Introduction

Salinity is one of the major factors limiting agricultural production in arid and semiarid regions all over the world (Acosta et al. 2011). In these environments, salt stress triggers cell damage, affecting plant growth, development and productivity (Chang et al. 2011). The root growth inhibition is one of the first effects caused by the osmotic and ionic effects of salt stress. As a consequence, the plants present damages in the processes of absorption, accumulation and distribution of water and mineral nutrients, triggering changes in water potential, nutritional status, and photosynthetic efficiency (Imada et al. 2009). The osmotic effects of salt stress leads to cell dehydration and loss of turgor induced by the external hyperosmolarity (D’Souza and Devaraj 2010). A strategy to maintain the water status under salt stress is osmotic adjustment by the synthesis and accumulation of different organic solutes into the plant cells (Ashraf and Harris 2004; Buchanan et al. 2009; Oueslati et al. 2010).

Plants display some mechanisms to cope with the damaging effects of ionic toxicity, as: (1) minimize Na+ entry into the cells and maximize its efflux; (2) minimize Na+ translocation to the shoot; (3) enhance Na+ recirculation by the phloem and (4) compartmentalize Na+ or direct it to basal and/or older plant parts (Tester and Davenport 2003). The ionic effects of salt stress involve the interference of saline ions in the absorption, distribution and utilization of mineral nutrients causing disturbances in the cell ionic homeostasis (Maathius et al. 1996). These disturbances affect the activity of enzymes involved in fundamental metabolic processes such as protein synthesis, respiration and photosynthesis (Ellouzi et al. 2011; Shabala et al. 2012).

The relative contribution of different solutes in the salt-induced osmoregulation as well as their accumulation and distribution in different organs and the mechanisms used to minimize the damages caused by osmotic and ionic effects vary with the species (Chaves et al. 2009). In this context, the knowledge about the accumulation and distribution of ions, as well as the contribution of osmotic adjustment in the productivity of native species of arid and semiarid regions in response to water and salt stress is very important because these species can be used for phytoremediation of areas degraded by salinity (Sousa et al. 2011; Fini et al. 2013).

Faveleira [Cnidoscolus phyllacanthus (M. Arg.) Pax et K. Hoffm] is a native woody Euphorbiaceae from the semiarid areas of the Northeast of Brazil (Ribeiro Filho et al. 2011), well adapted to the limiting conditions of reduced precipitation, high temperatures, and poor soil fertility (Silva et al. 2010). Previous work showed that Jatropha curcas L. exhibit a moderate tolerance to salinity and the negative influences of this stress are mainly due to Cl− and/or Na+ toxicity and to a nutritional imbalance caused by an increase in the Na+/K+ ratio (Díaz-López et al. 2012). However, faveleira displays great potential for the socio-economic development of the Northeast of Brazil as an alternative source of raw material for biodiesel production, taking into account the high seed oil content and quality.

Although the seeds of faveleira represent a promising source of bioenergy, the knowledge about its physiological responses to salinity is still scarce. It is known that faveleira, as well as other species of the Euphorbiaceae, presents xylopodium as a reserve organ. In addition, it has been largely studied that different organs or structures play a role in Na+ retention (Munns 2002; Davenport et al. 2005). However, little is known about the role of the xylopodium in the water and ionic homeostasis as a strategy for plant survival in regions characterized by water scarcity and/or excessive salts in the soil. In this sense, the elucidation of these mechanisms may provide new insights to improve the exploitation of this species in arid and semiarid regions. Thus, the aim of this study was to evaluate the physiological responses associated with plant growth, water status and the distribution of inorganic and organic solutes in different vegetative organs of faveleira.

Materials and methods

Plant material and experimental conditions

Faveleira seeds were disinfected with 0.2 % (w/v) sodium hypochlorite followed by three washes in distilled water. These seeds were sown in germination paper (dimensions of 28 × 38 cm) moistened with distilled water (1.5 times the mass of paper) and kept in a growth chamber at 25 ± 2 °C, 300 µmol m−2 s−1 (using Grolux® lamp) and under a photoperiod of 12 h. Thirty-day-old seedlings were standardized according to the number of leaves, height and weight, determining the initial length of the shoot (SL0) and root (RL0), the initial total fresh weight (TFW0), and the initial leaf number (LN0). These seedlings were transferred to nutrient solution of Hoagland supplemented with 0 (control), 50, 100, or 150 mM NaCl. The hydroponic cultures were maintained in a greenhouse at ambient temperature of 30 ± 5 °C under a photoperiod of 12 h, and relative humidity of approximately 50 %. The cultures were aerated continuously with filtered air provided by aquarium pumps and the nutrient solution was replaced by recently prepared solution after 7 days.

Eight days after the beginning of stress imposition, the final length of the shoot (SL1) and root (RL1), the final total fresh weight (TFW1) and the final leaf number (LN1) were evaluated. Then, the plants were harvested and separated into roots, xylopodium, stem + petiole (SP), basal leaves (closer to the stem base), median leaves, and apical leaves (closer to the stem apex). After that, we evaluated the effect of salinity on indicators of plant growth (shoot and root length, number of leaves and relative growth rate), water stress (relative water content and percentage of humidity), ion homeostasis (Na+ and K+ content), and osmotic adjustment (total soluble sugars, soluble proteins, total free amino acids and proline).

A completely randomized design was used with four treatments (0, 50, 100 and 150 mM NaCl) and five plants for replication and per treatment.

Indicators of plant growth and water stress

The symptoms of toxicity were verified by the presence of chlorotic and/or necrotic leaves. The growth ratio of the shoot and root, as well as the number of leaves were determined according to Macêdo et al. (2009), as follows: shoot growth rate (SGR) = (SL1/SL0) × 100, root growth rate (RGR) = (RL1/RL0) × 100, and leaf number rate (LNR) = (LN1/LN0) × 100. The initial and final total fresh mass was also used to determine the relative growth rate (RGR) according to the formula RGR = (TFW1/TFW0) × 100. For the salt-treated plants, all these rates were expressed as a percentage of the rate determined for the plants that were not exposed to salt (control).

For the determination of the relative water content (RWC), samples of roots, xylopodium, SP, basal leaves, median leaves and apical leaves were weighed to obtain the fresh weight (FW) and were immersed in deionized water at room temperature for 6 h. After that, the samples were dried with paper towels and the turgid weight (TW) was measured. To obtain the dry weight (DW), the different plant parts were dried at 75 °C for 72 h. The RWC was calculated according to Irigoyen et al. (1992), as follows: RWC = (FW − DW/TW − DW) × 100. The percentage of moisture was determined according to Slavick (1974) using the following formula: M % = [(FW − DW)/FW] × 100.

Na+ and K+

Samples of roots, xylopodium, SP, basal leaves, median leaves and apical leaves were dried at 75 °C for 48 h and the dry weight was determined. Then, these samples were pulverized and the determination of Na+ and K+ were performed according to Silva et al. (2009). Samples of 50 mg of dry plant tissue were extracted with 20 mL of boiling deionized water for 1 h. The extracts obtained were centrifuged and the supernatants were analyzed in a flame photometer (Micronal B462). Following the determination of the Na+ and K+ content in each plant part, the K+/Na+ ratio was calculated.

Organic solutes

For the determination of organic solutes, samples of different plant parts were distributed into vials containing 100 mL of deionized water. The vials were hermetically sealed and heated at 100 °C for 1 h (Maia et al. 2010; Maia et al. 2013). The remaining liquid solution in the vial was filtered and stored at −18 °C for later measurement of total soluble sugars (TSS), total free amino acids (TFAA) and proline (PRO).

The TSS content was determined by the method of “phenol–sulfuric” described by Dubois et al. (1956). Aliquots of 100 µL of the samples were added to 400 µL of deionized water, 500 µL of 5 % (w/v) phenol and stirred vigorously. To start the reaction, 2500 µL of concentrated H2SO4 were added. The homogenization was performed in a vortex mixer, and the mixture was incubated for 20 min at room temperature. The absorbance was measured at 490 nm and the results were expressed in µmol of glucose per gram of dry weight.

The content of total soluble protein (TSP) was determined according to the method of Bradford (1976) using bovine serum albumin as a standard.

The content of TFAA was determined according to the method described by Peoples et al. (1989). Aliquots of 100 µL of the extracts were added to 400 µL of deionized water, 250 µL of 200 mM citrate buffer (pH = 5.0) and 250 µL of ninhydrin reagent (0.1 mM KCN and 5 % (w/v) ninhydrin in methoxy-ethanol). The tubes were shaken in a vortex and incubated at 100 °C for 15 min. The reaction was stopped by cooling on ice, and 1.5 mL of 50 % (v/v) ethanol was added to each sample. After further stirring, the mixtures remained for 20 min at room temperature and the absorbance was read at 570 nm. The results are expressed in µmol l-glutamine per gram of dry weight.

The PRO content was determined following the method of Bates et al. (1973). Aliquots of 1 mL of the extracts were mixed with 1 mL of acidic ninhydrin reagent (1 g of ninhydrin dissolved in 24 mL of glacial acetic acid and 16 mL of 6 M H3PO4), and 1 mL of glacial acetic acid and incubated at 100 °C for 1 h. The reaction was stopped by cooling on ice, 2 mL of toluene were added and the mixtures were stirred vigorously for 20 s. After stabilization, the organic phase containing the reddish chromophore was collected, the absorbance was determined at 520 nm using toluene as the blank and the results were expressed as µmol proline per gram of dry weight.

Experimental design and data analysis

All parameters were submitted to variance analysis (ANOVA) and the means were compared by the Tukey test at a confidence level of 0.05. For all evaluated parameters, five plants for replication and per treatment were used.

Results

Effect of salinity on plant growth and water stress indicators

Visual symptoms of toxicity were observed in older leaves (basal and median) of faveleira plants subjected to the higher doses of NaCl (100 and 150 mM). Such symptoms, when compared with the control leaf blades (Fig. 1a) were characterized by the presence of chlorosis and necrosis initially in localized areas of the leaf blade (Fig. 1b) with progressive expansion (Fig. 1c, d). There were no symptoms of toxicity in young leaves (apical leaves) neither in the control nor in 50 mM NaCl treatments (data not shown).

Fig. 1
figure 1

Toxicity symptoms on leaves of faveleira exposed to different concentrations of NaCl (0, 50, 100 and 150 mM) for 8 days. a Basal leaf, control treatment, without toxicity symptoms; b basal leaf subjected to 100 mM NaCl, presenting localized areas of necrosis; c basal leaf subjected to 150 mM NaCl, presenting chlorosis; d median leaf submitted to 150 mM NaCl, presenting advanced necrosis

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Salinity affected the growth of faveleira plants, regardless of the external concentration of salt. Compared with the control, there was a reduction of the growth rates of the shoot and root, leaf number and relative growth rate, as indicated by the total fresh weight of the plants (Fig. 2a–d). The shoot length of plants cultivated for 8 days in the presence of 150 mM NaCl was about 20 % lower than that of the control, whereas under 50 and 100 mM NaCl the reduction was 14 and 13 %, respectively (Fig. 2a). The reduction in root length was between 14 and 22 % when compared with the control and no significant differences were recorded between the NaCl treatments (Fig. 2b). The number of leaves was also affected by salinity. Indeed, in comparison to the control, there was a decrease, although not significant, under 50 (7 %) and 100 (5 %) mM NaCl. However, under 150 mM the reduction was about 13 % although not significant (Fig. 2c). The relative growth rate (RGR), obtained from the total fresh weight of the plant before and after exposure to stress, which reflects all growth parameters described above, was affected by the NaCl treatments in the culture solutions (Fig. 2d). There was a reduction in RGR of all salt treatments compared with RGR of the control, although such effect was more pronounced under 150 mM NaCl. The plants submitted to concentrations of 50, 100 and 150 mM showed growth reduction rates of 17, 23 and 32 %, respectively, with regard to plants cultivated in the absence of NaCl (Fig. 2d).

Fig. 2
figure 2

Growth parameters in faveleira plants under different salt concentrations (0, 50, 100 and 150 mM NaCl) for 8 days. a Rate of shoot length; b rate of root length; c number of leaves; and d relative growth rate. The values represent the average of six repetitions. Different letters between the values represent significant differences by the Tukey test (p ≤ 0.05) and the standard deviations were represented vertical bars

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The dry weight of the different organs of faveleira decreased with the increasing external concentration of NaCl, especially in roots, basal and apical leaves (Table 1). In the roots, a decrease of 60 % of the dry weight under 50 mM NaCl in comparison with the control observed, whereas at doses of 100 and 150 mM this reduction was of 46 and 47 %, respectively (Table 1). In xylopodium and SP, the reduction of the dry weight achieved greater percentage under 150 mM NaCl of approximately 37 and 48 %, respectively, when compared with the control. In leaf strata, significant reduction of dry weight was observed, especially in the presence of the highest salt dose (150 mM). In basal leaves, this reduction was more pronounced, especially under 50 and 150 mM NaCl, reaching 69 and 60 % of reduction, respectively. In the median and apical leaves, the reduction dropped to 38 and 51 %, respectively, under 150 mM NaCl (Table 1).

Table 1 Dry weight of roots, xylopodium, SP, and basal, median and apical leaves of faveleira plants exposed to different concentrations of NaCl (0, 50, 100 and 150 mM) for 8 days
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The presence of NaCl in the nutrient solution did not affect the water status indicators expressed by the relative water content (RWC) and moisture percentage (% M). In all plant parts, when compared with the control, the RWC and the % M did not decrease significantly in the presence of salt (Table 2).

Table 2 Indicators of water stress in roots, xylopodium, SP, and basal, median and apical leaves of faveleira subjected to different concentrations of NaCl (0, 50, 100 and 150 mM) for 8 days
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Na+, K+, and the K+/Na+ ratio

The increase in the external NaCl concentration caused a progressive increase of the content of Na+ in the different parts of faveleira, especially in roots, xylopodium and SP (Table 3). In the roots under 150 mM NaCl treatment, a sevenfold increase in the Na+ content in relation to the control was observed. In xylopodium and SP, the Na+ content under 150 mM NaCl was increased by ninefold when compared with the respective controls. In aerial parts (leaf strata) the content of Na+ also increased in relation to the controls and this increase was directly proportional to the increase of NaCl concentration in the nutritive solution. Among the three leaf strata analyzed in the presence of the highest dose of salt (150 mM), the most significant increase occurred in the apical leaves (youngest leaves) with about 3.5-fold followed by 2.5- and 2-fold in basal and median leaves, respectively, compared with their respective controls (Table 3).

Table 3 Contents of Na+ and K+ and the K+/Na+ ratio in roots, xylopodium, SP and basal, median and apical leaves of faveleira subjected to different concentrations of NaCl (0, 50, 100 and 150 mM) for 8 days
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The content of K+ was higher in the aerial parts of both salt-treated and untreated plants. Such accumulation was higher in younger leaves (apical), followed by the median leaves, basal leaves and SP (Table 3). The NaCl treatment reduced the K+ content in the different parts of plants exposed to salt, except in SP and median leaves, where an increase in the content of this ion was observed when compared with their respective controls. The reduction in the K+ content was more severe at higher concentrations of NaCl mainly in roots and xylopodium (Table 3). At the highest dose of NaCl (150 mM), the reduction of K+ content in roots and xylopodium were about 40 and 45 %, respectively. In the leaf strata, the apical and basal leaves presented a decrease of 15 and 3 %, respectively, although these reductions were not significant when compared with their controls. In the SP, there was an increase of approximately 45 % and in the median leaves the increase was of about 11 % when compared to their respective controls (Table 3).

The K+/Na+ ratio in plants treated with NaCl presented a decrease in relation to plants from control since the 50 mM NaCl dose (Table 3). This effect was more pronounced in roots, xylopodium and SP, whose K+/Na+ ratio was less than 1. Such decrease was directly proportional to the increase of the external concentration of NaCl. At the dose of 150 mM, the roots and xylopodium showed a decrease in the K+/Na+ ratio greater than 90 %, whereas in the SP this decrease was about 84 % when compared with the respective controls (Table 3). The same effect, although less severe, maintaining a ratio superior than 1, was observed in leaf strata analyzed, with exception of the 50 mM dose in basal and median leaves. In the presence of 150 mM, the basal, median and apical leaves showed reduction of the K+/Na+ ratio of 61, 45 and 76 %, respectively, in relation to their controls (Table 3).

Organic solutes

The content of total soluble sugars (TSS) showed significant increase in the roots, SP, basal and apical leaves with the salt treatment, but there was a decrease of 55 % in the TSS content in xylopodium in the presence of 100 mM NaCl (Table 4). In the roots and SP, the increase of TSS content was between 1- and 1.8-fold. In leaf strata, with the exception of the median leaves, in which the TSS content remained unchanged when compared with the control, the increase was more pronounced, ranging between 2.5- and 3.1-fold for basal leaves and 1.5- and 3.2-fold for the apical leaves (Table 4).

Table 4 Content of total soluble sugars in roots, xylopodium, SP, and basal, middle and apical leaves of faveleira subjected to different concentrations of NaCl (0, 50, 100 and 150 mM) for 8 days
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On the other hand, the content of total soluble protein (TSP) increased in the roots at approximately 4.5-fold in relation to the control in the presence of 50 mM NaCl while in the xylopodium there was an increment of 1.6-fold under 150 mM NaCl (Table 5). In the SP, the increase in the content of TSP was of 3.2-fold in the presence of the most elevated NaCl dose (150 mM) when compared with control plants. In the leaf strata, an increase in the TSP content was also observed, particularly in the apical leaves (5.1-fold) in plants treated with 150 mM NaCl. In the other leaf strata (basal and median leaves), the content was increased 3.5- and 1.5-fold, respectively, in comparison to their respective controls (Table 5).

Table 5 Content of total soluble protein (TSP), total free amino acids (TFAA) and Proline (PRO) in roots, xylopodium, SP, and basal, middle and apical leaves of faveleira subjected to different concentrations of NaCl (0, 50, 100 and 150 mM) for 8 days
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The content of total free amino acids (TFAA) decreased in the roots with approximately 41 % in relation to the control in the presence of 150 mM NaCl, whereas in xylopodium there was an increment of 18.5 % at the same dose (Table 5). In the SP of plants subjected to salt stress, there was an increase in the content of TFAA between 16.5 and 126 % when compared with the control plants. The most pronounced increase (235.6 %) was observed in the apical leaves of the plants treated with 150 mM NaCl. In the other leaf strata (basal and median leaves), the content remained unchanged, in relation to the respective controls (Table 5).

The increase in proline content (PRO) was observed in all organs of faveleira, except the xylopodium which presented a decrease of PRO content ranging from 36 to 57 % compared with untreated plants (Table 5). In the roots, the increase was of PRO content was 2.7- to 3.9-fold and in the SP, this increase was between 1.7- and 2.7-fold in relation to control plants. However, the most pronounced increase of PRO content was observed in the leaf strata, mainly in basal leaves (2.9- to 5-fold) followed by apical (2.8- to 4.1-fold) and median (1.1- to 1.3-fold) (Table 5).

The contribution of proline (PRO) with respect to TFAA increased in plants treated with NaCl. In the roots, an increase of approximately 3.9, 3.8 and 5.3 % was observed in the presence of 50, 100, and 150 mM NaCl, respectively, with regard to the control, whereas in the xylopodium it was decreased up to 1.7 % under 50 and 150 mM NaCl (Table 6). In the stem, this increase was about 2.1 % under 100 mM NaCl compared with the respective control. In the leaf strata, this increase was more pronounced, especially in the basal and apical leaves. In the basal leaves of plants subjected to salt stress, there was an increase in the percentage of PRO of approximately 10 % under 50 and 150 mM NaCl. The most pronounced increase was observed in the apical leaves (with about 4.6 %) in the plants submitted to 100 mM NaCl (Table 6).

Table 6 % of PRO in relation to AALT in roots, xylopodium, SP, and basal, middle and apical leaves of faveleira subjected to different concentrations of NaCl (0, 50, 100 and 150 mM) for 8 days
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Discussion

The results of this study indicate that the salt treatments were able to induce significant physiological changes in both indicators of plant growth and the distribution of inorganic and organic solutes. However, the salt treatments did not alter the water status in different vegetative organs of faveleira plants.

Effect of NaCl salinity on plant growth

Visual symptoms characterized by chlorosis followed by necrosis (Fig. 1) were observed in basal and median leaves of faveleira which may be a consequence of toxicity caused by salt stress. Necrosis in basal leaves have appeared from the 6th day of salt stress, besides a precocious senescence have been described in the literature in species such as Myracrodrum urundeuva (Silva et al. 2000) and Anacardium occidentale (Morais et al. 2007). As faveleira plants remained 8 days in different salt treatments, it is possible that the symptoms observed in this species are due to the occurrence of ionic toxicity caused by toxic levels of saline ions in the leaf plant tissue.

Taking into account that the growth process is particularly sensitive to the effect of osmotic and ionic stress, plant growth indicators as height and number of leaves are good criteria for evaluation of the severity of stress and the ability of the plant to overcome it (Esteves and Suzuki 2008). In faveleira, the reduction of the relative growth rate (RGR), expressed on the basis of total fresh weight of the plant before and after the exposure to stress was proportional to the increase in salt concentration in the nutrient solution (Fig. 2a–d). The same results were obtained by Silva et al. (2005), when the Faveleira plants were exposed under salt stress.

The reduction and/or inhibition of growth and phytomass production caused by salinity has been widely reported in other species, such as Schinopsis quebracho (Meloni et al. 2008), Populus alba L. (Imada et al. 2009), Mentha pulegium (Oueslati et al. 2010) and Jatropha curcas (Silva et al. 2010). Such phenomenon has been attributed to: (1) drastic changes in water status of the plant due to disruption of homeostasis in water potential caused by osmotic effects, (2) ionic toxicity due to ionic imbalance, causing metabolic damage, physiological and oxidative stress, and (3) nutritional imbalance caused by interference in the absorption of essential nutrients (Munns et al. 2006; Munns and Tester 2008). In this work, it is likely that the reduction in faveleira plants growth was due to the occurrence of toxic levels of saline ions and/or a nutritional imbalance considering that NaCl did not affect the water status indicators expressed by the relative content of water and percentage of moisture in the different parts of the plant.

Ionic homeostasis

The increase in the external NaCl concentration led to the increase in the Na+ content in different organs of faveleira, especially in xylopodium, SP and roots. The xylopodium of salt-treated plants accumulated from eight to ninefold more Na+ in relation to the control, followed by the stem (seven to eightfold) and roots (six to sevenfold) (Table 3). However, the different leaf strata accumulated lower levels of Na+ than the organs mentioned above (two to threefold). Similar results were obtained with other tree species such as Anacardium occidentale L. (Morais et al. 2007), Schinopsis quebracho (Meloni et al. 2008) and Populus alba L. (Imada et al. 2009).

In faveleira, it is likely that the higher accumulation of Na+ observed in the basal parts of the plant is associated with a mechanism that control ion translocation to the shoot. It is also possible to infer that the xylopodium can act as Na+ accumulator organ, probably compartmentalizing it in the cell vacuoles and controlling the Na+ flow to the upper parts of the plant. It is known that plants can minimize the damage caused by compartmentalizing Na+ in vacuoles by Na+/H+ exchangers located in the tonoplast or even redirecting it to the basal and/or older plant parts (Ashraf 2002; Tester and Davenport 2003).

K+ is one of the most required plant macronutrient and it is also the most abundant cation in the plant cells (Gierth and Maser 2007). The elevated soil salinity compromises K+ absorption by plants, inducing the deficiency of this ion and generating cell metabolic disturbances due to the competition between Na+ and K+ in the active sites of the K+-dependent enzymes (Maathius and Amtmann 1999). In faveleira, a significant reduction of the content of K+ was observed under the more elevated dose of NaCl (150 mM) only in the roots and xylopodium. This effect could be attributed to the increased competition between Na+ and K+ in the sites of absorption and/or a higher K+ loading in the xylem and partitioning to the shoot. In faveleira, the three leaf strata analyzed, the median fully expanded leaves showed an increase in the content of K+ since the 100 mM NaCl treatment. It is known that, at least in part, the reduction of the plant growth rate involves energy allocation to maintain an efficient cell osmotic adjustment (Silveira et al. 2003) allowed by the accumulation of inorganic and organic solutes such as K+. Thus, the maintenance and/or accumulation of K+ observed in leaf strata of faveleira may indicate beneficial effects on water status, probably inducing decrease cell osmotic potential, as NaCl did not affect neither the relative water content nor the percentage of moisture of the plants. These results highlight the importance of K+ in the osmotic adjustment, as observed in Vitis vinifera (Patakas et al. 2002) and Anacardium occidentale (Morais et al. 2007). Additionally, in species such as Schinopsis quebracho colorado (Meloni et al. 2008) and Zea mays (Kholova et al. 2010), K+ and Cl− were identified as the main solutes that contribute into the cell water balance.

In general, salinity may cause two types of stress in higher plants: water deficit, as a result of the hyperosmolarity in the root environment, and ionic stress, which reflects, in large part, changes in the K+/Na+ ratio and excessive accumulation of saline ions, especially in leaves, which are harmful to the cell metabolism (Horie and Schroeder 2004). Thus, under conditions of salt stress the maintenance of the cell ion balance, the so-called “ionic homeostasis”, becomes even more relevant (Zhu 2003). Then, the K+/Na+ ratio could provide important information about the dynamics of ion accumulation under saline conditions.

In faveleira, the K+/Na+ ratio decreased in all organs, but the strongest effects were observed in the roots, xylopodium and SP, even under 50 mM NaCl (Table 3). According to Maathius and Amtmann (1999) the K+/Na+ ratio is an indicator of ionic toxicity, where values less than 1 indicate the preferential accumulation of Na+ in the plant tissues instead of K+. In faveleira, the K+/Na+ ratio below 1.0 in the roots, xylopodium, and SP and superior to 1.0 in the leaf strata suggests that Na+ retention in the basal plant parts may be an effective mechanism to avoid excessive Na+ accumulation in the shoots up to 100 mM NaCl, ensuring the maintenance of the K+/Na+ ratio compatible with the metabolic requirements of the plant. As discussed previously, it is likely that xylopodium plays a part in ion homeostasis in faveleira under salt stress, compartmentalizing Na+ within the cell vacuole and simultaneously contributing to higher K+ loading into the xylem.

Organic solutes in faveleira

Osmotic adjustment is a mechanism of tolerance to salt stress associated with the synthesis and accumulation of organic solutes such as sugars, amino acids, proline, and proteins into the plant cells (Heidari-Sharifabad and Mirzaie-Nodoushan 2006). In Plantago crassifolia (Vicente et al. 2004), Anacardium occidentale L. (Silveira et al. 2012), Reaumuria vermiculata (L.) (Gorai and Neffati 2011) and Zea mays (Köskerosglu and Tuna 2010) osmoregulation occurs through the accumulation of proline. Reducing sugars and ammonium quaternary compounds such as glycine-betaine are synthesized and accumulated in Lablab purpureus (D’Souza and Devaraj 2010) and Excoecaria agallocha (Jenci and Natarajan 2009) in response to salinity.

Sugars could contribute up to 50 % of the osmotic potential under abiotic stress (Ashraf and Harris 2004), besides preventing dehydration and being a preferential energy source (Elavumootil et al. 2003). In species such as Zea mays (Kholova et al. 2010), Ricinus communis (Babita et al. 2010), and Triticum aestivum (Nio et al. 2011), the increase of this osmolyte was considered as indicator of osmoregulation. In faveleira, it is possible that the increase of TSS observed in apical leaves (3.3-fold) may be related to osmoregulation since the water status of the leaves remained unchanged (Table 4). On the other hand, this response could be due to impaired sugar consumption by the leaf tissues. According to Munns and Weir (1981) the accumulation of sugars such as fructose and sucrose, which are part of the pool of TSS, may be related to stress-induced growth inhibition or changes in the activity of sucrose synthase or invertase (Sturm and Tang 1999). In relation to faveleira roots, it is suggested that the accumulation of TSS could reflect changes in sugar partitioning involving the export of carbohydrates from the shoot to the roots to allow root growth in spite of the adverse conditions in which the faveleira plants were grown (Silveira et al. 2003).

The accumulation of nitrogenous compounds as amino acids, amides, soluble proteins and polyamines is commonly related to salinity tolerance (Meloni et al. 2008). In several plants, these compounds play a part in osmotic adjustment, protection of cellular macromolecules, nutrient storage, maintenance of cellular pH, and detoxification of reactive oxygen species (Ashraf and Harris 2004). The plant cells could synthesize proteins to enable membrane stabilization and stress signalization (Tester and Davenport 2003). Moreover, the accumulation of total free amino acids (TFAA) and proline (PRO) could be caused either by increased protein hydrolysis driven by salt-induced metabolic damage or by de novo synthesis of compatible solutes in the intracellular environment (Alves and Setter 2004; Silva et al. 2009).

In faveleira, the changes in the content of nitrogenous compounds in response to salinity varied according to the plant part, although in the xylopodium and median leaves no significant effects were observed. In the SP and apical leaves, a significant increase in the content of TSP, TFAA and PRO and in the roots and basal leaves was observed. There was an increase in the content of total soluble protein (TSP) and PRO and decreased accumulation of TFAA (Table 5). Such results suggest that there was protein synthesis in both roots and basal leaves. Comparing the data on the content of TFAA and PRO with the RWC and % M, two hypotheses can be inferred. First, as salt stress did not alter the RWC and the % M neither in the SP nor in the apical leaves, it is possible that the increased content of TFAA in these organs could be a physiological response of the plant in attempt to osmotic adjustment through the activation of amino acid synthesis pathways. It could still be suggested that PRO is an amino acid responsible for this adjustment. Additionally, the increased content of TSP could also be related to the synthesis of specific proteins under adverse conditions, contributing to salt stress tolerance (Mohammdkani and Heidari 2008).

Second, in the basal leaves and roots, it is likely that protein synthesis is related to the decrease of the content of TFAA, although the data on PRO content do not reinforce this statement. However, the observed increase in the content of PRO may be due to a restricted incorporation of this amino acid into proteins. In this way, the increase of PRO content appears to be due to a metabolism imbalance, as already observed in cashew plants (Silveira et al. 2003; Morais et al. 2007). We cannot exclude the possibility of osmotic adjustment by PRO, considering that the salt treatments did not alter the RWC and the % M in these organs.

Finally, the accumulation of TSS, TSP, TFAA and PRO in the different faveleira organs could suggest metabolic alterations, but the involvement of these compounds in osmotic adjustment could not be ruled out as the water status of plant parts was not changed.

Conclusion

The results of this study indicate that treatments with NaCl were able to induce significant physiological and biochemical alterations in indicators of stress associated with plant growth and the distribution of inorganic and organic solutes in different vegetative organs of faveleira plants. The saline ions provoked symptoms of chlorosis and necrosis in basal and median leaves and affected plant growth, as observed by the relative growth rate, shoot and root growth, and the number of leaves. The ionic effect was preponderant, since the saline treatments did not alter the water status of the plant determined by the relative water content and humidity. Salt stress caused Na+ accumulation in all plant organs, especially in xylopodium, which suggests the involvement of this organ in Na+ compartmentalization and the control of Na+ flow to upper parts of the plant to avoid ionic toxicity in the leaves. The maintenance and/or accumulation of K+ in the different leaf strata of faveleira may indicate beneficial effects over the water status. Finally, different responses in the organs of faveleira in relation to organic solutes (TSS, TSP, TFAA, and PRO) were observed, evidencing possible metabolic alterations related to osmotic adjustment and possibly xylopodium plays an essential role in ionic homeostasis.

Author contributions

M. D. M. Oliveira carried out the experiment, analyzed the data and drafted the manuscript. L. L. Bezerra and C. V. S. Dantas assisted in the experiments. E. L. Voigt and J. M. Maia supervised the work and edited the manuscript together with C. E. C. Macedo. All authors read and approved the final version of this manuscript.