventional vegetable (waterleaf) consumed in some parts of Venezuela and more widely in Indonesia and Nigeria; plants are extensively cultivated in Nigeria in garden soils composed of up to 77 % sand containing 2.3 mmol kg−1 Na (Ukpong and Moses, 2001). Plants of T. triangulare are perennial sub-shrubs approx. 50 cm tall with partly succulent leaves. In Venezuela, plants grow equally well under full sun exposure or in the forest understorey, from 0 to 100 m, in humid as well as semi-arid regions. They may occasionally be found 25 m inland but this is relatively uncommon. Plants of T. triangulare under drought with CAM induced were shown to achieve osmotic adjustment (OA) using malate as osmoticum (Herrera et al., 2015). This OA would only be effective in improving water absorption by the roots during phases I and II of CAM, before all malate became decarboxylated. In halophytes, OA is not transitory, as would be the case in CAM plants; in a CAM halophyte, OA could be achieved through accumulation of other osmotica in the vacuole which might not be reverted during the day–night cycle. Under salinity, leaves tend to become more succulent because of increased cell volume or number (Suárez and Sobrado, 2000; González et al., 2012). Changes in stomatal characteristics have been reported in barley (Zhu et al., 2015) and quinoa (Shabala et al., 2013). Medina et al. (2008) separated species growing naturally in saline soils on the basis of their leaf K/Na ratio into halophytes (K/Na < 0.1) and non-halophytes and, among the latter, into halo-resistant (K/Na < 1) and halo-tolerant (K/Na between 0.1 and 1.0). This separation correlated well with succulence [Suc = (FM − DM)/FM], halophytes having values of 88 ± 1 % and non-halophytes, 76 ± 4 %. We hypothesized that CAM may be induced by salinity in T. triangulare. To determine whether plants of T. triangulare can grow in saline soils and whether CAM is induced in them by salinity, we subjected plants to a range of concentrations of NaCl and determined daily gas exchange, δ13C, ΔH+, water relations, leaf anatomical characteristics, growth analysis variables and mineral contents. MATERIALS AND METHODS Plant material and growth conditions Adult plants of T. triangulare Jacq. Willd. [syn. Talinum fruticosum (L.) Juss.], similar in height and foliage, were grown in 8-L plastic pots filled with 6.0 ± 0.5 kg silty-clay loamy soil purchased at a nursery (Viveros Exotica Raphia, S.R.L., Caracas, Venezuela) in a transparent polythene-roofed shed at the Instituto de Biología Experimental in Caracas. Plants were watered daily with tap water and weekly with nutrient solution (1 g L−1, Campos Green Multi-Plants-18-18-18 soluble fertilizer, Inversiones Green Valley C.A., Caracas, Venezuela). After 2 months, five control plants were watered every other day with tap water and weekly with nutrient solution, and three groups of five plants each watered every other day with 150, 300 and 400 mm NaCl and once a week with these solutions plus fertilizer; water or solutions were added until a few millilitres leached from the bottom. After 3 d of irrigation with each of the saline solutions, the water potential of the leachate, determined as for leaf water potential, ψ (see below), was very similar to that of the corresponding irrigation solution. After 21 d of treatment, plants were harvested and organs dried for measurement of DM. For measurements, only the youngest, fully expanded leaves were used. Microclimatic variables inside the greenhouse Day length was 12 h (0600–18:00 h). Photosynthetic photon flux density (PFD) was measured with a 190-S quantum sensor connected to an LI-185 meter (LI-COR Inc., Lincoln, NE, USA). Air temperature and relative humidity were measured using a HOBO Pro V2 logger and data dumped with a HOBO Waterproof Shuttle (Onset Computer Corporation, Pocasset, MA, USA). Microclimatic conditions in the greenhouse were (min/max): air temperature 15 ± 2/34 ± 2 °C, relative humidity 45 ± 4/83 ± 2 %, and maximum PFD 1035 ± 65 µmol m−2 s−1. Gas exchange Assimilation rate (PN), and transpiration rate (E) were measured in intact leaves with a CIRAS 2 infrared CO2 and H2O analyser connected to a PLC(B) assimilation chamber (PP Systems Inc., Amesbury, MA, USA). The source of CO2 was ambient air; [CO2] = 403 ± 1 µmol mol−1, leaf temperature 24.4 ± 0.1 °C and PFD 1000 µmol m−2 s−1, previously found to saturate PN. Water use efficiency was calculated as WUE = PN/E. Daily courses in one leaf per plant per treatment were done, with readings recorded automatically every 30 min. Different plants were used for measurements every week of treatment. The amount of water lost by transpiration per day was calculated by integrating the area below courses of E. Diurnal courses of integrated WUE were calculated as integrated PN/integrated diurnal E. Nocturnal acid accumulation On each sampling date one leaf from the third or fourth node of each of the five plants per group was collected in the morning and afternoon. Leaves were weighed and their length and width measured to calculate leaf area using an allometric relationship. Leaves were cut into small segments and boiled in 20 mL distilled water for 3 min in a microwave oven at full power. Extracts were passed through a colander, which was rinsed into a beaker, and titrated with 10 mm KOH to reach pH 7.0 for malate equivalents and 8.4 for citrate equivalents (details in Herrera et al., 2015), measured with an Orion pH meter (Thermo Fisher Scientific, Waltham, MA, USA). ΔH+ was calculated as the difference between morning and afternoon H+ contents at the two pH values (ΔH+malate eq. and ΔH+citrate eq). To determine how early in the afternoon and how late in the morning samples could be collected for the correct determination of ΔH+, a time-course of decarboxylation was previously done on plants that had been subjected to 7 d of drought. The latest morning and earliest afternoon times for leaf collection were found to be 0800 and 1600 h. Leaf carbon isotopic composition δ13C was determined in leaf dry ground samples using a ThermoFinnigan DeltaPlusXL mass spectrometer (Isotope Ratio Mass Spectrometer, San Jose, CA, USA) with an accuracy of 0.15 ‰ and Pee Dee Belemnite as standard. Water relations Leaf FM and area were determined in one leaf per plant and dried for 48 h at 60 °C to determine DM. Leaf water content was calculated as LWC = (FM − DM)/area (mg cm−2) or LWC = (FM − DM)/DM. The value of ψ was determined at 0800 0830 and 1200 1230 h in leaf discs placed in C-52 chambers connected to an HR-33T micro-voltmeter (Wescor Inc., Logan, UT, USA) operated in the psychrometric mode. The ψs was measured in the same discs frozen at −20 °C for 72 h and placed without thawing in the chambers. The change in ψs (Δψs) was determined for both morning and noon values of ψs as the difference between ψs of control plants and plants under salinity, without previous rehydration. Leaf pigment contents and anatomy Chlorophyll and carotenoid contents were determined in extracts obtained by submersing discs in 90 % ethanol and a small amount of CaCO3 for 3 d at 4 °C; absorbance was measured at 470, 649 and 665 nm according to Wellburn (1994). Tissue thickness and number of tissue cells were determined in leaf freehand sections fixed in formalin–acetic acid–alcohol and stained with toluidine blue; stomata were observed on nail-varnish imprints of leaf epidermes. Photographs were taken under the microscope at 40× (leaf sections) and 100× (stomata) and processed with the ImageJ software. Stomatal index was calculated after Salisbury (1927) as SI = number of stomata/(number of stomata + epidermal cells). Growth analysis and plant mineral contents At the end of treatment plants were harvested and their organs separated and dried at 60 °C for 72 h. Reproductive effort was calculated as RE = DM of reproductive structures/leaf DM, after Taisma and Herrera (1998). Reproductive structures comprised inflorescence peduncles, flowers, fruits and seeds from open mature fruits. Total cation contents were determined in powdered leaves, stems and roots by digestion with 4: 1 sulphuric acid/perchloric acid, and soluble cations in hot-water digestions, using a flame atomic absorption spectrometer (Varian Spectra AA mod. 55B). Total P content was determined in acid digestions as above after Murphy and Riley (1962). Standards used were: Na, Merck CertiPUR 1.19507.0500 1000 mg; Ca, Merck CertiPUR 1.19778.0500 1000 mg; K, Merck Titrisol 1.09924 1000 mg; and Mg, Merck Titrisol 1.09949 1000 mg. Leaf C and N contents were determined as for δ13C. The contribution to morning ψs of soluble Na and K was calculated based on values of LWC, FM/DM and ion contents per DM. Soil Na and K contents were measured in aqueous solutions (0.1 g in 0.025 mL distilled water). Soil electrical conductivity (EC) was measured in the same solutions using an EC Meter 19101-00 with a Pt cell (Cole Parmer, Vernon Hills, IL, USA). Statistics Values are given as mean ± s.e. (n = 5). Statistical significance was assessed where indicated through one- or two-way analysis of variance (P < 0.05) with the Statistica package. RESULTS Gas exchange and nocturnal acid accumulation Daily courses of PN and E (Fig. 1) remained relatively similar during treatment at 0 mm NaCl and on day 1 of treatment with 150, 300 and 400 mm NaCl, and after 8 d of treatment with 150 mm NaCl. For the rest of the time and the other salinities, significant reductions in both PN and E were found, the most drastic occurring after 21 d under 300 and 400 mm NaCl. After 21 d nocturnal CO2 compensation was measured for all three salinities. A distinct Phase II CO2 exchange pattern was found in the morning at lights-on under all salinities after 8 and 21 d. Daily courses of E showed the same trend as PN. Carbon gain diminished after 21 d to 8 % of control for all salinities. Fig. 1. View largeDownload slide Changes under treatment with increasing soil salinity in assimilation rate (PN, closed circles) and transpiration rate (E, open circles). The [NaCl] and time under treatment are indicated in each panel. Values are data points measured in a different plant per treatment every day. The filled bar on the abscissa indicates the duration of the night. Fig. 1. View largeDownload slide Changes under treatment with increasing soil salinity in assimilation rate (PN, closed circles) and transpiration rate (E, open circles). The [NaCl] and time under treatment are indicated in each panel. Values are data points measured in a different plant per treatment every day. The filled bar on the abscissa indicates the duration of the night. Diurnal integrated WUE decreased after 21 d of treatment; values of δ13C were lowest under 0 and 400 mm NaCl, and highest under 150 and 300 mm (Table 1). The mass of water transpired during the light period decreased from more than 100 % of LWC to an average of 25 % (Table 1). Table 1. Values of the proportion of leaf water content (LWC) transpired diurnally (E), integrated diurnal water use efficiency (WUE = integrated PN/integrated E) and carbon isotopic composition (δ13C) in leaves of plants of Talinum triangulare subjected for 1 and 21 d to irrigation with solutions of different [NaCl]. [NaCl] (mm) E (% of LWC) WUE (mmol mol−1 d−1) δ13C (‰) Day 1 Day 21 Day 1 Day 21 0 151 134 2.6 4.6 −27.73 ± 0.37a 150 102 29 2.2 3.4 −26.12 ± 0.23b 300 138 25 2.7 0.5 −25.49 ± 0.41b 400 156 18 4.2 1.0 −28.20 ± 0.40a [NaCl] (mm) E (% of LWC) WUE (mmol mol−1 d−1) δ13C (‰) Day 1 Day 21 Day 1 Day 21 0 151 134 2.6 4.6 −27.73 ± 0.37a 150 102 29 2.2 3.4 −26.12 ± 0.23b 300 138 25 2.7 0.5 −25.49 ± 0.41b 400 156 18 4.2 1.0 −28.20 ± 0.40a Values of E and WUE are data points; values of δ13C are mean ± s.e. (n = 5). Different letters indicate significant differences after a one-way ANOVA (P < 0.05). View Large Table 1. Values of the proportion of leaf water content (LWC) transpired diurnally (E), integrated diurnal water use efficiency (WUE = integrated PN/integrated E) and carbon isotopic composition (δ13C) in leaves of plants of Talinum triangulare subjected for 1 and 21 d to irrigation with solutions of different [NaCl]. [NaCl] (mm) E (% of LWC) WUE (mmol mol−1 d−1) δ13C (‰) Day 1 Day 21 Day 1 Day 21 0 151 134 2.6 4.6 −27.73 ± 0.37a 150 102 29 2.2 3.4 −26.12 ± 0.23b 300 138 25 2.7 0.5 −25.49 ± 0.41b 400 156 18 4.2 1.0 −28.20 ± 0.40a [NaCl] (mm) E (% of LWC) WUE (mmol mol−1 d−1) δ13C (‰) Day 1 Day 21 Day 1 Day 21 0 151 134 2.6 4.6 −27.73 ± 0.37a 150 102 29 2.2 3.4 −26.12 ± 0.23b 300 138 25 2.7 0.5 −25.49 ± 0.41b 400 156 18 4.2 1.0 −28.20 ± 0.40a Values of E and WUE are data points; values of δ13C are mean ± s.e. (n = 5). Different letters indicate significant differences after a one-way ANOVA (P < 0.05). View Large Salinity produced morning values of H+ content (malate eq.) significantly higher than afternoon values at 150 and 300 mm NaCl after 8 and 16 d of treatment, resulting after 8 d in maximum ΔH+malate eq. of 35.9 ± 8.7 and 50.2 ± 14.7 µmol H+ g−1 for 150 and 300 mm NaCl, respectively. There were no significant differences between morning and afternoon H+citrate eq. contents (Fig. 2). Fig. 2. View largeDownload slide Changes with time under treatment with increasing soil salinity in morning and afternoon H+ contents corresponding to malate and citrate equivalents. Open symbols, afternoon; closed symbols, morning. The acid is indicated above the uppermost panels, and [NaCl] in each panel. Values are mean ± s.e. (n = 5). Asterisks indicate significant differences between morning and afternoon values after a one-way ANOVA (time of day; P < 0.05). Fig. 2. View largeDownload slide Changes with time under treatment with increasing soil salinity in morning and afternoon H+ contents corresponding to malate and citrate equivalents. Open symbols, afternoon; closed symbols, morning. The acid is indicated above the uppermost panels, and [NaCl] in each panel. Values are mean ± s.e. (n = 5). Asterisks indicate significant differences between morning and afternoon values after a one-way ANOVA (time of day; P < 0.05). Water relations Figure 3 shows the time-course of changes in morning and noon values of ψ and ψs. Νo significant differences in ψ or ψs were found between morning and noon values. After 21 d of treatment, values of morning ψs were significantly lower than morning ψ in some, but not all, salinities relative to their control values. Fig. 3. View largeDownload slide Changes with time under treatment with increasing soil salinity in morning and noon leaf water (ψ) and osmotic (ψs) potential. Each time course for every salinity and time of day has its corresponding control course. Time of day is indicated above the uppermost panels, and [NaCl] in each panel. Circles, ψ; triangles, ψs. Open symbols, control (0 mm NaCl); closed symbols, salinity. Values are mean ± s.e. (n = 5). Different letters indicate significant differences at the end of treatment after a two-way ANOVA done separately for morning and noon values ([NaCl] × ψ or ψs; P < 0.05). Fig. 3. View largeDownload slide Changes with time under treatment with increasing soil salinity in morning and noon leaf water (ψ) and osmotic (ψs) potential. Each time course for every salinity and time of day has its corresponding control course. Time of day is indicated above the uppermost panels, and [NaCl] in each panel. Circles, ψ; triangles, ψs. Open symbols, control (0 mm NaCl); closed symbols, salinity. Values are mean ± s.e. (n = 5). Different letters indicate significant differences at the end of treatment after a two-way ANOVA done separately for morning and noon values ([NaCl] × ψ or ψs; P < 0.05). At the end of treatment, turgor potential was 0.21 ± 0.07, 0.28 ± 0.09, 0.35 ± 0.11 and 0.23 ± 0.10 MPa for 0, 150, 300 and 400 mm NaCl, respectively (P = 0.80). There were no significant differences among the three salinity treatments in Δψs calculated after 21 d of treatment with either morning or noon values of ψs, Δψs averaging 0.45 ± 0.04 MPa. The proportional contribution of malate and citrate equivalents to ψs was the same for all salinity treatments (P = 0.65), averaging 0.6 ± 0.1 %. Pigments, leaf anatomy and succulence Figure 4 shows changes in photosynthetic pigment contents with treatment. At the end of the experiment, total chlorophyll content decreased slightly or not at all in control and at all salinities, with no significant differences between control and salinity. Carotenoid content was not affected by either treatment or time under it, while total chlorophyll/carotenoids decreased slightly under all [NaCl] values except for 400 mm, where the reduction was more marked. Fig. 4. View largeDownload slide Changes in plants under treatment with increasing soil salinity in: A, total chlorophyll content; B, total carotenoid content; and C, chlorophyll/carotenoids. Values are mean ± s.e. (n = 5). Legend is given in panel A: empty bars, day 1 of treatment; striped bars, day 22 of treatment. Different letters indicate significant differences after a two-way ANOVA ([NaCl] × time; P < 0.05). Fig. 4. View largeDownload slide Changes in plants under treatment with increasing soil salinity in: A, total chlorophyll content; B, total carotenoid content; and C, chlorophyll/carotenoids. Values are mean ± s.e. (n = 5). Legend is given in panel A: empty bars, day 1 of treatment; striped bars, day 22 of treatment. Different letters indicate significant differences after a two-way ANOVA ([NaCl] × time; P < 0.05). Table 2 summarizes changes in leaf anatomical characteristics due to treatment. The sole significant differences were an increase in whole-leaf thickness under 150, 300 and 400 mm NaCl, and a slight increase in palisade parenchyma thickness under 300 mm NaCl and spongy mesophyll thickness under 150 and 300 mm NaCl. The other tissues and the number of cells per parenchyma (data not shown) suffered no significant changes due to the treatment × time interaction. Morning LWC was highest at the end of the experiment under any salinity, the interaction between [NaCl] and time under treatment not being significant. Values of integrated E showed that leaves transpired 39 % their own water at the end of treatment. Values of Suc at any time and salinity were 94 ± 0 %, with no significant differences between treatments. Stomatal density (SD) of the abaxial face showed no significant differences due to treatment at the end of the experiment, and adaxial SD was lower than abaxial SD, showing slight changes with treatment. At the end of the experiment, SI in both leaf faces of plants under saline treatments remained generally unchanged by treatment, except for a significant decrease in both faces relative to control plants. Table 2. Changes with time under treatment with NaCl solutions in whole-leaf and tissue thickness, morning leaf water content (LWC) and stomatal density (SD) and index (SI) [NaCl] (mm) 0 150 300 400 Initial Final Initial Final Initial Final Initial Final P Thickness (µm)  Whole leaf 755 ± 92abc 771 ± 58bc 569 ± 69a 927 ± 32cd 624 ± 50ab 971 ± 76d 566 ± 35ab 643 ± 70ab 0.02  Upper epidermis 37 ± 4a 37 ± 1a 32 ± 2a 37 ± 6a 30 ± 2a 30 ± 3a 29 ± 3a 33 ± 5a 0.92  Palisade parenchyma 362 ± 45ab 372 ± 39ab 268 ± 39ab 477 ± 17ab 288 ± 31ab 465 ± 35b 271 ± 20ab 297 ± 48a 0.01  Spongy parenchyma 314 ± 50ab 316 ± 19ab 247 ± 40a 373 ± 16bc 275 ± 23a 451 ± 41c 225 ± 13a 273 ± 21a 0.04  Lower epidermis 29 ± 4a 35 ± 2b 19 ± 2a 26 ± 3ab 22 ± 1a 25 ± 4a 28 ± 3ab 28 ± 4ab 0.65 LWC (mg cm−2) 89 ± 8ab 107 ± 9ab 95 ± 9ab 115 ± 6bc 95 ± 14ab 138 ± 12c 79 ± 6a 107 ± 12abc 0.61  (g water g−1 DM) 16.0 ± 1.0 14.3 ± 1.3 15.3 ± 0.6 14.8 ± 0.7 14.5 ± 1.8 17.0 ± 0.9 15.0 ± 0.9 15.8 ± 0.6 0.57 SD - abaxial (mm−2) 85 ± 19a 151 ± 11b 133 ± 23ab 106 ± 17ab 97 ± 22ab 92 ± 7a 121 ± 4ab 133 ± 25ab 0.06  SD - adaxial (mm−2) 36 ± 5a 72 ± 5b 69 ± 13b 50 ± 3ab 49 ± 10ab 40 ± 3a 53 ± 5ab 52 ± 13ab 0.09 SI - abaxial (%) 17 ± 1ab 29 ± 2c 19 ± 1ab 20 ± 2ab 16 ± 2ab 21 ± 1b 17 ± 1ab 16 ± 2a 0.00  SI - adaxial (%) 13 ± 0a 25 ± 1b 14 ± 1a 18 ± 1a 15 ± 2a 14 ± 2a 13 ± 1a 13 ± 3a 0.00 [NaCl] (mm) 0 150 300 400 Initial Final Initial Final Initial Final Initial Final P Thickness (µm)  Whole leaf 755 ± 92abc 771 ± 58bc 569 ± 69a 927 ± 32cd 624 ± 50ab 971 ± 76d 566 ± 35ab 643 ± 70ab 0.02  Upper epidermis 37 ± 4a 37 ± 1a 32 ± 2a 37 ± 6a 30 ± 2a 30 ± 3a 29 ± 3a 33 ± 5a 0.92  Palisade parenchyma 362 ± 45ab 372 ± 39ab 268 ± 39ab 477 ± 17ab 288 ± 31ab 465 ± 35b 271 ± 20ab 297 ± 48a 0.01  Spongy parenchyma 314 ± 50ab 316 ± 19ab 247 ± 40a 373 ± 16bc 275 ± 23a 451 ± 41c 225 ± 13a 273 ± 21a 0.04  Lower epidermis 29 ± 4a 35 ± 2b 19 ± 2a 26 ± 3ab 22 ± 1a 25 ± 4a 28 ± 3ab 28 ± 4ab 0.65 LWC (mg cm−2) 89 ± 8ab 107 ± 9ab 95 ± 9ab 115 ± 6bc 95 ± 14ab 138 ± 12c 79 ± 6a 107 ± 12abc 0.61  (g water g−1 DM) 16.0 ± 1.0 14.3 ± 1.3 15.3 ± 0.6 14.8 ± 0.7 14.5 ± 1.8 17.0 ± 0.9 15.0 ± 0.9 15.8 ± 0.6 0.57 SD - abaxial (mm−2) 85 ± 19a 151 ± 11b 133 ± 23ab 106 ± 17ab 97 ± 22ab 92 ± 7a 121 ± 4ab 133 ± 25ab 0.06  SD - adaxial (mm−2) 36 ± 5a 72 ± 5b 69 ± 13b 50 ± 3ab 49 ± 10ab 40 ± 3a 53 ± 5ab 52 ± 13ab 0.09 SI - abaxial (%) 17 ± 1ab 29 ± 2c 19 ± 1ab 20 ± 2ab 16 ± 2ab 21 ± 1b 17 ± 1ab 16 ± 2a 0.00  SI - adaxial (%) 13 ± 0a 25 ± 1b 14 ± 1a 18 ± 1a 15 ± 2a 14 ± 2a 13 ± 1a 13 ± 3a 0.00 Values are mean ± s.e. (n = 5). Different letters indicate significant differences after a two-way ANOVA ([NaCl] × time; P < 0.05). The value of P for the interaction time × [NaCl] is indicated. Values of P < 0.05 are shown in bold type. View Large Table 2. Changes with time under treatment with NaCl solutions in whole-leaf and tissue thickness, morning leaf water content (LWC) and stomatal density (SD) and index (SI) [NaCl] (mm) 0 150 300 400 Initial Final Initial Final Initial Final Initial Final P Thickness (µm)  Whole leaf 755 ± 92abc 771 ± 58bc 569 ± 69a 927 ± 32cd 624 ± 50ab 971 ± 76d 566 ± 35ab 643 ± 70ab 0.02  Upper epidermis 37 ± 4a 37 ± 1a 32 ± 2a 37 ± 6a 30 ± 2a 30 ± 3a 29 ± 3a 33 ± 5a 0.92  Palisade parenchyma 362 ± 45ab 372 ± 39ab 268 ± 39ab 477 ± 17ab 288 ± 31ab 465 ± 35b 271 ± 20ab 297 ± 48a 0.01  Spongy parenchyma 314 ± 50ab 316 ± 19ab 247 ± 40a 373 ± 16bc 275 ± 23a 451 ± 41c 225 ± 13a 273 ± 21a 0.04  Lower epidermis 29 ± 4a 35 ± 2b 19 ± 2a 26 ± 3ab 22 ± 1a 25 ± 4a 28 ± 3ab 28 ± 4ab 0.65 LWC (mg cm−2) 89 ± 8ab 107 ± 9ab 95 ± 9ab 115 ± 6bc 95 ± 14ab 138 ± 12c 79 ± 6a 107 ± 12abc 0.61  (g water g−1 DM) 16.0 ± 1.0 14.3 ± 1.3 15.3 ± 0.6 14.8 ± 0.7 14.5 ± 1.8 17.0 ± 0.9 15.0 ± 0.9 15.8 ± 0.6 0.57 SD - abaxial (mm−2) 85 ± 19a 151 ± 11b 133 ± 23ab 106 ± 17ab 97 ± 22ab 92 ± 7a 121 ± 4ab 133 ± 25ab 0.06  SD - adaxial (mm−2) 36 ± 5a 72 ± 5b 69 ± 13b 50 ± 3ab 49 ± 10ab 40 ± 3a 53 ± 5ab 52 ± 13ab 0.09 SI - abaxial (%) 17 ± 1ab 29 ± 2c 19 ± 1ab 20 ± 2ab 16 ± 2ab 21 ± 1b 17 ± 1ab 16 ± 2a 0.00  SI - adaxial (%) 13 ± 0a 25 ± 1b 14 ± 1a 18 ± 1a 15 ± 2a 14 ± 2a 13 ± 1a 13 ± 3a 0.00 [NaCl] (mm) 0 150 300 400 Initial Final Initial Final Initial Final Initial Final P Thickness (µm)  Whole leaf 755 ± 92abc 771 ± 58bc 569 ± 69a 927 ± 32cd 624 ± 50ab 971 ± 76d 566 ± 35ab 643 ± 70ab 0.02  Upper epidermis 37 ± 4a 37 ± 1a 32 ± 2a 37 ± 6a 30 ± 2a 30 ± 3a 29 ± 3a 33 ± 5a 0.92  Palisade parenchyma 362 ± 45ab 372 ± 39ab 268 ± 39ab 477 ± 17ab 288 ± 31ab 465 ± 35b 271 ± 20ab 297 ± 48a 0.01  Spongy parenchyma 314 ± 50ab 316 ± 19ab 247 ± 40a 373 ± 16bc 275 ± 23a 451 ± 41c 225 ± 13a 273 ± 21a 0.04  Lower epidermis 29 ± 4a 35 ± 2b 19 ± 2a 26 ± 3ab 22 ± 1a 25 ± 4a 28 ± 3ab 28 ± 4ab 0.65 LWC (mg cm−2) 89 ± 8ab 107 ± 9ab 95 ± 9ab 115 ± 6bc 95 ± 14ab 138 ± 12c 79 ± 6a 107 ± 12abc 0.61  (g water g−1 DM) 16.0 ± 1.0 14.3 ± 1.3 15.3 ± 0.6 14.8 ± 0.7 14.5 ± 1.8 17.0 ± 0.9 15.0 ± 0.9 15.8 ± 0.6 0.57 SD - abaxial (mm−2) 85 ± 19a 151 ± 11b 133 ± 23ab 106 ± 17ab 97 ± 22ab 92 ± 7a 121 ± 4ab 133 ± 25ab 0.06  SD - adaxial (mm−2) 36 ± 5a 72 ± 5b 69 ± 13b 50 ± 3ab 49 ± 10ab 40 ± 3a 53 ± 5ab 52 ± 13ab 0.09 SI - abaxial (%) 17 ± 1ab 29 ± 2c 19 ± 1ab 20 ± 2ab 16 ± 2ab 21 ± 1b 17 ± 1ab 16 ± 2a 0.00  SI - adaxial (%) 13 ± 0a 25 ± 1b 14 ± 1a 18 ± 1a 15 ± 2a 14 ± 2a 13 ± 1a 13 ± 3a 0.00 Values are mean ± s.e. (n = 5). Different letters indicate significant differences after a two-way ANOVA ([NaCl] × time; P < 0.05). The value of P for the interaction time × [NaCl] is indicated. Values of P < 0.05 are shown in bold type. View Large Growth analysis and mineral contents Only plants irrigated with 400 mm NaCl solution showed significant reductions in biomass, which took place solely in stems (41 %) and reproductive structures (78 %), leaf mass remaining unchanged; the root/shoot ratio was unaffected by treatment, and RE decreased only under 400 mm NaCl (Fig. 5). Fig. 5. View largeDownload slide Changes in plants after 22 d of treatment with increasing soil salinity in: A, biomass distribution among organs; B, root/shoot ratio; and C, reproductive effort (RE). Legend is given in panel A. Values are mean ± s.e. (n = 5). Asterisks indicate significant differences after a one-way ANOVA ([NaCl] per organ; P < 0.05). Fig. 5. View largeDownload slide Changes in plants after 22 d of treatment with increasing soil salinity in: A, biomass distribution among organs; B, root/shoot ratio; and C, reproductive effort (RE). Legend is given in panel A. Values are mean ± s.e. (n = 5). Asterisks indicate significant differences after a one-way ANOVA ([NaCl] per organ; P < 0.05). Changes with treatment in soluble Na and K contents and their contribution to morning ψs are shown in Table 3. Leaf Na content increased with salinity in stems, was highest under 150 and 300 mm and, under 400 mm, it was higher in roots than under the other two [NaCl] values. Soil Na content of control plants and tap water were 183 ± 30 and 0.4 ± 0.0 mmol kg−1 (0.58 and 0.30 dS m−1), respectively. The K/Na molar ratio in soils was 0.11 ± 0.01, and pH in a 1: 5 solution (w/w) was 7.3 ± 0.1 (n = 6). Contents of K remained unchanged in leaves, increased in stems and showed no differences in roots. The contribution of Na to morning ψs increased with salinity, whereas that of K remained relatively unchanged; both cations contributed on average 52 % to ψs and 50 % (Na) and 24 % (K) on average to the observed Δψs. The total K/total Na molar ratio in control plants was five times higher than the average under salinity (Table 3). The contribution of soluble Mg, whose content was 26 % of the total Mg content, was constant and amounted on average to only 5 %. Table 3. Changes with treatment for 21 d in organ soluble Na and K contents, their contribution to morning osmotic potential (ψs) and the leaf total K/total Na molar ratio in leaves of plants of Talinum triangulare [NaCl] (mm) Mineral content (mmol kg−1 DM) Na K % ψs K/Na Leaves Stems Roots Leaves Stems Roots Na K (mol mol−1) 0 328 ± 26a 761 ± 58a 54 ± 4a 1222 ± 69b 776 ± 45 825 ± 103 12 ± 1a 30 ± 3b 2.0 ± 0.4b 150 1569 ± 155b 1951 ± 194b 93 ± 21ab 914 ± 82a 724 ± 59 1027 ± 55 32 ± 4a 23 ± 6ab 0.5 ± 0.0a 300 2313 ± 189bc 1441 ± 280b 124 ± 14b 1116 ± 55ab 808 ± 64 760 ± 112 34 ± 4ab 15 ± 2a 0.4 ± 0.0a 400 3146 ± 633c 848 ± 288a 265 ± 12c 1096 ± 118ab 912 ± 123 942 ± 85 69 ± 23b 16 ± 2ab 0.3 ± 0.0a [NaCl] (mm) Mineral content (mmol kg−1 DM) Na K % ψs K/Na Leaves Stems Roots Leaves Stems Roots Na K (mol mol−1) 0 328 ± 26a 761 ± 58a 54 ± 4a 1222 ± 69b 776 ± 45 825 ± 103 12 ± 1a 30 ± 3b 2.0 ± 0.4b 150 1569 ± 155b 1951 ± 194b 93 ± 21ab 914 ± 82a 724 ± 59 1027 ± 55 32 ± 4a 23 ± 6ab 0.5 ± 0.0a 300 2313 ± 189bc 1441 ± 280b 124 ± 14b 1116 ± 55ab 808 ± 64 760 ± 112 34 ± 4ab 15 ± 2a 0.4 ± 0.0a 400 3146 ± 633c 848 ± 288a 265 ± 12c 1096 ± 118ab 912 ± 123 942 ± 85 69 ± 23b 16 ± 2ab 0.3 ± 0.0a Values are mean ± s.e. (n = 5). Different letters indicate significant differences after a one-way ANOVA ([NaCl] for each cation per organ; P < 0.05). View Large Table 3. Changes with treatment for 21 d in organ soluble Na and K contents, their contribution to morning osmotic potential (ψs) and the leaf total K/total Na molar ratio in leaves of plants of Talinum triangulare [NaCl] (mm) Mineral content (mmol kg−1 DM) Na K % ψs K/Na Leaves Stems Roots Leaves Stems Roots Na K (mol mol−1) 0 328 ± 26a 761 ± 58a 54 ± 4a 1222 ± 69b 776 ± 45 825 ± 103 12 ± 1a 30 ± 3b 2.0 ± 0.4b 150 1569 ± 155b 1951 ± 194b 93 ± 21ab 914 ± 82a 724 ± 59 1027 ± 55 32 ± 4a 23 ± 6ab 0.5 ± 0.0a 300 2313 ± 189bc 1441 ± 280b 124 ± 14b 1116 ± 55ab 808 ± 64 760 ± 112 34 ± 4ab 15 ± 2a 0.4 ± 0.0a 400 3146 ± 633c 848 ± 288a 265 ± 12c 1096 ± 118ab 912 ± 123 942 ± 85 69 ± 23b 16 ± 2ab 0.3 ± 0.0a [NaCl] (mm) Mineral content (mmol kg−1 DM) Na K % ψs K/Na Leaves Stems Roots Leaves Stems Roots Na K (mol mol−1) 0 328 ± 26a 761 ± 58a 54 ± 4a 1222 ± 69b 776 ± 45 825 ± 103 12 ± 1a 30 ± 3b 2.0 ± 0.4b 150 1569 ± 155b 1951 ± 194b 93 ± 21ab 914 ± 82a 724 ± 59 1027 ± 55 32 ± 4a 23 ± 6ab 0.5 ± 0.0a 300 2313 ± 189bc 1441 ± 280b 124 ± 14b 1116 ± 55ab 808 ± 64 760 ± 112 34 ± 4ab 15 ± 2a 0.4 ± 0.0a 400 3146 ± 633c 848 ± 288a 265 ± 12c 1096 ± 118ab 912 ± 123 942 ± 85 69 ± 23b 16 ± 2ab 0.3 ± 0.0a Values are mean ± s.e. (n = 5). Different letters indicate significant differences after a one-way ANOVA ([NaCl] for each cation per organ; P < 0.05). View Large Salinity had varying effects on total mineral contents (Fig. 6). It did not affect Mg or Ca contents in leaves and stems, while causing a significant increase in P content of stems at 300 mm NaCl, and stems and roots at 400 mm NaCl. A decrease with [NaCl] in leaf C/N was due to a significant decrease in C with increasing salinity and an increase in N at 400 mm NaCl. Fig. 6. View largeDownload slide Changes in plants after 22 d of treatment with increasing soil salinity in total Mg, Ca and P contents per organ, and leaf C/N ratio. Legends inserted. Values are mean ± s.e. (n = 5). Different letters indicate significant differences after a two-way ANOVA ([NaCl] × organ per mineral; P < 0.05). Fig. 6. View largeDownload slide Changes in plants after 22 d of treatment with increasing soil salinity in total Mg, Ca and P contents per organ, and leaf C/N ratio. Legends inserted. Values are mean ± s.e. (n = 5). Different letters indicate significant differences after a two-way ANOVA ([NaCl] × organ per mineral; P < 0.05). DISCUSSION Plants of T. triangulare tolerated treatment with up to 400 mm NaCl, PN and E decreasing markedly under 300 and 400 mm NaCl, whereas growth became significantly reduced only by 400 mm NaCl. Plants under 150 and 300 mm NaCl had values of ΔH+malate eq. significantly higher than the control, which is evidence of the operation of CAM. Nocturnal CO2 fixation was not found under any [NaCl], contrasting with previous reports on the operation of CAM in T. triangulare (Herrera et al., 1991, 2015; Pieters et al., 2003). Therefore, the occurrence of significant ΔH+ must have been due to internal recycling of night-time respiratory CO2, as previously shown in T. calycinum by Martin and Zee (1983). In contrast to previous reports (Martin and Zee, 1983; Harris and Martin, 1991), cycling, i.e. ΔH+ in watered plants without nocturnal CO2 fixation, was not observed; therefore, the correct way to designate the operation of CAM in the present work would be recycling. The increase in δ13C found in plants irrigated with 150 and 300 mm NaCl was related to higher LWC, but not increased integrated WUE. In contrast, in four out of five species of Talinum considered to be cycling CAM species, the correlation between ΔH+ and water content was higher in plants subjected to drought than in watered plants; nevertheless, no relationship was detected between ΔH+ and δ13C, which ranged from approx. −31 to −24 ‰ (Harris and Martin, 1991). Increased WUE may increase δ13C in C3 plants due to stomatal closure, as shown in wheat cultivars by Farquhar and Richards (1984). Because recycling does not contribute to C gain, it is difficult to see why recycling could change δ13C, unless part of the refixed respiratory CO2 came from non-autotrophic tissues, as these have been shown to be enriched in 13C by about 1–3 ‰ relative to autotrophic organs in C3 plants (Cernusak et al., 2009). Values of ΔH+malate eq. after 8 d of treatment with 300 mm NaCl were within the range of those reported for plants of T. triangulare under drought (50–100 µmol g−1; Herrera et al., 1991; Taisma and Herrera, 1998; Herrera, 1999; Pieters et al., 2003). The significance of CAM recycling as an adaptive response to salinity is not clear because, consistent with previous observations in plants of T. triangulare under drought (Herrera et al., 1991; Pieters et al., 2003), a high ΔH+ was not maintained in time but decreased after 8–16 d. The time under treatment with 300 mm NaCl when an increase in ΔH+ was found falls within the time range, 1–2 weeks, necessary for CAM induction by salinity in M. crystallinum, suggesting that delayed CAM induction is a long-term adaptation to diminished water availability, rather than a tolerance mechanism against high salt concentration in the cytoplasm (Lüttge, 1993). Because a high ΔH+ in the present research was not maintained over time, we conclude that CAM is a protective mechanism against the salinity shock but the actual tolerance mechanism may lie in the accumulation of ions into leaf vacuoles for osmotic adjustment, as shown in Avicennia germinans (Suárez and Sobrado, 2000). ΔH+citrate eq. was nil under any salinity, which coincides with previous observations in plants of T. triangulare under drought (Herrera et al., 2015) but contrasts with a marked diurnal rhythm in citrate found in plants of M. crystallinum under 300 mm NaCl (Herppich et al., 1995). In this example, citrate accumulation occurred previous to malate accumulation and was adjudicated a role in energy conservation before a diurnal cycle of malate became established once plants were old enough. Similarly to our results, citrate in M. crystallinum amounted to a very small proportion of the mass of total inorganic ions accumulated in the leaves under salinity. Changes in ΔH+malate eq. observed in plants of T. triangulare subjected to salinity were not caused by a decrease in leaf water status, because LWC remained fairly constant during treatment and was highest under 300 mm NaCl, where nocturnal acid accumulation was found, and ψ was not different from the control value after 8 d of treatment. Similarly, leaf water status apparently was not the trigger of CAM operation in plants of the same species under drought (Herrera et al., 2015). In plants of the inducible CAM species Guzmania monostachia under water deficit, CAM operated only in the apical leaf region, which showed no reduction in relative water content compared to the same region in watered plants performing only C3 photosynthesis (Freschi et al., 2010). An experiment in which one half of a split-root system was subjected to water deficit suggested that a root signal, possibly abscisic acid, not leaf water status, was the inducing factor of CAM in M. crystallinum (Eastmond and Ross, 1997). Increased LWC in plants under irrigation with 300 mm NaCl correlated with an increase in whole-leaf and parenchyma thickness, as found in plants of N. glauca under salinity (González et al., 2012). A similar increase in LWC was found in the mangrove A. germinans under 32 ‰ (547 mm) NaCl but in this case not only were mesophyll cells larger, but also the hypodermis duplicated its cell layers (Suárez and Sobrado, 2000). In the present study, a decrease in SI due to salt suggested that differentiation of epidermal cells into stomata became inhibited. Under salinity, SD in 14 genotypes of quinoa differing in salt tolerance diminished by up to 30 %, which was interpreted as a mechanism to deal with reduced water availability (Shabala et al., 2013). In contrast, increased SD was associated with increased tolerance to salinity in 46 genotypes of barley (Zhu et al., 2015). In view of the controversial results and interpretations given to changes in SD under salinity, the role of decreased SD or SI in the tolerance of plants is arguable. The Δψs observed was partly achieved by leaf accumulation of Na and K. In plants of T. paniculatum irrigated with saline solutions, in which salt was accumulated mainly in the root instead of leaves, increased proline content was associated with a value of OA of approx. 0.3 MPa (Assaha et al., 2017). The average Δψs by plants of T. triangulare at the end of the experiment in the three salinities was similar to the values of OA reported in plants of T. triangulare after 27 d of drought (Herrera et al., 2015) but much lower than in the mangrove A. germinans under salinity, 1.2 MPa, calculated with values of ψs at full turgor, i.e. OA (Suárez and Sobrado, 2000). In plants of N. glauca, OA was higher under salinity than under drought, but OA did not prevent PN decreasing with salinity (González et al., 2012). In contrast, plants of Lycium nodosum showed after 15 d of saline treatment an OA of 0.94 MPa with PN remaining unaltered (Tezara et al., 2003). The latter results support the hypothesis that OA contributes to the maintenance of photosynthetic activity (Munns and Tester, 2008), which, if Δψs without change in LWC is taken as a surrogate for OA, was not the case in the present study. The significance of an increase in Δψs as a means to guarantee water absorption by roots for plants of T. triangulare under salinity was uncertain, because a simple calculation using the van’t Hoff relationship indicates that plants would not be able to absorb water from the soil under any salinity, as ψ would be higher than soil water potential, which would be at most −0.7, −1.5 and −2.0 MPa for 150, 300 and 400 mm, respectively, and the value of Δψs did not contribute to reduce ψs to values lower than the soil water potential in any of the salt treatments. Given that Δψs was determined in unsaturated plants and relative water content was not measured, it would not be correct to state that that variable is the same as OA. Additionally, Δψs was measured solely in leaves. In roots of the halophyte Atriplex nummularia, a significant OA of 0.5 MPa was found when plants were irrigated with 450 and 600 mm NaCl (Silveira et al., 2009), which suggests that roots may actively contribute to increase the capacity of the plant to absorb water from the substrate. The observed reduction in ψs relative to the controls in T. triangulare apparently favoured the maintenance of values of turgor potential similar to those in controls. Our interpretation of the occurrence of Δψs is that this was caused only as a response to increased NaCl content in vacuoles, not as a mechanism to increase root water absorption. Because transpiration continued, although at a very low rate, after 21 d of treatment, we assume that E was sustained by leaf water. Salinity did not alter chlorophyll content, coinciding with observations on plants of Zygophyllum xanthoxylum, in which chlorophyll content remained unchanged although at a much lower salinity (50 mm; Ma et al., 2011). Total chlorophyll content remained unchanged after 23–24 d of drought in plants of T. triangulare (Herrera et al., 1991) and T. paniculatum (Güerere et al., 1996). In T. paniculatum total chlorophyll content even increased after irrigation with 200 and 300 mm NaCl (Assaha et al., 2017). Additional determinations, such as chlorophyll a fluorescence, should help ascertain whether salinity affected photosynthetic capacity. Unchanged chlorophyll content in the present experiment was associated with unchanged carotenoid content, supporting the antioxidative role given to carotenoids in plants under abiotic stress (Gill and Tuteja, 2010; Assaha et al., 2017). The exception to the general trend was a marked decrease in chlorophyll/carotenoids at 400 mm, the sole concentration which affected growth presumably by damage caused by salt. The high K/Na molar ratio in control plants makes T. triangulare a non-halophyte, whereas the value in plants under salinity suggests considering this a halo-tolerant species, i.e. one which, while not requiring salt to grow, can tolerate high concentration in its substrate (after Medina et al., 2008). The succulence in T. triangulare was much higher than that of halophytes, as reported by Medina et al. (2008), which surely results from its CAM characteristics. Additionally, the high Na content in control plants suggests that this species tends to concentrate Na from the substrate. In our case, soil Na conductivity in pots watered with tap water is considered to cause negligible effects on crops (0–2 dS m−1, Abrol et al., 1988). In control plants of the halophytes Atriplex portulacoides (Redondo-Gómez et al., 2007), Atriplex centralasiatica (Qiu et al., 2003) and Halostachys caspica (Zeng et al., 2015), leaf Na content was 1000, 1500 and 2600 mmol kg−1 DM, respectively. Irrigation of plants of T. triangulare with NaCl produced a significant decrease in stem biomass only at 400 mm, similarly to plants of T. paniculatum, in which 300 mm NaCl produced a 53 % reduction (Assaha et al., 2017). The biomass of the remaining organs in T. triangulare, except for the reproductive structures, was unaffected by any of the salinities, contrary to the case of T. paniculatum, in which all organs were affected (Assaha et al., 2017). Salinity had little effect on mineral contents except for increased leaf Na with [NaCl], and increased P in stems and roots at 300 and 400 mm, which indicates that the general nutritional status of plants for the duration of the experiment was adequate. The negative effects of salt on gas exchange in general and growth under 400 mm NaCl may have been due to the frequently reported effects of salt on gas exchange, and hence growth (Cheeseman, 1988), rather than nutrient deficiency. CONCLUSIONS Plants of T. triangulare tolerated salinity treatment up to 400 mm NaCl, the latter concentration decreasing growth and reproductive effort. Plants under 150 and 300 mm NaCl showed values of ΔH+ significantly higher than the control without nocturnal CO2 fixation, which is evidence of the operation of the CAM in the recycling mode. Leaf water status bore no obvious relationship with CAM induction by salinity. A low K/Na ratio in plants under salinity makes T. triangulare a halo-tolerant species. ACKNOWLEDGEMENTS This work was partly supported by grant PG-03-7983-2011/2 (Consejo de Desarrollo Científico y Humanístico, Universidad Central de Venezuela). Alejandro Pieters (IVIC, Venezuela) facilitated the use of his equipment for measurement of water relations. Jan Tillet (IEJB, Venezuela) kindly built a plastic shed for performing initial experiments. E. A. Goddard, of the Stable Isotope Biogeochemistry Laboratory, College of Marine Science, USF, performed the δ13C determinations, for which we are much obliged. LITERATURE CITED Abrol IP , Yadav JSP , Massoud FI . 1988 . Salt-affected soils and their management . Rome : Food & Agriculture Organization of the United Nations , No. 39 . Assaha DVM , Mekawy AMM , Liu L et al. 2017 . Na+ retention in the root is a key adaptive mechanism to low and high salinity in the glycophyte, Talinum paniculatum (Jacq.) Gaertn. (Portulacaceae) . Journal of Agronomy and Crop Science 203 : 56 – 67 . Google Scholar CrossRef Search ADS Bamidele JF , Egharevba RKA , Okpoh IM . 2007 . Physiological changes in seedlings of Talinum triangulare (water leaf) grown in saline conditions . Asian Journal of Plant Sciences 6 : 56 – 60 . Google Scholar CrossRef Search ADS Cernusak LA , Tcherkez G , Keitel C et al. 2009 . Why are non-photosynthetic tissues generally 13C enriched compared with leaves in C3 plants? Review and synthesis of current hypotheses . Functional Plant Biology 36 : 199 – 213 . Google Scholar CrossRef Search ADS Cheeseman JM . 1988 . Mechanisms of salinity tolerance in plants . Plant Physiology 87 : 547 – 550 . Google Scholar CrossRef Search ADS PubMed Eastmond PJ , Ross JD . 1997 . Evidence that the induction of Crassulacean acid metabolism by water stress in Mesembryanthemum crystallinum (L.) involves root signalling . Plant, Cell & Environment 20 : 1559 – 1565 . Google Scholar CrossRef Search ADS Farquhar GD , Richards RA . 1984 . Isotopic composition of plant carbon correlates with water-use efficiency of wheat genotypes . Australian Journal of Plant Physiology 11 : 539 – 552 . Google Scholar CrossRef Search ADS Freschi L , Takahashi CA , Cambui CA et al. 2010 . Specific leaf areas of the tank bromeliad Guzmania monostachia perform distinct functions in response to water shortage . Journal of Plant Physiology 167 : 526 – 533 . Google Scholar CrossRef Search ADS PubMed Gill SS , Tuteja N . 2010 . Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants . Plant Physiology and Biochemistry 48 : 909 – 930 . Google Scholar CrossRef Search ADS PubMed González A , Tezara W , Rengifo E , Herrera A . 2012 . Ecophysiological responses to drought and salinity in the cosmopolitan invader Nicotiana glauca . Brazilian Journal of Plant Physiology 24 : 212 – 222 . Google Scholar CrossRef Search ADS Güerere I , Tezara W , Herrera C , Fernández M , Herrera A . 1996 . Recycling of CO2 during induction of CAM by drought in Talinum paniculatum (Portulacaceae) . Physiologia Plantarum 98 : 471 – 476 . Google Scholar CrossRef Search ADS Harris FS , Martin CE . 1991 . Correlation between CAM-cycling and photosynthetic gas exchange in five species of Talinum (Portulacaceae) . Plant Physiology 96 : 1118 – 1124 . Google Scholar CrossRef Search ADS PubMed Herrera A . 1999 . Effects of photoperiod and drought on the induction of CAM and the reproduction of plants of Talinum triangulare . Canadian Journal of Botany 77 : 1 – 6 . Google Scholar CrossRef Search ADS Herrera A . 2009 . Crassulacean acid metabolism and fitness under water deficit stress: if not for carbon gain, what is facultative CAM good for ? Annals of Botany 103 : 645 – 653 . Google Scholar CrossRef Search ADS PubMed Herrera A , Delgado J , Paraguatey I . 1991 . Occurrence of inducible crassulacean acid metabolism in leaves of Talinum triangulare (Portulacaceae) . Journal of Experimental Botany 42 : 493 – 499 . Google Scholar CrossRef Search ADS Herrera A , Ballestrini C , Montes E . 2015 . What is the potential for dark CO2 fixation in the facultative crassulacean acid metabolism species Talinum triangulare ? Journal of Plant Physiology 174 : 55 – 61 . Google Scholar CrossRef Search ADS PubMed Herppich M , von Willert DJ , Herppich WB . 1995 . Diurnal rhythm in citric acid content preceded the onset of nighttime malic acid accumulation during metabolic changes from C3 to CAM in salt-stressed plants of Mesembryanthemum crystallinum . Journal of Plant Physiology 147 : 38 – 42 . Google Scholar CrossRef Search ADS Lüttge U . 1993 . The role of crassulacean acid metabolism (CAM) in the adaptation of plants to salinity . New Phytologist 125 : 59 – 71 . Google Scholar CrossRef Search ADS Ma Q , Yue LJ , Zhang JL , Wu GQ , Bao AK , Wang SM . 2011 . Sodium chloride improves photosynthesis and water status in the succulent xerophyte Zygophyllum xanthoxylum . Tree Physiology 32 : 4 – 13 . Google Scholar CrossRef Search ADS PubMed Martin C , Zee A . 1983 . C3 photosynthesis and CAM in a Kansas rock outcrop succulent, Talinum calycinum (Portulacaceae) . Plant Physiology 73 : 718 – 723 . Google Scholar CrossRef Search ADS PubMed Medina E , Francisco AM , Wingfield R , Casañas OL . 2008 . Halophytism in plants of the Caribbean coast of Venezuela: halophytes and halotolerants . Acta Botanica Venezuelica 31 : 49 – 80 . Munns R , Tester M . 2008 . Mechanism of salinity tolerance . Annual Review of Plant Biology 59 : 651 – 681 . Google Scholar CrossRef Search ADS PubMed Murphy J , Riley JP . 1962 . A modified single solution method for the determination of phosphate in natural waters . Analytica Chimica Acta 27 : 31 – 36 . Google Scholar CrossRef Search ADS Niewiadomska E , Karpinska B , Romanowska E , Slesak I , Karpinski S . 2004 . A salinity-induced C3-CAM transition increases energy conservation in the halophyte Mesembryanthemum crystallinum . Plant and Cell Physiology 45 : 789 – 794 . Google Scholar CrossRef Search ADS PubMed Osmond CB . 1978 . Crassulacean acid metabolism: a curiosity in context . Annual Review of Plant Physiology 29 : 379 – 414 . Google Scholar CrossRef Search ADS Pieters AJ , Tezara W , Herrera A . 2003 . Operation of the xanthophyll cycle and degradation of D1 protein in the inducible CAM plant, Talinum triangulare, under water deficit . Annals of Botany 92 : 393 – 399 . Google Scholar CrossRef Search ADS PubMed Qiu N , Lu Q , Lu C . 2003 . Photosynthesis, photosystem II efficiency and the xanthophyll cycle in the salt‐adapted halophyte Atriplex centralasiatica . New Phytologist 159 : 479 – 486 . Google Scholar CrossRef Search ADS Redondo-Gómez S , Mateos-Naranjo E , Davy AJ et al. 2007 . Growth and photosynthetic responses to salinity of the salt-marsh shrub Atriplex portulacoides . Annals of Botany 100 : 555 – 563 . Google Scholar CrossRef Search ADS PubMed Salisbury EJ . 1927 . On the causes and ecological significance of stomatal frequency, with special reference to the woodland flora . Proceedings of the Royal Society of London, Series B 216 : 303 – 309 . Shabala S , Hariadi Y , Jacobsen SE . 2013 . Genotypic difference in salinity tolerance in quinoa is determined by differential control of xylem Na+ loading and stomatal density . Journal of Plant Physiology 170 : 906 – 914 . Google Scholar CrossRef Search ADS PubMed Silveira JAG , Araújo SAM , Lima JPMS , Viégas RA . 2009 . Roots and leaves display contrasting osmotic adjustment mechanisms in response to NaCl-salinity in Atriplex nummularia . Environmental and Experimental Botany 66 : 1 – 8 . Google Scholar CrossRef Search ADS Suárez N , Sobrado MA . 2000 . Adjustments in leaf water relations of mangrove (Avicennia germinans) seedlings grown in a salinity gradient . Tree Physiology 20 : 277 – 282 . Google Scholar CrossRef Search ADS PubMed Taisma MA , Herrera A . 1998 . A relationship between fecundity, survival and the operation of CAM in Talinum triangulare . Canadian Journal of Botany 76 : 1 – 8 . Google Scholar CrossRef Search ADS Tezara W , Martinez D , Rengifo E , Herrera A . 2003 . Photosynthetic responses of the tropical spiny shrub Lycium nodosum (Solanaceae) to drought, soil salinity and saline spray . Annals of Botany 92 : 757 – 765 . Google Scholar CrossRef Search ADS PubMed Ukpong IE , Moses JO . 2001 . Nutrient requirements for the growth of waterleaf (Talinum triangulare) in Uyo metropolis, Nigeria . The Environmentalist 21 : 153 – 159 . Google Scholar CrossRef Search ADS Wellburn A . 1994 . The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution . Journal of Plant Physiology 144 : 307 – 313 . Google Scholar CrossRef Search ADS Winter K , Holtum JAM . 2002a . The effects of salinity, crassulacean acid metabolism and plant age on the carbon isotopic composition of Mesembryanthemum crystallinum L., a halophytic C3-CAM species . Planta 222 : 201 – 209 . Google Scholar CrossRef Search ADS Winter K , Holtum JAM . 2002b . How closely do the δ13C values of crassulacean acid metabolism plants reflect the proportion of CO2 fixed during day and night ? Plant Physiology 129 : 1843 – 1851 . Google Scholar CrossRef Search ADS Zeng Y , Li L , Yan R , Yi X , Zhang B . 2015 . Contribution and distribution of inorganic ions and organic compounds to the osmotic adjustment in Halostachys caspica response to salt stress . Scientific Reports 5 : 13639 . Google Scholar CrossRef Search ADS PubMed Zhu M , Zhou M , Shabala L , Shabala S . 2015 . Linking osmotic adjustment and stomatal characteristics with salinity stress tolerance in contrasting barley accessions . Functional Plant Biology 42 : 252 – 263 . Google Scholar CrossRef Search ADS © The Author(s) 2018. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. 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Salinity induction of recycling Crassulacean acid metabolism and salt tolerance in plants of Talinum triangulare

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
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10.1093/aob/mcy030
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Abstract

Abstract Background and Aims Crassulacean acid metabolism (CAM) can be induced by salinity, thus conferring the plant higher water-use efficiency. Talinum triangulare does not frequently encounter salt in its natural habitat but is cultivated in soils that may become salinized. Here we examined whether plants of T. triangulare can grow in saline soils and show salt-induced CAM. Methods Leaf gas exchange, carbon isotopic ratio (δ13C), nocturnal acid accumulation (ΔH+), water relations, photosynthetic pigment and mineral contents, leaf anatomy and growth were determined in greenhouse in plants irrigated with 0, 150, 300 and 400 mm NaCl. Key Results Salinity reduced gas exchange and induced CAM, ΔH+ reaching 50.2 μmol H+ g−1 fresh mass under 300 mm NaCl. No nocturnal CO2 uptake, but compensation, was observed. Values of δ13C were lowest under 0 and 400 mm NaCl, and highest under 150 and 300 mm. The difference in osmotic potential (ψs) between control and treated plants averaged 0.45 MPa for the three [NaCl] values, the decrease in ψs being accounted for by up to 63 % by Na+ and K+. Pigment contents were unaffected by treatment, suggesting lack of damage to the photosynthetic machinery. Changes in stomatal index with unchanged stomatal density in newly expanded leaves suggested inhibited differentiation of epidermal cells into stomata. Whole-leaf and parenchymata thickness increased under 150 and 300 mm NaCl. Only plants irrigated with 400 mm NaCl showed reductions in biomass (stems, 41 %; reproductive structures, 78 %). The K/Na molar ratio decreased with [NaCl] from 2.0 to 0.4. Conclusions The operation of CAM in the recycling mode was evidenced by increased ΔH+ with no nocturnal CO2 uptake. Talinum triangulare can be classified as a halo-tolerant species based on its low K/Na molar ratio under salinity and the relatively small reduction in growth only at the highest [NaCl]. CAM, Crassulacean acid metabolism, Talinum triangulare, mineral content, salinity INTRODUCTION Plants can respond to salinity by either tolerating osmotic stress, excluding Na+ or Cl−, or accumulating Na+ or Cl−; these responses can vary in time and work in combination with each other (Munns and Tester, 2008). Osmotic adjustment is a mechanism which some plants employ to reduce osmotic potential (ψs) and increase turgor potential against water deficit conditions, including salinity (Munns and Tester, 2008). Osmotic adjustment has been shown to occur under salinity in many glycophytes, such as Nicotiana glauca, which are not usually subjected to saline soils (González et al., 2012). Induction by drought of Crassulacean acid metabolism (CAM) has been reported in Talinum triangulare by, among others, Herrera et al. (1991, 2015), Taisma and Herrera (1998), and Pieters et al. (2003). The possibility exists that salinity may be an inducer of CAM in T. triangulare, as in Mesembryanthemum crystallinum (Winter and Holtum, 2002a). In plants of M. crystallinum under exposure to 400 mm NaCl, δ13C values were higher (−21.0 ‰) than without salt (between −28.3 and −29.5 ‰; Winter and Holtum, 2002a), evidencing a switch from C3 metabolism to CAM. During a daily cycle of gas exchange in a CAM plant, four phases can be distinguished, phase I taking place during the night, phase II at dawn and phases III and IV during the day; for a description of the four phases, see Niewiadomska et al. (2004). CAM can occur in several modes, which include the constitutive or obligate mode, the cycling mode, with nocturnal acid accumulation but no dark CO2 fixation in watered plants, and inducible/facultative or C3-CAM (Herrera, 2009), with moderate ΔH+ and low dark CO2 fixation. CAM induction is considered an adaptive strategy that increases water use efficiency (Herrera, 2009), which is particularly important for plants living in arid and/or saline environments (Niewiadomska et al., 2004). The main diagnostic characters of CAM are nocturnal acid accumulation (ΔH+) and nocturnal stomatal opening; mesophyll succulence apparently is a requisite for CAM performance (Osmond, 1978; but see Herrera, 2009). Values of δ13C of photosynthetic organs may help in assessing the operation of CAM (Herrera, 2009). The δ13C of plants that acquire one-third or less of their carbon in the dark may be confused with that of C3 plants when the carbon fixation pathway is assigned solely on the basis of δ13C (Winter and Holtum, 2002b). Values of δ13C between −30 and −21 ‰ suggest operation of weak CAM in two of its modes, i.e. cycling or facultative (Herrera, 2009). Plants of T. triangulare and T. paniculatum do not frequently grow in saline soils. Plants of T. triangulare tolerated up to 560 mm NaCl (Bamidele et al., 2007) and those of T. paniculatum, up to 300 mm NaCl (Assaha et al., 2017). In T. triangulare decreases in average fresh mass (FM) of leaves, stems and roots of 87 % and dry mass (DM) of 82 % were noted, leaf succulence remaining unchanged at up to 280 mm NaCl (Bamidele et al., 2007). Thus far, no report of CAM induction by salinity has been published for either species. Leaves of T. triangulare are an unconventional vegetable (waterleaf) consumed in some parts of Venezuela and more widely in Indonesia and Nigeria; plants are extensively cultivated in Nigeria in garden soils composed of up to 77 % sand containing 2.3 mmol kg−1 Na (Ukpong and Moses, 2001). Plants of T. triangulare are perennial sub-shrubs approx. 50 cm tall with partly succulent leaves. In Venezuela, plants grow equally well under full sun exposure or in the forest understorey, from 0 to 100 m, in humid as well as semi-arid regions. They may occasionally be found 25 m inland but this is relatively uncommon. Plants of T. triangulare under drought with CAM induced were shown to achieve osmotic adjustment (OA) using malate as osmoticum (Herrera et al., 2015). This OA would only be effective in improving water absorption by the roots during phases I and II of CAM, before all malate became decarboxylated. In halophytes, OA is not transitory, as would be the case in CAM plants; in a CAM halophyte, OA could be achieved through accumulation of other osmotica in the vacuole which might not be reverted during the day–night cycle. Under salinity, leaves tend to become more succulent because of increased cell volume or number (Suárez and Sobrado, 2000; González et al., 2012). Changes in stomatal characteristics have been reported in barley (Zhu et al., 2015) and quinoa (Shabala et al., 2013). Medina et al. (2008) separated species growing naturally in saline soils on the basis of their leaf K/Na ratio into halophytes (K/Na < 0.1) and non-halophytes and, among the latter, into halo-resistant (K/Na < 1) and halo-tolerant (K/Na between 0.1 and 1.0). This separation correlated well with succulence [Suc = (FM − DM)/FM], halophytes having values of 88 ± 1 % and non-halophytes, 76 ± 4 %. We hypothesized that CAM may be induced by salinity in T. triangulare. To determine whether plants of T. triangulare can grow in saline soils and whether CAM is induced in them by salinity, we subjected plants to a range of concentrations of NaCl and determined daily gas exchange, δ13C, ΔH+, water relations, leaf anatomical characteristics, growth analysis variables and mineral contents. MATERIALS AND METHODS Plant material and growth conditions Adult plants of T. triangulare Jacq. Willd. [syn. Talinum fruticosum (L.) Juss.], similar in height and foliage, were grown in 8-L plastic pots filled with 6.0 ± 0.5 kg silty-clay loamy soil purchased at a nursery (Viveros Exotica Raphia, S.R.L., Caracas, Venezuela) in a transparent polythene-roofed shed at the Instituto de Biología Experimental in Caracas. Plants were watered daily with tap water and weekly with nutrient solution (1 g L−1, Campos Green Multi-Plants-18-18-18 soluble fertilizer, Inversiones Green Valley C.A., Caracas, Venezuela). After 2 months, five control plants were watered every other day with tap water and weekly with nutrient solution, and three groups of five plants each watered every other day with 150, 300 and 400 mm NaCl and once a week with these solutions plus fertilizer; water or solutions were added until a few millilitres leached from the bottom. After 3 d of irrigation with each of the saline solutions, the water potential of the leachate, determined as for leaf water potential, ψ (see below), was very similar to that of the corresponding irrigation solution. After 21 d of treatment, plants were harvested and organs dried for measurement of DM. For measurements, only the youngest, fully expanded leaves were used. Microclimatic variables inside the greenhouse Day length was 12 h (0600–18:00 h). Photosynthetic photon flux density (PFD) was measured with a 190-S quantum sensor connected to an LI-185 meter (LI-COR Inc., Lincoln, NE, USA). Air temperature and relative humidity were measured using a HOBO Pro V2 logger and data dumped with a HOBO Waterproof Shuttle (Onset Computer Corporation, Pocasset, MA, USA). Microclimatic conditions in the greenhouse were (min/max): air temperature 15 ± 2/34 ± 2 °C, relative humidity 45 ± 4/83 ± 2 %, and maximum PFD 1035 ± 65 µmol m−2 s−1. Gas exchange Assimilation rate (PN), and transpiration rate (E) were measured in intact leaves with a CIRAS 2 infrared CO2 and H2O analyser connected to a PLC(B) assimilation chamber (PP Systems Inc., Amesbury, MA, USA). The source of CO2 was ambient air; [CO2] = 403 ± 1 µmol mol−1, leaf temperature 24.4 ± 0.1 °C and PFD 1000 µmol m−2 s−1, previously found to saturate PN. Water use efficiency was calculated as WUE = PN/E. Daily courses in one leaf per plant per treatment were done, with readings recorded automatically every 30 min. Different plants were used for measurements every week of treatment. The amount of water lost by transpiration per day was calculated by integrating the area below courses of E. Diurnal courses of integrated WUE were calculated as integrated PN/integrated diurnal E. Nocturnal acid accumulation On each sampling date one leaf from the third or fourth node of each of the five plants per group was collected in the morning and afternoon. Leaves were weighed and their length and width measured to calculate leaf area using an allometric relationship. Leaves were cut into small segments and boiled in 20 mL distilled water for 3 min in a microwave oven at full power. Extracts were passed through a colander, which was rinsed into a beaker, and titrated with 10 mm KOH to reach pH 7.0 for malate equivalents and 8.4 for citrate equivalents (details in Herrera et al., 2015), measured with an Orion pH meter (Thermo Fisher Scientific, Waltham, MA, USA). ΔH+ was calculated as the difference between morning and afternoon H+ contents at the two pH values (ΔH+malate eq. and ΔH+citrate eq). To determine how early in the afternoon and how late in the morning samples could be collected for the correct determination of ΔH+, a time-course of decarboxylation was previously done on plants that had been subjected to 7 d of drought. The latest morning and earliest afternoon times for leaf collection were found to be 0800 and 1600 h. Leaf carbon isotopic composition δ13C was determined in leaf dry ground samples using a ThermoFinnigan DeltaPlusXL mass spectrometer (Isotope Ratio Mass Spectrometer, San Jose, CA, USA) with an accuracy of 0.15 ‰ and Pee Dee Belemnite as standard. Water relations Leaf FM and area were determined in one leaf per plant and dried for 48 h at 60 °C to determine DM. Leaf water content was calculated as LWC = (FM − DM)/area (mg cm−2) or LWC = (FM − DM)/DM. The value of ψ was determined at 0800 0830 and 1200 1230 h in leaf discs placed in C-52 chambers connected to an HR-33T micro-voltmeter (Wescor Inc., Logan, UT, USA) operated in the psychrometric mode. The ψs was measured in the same discs frozen at −20 °C for 72 h and placed without thawing in the chambers. The change in ψs (Δψs) was determined for both morning and noon values of ψs as the difference between ψs of control plants and plants under salinity, without previous rehydration. Leaf pigment contents and anatomy Chlorophyll and carotenoid contents were determined in extracts obtained by submersing discs in 90 % ethanol and a small amount of CaCO3 for 3 d at 4 °C; absorbance was measured at 470, 649 and 665 nm according to Wellburn (1994). Tissue thickness and number of tissue cells were determined in leaf freehand sections fixed in formalin–acetic acid–alcohol and stained with toluidine blue; stomata were observed on nail-varnish imprints of leaf epidermes. Photographs were taken under the microscope at 40× (leaf sections) and 100× (stomata) and processed with the ImageJ software. Stomatal index was calculated after Salisbury (1927) as SI = number of stomata/(number of stomata + epidermal cells). Growth analysis and plant mineral contents At the end of treatment plants were harvested and their organs separated and dried at 60 °C for 72 h. Reproductive effort was calculated as RE = DM of reproductive structures/leaf DM, after Taisma and Herrera (1998). Reproductive structures comprised inflorescence peduncles, flowers, fruits and seeds from open mature fruits. Total cation contents were determined in powdered leaves, stems and roots by digestion with 4: 1 sulphuric acid/perchloric acid, and soluble cations in hot-water digestions, using a flame atomic absorption spectrometer (Varian Spectra AA mod. 55B). Total P content was determined in acid digestions as above after Murphy and Riley (1962). Standards used were: Na, Merck CertiPUR 1.19507.0500 1000 mg; Ca, Merck CertiPUR 1.19778.0500 1000 mg; K, Merck Titrisol 1.09924 1000 mg; and Mg, Merck Titrisol 1.09949 1000 mg. Leaf C and N contents were determined as for δ13C. The contribution to morning ψs of soluble Na and K was calculated based on values of LWC, FM/DM and ion contents per DM. Soil Na and K contents were measured in aqueous solutions (0.1 g in 0.025 mL distilled water). Soil electrical conductivity (EC) was measured in the same solutions using an EC Meter 19101-00 with a Pt cell (Cole Parmer, Vernon Hills, IL, USA). Statistics Values are given as mean ± s.e. (n = 5). Statistical significance was assessed where indicated through one- or two-way analysis of variance (P < 0.05) with the Statistica package. RESULTS Gas exchange and nocturnal acid accumulation Daily courses of PN and E (Fig. 1) remained relatively similar during treatment at 0 mm NaCl and on day 1 of treatment with 150, 300 and 400 mm NaCl, and after 8 d of treatment with 150 mm NaCl. For the rest of the time and the other salinities, significant reductions in both PN and E were found, the most drastic occurring after 21 d under 300 and 400 mm NaCl. After 21 d nocturnal CO2 compensation was measured for all three salinities. A distinct Phase II CO2 exchange pattern was found in the morning at lights-on under all salinities after 8 and 21 d. Daily courses of E showed the same trend as PN. Carbon gain diminished after 21 d to 8 % of control for all salinities. Fig. 1. View largeDownload slide Changes under treatment with increasing soil salinity in assimilation rate (PN, closed circles) and transpiration rate (E, open circles). The [NaCl] and time under treatment are indicated in each panel. Values are data points measured in a different plant per treatment every day. The filled bar on the abscissa indicates the duration of the night. Fig. 1. View largeDownload slide Changes under treatment with increasing soil salinity in assimilation rate (PN, closed circles) and transpiration rate (E, open circles). The [NaCl] and time under treatment are indicated in each panel. Values are data points measured in a different plant per treatment every day. The filled bar on the abscissa indicates the duration of the night. Diurnal integrated WUE decreased after 21 d of treatment; values of δ13C were lowest under 0 and 400 mm NaCl, and highest under 150 and 300 mm (Table 1). The mass of water transpired during the light period decreased from more than 100 % of LWC to an average of 25 % (Table 1). Table 1. Values of the proportion of leaf water content (LWC) transpired diurnally (E), integrated diurnal water use efficiency (WUE = integrated PN/integrated E) and carbon isotopic composition (δ13C) in leaves of plants of Talinum triangulare subjected for 1 and 21 d to irrigation with solutions of different [NaCl]. [NaCl] (mm) E (% of LWC) WUE (mmol mol−1 d−1) δ13C (‰) Day 1 Day 21 Day 1 Day 21 0 151 134 2.6 4.6 −27.73 ± 0.37a 150 102 29 2.2 3.4 −26.12 ± 0.23b 300 138 25 2.7 0.5 −25.49 ± 0.41b 400 156 18 4.2 1.0 −28.20 ± 0.40a [NaCl] (mm) E (% of LWC) WUE (mmol mol−1 d−1) δ13C (‰) Day 1 Day 21 Day 1 Day 21 0 151 134 2.6 4.6 −27.73 ± 0.37a 150 102 29 2.2 3.4 −26.12 ± 0.23b 300 138 25 2.7 0.5 −25.49 ± 0.41b 400 156 18 4.2 1.0 −28.20 ± 0.40a Values of E and WUE are data points; values of δ13C are mean ± s.e. (n = 5). Different letters indicate significant differences after a one-way ANOVA (P < 0.05). View Large Table 1. Values of the proportion of leaf water content (LWC) transpired diurnally (E), integrated diurnal water use efficiency (WUE = integrated PN/integrated E) and carbon isotopic composition (δ13C) in leaves of plants of Talinum triangulare subjected for 1 and 21 d to irrigation with solutions of different [NaCl]. [NaCl] (mm) E (% of LWC) WUE (mmol mol−1 d−1) δ13C (‰) Day 1 Day 21 Day 1 Day 21 0 151 134 2.6 4.6 −27.73 ± 0.37a 150 102 29 2.2 3.4 −26.12 ± 0.23b 300 138 25 2.7 0.5 −25.49 ± 0.41b 400 156 18 4.2 1.0 −28.20 ± 0.40a [NaCl] (mm) E (% of LWC) WUE (mmol mol−1 d−1) δ13C (‰) Day 1 Day 21 Day 1 Day 21 0 151 134 2.6 4.6 −27.73 ± 0.37a 150 102 29 2.2 3.4 −26.12 ± 0.23b 300 138 25 2.7 0.5 −25.49 ± 0.41b 400 156 18 4.2 1.0 −28.20 ± 0.40a Values of E and WUE are data points; values of δ13C are mean ± s.e. (n = 5). Different letters indicate significant differences after a one-way ANOVA (P < 0.05). View Large Salinity produced morning values of H+ content (malate eq.) significantly higher than afternoon values at 150 and 300 mm NaCl after 8 and 16 d of treatment, resulting after 8 d in maximum ΔH+malate eq. of 35.9 ± 8.7 and 50.2 ± 14.7 µmol H+ g−1 for 150 and 300 mm NaCl, respectively. There were no significant differences between morning and afternoon H+citrate eq. contents (Fig. 2). Fig. 2. View largeDownload slide Changes with time under treatment with increasing soil salinity in morning and afternoon H+ contents corresponding to malate and citrate equivalents. Open symbols, afternoon; closed symbols, morning. The acid is indicated above the uppermost panels, and [NaCl] in each panel. Values are mean ± s.e. (n = 5). Asterisks indicate significant differences between morning and afternoon values after a one-way ANOVA (time of day; P < 0.05). Fig. 2. View largeDownload slide Changes with time under treatment with increasing soil salinity in morning and afternoon H+ contents corresponding to malate and citrate equivalents. Open symbols, afternoon; closed symbols, morning. The acid is indicated above the uppermost panels, and [NaCl] in each panel. Values are mean ± s.e. (n = 5). Asterisks indicate significant differences between morning and afternoon values after a one-way ANOVA (time of day; P < 0.05). Water relations Figure 3 shows the time-course of changes in morning and noon values of ψ and ψs. Νo significant differences in ψ or ψs were found between morning and noon values. After 21 d of treatment, values of morning ψs were significantly lower than morning ψ in some, but not all, salinities relative to their control values. Fig. 3. View largeDownload slide Changes with time under treatment with increasing soil salinity in morning and noon leaf water (ψ) and osmotic (ψs) potential. Each time course for every salinity and time of day has its corresponding control course. Time of day is indicated above the uppermost panels, and [NaCl] in each panel. Circles, ψ; triangles, ψs. Open symbols, control (0 mm NaCl); closed symbols, salinity. Values are mean ± s.e. (n = 5). Different letters indicate significant differences at the end of treatment after a two-way ANOVA done separately for morning and noon values ([NaCl] × ψ or ψs; P < 0.05). Fig. 3. View largeDownload slide Changes with time under treatment with increasing soil salinity in morning and noon leaf water (ψ) and osmotic (ψs) potential. Each time course for every salinity and time of day has its corresponding control course. Time of day is indicated above the uppermost panels, and [NaCl] in each panel. Circles, ψ; triangles, ψs. Open symbols, control (0 mm NaCl); closed symbols, salinity. Values are mean ± s.e. (n = 5). Different letters indicate significant differences at the end of treatment after a two-way ANOVA done separately for morning and noon values ([NaCl] × ψ or ψs; P < 0.05). At the end of treatment, turgor potential was 0.21 ± 0.07, 0.28 ± 0.09, 0.35 ± 0.11 and 0.23 ± 0.10 MPa for 0, 150, 300 and 400 mm NaCl, respectively (P = 0.80). There were no significant differences among the three salinity treatments in Δψs calculated after 21 d of treatment with either morning or noon values of ψs, Δψs averaging 0.45 ± 0.04 MPa. The proportional contribution of malate and citrate equivalents to ψs was the same for all salinity treatments (P = 0.65), averaging 0.6 ± 0.1 %. Pigments, leaf anatomy and succulence Figure 4 shows changes in photosynthetic pigment contents with treatment. At the end of the experiment, total chlorophyll content decreased slightly or not at all in control and at all salinities, with no significant differences between control and salinity. Carotenoid content was not affected by either treatment or time under it, while total chlorophyll/carotenoids decreased slightly under all [NaCl] values except for 400 mm, where the reduction was more marked. Fig. 4. View largeDownload slide Changes in plants under treatment with increasing soil salinity in: A, total chlorophyll content; B, total carotenoid content; and C, chlorophyll/carotenoids. Values are mean ± s.e. (n = 5). Legend is given in panel A: empty bars, day 1 of treatment; striped bars, day 22 of treatment. Different letters indicate significant differences after a two-way ANOVA ([NaCl] × time; P < 0.05). Fig. 4. View largeDownload slide Changes in plants under treatment with increasing soil salinity in: A, total chlorophyll content; B, total carotenoid content; and C, chlorophyll/carotenoids. Values are mean ± s.e. (n = 5). Legend is given in panel A: empty bars, day 1 of treatment; striped bars, day 22 of treatment. Different letters indicate significant differences after a two-way ANOVA ([NaCl] × time; P < 0.05). Table 2 summarizes changes in leaf anatomical characteristics due to treatment. The sole significant differences were an increase in whole-leaf thickness under 150, 300 and 400 mm NaCl, and a slight increase in palisade parenchyma thickness under 300 mm NaCl and spongy mesophyll thickness under 150 and 300 mm NaCl. The other tissues and the number of cells per parenchyma (data not shown) suffered no significant changes due to the treatment × time interaction. Morning LWC was highest at the end of the experiment under any salinity, the interaction between [NaCl] and time under treatment not being significant. Values of integrated E showed that leaves transpired 39 % their own water at the end of treatment. Values of Suc at any time and salinity were 94 ± 0 %, with no significant differences between treatments. Stomatal density (SD) of the abaxial face showed no significant differences due to treatment at the end of the experiment, and adaxial SD was lower than abaxial SD, showing slight changes with treatment. At the end of the experiment, SI in both leaf faces of plants under saline treatments remained generally unchanged by treatment, except for a significant decrease in both faces relative to control plants. Table 2. Changes with time under treatment with NaCl solutions in whole-leaf and tissue thickness, morning leaf water content (LWC) and stomatal density (SD) and index (SI) [NaCl] (mm) 0 150 300 400 Initial Final Initial Final Initial Final Initial Final P Thickness (µm)  Whole leaf 755 ± 92abc 771 ± 58bc 569 ± 69a 927 ± 32cd 624 ± 50ab 971 ± 76d 566 ± 35ab 643 ± 70ab 0.02  Upper epidermis 37 ± 4a 37 ± 1a 32 ± 2a 37 ± 6a 30 ± 2a 30 ± 3a 29 ± 3a 33 ± 5a 0.92  Palisade parenchyma 362 ± 45ab 372 ± 39ab 268 ± 39ab 477 ± 17ab 288 ± 31ab 465 ± 35b 271 ± 20ab 297 ± 48a 0.01  Spongy parenchyma 314 ± 50ab 316 ± 19ab 247 ± 40a 373 ± 16bc 275 ± 23a 451 ± 41c 225 ± 13a 273 ± 21a 0.04  Lower epidermis 29 ± 4a 35 ± 2b 19 ± 2a 26 ± 3ab 22 ± 1a 25 ± 4a 28 ± 3ab 28 ± 4ab 0.65 LWC (mg cm−2) 89 ± 8ab 107 ± 9ab 95 ± 9ab 115 ± 6bc 95 ± 14ab 138 ± 12c 79 ± 6a 107 ± 12abc 0.61  (g water g−1 DM) 16.0 ± 1.0 14.3 ± 1.3 15.3 ± 0.6 14.8 ± 0.7 14.5 ± 1.8 17.0 ± 0.9 15.0 ± 0.9 15.8 ± 0.6 0.57 SD - abaxial (mm−2) 85 ± 19a 151 ± 11b 133 ± 23ab 106 ± 17ab 97 ± 22ab 92 ± 7a 121 ± 4ab 133 ± 25ab 0.06  SD - adaxial (mm−2) 36 ± 5a 72 ± 5b 69 ± 13b 50 ± 3ab 49 ± 10ab 40 ± 3a 53 ± 5ab 52 ± 13ab 0.09 SI - abaxial (%) 17 ± 1ab 29 ± 2c 19 ± 1ab 20 ± 2ab 16 ± 2ab 21 ± 1b 17 ± 1ab 16 ± 2a 0.00  SI - adaxial (%) 13 ± 0a 25 ± 1b 14 ± 1a 18 ± 1a 15 ± 2a 14 ± 2a 13 ± 1a 13 ± 3a 0.00 [NaCl] (mm) 0 150 300 400 Initial Final Initial Final Initial Final Initial Final P Thickness (µm)  Whole leaf 755 ± 92abc 771 ± 58bc 569 ± 69a 927 ± 32cd 624 ± 50ab 971 ± 76d 566 ± 35ab 643 ± 70ab 0.02  Upper epidermis 37 ± 4a 37 ± 1a 32 ± 2a 37 ± 6a 30 ± 2a 30 ± 3a 29 ± 3a 33 ± 5a 0.92  Palisade parenchyma 362 ± 45ab 372 ± 39ab 268 ± 39ab 477 ± 17ab 288 ± 31ab 465 ± 35b 271 ± 20ab 297 ± 48a 0.01  Spongy parenchyma 314 ± 50ab 316 ± 19ab 247 ± 40a 373 ± 16bc 275 ± 23a 451 ± 41c 225 ± 13a 273 ± 21a 0.04  Lower epidermis 29 ± 4a 35 ± 2b 19 ± 2a 26 ± 3ab 22 ± 1a 25 ± 4a 28 ± 3ab 28 ± 4ab 0.65 LWC (mg cm−2) 89 ± 8ab 107 ± 9ab 95 ± 9ab 115 ± 6bc 95 ± 14ab 138 ± 12c 79 ± 6a 107 ± 12abc 0.61  (g water g−1 DM) 16.0 ± 1.0 14.3 ± 1.3 15.3 ± 0.6 14.8 ± 0.7 14.5 ± 1.8 17.0 ± 0.9 15.0 ± 0.9 15.8 ± 0.6 0.57 SD - abaxial (mm−2) 85 ± 19a 151 ± 11b 133 ± 23ab 106 ± 17ab 97 ± 22ab 92 ± 7a 121 ± 4ab 133 ± 25ab 0.06  SD - adaxial (mm−2) 36 ± 5a 72 ± 5b 69 ± 13b 50 ± 3ab 49 ± 10ab 40 ± 3a 53 ± 5ab 52 ± 13ab 0.09 SI - abaxial (%) 17 ± 1ab 29 ± 2c 19 ± 1ab 20 ± 2ab 16 ± 2ab 21 ± 1b 17 ± 1ab 16 ± 2a 0.00  SI - adaxial (%) 13 ± 0a 25 ± 1b 14 ± 1a 18 ± 1a 15 ± 2a 14 ± 2a 13 ± 1a 13 ± 3a 0.00 Values are mean ± s.e. (n = 5). Different letters indicate significant differences after a two-way ANOVA ([NaCl] × time; P < 0.05). The value of P for the interaction time × [NaCl] is indicated. Values of P < 0.05 are shown in bold type. View Large Table 2. Changes with time under treatment with NaCl solutions in whole-leaf and tissue thickness, morning leaf water content (LWC) and stomatal density (SD) and index (SI) [NaCl] (mm) 0 150 300 400 Initial Final Initial Final Initial Final Initial Final P Thickness (µm)  Whole leaf 755 ± 92abc 771 ± 58bc 569 ± 69a 927 ± 32cd 624 ± 50ab 971 ± 76d 566 ± 35ab 643 ± 70ab 0.02  Upper epidermis 37 ± 4a 37 ± 1a 32 ± 2a 37 ± 6a 30 ± 2a 30 ± 3a 29 ± 3a 33 ± 5a 0.92  Palisade parenchyma 362 ± 45ab 372 ± 39ab 268 ± 39ab 477 ± 17ab 288 ± 31ab 465 ± 35b 271 ± 20ab 297 ± 48a 0.01  Spongy parenchyma 314 ± 50ab 316 ± 19ab 247 ± 40a 373 ± 16bc 275 ± 23a 451 ± 41c 225 ± 13a 273 ± 21a 0.04  Lower epidermis 29 ± 4a 35 ± 2b 19 ± 2a 26 ± 3ab 22 ± 1a 25 ± 4a 28 ± 3ab 28 ± 4ab 0.65 LWC (mg cm−2) 89 ± 8ab 107 ± 9ab 95 ± 9ab 115 ± 6bc 95 ± 14ab 138 ± 12c 79 ± 6a 107 ± 12abc 0.61  (g water g−1 DM) 16.0 ± 1.0 14.3 ± 1.3 15.3 ± 0.6 14.8 ± 0.7 14.5 ± 1.8 17.0 ± 0.9 15.0 ± 0.9 15.8 ± 0.6 0.57 SD - abaxial (mm−2) 85 ± 19a 151 ± 11b 133 ± 23ab 106 ± 17ab 97 ± 22ab 92 ± 7a 121 ± 4ab 133 ± 25ab 0.06  SD - adaxial (mm−2) 36 ± 5a 72 ± 5b 69 ± 13b 50 ± 3ab 49 ± 10ab 40 ± 3a 53 ± 5ab 52 ± 13ab 0.09 SI - abaxial (%) 17 ± 1ab 29 ± 2c 19 ± 1ab 20 ± 2ab 16 ± 2ab 21 ± 1b 17 ± 1ab 16 ± 2a 0.00  SI - adaxial (%) 13 ± 0a 25 ± 1b 14 ± 1a 18 ± 1a 15 ± 2a 14 ± 2a 13 ± 1a 13 ± 3a 0.00 [NaCl] (mm) 0 150 300 400 Initial Final Initial Final Initial Final Initial Final P Thickness (µm)  Whole leaf 755 ± 92abc 771 ± 58bc 569 ± 69a 927 ± 32cd 624 ± 50ab 971 ± 76d 566 ± 35ab 643 ± 70ab 0.02  Upper epidermis 37 ± 4a 37 ± 1a 32 ± 2a 37 ± 6a 30 ± 2a 30 ± 3a 29 ± 3a 33 ± 5a 0.92  Palisade parenchyma 362 ± 45ab 372 ± 39ab 268 ± 39ab 477 ± 17ab 288 ± 31ab 465 ± 35b 271 ± 20ab 297 ± 48a 0.01  Spongy parenchyma 314 ± 50ab 316 ± 19ab 247 ± 40a 373 ± 16bc 275 ± 23a 451 ± 41c 225 ± 13a 273 ± 21a 0.04  Lower epidermis 29 ± 4a 35 ± 2b 19 ± 2a 26 ± 3ab 22 ± 1a 25 ± 4a 28 ± 3ab 28 ± 4ab 0.65 LWC (mg cm−2) 89 ± 8ab 107 ± 9ab 95 ± 9ab 115 ± 6bc 95 ± 14ab 138 ± 12c 79 ± 6a 107 ± 12abc 0.61  (g water g−1 DM) 16.0 ± 1.0 14.3 ± 1.3 15.3 ± 0.6 14.8 ± 0.7 14.5 ± 1.8 17.0 ± 0.9 15.0 ± 0.9 15.8 ± 0.6 0.57 SD - abaxial (mm−2) 85 ± 19a 151 ± 11b 133 ± 23ab 106 ± 17ab 97 ± 22ab 92 ± 7a 121 ± 4ab 133 ± 25ab 0.06  SD - adaxial (mm−2) 36 ± 5a 72 ± 5b 69 ± 13b 50 ± 3ab 49 ± 10ab 40 ± 3a 53 ± 5ab 52 ± 13ab 0.09 SI - abaxial (%) 17 ± 1ab 29 ± 2c 19 ± 1ab 20 ± 2ab 16 ± 2ab 21 ± 1b 17 ± 1ab 16 ± 2a 0.00  SI - adaxial (%) 13 ± 0a 25 ± 1b 14 ± 1a 18 ± 1a 15 ± 2a 14 ± 2a 13 ± 1a 13 ± 3a 0.00 Values are mean ± s.e. (n = 5). Different letters indicate significant differences after a two-way ANOVA ([NaCl] × time; P < 0.05). The value of P for the interaction time × [NaCl] is indicated. Values of P < 0.05 are shown in bold type. View Large Growth analysis and mineral contents Only plants irrigated with 400 mm NaCl solution showed significant reductions in biomass, which took place solely in stems (41 %) and reproductive structures (78 %), leaf mass remaining unchanged; the root/shoot ratio was unaffected by treatment, and RE decreased only under 400 mm NaCl (Fig. 5). Fig. 5. View largeDownload slide Changes in plants after 22 d of treatment with increasing soil salinity in: A, biomass distribution among organs; B, root/shoot ratio; and C, reproductive effort (RE). Legend is given in panel A. Values are mean ± s.e. (n = 5). Asterisks indicate significant differences after a one-way ANOVA ([NaCl] per organ; P < 0.05). Fig. 5. View largeDownload slide Changes in plants after 22 d of treatment with increasing soil salinity in: A, biomass distribution among organs; B, root/shoot ratio; and C, reproductive effort (RE). Legend is given in panel A. Values are mean ± s.e. (n = 5). Asterisks indicate significant differences after a one-way ANOVA ([NaCl] per organ; P < 0.05). Changes with treatment in soluble Na and K contents and their contribution to morning ψs are shown in Table 3. Leaf Na content increased with salinity in stems, was highest under 150 and 300 mm and, under 400 mm, it was higher in roots than under the other two [NaCl] values. Soil Na content of control plants and tap water were 183 ± 30 and 0.4 ± 0.0 mmol kg−1 (0.58 and 0.30 dS m−1), respectively. The K/Na molar ratio in soils was 0.11 ± 0.01, and pH in a 1: 5 solution (w/w) was 7.3 ± 0.1 (n = 6). Contents of K remained unchanged in leaves, increased in stems and showed no differences in roots. The contribution of Na to morning ψs increased with salinity, whereas that of K remained relatively unchanged; both cations contributed on average 52 % to ψs and 50 % (Na) and 24 % (K) on average to the observed Δψs. The total K/total Na molar ratio in control plants was five times higher than the average under salinity (Table 3). The contribution of soluble Mg, whose content was 26 % of the total Mg content, was constant and amounted on average to only 5 %. Table 3. Changes with treatment for 21 d in organ soluble Na and K contents, their contribution to morning osmotic potential (ψs) and the leaf total K/total Na molar ratio in leaves of plants of Talinum triangulare [NaCl] (mm) Mineral content (mmol kg−1 DM) Na K % ψs K/Na Leaves Stems Roots Leaves Stems Roots Na K (mol mol−1) 0 328 ± 26a 761 ± 58a 54 ± 4a 1222 ± 69b 776 ± 45 825 ± 103 12 ± 1a 30 ± 3b 2.0 ± 0.4b 150 1569 ± 155b 1951 ± 194b 93 ± 21ab 914 ± 82a 724 ± 59 1027 ± 55 32 ± 4a 23 ± 6ab 0.5 ± 0.0a 300 2313 ± 189bc 1441 ± 280b 124 ± 14b 1116 ± 55ab 808 ± 64 760 ± 112 34 ± 4ab 15 ± 2a 0.4 ± 0.0a 400 3146 ± 633c 848 ± 288a 265 ± 12c 1096 ± 118ab 912 ± 123 942 ± 85 69 ± 23b 16 ± 2ab 0.3 ± 0.0a [NaCl] (mm) Mineral content (mmol kg−1 DM) Na K % ψs K/Na Leaves Stems Roots Leaves Stems Roots Na K (mol mol−1) 0 328 ± 26a 761 ± 58a 54 ± 4a 1222 ± 69b 776 ± 45 825 ± 103 12 ± 1a 30 ± 3b 2.0 ± 0.4b 150 1569 ± 155b 1951 ± 194b 93 ± 21ab 914 ± 82a 724 ± 59 1027 ± 55 32 ± 4a 23 ± 6ab 0.5 ± 0.0a 300 2313 ± 189bc 1441 ± 280b 124 ± 14b 1116 ± 55ab 808 ± 64 760 ± 112 34 ± 4ab 15 ± 2a 0.4 ± 0.0a 400 3146 ± 633c 848 ± 288a 265 ± 12c 1096 ± 118ab 912 ± 123 942 ± 85 69 ± 23b 16 ± 2ab 0.3 ± 0.0a Values are mean ± s.e. (n = 5). Different letters indicate significant differences after a one-way ANOVA ([NaCl] for each cation per organ; P < 0.05). View Large Table 3. Changes with treatment for 21 d in organ soluble Na and K contents, their contribution to morning osmotic potential (ψs) and the leaf total K/total Na molar ratio in leaves of plants of Talinum triangulare [NaCl] (mm) Mineral content (mmol kg−1 DM) Na K % ψs K/Na Leaves Stems Roots Leaves Stems Roots Na K (mol mol−1) 0 328 ± 26a 761 ± 58a 54 ± 4a 1222 ± 69b 776 ± 45 825 ± 103 12 ± 1a 30 ± 3b 2.0 ± 0.4b 150 1569 ± 155b 1951 ± 194b 93 ± 21ab 914 ± 82a 724 ± 59 1027 ± 55 32 ± 4a 23 ± 6ab 0.5 ± 0.0a 300 2313 ± 189bc 1441 ± 280b 124 ± 14b 1116 ± 55ab 808 ± 64 760 ± 112 34 ± 4ab 15 ± 2a 0.4 ± 0.0a 400 3146 ± 633c 848 ± 288a 265 ± 12c 1096 ± 118ab 912 ± 123 942 ± 85 69 ± 23b 16 ± 2ab 0.3 ± 0.0a [NaCl] (mm) Mineral content (mmol kg−1 DM) Na K % ψs K/Na Leaves Stems Roots Leaves Stems Roots Na K (mol mol−1) 0 328 ± 26a 761 ± 58a 54 ± 4a 1222 ± 69b 776 ± 45 825 ± 103 12 ± 1a 30 ± 3b 2.0 ± 0.4b 150 1569 ± 155b 1951 ± 194b 93 ± 21ab 914 ± 82a 724 ± 59 1027 ± 55 32 ± 4a 23 ± 6ab 0.5 ± 0.0a 300 2313 ± 189bc 1441 ± 280b 124 ± 14b 1116 ± 55ab 808 ± 64 760 ± 112 34 ± 4ab 15 ± 2a 0.4 ± 0.0a 400 3146 ± 633c 848 ± 288a 265 ± 12c 1096 ± 118ab 912 ± 123 942 ± 85 69 ± 23b 16 ± 2ab 0.3 ± 0.0a Values are mean ± s.e. (n = 5). Different letters indicate significant differences after a one-way ANOVA ([NaCl] for each cation per organ; P < 0.05). View Large Salinity had varying effects on total mineral contents (Fig. 6). It did not affect Mg or Ca contents in leaves and stems, while causing a significant increase in P content of stems at 300 mm NaCl, and stems and roots at 400 mm NaCl. A decrease with [NaCl] in leaf C/N was due to a significant decrease in C with increasing salinity and an increase in N at 400 mm NaCl. Fig. 6. View largeDownload slide Changes in plants after 22 d of treatment with increasing soil salinity in total Mg, Ca and P contents per organ, and leaf C/N ratio. Legends inserted. Values are mean ± s.e. (n = 5). Different letters indicate significant differences after a two-way ANOVA ([NaCl] × organ per mineral; P < 0.05). Fig. 6. View largeDownload slide Changes in plants after 22 d of treatment with increasing soil salinity in total Mg, Ca and P contents per organ, and leaf C/N ratio. Legends inserted. Values are mean ± s.e. (n = 5). Different letters indicate significant differences after a two-way ANOVA ([NaCl] × organ per mineral; P < 0.05). DISCUSSION Plants of T. triangulare tolerated treatment with up to 400 mm NaCl, PN and E decreasing markedly under 300 and 400 mm NaCl, whereas growth became significantly reduced only by 400 mm NaCl. Plants under 150 and 300 mm NaCl had values of ΔH+malate eq. significantly higher than the control, which is evidence of the operation of CAM. Nocturnal CO2 fixation was not found under any [NaCl], contrasting with previous reports on the operation of CAM in T. triangulare (Herrera et al., 1991, 2015; Pieters et al., 2003). Therefore, the occurrence of significant ΔH+ must have been due to internal recycling of night-time respiratory CO2, as previously shown in T. calycinum by Martin and Zee (1983). In contrast to previous reports (Martin and Zee, 1983; Harris and Martin, 1991), cycling, i.e. ΔH+ in watered plants without nocturnal CO2 fixation, was not observed; therefore, the correct way to designate the operation of CAM in the present work would be recycling. The increase in δ13C found in plants irrigated with 150 and 300 mm NaCl was related to higher LWC, but not increased integrated WUE. In contrast, in four out of five species of Talinum considered to be cycling CAM species, the correlation between ΔH+ and water content was higher in plants subjected to drought than in watered plants; nevertheless, no relationship was detected between ΔH+ and δ13C, which ranged from approx. −31 to −24 ‰ (Harris and Martin, 1991). Increased WUE may increase δ13C in C3 plants due to stomatal closure, as shown in wheat cultivars by Farquhar and Richards (1984). Because recycling does not contribute to C gain, it is difficult to see why recycling could change δ13C, unless part of the refixed respiratory CO2 came from non-autotrophic tissues, as these have been shown to be enriched in 13C by about 1–3 ‰ relative to autotrophic organs in C3 plants (Cernusak et al., 2009). Values of ΔH+malate eq. after 8 d of treatment with 300 mm NaCl were within the range of those reported for plants of T. triangulare under drought (50–100 µmol g−1; Herrera et al., 1991; Taisma and Herrera, 1998; Herrera, 1999; Pieters et al., 2003). The significance of CAM recycling as an adaptive response to salinity is not clear because, consistent with previous observations in plants of T. triangulare under drought (Herrera et al., 1991; Pieters et al., 2003), a high ΔH+ was not maintained in time but decreased after 8–16 d. The time under treatment with 300 mm NaCl when an increase in ΔH+ was found falls within the time range, 1–2 weeks, necessary for CAM induction by salinity in M. crystallinum, suggesting that delayed CAM induction is a long-term adaptation to diminished water availability, rather than a tolerance mechanism against high salt concentration in the cytoplasm (Lüttge, 1993). Because a high ΔH+ in the present research was not maintained over time, we conclude that CAM is a protective mechanism against the salinity shock but the actual tolerance mechanism may lie in the accumulation of ions into leaf vacuoles for osmotic adjustment, as shown in Avicennia germinans (Suárez and Sobrado, 2000). ΔH+citrate eq. was nil under any salinity, which coincides with previous observations in plants of T. triangulare under drought (Herrera et al., 2015) but contrasts with a marked diurnal rhythm in citrate found in plants of M. crystallinum under 300 mm NaCl (Herppich et al., 1995). In this example, citrate accumulation occurred previous to malate accumulation and was adjudicated a role in energy conservation before a diurnal cycle of malate became established once plants were old enough. Similarly to our results, citrate in M. crystallinum amounted to a very small proportion of the mass of total inorganic ions accumulated in the leaves under salinity. Changes in ΔH+malate eq. observed in plants of T. triangulare subjected to salinity were not caused by a decrease in leaf water status, because LWC remained fairly constant during treatment and was highest under 300 mm NaCl, where nocturnal acid accumulation was found, and ψ was not different from the control value after 8 d of treatment. Similarly, leaf water status apparently was not the trigger of CAM operation in plants of the same species under drought (Herrera et al., 2015). In plants of the inducible CAM species Guzmania monostachia under water deficit, CAM operated only in the apical leaf region, which showed no reduction in relative water content compared to the same region in watered plants performing only C3 photosynthesis (Freschi et al., 2010). An experiment in which one half of a split-root system was subjected to water deficit suggested that a root signal, possibly abscisic acid, not leaf water status, was the inducing factor of CAM in M. crystallinum (Eastmond and Ross, 1997). Increased LWC in plants under irrigation with 300 mm NaCl correlated with an increase in whole-leaf and parenchyma thickness, as found in plants of N. glauca under salinity (González et al., 2012). A similar increase in LWC was found in the mangrove A. germinans under 32 ‰ (547 mm) NaCl but in this case not only were mesophyll cells larger, but also the hypodermis duplicated its cell layers (Suárez and Sobrado, 2000). In the present study, a decrease in SI due to salt suggested that differentiation of epidermal cells into stomata became inhibited. Under salinity, SD in 14 genotypes of quinoa differing in salt tolerance diminished by up to 30 %, which was interpreted as a mechanism to deal with reduced water availability (Shabala et al., 2013). In contrast, increased SD was associated with increased tolerance to salinity in 46 genotypes of barley (Zhu et al., 2015). In view of the controversial results and interpretations given to changes in SD under salinity, the role of decreased SD or SI in the tolerance of plants is arguable. The Δψs observed was partly achieved by leaf accumulation of Na and K. In plants of T. paniculatum irrigated with saline solutions, in which salt was accumulated mainly in the root instead of leaves, increased proline content was associated with a value of OA of approx. 0.3 MPa (Assaha et al., 2017). The average Δψs by plants of T. triangulare at the end of the experiment in the three salinities was similar to the values of OA reported in plants of T. triangulare after 27 d of drought (Herrera et al., 2015) but much lower than in the mangrove A. germinans under salinity, 1.2 MPa, calculated with values of ψs at full turgor, i.e. OA (Suárez and Sobrado, 2000). In plants of N. glauca, OA was higher under salinity than under drought, but OA did not prevent PN decreasing with salinity (González et al., 2012). In contrast, plants of Lycium nodosum showed after 15 d of saline treatment an OA of 0.94 MPa with PN remaining unaltered (Tezara et al., 2003). The latter results support the hypothesis that OA contributes to the maintenance of photosynthetic activity (Munns and Tester, 2008), which, if Δψs without change in LWC is taken as a surrogate for OA, was not the case in the present study. The significance of an increase in Δψs as a means to guarantee water absorption by roots for plants of T. triangulare under salinity was uncertain, because a simple calculation using the van’t Hoff relationship indicates that plants would not be able to absorb water from the soil under any salinity, as ψ would be higher than soil water potential, which would be at most −0.7, −1.5 and −2.0 MPa for 150, 300 and 400 mm, respectively, and the value of Δψs did not contribute to reduce ψs to values lower than the soil water potential in any of the salt treatments. Given that Δψs was determined in unsaturated plants and relative water content was not measured, it would not be correct to state that that variable is the same as OA. Additionally, Δψs was measured solely in leaves. In roots of the halophyte Atriplex nummularia, a significant OA of 0.5 MPa was found when plants were irrigated with 450 and 600 mm NaCl (Silveira et al., 2009), which suggests that roots may actively contribute to increase the capacity of the plant to absorb water from the substrate. The observed reduction in ψs relative to the controls in T. triangulare apparently favoured the maintenance of values of turgor potential similar to those in controls. Our interpretation of the occurrence of Δψs is that this was caused only as a response to increased NaCl content in vacuoles, not as a mechanism to increase root water absorption. Because transpiration continued, although at a very low rate, after 21 d of treatment, we assume that E was sustained by leaf water. Salinity did not alter chlorophyll content, coinciding with observations on plants of Zygophyllum xanthoxylum, in which chlorophyll content remained unchanged although at a much lower salinity (50 mm; Ma et al., 2011). Total chlorophyll content remained unchanged after 23–24 d of drought in plants of T. triangulare (Herrera et al., 1991) and T. paniculatum (Güerere et al., 1996). In T. paniculatum total chlorophyll content even increased after irrigation with 200 and 300 mm NaCl (Assaha et al., 2017). Additional determinations, such as chlorophyll a fluorescence, should help ascertain whether salinity affected photosynthetic capacity. Unchanged chlorophyll content in the present experiment was associated with unchanged carotenoid content, supporting the antioxidative role given to carotenoids in plants under abiotic stress (Gill and Tuteja, 2010; Assaha et al., 2017). The exception to the general trend was a marked decrease in chlorophyll/carotenoids at 400 mm, the sole concentration which affected growth presumably by damage caused by salt. The high K/Na molar ratio in control plants makes T. triangulare a non-halophyte, whereas the value in plants under salinity suggests considering this a halo-tolerant species, i.e. one which, while not requiring salt to grow, can tolerate high concentration in its substrate (after Medina et al., 2008). The succulence in T. triangulare was much higher than that of halophytes, as reported by Medina et al. (2008), which surely results from its CAM characteristics. Additionally, the high Na content in control plants suggests that this species tends to concentrate Na from the substrate. In our case, soil Na conductivity in pots watered with tap water is considered to cause negligible effects on crops (0–2 dS m−1, Abrol et al., 1988). In control plants of the halophytes Atriplex portulacoides (Redondo-Gómez et al., 2007), Atriplex centralasiatica (Qiu et al., 2003) and Halostachys caspica (Zeng et al., 2015), leaf Na content was 1000, 1500 and 2600 mmol kg−1 DM, respectively. Irrigation of plants of T. triangulare with NaCl produced a significant decrease in stem biomass only at 400 mm, similarly to plants of T. paniculatum, in which 300 mm NaCl produced a 53 % reduction (Assaha et al., 2017). The biomass of the remaining organs in T. triangulare, except for the reproductive structures, was unaffected by any of the salinities, contrary to the case of T. paniculatum, in which all organs were affected (Assaha et al., 2017). Salinity had little effect on mineral contents except for increased leaf Na with [NaCl], and increased P in stems and roots at 300 and 400 mm, which indicates that the general nutritional status of plants for the duration of the experiment was adequate. The negative effects of salt on gas exchange in general and growth under 400 mm NaCl may have been due to the frequently reported effects of salt on gas exchange, and hence growth (Cheeseman, 1988), rather than nutrient deficiency. CONCLUSIONS Plants of T. triangulare tolerated salinity treatment up to 400 mm NaCl, the latter concentration decreasing growth and reproductive effort. Plants under 150 and 300 mm NaCl showed values of ΔH+ significantly higher than the control without nocturnal CO2 fixation, which is evidence of the operation of the CAM in the recycling mode. Leaf water status bore no obvious relationship with CAM induction by salinity. A low K/Na ratio in plants under salinity makes T. triangulare a halo-tolerant species. ACKNOWLEDGEMENTS This work was partly supported by grant PG-03-7983-2011/2 (Consejo de Desarrollo Científico y Humanístico, Universidad Central de Venezuela). Alejandro Pieters (IVIC, Venezuela) facilitated the use of his equipment for measurement of water relations. Jan Tillet (IEJB, Venezuela) kindly built a plastic shed for performing initial experiments. E. A. Goddard, of the Stable Isotope Biogeochemistry Laboratory, College of Marine Science, USF, performed the δ13C determinations, for which we are much obliged. LITERATURE CITED Abrol IP , Yadav JSP , Massoud FI . 1988 . Salt-affected soils and their management . Rome : Food & Agriculture Organization of the United Nations , No. 39 . Assaha DVM , Mekawy AMM , Liu L et al. 2017 . Na+ retention in the root is a key adaptive mechanism to low and high salinity in the glycophyte, Talinum paniculatum (Jacq.) Gaertn. (Portulacaceae) . Journal of Agronomy and Crop Science 203 : 56 – 67 . Google Scholar CrossRef Search ADS Bamidele JF , Egharevba RKA , Okpoh IM . 2007 . Physiological changes in seedlings of Talinum triangulare (water leaf) grown in saline conditions . Asian Journal of Plant Sciences 6 : 56 – 60 . Google Scholar CrossRef Search ADS Cernusak LA , Tcherkez G , Keitel C et al. 2009 . Why are non-photosynthetic tissues generally 13C enriched compared with leaves in C3 plants? Review and synthesis of current hypotheses . Functional Plant Biology 36 : 199 – 213 . Google Scholar CrossRef Search ADS Cheeseman JM . 1988 . Mechanisms of salinity tolerance in plants . Plant Physiology 87 : 547 – 550 . Google Scholar CrossRef Search ADS PubMed Eastmond PJ , Ross JD . 1997 . Evidence that the induction of Crassulacean acid metabolism by water stress in Mesembryanthemum crystallinum (L.) involves root signalling . Plant, Cell & Environment 20 : 1559 – 1565 . Google Scholar CrossRef Search ADS Farquhar GD , Richards RA . 1984 . Isotopic composition of plant carbon correlates with water-use efficiency of wheat genotypes . Australian Journal of Plant Physiology 11 : 539 – 552 . Google Scholar CrossRef Search ADS Freschi L , Takahashi CA , Cambui CA et al. 2010 . Specific leaf areas of the tank bromeliad Guzmania monostachia perform distinct functions in response to water shortage . Journal of Plant Physiology 167 : 526 – 533 . Google Scholar CrossRef Search ADS PubMed Gill SS , Tuteja N . 2010 . Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants . Plant Physiology and Biochemistry 48 : 909 – 930 . Google Scholar CrossRef Search ADS PubMed González A , Tezara W , Rengifo E , Herrera A . 2012 . Ecophysiological responses to drought and salinity in the cosmopolitan invader Nicotiana glauca . Brazilian Journal of Plant Physiology 24 : 212 – 222 . Google Scholar CrossRef Search ADS Güerere I , Tezara W , Herrera C , Fernández M , Herrera A . 1996 . Recycling of CO2 during induction of CAM by drought in Talinum paniculatum (Portulacaceae) . Physiologia Plantarum 98 : 471 – 476 . Google Scholar CrossRef Search ADS Harris FS , Martin CE . 1991 . Correlation between CAM-cycling and photosynthetic gas exchange in five species of Talinum (Portulacaceae) . Plant Physiology 96 : 1118 – 1124 . Google Scholar CrossRef Search ADS PubMed Herrera A . 1999 . Effects of photoperiod and drought on the induction of CAM and the reproduction of plants of Talinum triangulare . Canadian Journal of Botany 77 : 1 – 6 . Google Scholar CrossRef Search ADS Herrera A . 2009 . Crassulacean acid metabolism and fitness under water deficit stress: if not for carbon gain, what is facultative CAM good for ? Annals of Botany 103 : 645 – 653 . Google Scholar CrossRef Search ADS PubMed Herrera A , Delgado J , Paraguatey I . 1991 . Occurrence of inducible crassulacean acid metabolism in leaves of Talinum triangulare (Portulacaceae) . Journal of Experimental Botany 42 : 493 – 499 . Google Scholar CrossRef Search ADS Herrera A , Ballestrini C , Montes E . 2015 . What is the potential for dark CO2 fixation in the facultative crassulacean acid metabolism species Talinum triangulare ? Journal of Plant Physiology 174 : 55 – 61 . Google Scholar CrossRef Search ADS PubMed Herppich M , von Willert DJ , Herppich WB . 1995 . Diurnal rhythm in citric acid content preceded the onset of nighttime malic acid accumulation during metabolic changes from C3 to CAM in salt-stressed plants of Mesembryanthemum crystallinum . Journal of Plant Physiology 147 : 38 – 42 . Google Scholar CrossRef Search ADS Lüttge U . 1993 . The role of crassulacean acid metabolism (CAM) in the adaptation of plants to salinity . New Phytologist 125 : 59 – 71 . Google Scholar CrossRef Search ADS Ma Q , Yue LJ , Zhang JL , Wu GQ , Bao AK , Wang SM . 2011 . Sodium chloride improves photosynthesis and water status in the succulent xerophyte Zygophyllum xanthoxylum . Tree Physiology 32 : 4 – 13 . Google Scholar CrossRef Search ADS PubMed Martin C , Zee A . 1983 . C3 photosynthesis and CAM in a Kansas rock outcrop succulent, Talinum calycinum (Portulacaceae) . Plant Physiology 73 : 718 – 723 . Google Scholar CrossRef Search ADS PubMed Medina E , Francisco AM , Wingfield R , Casañas OL . 2008 . Halophytism in plants of the Caribbean coast of Venezuela: halophytes and halotolerants . Acta Botanica Venezuelica 31 : 49 – 80 . Munns R , Tester M . 2008 . Mechanism of salinity tolerance . Annual Review of Plant Biology 59 : 651 – 681 . Google Scholar CrossRef Search ADS PubMed Murphy J , Riley JP . 1962 . A modified single solution method for the determination of phosphate in natural waters . Analytica Chimica Acta 27 : 31 – 36 . Google Scholar CrossRef Search ADS Niewiadomska E , Karpinska B , Romanowska E , Slesak I , Karpinski S . 2004 . A salinity-induced C3-CAM transition increases energy conservation in the halophyte Mesembryanthemum crystallinum . Plant and Cell Physiology 45 : 789 – 794 . Google Scholar CrossRef Search ADS PubMed Osmond CB . 1978 . Crassulacean acid metabolism: a curiosity in context . Annual Review of Plant Physiology 29 : 379 – 414 . Google Scholar CrossRef Search ADS Pieters AJ , Tezara W , Herrera A . 2003 . Operation of the xanthophyll cycle and degradation of D1 protein in the inducible CAM plant, Talinum triangulare, under water deficit . Annals of Botany 92 : 393 – 399 . Google Scholar CrossRef Search ADS PubMed Qiu N , Lu Q , Lu C . 2003 . Photosynthesis, photosystem II efficiency and the xanthophyll cycle in the salt‐adapted halophyte Atriplex centralasiatica . New Phytologist 159 : 479 – 486 . Google Scholar CrossRef Search ADS Redondo-Gómez S , Mateos-Naranjo E , Davy AJ et al. 2007 . Growth and photosynthetic responses to salinity of the salt-marsh shrub Atriplex portulacoides . Annals of Botany 100 : 555 – 563 . Google Scholar CrossRef Search ADS PubMed Salisbury EJ . 1927 . On the causes and ecological significance of stomatal frequency, with special reference to the woodland flora . Proceedings of the Royal Society of London, Series B 216 : 303 – 309 . Shabala S , Hariadi Y , Jacobsen SE . 2013 . Genotypic difference in salinity tolerance in quinoa is determined by differential control of xylem Na+ loading and stomatal density . Journal of Plant Physiology 170 : 906 – 914 . Google Scholar CrossRef Search ADS PubMed Silveira JAG , Araújo SAM , Lima JPMS , Viégas RA . 2009 . Roots and leaves display contrasting osmotic adjustment mechanisms in response to NaCl-salinity in Atriplex nummularia . Environmental and Experimental Botany 66 : 1 – 8 . Google Scholar CrossRef Search ADS Suárez N , Sobrado MA . 2000 . Adjustments in leaf water relations of mangrove (Avicennia germinans) seedlings grown in a salinity gradient . Tree Physiology 20 : 277 – 282 . Google Scholar CrossRef Search ADS PubMed Taisma MA , Herrera A . 1998 . A relationship between fecundity, survival and the operation of CAM in Talinum triangulare . Canadian Journal of Botany 76 : 1 – 8 . Google Scholar CrossRef Search ADS Tezara W , Martinez D , Rengifo E , Herrera A . 2003 . Photosynthetic responses of the tropical spiny shrub Lycium nodosum (Solanaceae) to drought, soil salinity and saline spray . Annals of Botany 92 : 757 – 765 . Google Scholar CrossRef Search ADS PubMed Ukpong IE , Moses JO . 2001 . Nutrient requirements for the growth of waterleaf (Talinum triangulare) in Uyo metropolis, Nigeria . The Environmentalist 21 : 153 – 159 . Google Scholar CrossRef Search ADS Wellburn A . 1994 . The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution . Journal of Plant Physiology 144 : 307 – 313 . Google Scholar CrossRef Search ADS Winter K , Holtum JAM . 2002a . The effects of salinity, crassulacean acid metabolism and plant age on the carbon isotopic composition of Mesembryanthemum crystallinum L., a halophytic C3-CAM species . Planta 222 : 201 – 209 . Google Scholar CrossRef Search ADS Winter K , Holtum JAM . 2002b . How closely do the δ13C values of crassulacean acid metabolism plants reflect the proportion of CO2 fixed during day and night ? Plant Physiology 129 : 1843 – 1851 . Google Scholar CrossRef Search ADS Zeng Y , Li L , Yan R , Yi X , Zhang B . 2015 . Contribution and distribution of inorganic ions and organic compounds to the osmotic adjustment in Halostachys caspica response to salt stress . Scientific Reports 5 : 13639 . Google Scholar CrossRef Search ADS PubMed Zhu M , Zhou M , Shabala L , Shabala S . 2015 . Linking osmotic adjustment and stomatal characteristics with salinity stress tolerance in contrasting barley accessions . Functional Plant Biology 42 : 252 – 263 . Google Scholar CrossRef Search ADS © The Author(s) 2018. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Annals of BotanyOxford University Press

Published: Mar 26, 2018

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