Plant Biology ISSN 1435-8603
RESEARCH PAPER
Physiological adjustment to salt stress in Jatropha curcas is associated with accumulation of salt ions, transport and selectivity of K+, osmotic adjustment and K+/Na+ homeostasis gas5 E. N. Silva1, J. A. G. Silveira2, C. R. F. Rodrigues3,4 & R. A. Vie 1 Faculdade de Educacß~ ao, Ci^ encias e Letras do Sert~ ao, Central, Universidade Estadual do Ceara, Quixada, Ceara, Brazil 2 Departamento de Bioquımica e Biologia Molecular/Instituto Nacional de Ci^encia e Tecnologia em Salinidade (INCTsal/CNPq), Universidade Federal do Ceara, Fortaleza, Cear a, Brazil 3 Departamento de Ecologia, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil 4 Faculdade Le~ ao Sampaio, Juazeiro do Norte, CE, Brazil 5 Departamento de Engenharia Florestal da UFPB, Universidade Federal de Campina Grande, Patos, Paraıba, Brazil
Keywords Ion accumulation; Jatropha curcas; organic solutes; osmotic adjustment; salinity. Correspondence E.N. Silva, Faculdade de Educacß~ ao, Ci^ encias e Letras do Sert~ ao, Central, Universidade Estadual do Cear a, CEP 63900-000, Quixad a, Cear a, Brazil. E-mail: [email protected] Editor G. Thiel Received: 14 October 2014; Accepted: 8 April 2015 doi:10.1111/plb.12337
ABSTRACT This study assessed the capacity of Jatropha curcas to physiologically adjust to salinity. Seedlings were exposed to increasing NaCl concentrations (25, 50, 75 and 100 mM) for 15 days. Treatment without NaCl was adopted as control. Shoot dry weight was strongly reduced by NaCl, reaching values of 35% to 65% with 25 to 100 mM NaCl. The shoot/root ratio was only affected with 100 mM NaCl. Relative water content (RWC) increased only with 100 mM NaCl, while electrolyte leakage (EL) was much enhanced with 50 mM NaCl. The Na+ transport rate to the shoot was more affected with 50 and 100 mM NaCl. In parallel, Cl transport rate increased with 75 and 100 mM NaCl, while K+ transport rate fell from 50 mM to 100 mM NaCl. In roots, Na+ and Cl transport rates fell slightly only in 50 mM (to Na+) and 50 and 100 mM (to Cl) NaCl, while K+ transport rate fell significantly with increasing NaCl. In general, our data demonstrate that J. curcas seedlings present changes in key physiological processes that allow this species to adjust to salinity. These responses are related to accumulation of Na+ and Cl in leaves and roots, K+/Na+ homeostasis, transport of K+ and selectivity (K–Na) in roots, and accumulation of organic solutes contributing to osmotic adjustment of the species.
INTRODUCTION Salinity is one of the abiotic stresses that can affect worldwide crop productivity. It can inhibit plant growth as well as induce low external water potential, ion toxicity and interferes with uptake of nutrients, particularly K+ (Munns & Tester 2008; Zhang et al. 2010). This problem is most severe in arid and semi-arid regions, where soils containing a large amount of salts are often found (Ferreira-Silva et al. 2009). In these regions, soil conditions and climate favour Na+ accumulation, which causes unfavourable changes in soil chemical characteristics, e.g. decreased K+ availability (Chen et al. 2007; Benderradji et al. 2011). The main saline ions, Na+ and Cl, can affect nutrient uptake through competitive interaction or by affecting membrane selectivity. For example, a high level of Na+ frequently induces Ca2+ and K+ deficiencies (Tester & Davenport 2003). High salinity induces both osmotic stress (caused by a high concentration of salt in soil that reduces water uptake by plants) and ionic stress in plant tissues. The latter stress is associated with alterations in the Na+/K+ and Na+/Ca2+ ratios due to accumulation of ions (Na+ and Cl), which can be harmful to plant metabolism (Apse & Blumwald 2007). Moreover, under salt stress, maintenance of K+ and Na+ homeostasis and plant metabolism become even more important (Maathuis & Amtmann 1999).
Salinity resistance has been defined as ability to maintain adequate growth and metabolism under stress conditions (Munns & Tester 2008). The uptake mechanisms and ion accumulation patterns in different plant organs are important in distinguishing between salt-tolerant and salt-sensitive genotypes (Paranychianakis & Angelakis 2008). However, the complexity and diversity of the mechanisms involved in salt–plant interactions, which are associated with plant plasticity, make it difficult to determine the basic processes that account for tolerance to a specific abiotic stress factor (Flowers 2004). In glycophytes, diverse physiological strategies have been reported in the literature as mechanisms that confer protection and tolerance to the presence of excess n et al. 2005; MartiNa+ and Cl- in the root medium (Esta~ nez-Rodriguez et al. 2008). Among all of the salt tolerance mechanisms, exclusion of Na+ and Cl- from the cytosol via compartmentalisation into vacuoles has frequently been observed in glycophyte species (Zhu 2003). To maintain acceptable levels of Na+ in the cytosol, maintaining a high concentration of K+ is essential for normal metabolism, enzyme activity and cytosolic osmotic adjustment (Apse & Blumwald 2007). Despite the importance of Na+/K+ homoeostasis, less attention has been given to the mechanism of Cl- toxicity. This anion is extremely toxic for several plant species because it affects important metabolic processes (Prior et al. 2007).
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Another major factor associated with salt tolerance mechanisms is the ability to adjust osmotic pressure in the cytosol, which is mediated by de novo synthesis of organic solutes, such as proline, glycine-betaine, polyols and sugars (Ashraf & Foolad 2007; Carillo et al. 2008). The accumulation of such compounds during stress is important for osmoregulation and cell protection from salinity (Park et al. 2006; Molinari et al. 2007). In cashew rootstocks, the accumulation of proline is apparently associated with salt-induced injury rather than with an osmo-protector adaptive effect (Silveira et al. 2003; Ferreira-Silva et al. 2010). Jatropha curcas grows in marginal areas where important crop species are not able to survive (Silva et al. 2010a). Moreover, it has high economic potential due to its seed oil quality, which can be converted into biodiesel (Kheira & Atta 2009). Although this species had shown satisfactory yields under constraining conditions of semi-arid regions, such as drought and high temperature, its physiological adjustment mechanism has rarely been evaluated in terms of changes in growth, accumulation and transport of Na+, Cl- and K+ in shoot and root, K+/ Na+ homeostasis and osmotic adjustment under saline conditions. Thus, the aim of this study was to evaluate salt stressinduced changes in key physiological processes in J. curcas and its capacity to adjust to salinity. MATERIAL AND METHODS Plant material, growth conditions and harvest The experiment was carried out under greenhouse conditions, where the environmental conditions were: 24 °C minimum and 36 °C maximum mean air temperature; mean air relative humidity of 65%; maximum photosynthetic photon flux density (PPFD) of ca. 1500 lmolm2s1; and 12-h photoperiod. J. curcas cv. T1 seeds of homogeneous size and weight were surface sterilised for 1 min with sodium hypochlorite solution (5%, v/v), then germinated in sand. Eight days after germination, the seedlings were transferred to plastic pots (2 l) containing one-quarter strength Hoagland & Arnon (1950) nutrient solution (pH 6.0) in the first week and full strength solution thereafter. The seedlings were subjected to salt stress for 15 days by amending the nutrient solution with 25, 50, 75 or 100 mM NaCl added gradually (25 mmol NaCl) into the solution in order to avoid osmotic shock. The pH was monitored daily. The treatment without NaCl was adopted as control. At the end of experiment, the plants were collected and separated into leaves, stems, petioles and roots. Dry weights of these tissues were obtained after drying in an oven at 75 °C for 48 h. Relative water content, electrolyte leakage and osmotic adjustment The leaf relative water content (RWC) was calculated as RWC = [(FW – DW)/(TW – DW) 9 100, where FW is fresh weight, TW is turgid weight measured after 6 h of saturation in deionised water at 4 °C in the dark. Electrolyte leakage was assessed as described in Cavalcanti et al. (2007). Leaf discs were placed in closed tubes containing 10 ml deionised water and incubated at 25 °C in a water bath for 6 h; subsequently, electrical conductivity of the solution (L1) was determined. Samples were then boiled at 100 °C for 1 h, and after equilibration at 25 °C, a second electrical conductivity (L2) measurement was 1024
obtained. Electrolyte leakage (EL) was calculated as EL (%) = (L1/L2) 9100. To determine osmolality, small segments from fully expanded leaves were macerated in a mortar. After filtration through a Miracloth membrane, the sap was centrifuged at 10,000 g for 10 min at 4 °C. The resultant supernatant was used to determine osmolality (c) with a vapour pressure osmometer (Vapro 5520; Wescor, Logan, UT, USA). Osmotic potential was determined using the formula: Ψs (MPa) = c (mosmolkg1) 9 2.58 9 10-3, according to the Van’t Hoff equation. For measurement of osmotic potential at full turgor (Ψs100), intact leaves and root segments of stressed and control plants were fully hydrated on moistened filter paper in Petri dishes for 24 h at 4 °C in the dark. The total OA was calculated as the difference in osmotic potential at full turgor between control (Ψsc100) and salt stress (Ψss100) conditions (MartinezBallesta et al. 2004). Organic and inorganic solutes Lyophilised leaf samples were transferred to hermetically closed tubes containing deionised water and placed in a water bath at 100 °C for 1 h. After supernatant extraction, total soluble sugar concentration was determined using the phenol–sulphuric acid method (Dubois et al. 1956). Total free amino acids, proline and glycine-betaine (GB) concentrations in leaves were determined as previously described (Silveira et al. 2009; Silva et al. 2010b). The Na+ and K+ concentrations were determined using flame photometry, and Cl- concentration determined through titration with AgNO3 (Silveira et al. 2009). Accumulation rates and selectivity of ions in the shoot and root The accumulation rates (JS) of Na+ (JNa), Cl- (JCl) and K+ (JK) in the shoot and root were calculated from changes in the content of Na+, Cl- and K+ from the onset to the end of the experiment, and were calculated according to Welbank (1962): Js ¼
ðM2 M1 Þ ln ðW2 W1 Þ T2 T1 W2 W1
ð1Þ
Js was calculated after determination of the ion content in the shoot and root (mmolkg1 DW) and expressed on the basis of root dry weight (DW) as described below: M1 and M2 = ion content in shoot and root determined at the onset (M1) and at the end (M2) of the experimental period; T2T1 = experimental period (15 days); and W2W1 = difference between root DW at the end (W2) and at the beginning (W1) of the experimental period. The results are expressed as mmol (kg root DW day)1. Shoot and root K+ selectivity in relation to Na+ (SK,Na) were calculated as described in Jeschke & Stelter (1983): SK;Na ¼
JK ½Naþ ext þ ½K ext JNa
ð2Þ
JK and JNa represent K+ and Na+ accumulation rates, respectively, in a specific organ; [Na+]ext and [K+]ext correspond to Na+ and K+ concentrations in the nutrient solution, respec-
Plant Biology 17 (2015) 1023–1029 © 2015 German Botanical Society and The Royal Botanical Society of the Netherlands
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Physiological adjustment to salt stress in J. curcas
tively. The selectivity was expressed in terms of mmol mmol1 (dimensionless). Physiological response analysis and statistical analysis The physiological responses analysis (PRA) to salinity for each trait and all traits together was calculated as the difference between minimum and maximum mean values of control and stress treatments divided by maximum mean value, as performed in previous studies (Valladares et al. 2000; Rodrigues et al. 2014). The experiment followed a completely randomised design, with five treatments (0, 25, 50 75 and 100 mM NaCl) and four replications. The experimental unit was one plant in one pot. The data were subjected to ANOVA, and means were compared with Tukey’s test at 0.05 confidence level. RESULTS Effect of salt stress on growth, water status, membrane damage and osmotic adjustment Salt stress significantly decreased (P = 0.05) growth of J. curcas. The dry weights of leaves, stems, petioles and roots were reduced in all NaCl treatments (Table 1). For example, the reduction in leaf DW reached 25% to 50% in 25 to 100 mM NaCl. In stems, petioles and roots, the effect of NaCl was more accentuated. In the interval from 25 to 100 mM, reductions were 37% to 66% in stems, while in petioles and roots, the decreases were 30% to 69% and 35% to 58%, respectively. Shoot DW (DW of leaves + stems + petioles) was strongly reduced by NaCl, reaching 35% to 65% in 25 to 100 mM NaCl. The shoot/root ratio was only affected in 100 mM NaCl. Regarding RWC, in 25 to 75 mM NaCl, values remained near controls, but at 100 mM NaCl, RWC increased 37% (Table 2). Electrolyte leakage (EL) was intensely enhanced with 50 mM NaCl. Between 50 and 100 mM NaCl, EL increased 58% to 104% when compared to controls (Table 2). It is important to report that J. curcas seedlings can osmotically adjust to salt stress (Table 2). Effects of salt stress on accumulation of inorganic and organic solutes The Na+ concentrations in leaves and roots with 25–100 mM NaCl increased four- to seven-fold and three- to six-fold, respectively, above control plants (Table 3). In addition, ClTable 1. Dry weights of leaves, stems, petioles, roots and shoot and shoot/ root ratio in Jatropha curcas seedlings exposed to NaCl treatment for 15 days. leaf DW
stem DW
treatment (mM NaCl)
g per plant
0 25 50 75 100
0.806a 0.599b 0.462c 0.448c 0.406d
6.53a 4.07b 3.71b 2.45c 2.17c
petiole DW
1.0a 0.694b 0.692b 0.407c 0.312d
root DW
0.889a 0.559b 0.526b 0.385c 0.372c
shoot DW
8.33a 5.361b 4.866b 3.305c 2.888c
shoot/ root
9.37a 9.59a 9.25a 8.58b 7.76c
Values represent means of four replicates. Means followed by the same letter did not differ significantly according to Tukey’s test (P = 0.05)
concentrations strongly increased with 25–100 mM NaCl, reaching values seven- to 14-fold higher in leaves and ten- to 12-fold higher in roots, when compared to control plants (Table 3). In contrast, K+ content fell in leaves at all NaCl concentrations; between 25 and 100 mM NaCl, the reduction was 61% to 87% (Table 3). In roots there was a reduction of 22% to 35% with 50 to 100 mM NaCl. The K+/Na+ ratios progressively fell in leaves as in roots, at increasing NaCl concentrations (Table 3). Regarding organic solute accumulation, it is important to mention that leaf free amino acid content increased 46% with 50 mM NaCl and 92% with 100 mM NaCl when compared to control plants (Table 4). Total soluble sugars slightly increased with NaCl, with a maximum of 16% at 100 mM. In contrast, proline content was stimulated in the presence of salt by 55% (50 mM) and 150% (100 mM) (Table 4). The glycine-betaine content was also enhanced in this interval to16% and 46% (Table 4). Effect of salt stress on transport and selectivity of ions in shoot and root The Na+ transport to shoots was more affected with 50 and 100 mM NaCl (Fig 1a). In parallel, Cl- transport increased with 75 and 100 mM NaCl (Fig. 1b), while K+ transport fell with 50 mM to 100 mM (Fig. 1c). In roots, Na+ and Cl- transport fell slightly only in the 50 mM (to Na+) and 50 and 100 mM (to Cl-) NaCl concentrations (Fig. 2a, b), while K+ transport fell significantly with increasing NaCl concentration (Fig. 2c). In addition, the selectivity (K-Na) in both shoot and root was progressively enhanced with increasing NaCl concentration (Fig. 3a, b). Physiological responses analysis (PRA) The physiological responses to salt stress suggest physiological adjustment in this species, as shown in the radial plot (Fig. 4). While some physiological variables fell with increasing salt stress (negative PRA), others increased (positive PRA). The most important information in the PRA is the modular value, which represents the magnitude of the response. According to this response, there are three PRA groups: low (PRA < 0.5), moderate (0.5 < PRA < 0.8) and high (PRA > 0.8). The highest PRA values were found in relation to concentrations of Na+ and Cl- in leaves and roots, concentration of K+ in leaves, K+/
Table 2. Relative water content (RWC), electrolyte leakage (EL) and osmotic adjustment (OA) in leaves of Jatropha curcas seedlings exposed to NaCl treatment for 15 days. treatment (mM NaCl)
RWC %
EL % MD
OA
0 25 50 75 100
68.11b 71.19b 72.46b 74.9b 93.67a
10.48d 11.36d 16.57c 18.34b 21.38a
0.37d 0.64c 0.85b 1.21a
Values represent means of four replicates. Means followed by the same letter did not differ significantly according to Tukey’s test (P = 0.05)
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Table 3. Content of Na+, Cl, K+ and K+/Na+ ratio in leaves and roots of Jatropha curcas seedlings exposed to NaCl treatment for 15 days. treatment mM NaCl 0 25 50 75 100
leaf Cl
leaf Na+
leaf K+
root Na+
mmolkg1 DW 376.81e 1710.14d 2202.89c 2376.81a 2492.75a
70d 525c 770b 956.67a 980a
1435.9a 547.01b 329.79c 256.41d 188.03e
leaf K+/Na+
mmolkg1 DW
20.51a 1.04b 0.43c 0.27d 0.19e
289.86e 898.55d 1101.45c 1391.3b 1681.16a
root Cl
root K+ root K+/Na+
116.67c 1166.67b 1376.67a 1423.33a 1458.33a
1606.84a 1589.74a 1247.86b 1162.39b 1042.73c
13.77a 1.36b 0.91c 0.82d 0.72e
Values represent means of four replicates. Means followed by the same letter did not differ significantly according to Tukey’s test (P = 0.05)
Table 4. Content of total free amino acids (TFAA), total soluble sugars (TSS), proline (Pro) and glycine-betaine (GB) in leaves of Jatropha curcas seedlings exposed to NaCl treatment for 15 days. TFAA
treatment (mM NaCl)
(mmolkg
0 z25 50 75 100
164.697d 199.875c 241.449b 267.033b 317.668a
1
TSS
Pro
GB
204.48b 217.72a 229.04a 231.59a 238.93a
0.89d 1.11c 1.39b 1.50b 2.24a
127.15c 138.62b 147.70b 184.51a 186.10a
a
DW)
b
Values represent means of four replicates. Means followed by the same letter did not differ significantly according to Tukey’s test (P = 0.05)
Na+ ratios in leaves and roots, transport of K+ and selectivity (K–Na) in roots. c
DISCUSSION Jatropha curcas display physiological changes in response to salinity The results of this study suggest that salt stress-induced changes in J. curcas contribute to its capacity to adjust to a saline environment. In general, the ability of a plant to acclimate to constraining environmental conditions is genetically determined (Donohue et al. 2005; Miner et al. 2005). It has been documented that, while some species exhibit substantial capacity for acclimation, others show only a modest plastic response to such changes (Gonzalez & Gianoli 2004; Valladares et al. 2007). The NaCl treatments induced significant alterations in physiological indicators associated with growth, accumulation, transport and selectivity of ions, and osmotic adjustment in J. curcas. For example, there was a large reduction in growth of all organs studied in mild salinity conditions (25 to 50 mM NaCl). These results corroborate previous studies of our group that demonstrated that J. curcas is a species sensitive to mild or moderate salt stress (Silva et al. 2010c; Rodrigues et al. 2012). This response may be strongly associated with ionic disturbance due to excess Na+ and Cl- uptake in plant tissues under NaCl treatment (Ashraf & Harris 2004). The high uptake of salt ions indicates that J. curcas does not have Na+ and Cl- redistribution and efficient exclusion mechanisms to reduce excess accumulation of salt ions in shoots under salinity (Silva et al. 2009a, 2010c, 2011). This response 1026
Fig 1. Shoot uptake rates of Na+ (a), Cl (b) and K+ (c) in Jatropha curcas seedlings exposed to NaCl treatment for 15 days. Values represent means of four replicates. Means followed by the same letter did not differ significantly according to Tukey’s test (P = 0.05).
varies among and within species, i.e. some genotypes have efficient exclusion mechanisms for Na+ and Cl- to minimise deleterious effects (Esta~ n et al. 2005), while others have salt-includer characteristics, similar to J. curcas (Martinez-Rodriguez et al. 2008; : Silva et al. 2009a). In addition, some plants have preferential Cl- accumulation, such as Citrus (Garcıa-Sanchez et al. 2002; Prior et al. 2007) and cashew (Ferreira-Silva et al. 2008; Silveira et al. 2012). In this context, our group found that J. curcas has high uptake of Na+ and Cl- ions, which are the most important solutes for osmotic adjustment under salinity (Silva et al. 2009b). Hence, J. curcas can be characterised as a salt includer species, especially for Na+ and Cl-, even at low NaCl levels (Silva et al. 2009a, 2010c). This response is frequently found in some
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Silva, Silveira, Rodrigues & Vi egas
a
b
Physiological adjustment to salt stress in J. curcas
a
b
c
Fig 3. (a) Selectivity index for K+ in relation to Na+ in the shoot (SK,Na) and (b) selectivity index for K+ in relation to Na+ in roots (SK,Na) of Jatropha curcas seedlings exposed to NaCl treatment for 15 days. Values represent means of four replicates. Means followed by the same letter did not differ significantly according to Tukey’s test (P = 0.05).
Fig 2. Root uptake rates of Na+ (a), Cl (b) and K+ (c) in Jatropha curcas seedlings exposed to NaCl treatment for 15 days. Values represent means of four replicates. Means followed by the same letter did not differ significantly according to Tukey’s test (P = 0.05).
glycophytes (Munns 2005; Ottow et al. 2005) and halophytes (Vasquez et al. 2006; Silveira et al. 2009). On the other hand, K+ content in leaves and roots may fall due to high Na+ accumulation in these tissues through antagonism between the two ions (Carillo et al. 2011). Regarding K+/Na+ ratios in plant tissues, it has widely been reported that elevated K+ in the external medium exerts a beneficial effect on plants exposed to high levels of Na+, restricting salt toxicity. Moreover, maintenance of a high cytosolic K+/ Na+ ratio is key to salt tolerance. Under optimal conditions, the cytosolic K+ content is about 150 mM with a negligible Na+ content (Carden et al. 2003). However, excess Na+ accumulation in the cytosol is associated with salt-induced K+ efflux, and the cytosolic K+/Na+ ratio falls dramatically under saline conditions (Shabala et al. 2003). This salinity-induced K+ loss from cells is a result of NaCl-induced membrane depolarisation, leading to activation of depolarisation-activated outwardrectifying K+ channels (Cuin et al. 2008). Several authors have suggested that adequate K+ levels might alleviate the toxic effects of Na+ in plant tissues (Cakmak 2005; Chen et al. 2007; Wu et al. 2009). It is also widely accepted that adequate K+/Na+ ratios in plant tissues (generally above 1.0) in the presence of NaCl salinity are essential for K+–Na+ homeostasis and salinity tolerance (Apse & Blumwald 2007; Britto
et al. 2010). Nevertheless, this assumption is based on very little experimental evidence. Others have suggested the use of K+/ Na+ ratios in leaves as a good indicator for the selection of genotypes with salt tolerance (Flowers 2004; Munns & Tester 2008). Our data show that only in the 25 mM NaCl treatment was there an adequate K+/Na+ ratio in both leaves and roots. All other salt treatments had values of K+/Na+ below 1.0 in both organs, suggesting that J. curcas presents an ionic imbalance and low tolerance under moderate to severe salt stress. This response reinforces previous results obtained by our group that demonstrate that this species has high retention of Na+ even under low NaCl concentrations. Moreover is important to mention that the ionic disturbances in leaves were more severe than in roots, as indicated by higher K+/Na+ ratios. This response may be associated with higher transport of K+ for the root alleviating the toxic effects of Na+. Although the toxic effects of salinity on shoots and roots of J. curcas seedlings were examined in this study, the leaf RWC increased with increasing NaCl concentrations. This response indicates maintenance of the water status of the leaves where the excess accumulated ions contributed to water retention in the tissues. In parallel, the osmotic potential decreased (data not shown). A reduction of Ψs of woody plants can be related to increased Na+ and Cl- accumulation (Ottow et al. 2005; Silveira et al. 2009). Taken together, these responses contribute to a mechanism for osmotic adjustment under salinity. Physiological response analysis and its importance for J. curcas under salt stress The physiological responses of J. curcas to salt stress revealed a low PRA to shoot/root ratio, RWC, concentration of K+ in
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Fig 4. Overall representation of physiological response analysis (PRA) in Jatropha curcas seedlings exposed to NaCl treatment as a radial plot. Data refer to the following variables: Leaf DW, stem DW, petiole DW, root DW, shoot DW, shoot/root ratio, RWC, electrolyte leakage, osmotic adjustment, leaf Na+ content, root Na+ content, leaf Cl content, root Cl content, leaf K+ content, root K+ content, leaf K+/Na+ ratio, root K+/Na+ ratio, total free amino acids, total soluble sugars, proline, glycinebetaine, shoot Na+ transport rate, shoot Cl transport rate, shoot K+ transport rate, root Na+ transport rate, root Cl transport rate, root K+ transport rate, shoot selectivity for K–Na and root selectivity for K–Na.
root, amino acids, soluble sugars, glycine-betaine and transport of Na+ and Cl- to shoots and roots. Moderate values of PRA were found for stem, petiole, root and shoot DW, EL, OA, proline content, transport of K+ to shoots and selectivity (K-Na) for the shoot. The highest PRA was found for concentrations of Na+ and Cl- in the leaves and roots, concentration of K+ in leaves, K+/Na+ ratio in leaves and roots, transport of K+ and selectivity (K-Na) for roots (Fig. 4). According to these responses, our data support the idea that accumulation of Na+ and Cl- in leaves and roots is evidence that J. curcas can adjust osmotically to salt stress. Silva et al. (2009b) demonstrated that Na+ and Cl- were the major solutes that contribute to osmotic adjustment of species under salinity conditions. Recently, Rodrigues et al. (2014) demonstrated that R. communis, a species in the same family, has efficient osmotic adjustment to salt stress through extension of Na+ and Cluptake. These results are in concordance with those obtained here, showing that both species present similar mechanisms of salinity tolerance. Although organic solutes, such as amino acids, soluble sugars and glycine-betaine did not reach high values of PRA, their accumulation is associated with maintenance of a good leaf REFERENCES Apse M.P., Blumwald E. (2007) Na+ transport in plants. FEBS Letters, 581, 2247–2254. Ashraf M., Foolad M.R. (2007) Roles of glycinebetaine and proline in improving plant abiotic stress resistance. Environmental and Experimental Botany, 59, 206–216. Ashraf M., Harris P.J.C. (2004) Potential biochemical indicators of salinity tolerance in plants. Plant Science, 166, 3–16.
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water status, which suggests that these osmo-solutes may be involved in the mechanism of osmotic adjustment, as previously shown (Silva et al. 2009b). In fact, they are known to contribute to osmotic adjustment of plants under salt stress conditions (Silveira et al. 2009). Here, our data support the occurrence of osmotic adjustment (see OA values in Table 2) and the involvement of these organic solutes in this mechanism. In summary, we have demonstrated that J. curcas presents changes in key physiological processes that allow it to adjust to a saline environment. These responses are related to accumulation of Na+ and Cl in leaves and roots, K+/Na+ homeostasis, transport of K+ and selectivity (K-Na) for roots and accumulation of organic solutes contributing to osmotic adjustment. ACKNOWLEDGEMENTS We thank the Conselho Nacional de Desenvolvimento Cientıfico e Tecnol ogico (CNPq) and Fundacß~ao Cearense de Apoio ao Desenvolvimento Cientıfico e Tecnol ogico (FUNCAP) for financial support, and the Tamandua Farm, Patos, Paraıba, Brazil, for supplying J. curcas seeds.
Benderradji L., Brini F., Amar S.B., Kellou K., Azaza J., Masmoudi K., Bouzerzour H.M. (2011) Sodium transport in the plants of two bread wheat (Triticum aestivum L.) genotypes showing contrasting salt stress tolerance. Australian Journal of Crop Science, 5, 233–241. Britto D.T., Ebrahimi-Ardebili S., Hamam A.M., Coskun D., Kronzucker H.J. (2010) 42K analysis of sodium-induced potassium efflux in barley: mechanism and relevance to salt tolerance. New Phytologist, 186, 373–384.
Cakmak L. (2005) The role of potassium in alleviating detrimental effects of abiotic stresses in plants. Journal of Plant Nutrition and Soil Science, 168, 521– 530. Carden D.E., Walker D.J., Flowers T.J., Miller A.J. (2003) Single-cell measurements of the contributions of cytosolic Na+ and K+ to salt tolerance. Plant Physiology, 131, 676–683. Carillo P., Mastrolonardo G., Nacca F., Parisi D., Verlotta A., Fuggi A. (2008) Nitrogen metabolism in durum wheat under salinity: accumulation of pro-
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line and glycine-betaine. Functional Plant Biology, 35, 412–426. Carillo P., Parisi D., Woodrow P., Pontecorvo G., Massaro G., Annunziata M.G., Fuggi A., Sulpice R. (2011) Salt-induced accumulation of glycine-betaine is inhibited by high light in durum wheat. Functional Plant Biology, 38, 139–150. Cavalcanti F.R., Lima J.P.M.S., Ferreira-Silva S.L., Viegas R.A., Silveira J.A.G. (2007) Roots and leaves display contrasting oxidative response during salt stress and recovery in cowpea. Journal of Plant Physiology, 164, 591–600. Chen Z.H., Zhou M.X., Newman I.A., Mendham N.J., Zhang G.P., Shabala S. (2007) Potassium and sodium relations in salinised barley tissues as a basis of differential salt tolerance. Functional Plant Biology, 34, 150–162. Cuin T.A., Betts S.A., Chalmandrier R., Shabala S. (2008) A root’s ability to retain K+ correlates with salt tolerance in wheat. Journal of Experimental Botany, 59, 2697–2706. Donohue K., Polisetty C.R., Wender N.J. (2005) Genetic basis and consequences of niche construction: plasticity-induced genetic constraints on the evolution of seed dispersal in Arabidopsis thaliana. The American Naturalist, 165, 537–550. Dubois M., Gilles K.A., Hamilton J.K., Rebers P., Asmith F. (1956) Colorimetric method for determination of sugar and related substances. Analytical Chemistry, 28, 350–356. Esta~ n M.T., Martinez-Rodriguez M.M., Perez-Alfocea F., Flowers T.J., Bolarin C. (2005) Grafting raises the salt tolerance of tomato through limiting the transport of sodium and chloride to the shoot. Journal of Experimental Botany, 56, 703–712. Ferreira-Silva S.L., Silveira J.A.G., Voigt E.L., Soares L.S.P., Viegas R.A. (2008) Changes in physiological indicators associated with salt tolerance in two contrasting cashew rootstocks. Brazilian Journal of Plant Physiology, 20, 51–59. Ferreira-Silva S.L., Voigt E.L., Viegas R.A., Paiva J.R., Silveira J.A.G. (2009) Influence of rootstocks on the resistance of cashew plantlets to salt stress. Pesquisa Agropecuaria Brasileira, 44, 361–367. Ferreira-Silva S.L., Silva E.N., Carvalho F.E.L., Lima C.S., Alves F.A.L., Silveira J.A.G. (2010) Physiological alterations modulated by rootstock and scion combination in cashew under salinity. Scientia Horticulturae, 127, 39–45. Flowers T.J. (2004) Improving crop salt tolerance. Journal of Experimental Botany, 55, 307–319. Garcıa-Sanchez F., Jifon J.L., Carvajal M., Syvertsen J.P. (2002) Gas exchange, chlorophyll and nutrient contents in relation to Na+ and Cl– accumulation in Sunburst mandarin grafted on different rootstocks. Plant Science, 162, 705–712. Gonzalez A.V., Gianoli E. (2004) Morphological plasticity in response to shading in three Convolvulus species of different ecological breadth. Acta Oecologica, 26, 185–190. Hoagland D.R., Arnon D.I. (1950) The water-culture method for growing plants without soil. California Agriculture Experimental Station Circular, 347, 1–39. Jeschke W.D., Stelter W. (1983) Ionic relations of garden orache, Atriplex hortensis: growth and ion distribution at moderate salinity and the function of bladder hair. Journal of Experimental Botany, 34, 795–810. Kheira A.A.A., Atta N.M.M. (2009) Response of Jatropha curcas L. to water deficits: yield, water use effi-
Physiological adjustment to salt stress in J. curcas
ciency and oilseed characteristics. Biomass and Bioenergy, 33, 1343–1350. Maathuis F.J.M., Amtmann A. (1999) K+ nutrition and Na+ toxicity: the basis of cellular K+/Na+ ratios. Annals of Botany, 84, 123–133. Martinez-Ballesta M.C., Martinez V., Carvajal M. (2004) Osmotic adjustment, water relations and gas exchange in pepper plants grown under NaCl or KCl. Environmental and Experimental Botany, 52, 161–174. Martinez-Rodriguez M.M., Esta~ n M.T., Moyano E., Garcia-Abellan J.O., Flores F.B., Campos J.F., Al-Azzawi M.J., Flowers T.J., Boların M.C. (2008) The effectiveness of grafting to improve salt tolerance in tomato when an ‘excluder’ genotype is used as scion. Environmental and Experimental Botany, 63, 392–401. Miner B.G., Sultan S.E., Morgan S.G., Padilla D.K., Relyea R.A. (2005) Ecological consequences of phenotypic plasticity. Trends in Ecology & Evolution, 20, 685–692. Molinari H.B.C., Marur C.J., Daros E., Campos M.K.F., Carvalho J.F.R.P., Filho J.C.B. (2007) Evaluation of the stress-inducible production of proline in transgenic sugarcane (Saccharum spp.): osmotic adjustment, chlorophyll fluorescence and oxidative stress. Physiologia Plantarum, 29, 130–218. Munns R. (2005) Genes and salt tolerance: bringing them together. New Phytologist, 167, 645–663. Munns R., Tester M. (2008) Mechanisms of salinity tolerance. Annual Review of Plant Biology, 59, 651–681. Ottow E.A., Brinker M., Teichmann T., Fritz E., Kaiser W., Brosche M., Kangasj€arvi J., Jiang X., Polle A. (2005) Populus euphratica displays apoplastic sodium accumulation, osmotic adjustment by decreases in calcium and soluble carbohydrates, and develops leaf succulence under salt stress. Plant Physiology, 139, 1762–1772. Paranychianakis N.V., Angelakis A.N. (2008) The effect of water stress and rootstock on the development of leaf injuries in grapevines irrigated with saline effluent. Agricultural Water Management, 95, 375–382. Park E.J., Jeknic Z., Chen T.H.H. (2006) Exogenous application of glycine-betaine increases chilling tolerance in tomato plants. Plant and Cell Physiology, 47, 706–714. Prior L.D., Grieve A.M., Bevington K.B., Slavich P.G. (2007) Long-term effects of saline irrigation water on ‘Valencia’ orange trees: relationships between growth and yield, and salt levels in soil and leaves. Australian Journal of Agricultural Research, 58, 349–358. Rodrigues C.R.F., Silveira J.A.G., Silva E.N., Dutra A.T.B., Viegas R.A. (2012) Potassium transport and partitioning alleviates toxic effects of sodium on young physic nut plants. Revista Brasileira de Ci^encia do Solo, 36, 223–232. Rodrigues C.R.F., Silva E.N., Moura R.M., Anjos D.C., Hernandez F.F.F., Viegas R.A. (2014) Physiological adjustment to salt stress in R. communis seedlings is associated with a probable mechanism of osmotic adjustment and a reduction in water lost by transpiration. Industrial Crops and Products, 54, 233–239. Shabala S.N., Shabala I., van Volkenburgh E. (2003) Effect of calcium on root development and root ion fluxes in salinised barley seedlings. Functional Plant Biology, 30, 507–514. Silva E.N., Silveira J.A.G., Rodrigues C.R.F., Dutra A.T.B., Arag~ao R.M. (2009a) Ion uptake and growth of physic nut under different salinity levels. Revista Ci^encia Agron^omica, 40, 25–32.
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Silva E.N., Silveira J.A.G., Rodrigues C.R.F., Lima C.S., Viegas R.A. (2009b) Contribution of organic and inorganic solutes to osmotic adjustment of physic nut under salinity. Pesquisa Agropecuaria Brasileira, 44, 437–445. Silva E.N., Ferreira-Silva S.L., Fontenele A.V., Ribeiro R.V., Viegas R.A., Silveira J.A.G. (2010a) Photosynthetic changes and protective mechanisms against oxidative damage subjected to isolated and combined drought and heat stresses in Jatropha curcas plants. Journal of Plant Physiology, 167, 1157–1164. Silva E.N., Ferreira-Silva S.L., Viegas R.A., Silveira J.A.G. (2010b) The role of organic and inorganic solutes in the osmotic adjustment of drought-stressed Jatropha curcas plants. Environmental and Experimental Botany, 69, 279–285. Silva E.N., Ribeiro R.V., Ferreira-Silva S.L., Viegas R.A., Silveira J.A.G. (2010c) Comparative effects of salinity and water stress on photosynthesis, water relations and growth of Jatropha curcas plants. Journal of Arid Environments, 74, 1130–1137. Silva E.N., Ribeiro R.V., Ferreira-Silva S.L., Viegas R.A., Silveira J.A.G. (2011) Salt stress induced damages on the photosynthesis of physic nut young plants. Scientia Agricola, 68, 62–68. Silveira J.A.G., Viegas R.A., Rocha I.M.A., Oliveira T.J.A. (2003) Proline accumulation and glutamine synthetase activity are increased by salt-induced proteolysis in cashew leaves. Journal of Plant Physiology, 160, 115–123. Silveira J.A.G., Ara ujo S.A., Lima J.P.M.S., Viegas R.A. (2009) Roots and leaves display contrasting osmotic adjustment mechanisms in response to NaCl-salinity in Atriplex numularia. Environment and Experimental Botany, 66, 1–8. Silveira J.A.G., Junior J.M., Silva E.N., Ferreira-Silva S.L., Arag~ao R.M., Viegas R.A. (2012) Salt resistance in two cashew species is associated with accumulation of organic and inorganic solutes. Acta Physiologiae Plantarum, 34, 1629–1637. Tester M., Davenport R.J. (2003) Na+ transport and Na+ tolerance in higher plants. Annals of Botany, 91, 503–527. Valladares F., Wright S.J., Lasso E., Kitajima K., Pearcy R.W. (2000) Plastic phenotypic response to light of 16 congeneric shrubs from Panamanian rainforest. Ecology, 81, 1925–1936. Valladares F., Gianoli E., G omez J.M. (2007) Ecological limits to plant phenotypic plasticity. Tansley review. The New Phytologist, 176, 749–763. Vasquez E.A., Glenn E.P., Guntenspergen G.R., Brown J.J., Nelson S.G. (2006) Salt tolerance and osmotic adjustment of Spartina alterniflora (Poaceae) and the invasive M haplotype of Phragmites australis (Poaceae) along a salinity gradient. American Journal of Botany, 93, 1784–1790. Welbank P.J. (1962) The effects of competition with Agropyron repens and nitrogen and water on the nitrogen content of Impatiens parviflora. Annals of Botany, 26, 361–373. Wu Y., Hu Y., Xu G. (2009) Interactive effects of potassium and sodium on root growth and expression of K/Na transporter genes in rice. Plant Growth Regulation, 57, 271–280. Zhang J.L., Flowers T.J., Wang S.M. (2010) Mechanisms of sodium uptake by roots of higher plants. Plant and Soil, 326, 45–60. Zhu J. (2003) Regulation of ion homeostasis under salt stress. Current Opinion in Plant Biology, 6, 441–444.
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RESEARCH PAPER
Physiological adjustment to salt stress in Jatropha curcas is associated with accumulation of salt ions, transport and selectivity of K+, osmotic adjustment and K+/Na+ homeostasis gas5 E. N. Silva1, J. A. G. Silveira2, C. R. F. Rodrigues3,4 & R. A. Vie 1 Faculdade de Educacß~ ao, Ci^ encias e Letras do Sert~ ao, Central, Universidade Estadual do Ceara, Quixada, Ceara, Brazil 2 Departamento de Bioquımica e Biologia Molecular/Instituto Nacional de Ci^encia e Tecnologia em Salinidade (INCTsal/CNPq), Universidade Federal do Ceara, Fortaleza, Cear a, Brazil 3 Departamento de Ecologia, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil 4 Faculdade Le~ ao Sampaio, Juazeiro do Norte, CE, Brazil 5 Departamento de Engenharia Florestal da UFPB, Universidade Federal de Campina Grande, Patos, Paraıba, Brazil
Keywords Ion accumulation; Jatropha curcas; organic solutes; osmotic adjustment; salinity. Correspondence E.N. Silva, Faculdade de Educacß~ ao, Ci^ encias e Letras do Sert~ ao, Central, Universidade Estadual do Cear a, CEP 63900-000, Quixad a, Cear a, Brazil. E-mail: [email protected] Editor G. Thiel Received: 14 October 2014; Accepted: 8 April 2015 doi:10.1111/plb.12337
ABSTRACT This study assessed the capacity of Jatropha curcas to physiologically adjust to salinity. Seedlings were exposed to increasing NaCl concentrations (25, 50, 75 and 100 mM) for 15 days. Treatment without NaCl was adopted as control. Shoot dry weight was strongly reduced by NaCl, reaching values of 35% to 65% with 25 to 100 mM NaCl. The shoot/root ratio was only affected with 100 mM NaCl. Relative water content (RWC) increased only with 100 mM NaCl, while electrolyte leakage (EL) was much enhanced with 50 mM NaCl. The Na+ transport rate to the shoot was more affected with 50 and 100 mM NaCl. In parallel, Cl transport rate increased with 75 and 100 mM NaCl, while K+ transport rate fell from 50 mM to 100 mM NaCl. In roots, Na+ and Cl transport rates fell slightly only in 50 mM (to Na+) and 50 and 100 mM (to Cl) NaCl, while K+ transport rate fell significantly with increasing NaCl. In general, our data demonstrate that J. curcas seedlings present changes in key physiological processes that allow this species to adjust to salinity. These responses are related to accumulation of Na+ and Cl in leaves and roots, K+/Na+ homeostasis, transport of K+ and selectivity (K–Na) in roots, and accumulation of organic solutes contributing to osmotic adjustment of the species.
INTRODUCTION Salinity is one of the abiotic stresses that can affect worldwide crop productivity. It can inhibit plant growth as well as induce low external water potential, ion toxicity and interferes with uptake of nutrients, particularly K+ (Munns & Tester 2008; Zhang et al. 2010). This problem is most severe in arid and semi-arid regions, where soils containing a large amount of salts are often found (Ferreira-Silva et al. 2009). In these regions, soil conditions and climate favour Na+ accumulation, which causes unfavourable changes in soil chemical characteristics, e.g. decreased K+ availability (Chen et al. 2007; Benderradji et al. 2011). The main saline ions, Na+ and Cl, can affect nutrient uptake through competitive interaction or by affecting membrane selectivity. For example, a high level of Na+ frequently induces Ca2+ and K+ deficiencies (Tester & Davenport 2003). High salinity induces both osmotic stress (caused by a high concentration of salt in soil that reduces water uptake by plants) and ionic stress in plant tissues. The latter stress is associated with alterations in the Na+/K+ and Na+/Ca2+ ratios due to accumulation of ions (Na+ and Cl), which can be harmful to plant metabolism (Apse & Blumwald 2007). Moreover, under salt stress, maintenance of K+ and Na+ homeostasis and plant metabolism become even more important (Maathuis & Amtmann 1999).
Salinity resistance has been defined as ability to maintain adequate growth and metabolism under stress conditions (Munns & Tester 2008). The uptake mechanisms and ion accumulation patterns in different plant organs are important in distinguishing between salt-tolerant and salt-sensitive genotypes (Paranychianakis & Angelakis 2008). However, the complexity and diversity of the mechanisms involved in salt–plant interactions, which are associated with plant plasticity, make it difficult to determine the basic processes that account for tolerance to a specific abiotic stress factor (Flowers 2004). In glycophytes, diverse physiological strategies have been reported in the literature as mechanisms that confer protection and tolerance to the presence of excess n et al. 2005; MartiNa+ and Cl- in the root medium (Esta~ nez-Rodriguez et al. 2008). Among all of the salt tolerance mechanisms, exclusion of Na+ and Cl- from the cytosol via compartmentalisation into vacuoles has frequently been observed in glycophyte species (Zhu 2003). To maintain acceptable levels of Na+ in the cytosol, maintaining a high concentration of K+ is essential for normal metabolism, enzyme activity and cytosolic osmotic adjustment (Apse & Blumwald 2007). Despite the importance of Na+/K+ homoeostasis, less attention has been given to the mechanism of Cl- toxicity. This anion is extremely toxic for several plant species because it affects important metabolic processes (Prior et al. 2007).
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Physiological adjustment to salt stress in J. curcas
Another major factor associated with salt tolerance mechanisms is the ability to adjust osmotic pressure in the cytosol, which is mediated by de novo synthesis of organic solutes, such as proline, glycine-betaine, polyols and sugars (Ashraf & Foolad 2007; Carillo et al. 2008). The accumulation of such compounds during stress is important for osmoregulation and cell protection from salinity (Park et al. 2006; Molinari et al. 2007). In cashew rootstocks, the accumulation of proline is apparently associated with salt-induced injury rather than with an osmo-protector adaptive effect (Silveira et al. 2003; Ferreira-Silva et al. 2010). Jatropha curcas grows in marginal areas where important crop species are not able to survive (Silva et al. 2010a). Moreover, it has high economic potential due to its seed oil quality, which can be converted into biodiesel (Kheira & Atta 2009). Although this species had shown satisfactory yields under constraining conditions of semi-arid regions, such as drought and high temperature, its physiological adjustment mechanism has rarely been evaluated in terms of changes in growth, accumulation and transport of Na+, Cl- and K+ in shoot and root, K+/ Na+ homeostasis and osmotic adjustment under saline conditions. Thus, the aim of this study was to evaluate salt stressinduced changes in key physiological processes in J. curcas and its capacity to adjust to salinity. MATERIAL AND METHODS Plant material, growth conditions and harvest The experiment was carried out under greenhouse conditions, where the environmental conditions were: 24 °C minimum and 36 °C maximum mean air temperature; mean air relative humidity of 65%; maximum photosynthetic photon flux density (PPFD) of ca. 1500 lmolm2s1; and 12-h photoperiod. J. curcas cv. T1 seeds of homogeneous size and weight were surface sterilised for 1 min with sodium hypochlorite solution (5%, v/v), then germinated in sand. Eight days after germination, the seedlings were transferred to plastic pots (2 l) containing one-quarter strength Hoagland & Arnon (1950) nutrient solution (pH 6.0) in the first week and full strength solution thereafter. The seedlings were subjected to salt stress for 15 days by amending the nutrient solution with 25, 50, 75 or 100 mM NaCl added gradually (25 mmol NaCl) into the solution in order to avoid osmotic shock. The pH was monitored daily. The treatment without NaCl was adopted as control. At the end of experiment, the plants were collected and separated into leaves, stems, petioles and roots. Dry weights of these tissues were obtained after drying in an oven at 75 °C for 48 h. Relative water content, electrolyte leakage and osmotic adjustment The leaf relative water content (RWC) was calculated as RWC = [(FW – DW)/(TW – DW) 9 100, where FW is fresh weight, TW is turgid weight measured after 6 h of saturation in deionised water at 4 °C in the dark. Electrolyte leakage was assessed as described in Cavalcanti et al. (2007). Leaf discs were placed in closed tubes containing 10 ml deionised water and incubated at 25 °C in a water bath for 6 h; subsequently, electrical conductivity of the solution (L1) was determined. Samples were then boiled at 100 °C for 1 h, and after equilibration at 25 °C, a second electrical conductivity (L2) measurement was 1024
obtained. Electrolyte leakage (EL) was calculated as EL (%) = (L1/L2) 9100. To determine osmolality, small segments from fully expanded leaves were macerated in a mortar. After filtration through a Miracloth membrane, the sap was centrifuged at 10,000 g for 10 min at 4 °C. The resultant supernatant was used to determine osmolality (c) with a vapour pressure osmometer (Vapro 5520; Wescor, Logan, UT, USA). Osmotic potential was determined using the formula: Ψs (MPa) = c (mosmolkg1) 9 2.58 9 10-3, according to the Van’t Hoff equation. For measurement of osmotic potential at full turgor (Ψs100), intact leaves and root segments of stressed and control plants were fully hydrated on moistened filter paper in Petri dishes for 24 h at 4 °C in the dark. The total OA was calculated as the difference in osmotic potential at full turgor between control (Ψsc100) and salt stress (Ψss100) conditions (MartinezBallesta et al. 2004). Organic and inorganic solutes Lyophilised leaf samples were transferred to hermetically closed tubes containing deionised water and placed in a water bath at 100 °C for 1 h. After supernatant extraction, total soluble sugar concentration was determined using the phenol–sulphuric acid method (Dubois et al. 1956). Total free amino acids, proline and glycine-betaine (GB) concentrations in leaves were determined as previously described (Silveira et al. 2009; Silva et al. 2010b). The Na+ and K+ concentrations were determined using flame photometry, and Cl- concentration determined through titration with AgNO3 (Silveira et al. 2009). Accumulation rates and selectivity of ions in the shoot and root The accumulation rates (JS) of Na+ (JNa), Cl- (JCl) and K+ (JK) in the shoot and root were calculated from changes in the content of Na+, Cl- and K+ from the onset to the end of the experiment, and were calculated according to Welbank (1962): Js ¼
ðM2 M1 Þ ln ðW2 W1 Þ T2 T1 W2 W1
ð1Þ
Js was calculated after determination of the ion content in the shoot and root (mmolkg1 DW) and expressed on the basis of root dry weight (DW) as described below: M1 and M2 = ion content in shoot and root determined at the onset (M1) and at the end (M2) of the experimental period; T2T1 = experimental period (15 days); and W2W1 = difference between root DW at the end (W2) and at the beginning (W1) of the experimental period. The results are expressed as mmol (kg root DW day)1. Shoot and root K+ selectivity in relation to Na+ (SK,Na) were calculated as described in Jeschke & Stelter (1983): SK;Na ¼
JK ½Naþ ext þ ½K ext JNa
ð2Þ
JK and JNa represent K+ and Na+ accumulation rates, respectively, in a specific organ; [Na+]ext and [K+]ext correspond to Na+ and K+ concentrations in the nutrient solution, respec-
Plant Biology 17 (2015) 1023–1029 © 2015 German Botanical Society and The Royal Botanical Society of the Netherlands
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Physiological adjustment to salt stress in J. curcas
tively. The selectivity was expressed in terms of mmol mmol1 (dimensionless). Physiological response analysis and statistical analysis The physiological responses analysis (PRA) to salinity for each trait and all traits together was calculated as the difference between minimum and maximum mean values of control and stress treatments divided by maximum mean value, as performed in previous studies (Valladares et al. 2000; Rodrigues et al. 2014). The experiment followed a completely randomised design, with five treatments (0, 25, 50 75 and 100 mM NaCl) and four replications. The experimental unit was one plant in one pot. The data were subjected to ANOVA, and means were compared with Tukey’s test at 0.05 confidence level. RESULTS Effect of salt stress on growth, water status, membrane damage and osmotic adjustment Salt stress significantly decreased (P = 0.05) growth of J. curcas. The dry weights of leaves, stems, petioles and roots were reduced in all NaCl treatments (Table 1). For example, the reduction in leaf DW reached 25% to 50% in 25 to 100 mM NaCl. In stems, petioles and roots, the effect of NaCl was more accentuated. In the interval from 25 to 100 mM, reductions were 37% to 66% in stems, while in petioles and roots, the decreases were 30% to 69% and 35% to 58%, respectively. Shoot DW (DW of leaves + stems + petioles) was strongly reduced by NaCl, reaching 35% to 65% in 25 to 100 mM NaCl. The shoot/root ratio was only affected in 100 mM NaCl. Regarding RWC, in 25 to 75 mM NaCl, values remained near controls, but at 100 mM NaCl, RWC increased 37% (Table 2). Electrolyte leakage (EL) was intensely enhanced with 50 mM NaCl. Between 50 and 100 mM NaCl, EL increased 58% to 104% when compared to controls (Table 2). It is important to report that J. curcas seedlings can osmotically adjust to salt stress (Table 2). Effects of salt stress on accumulation of inorganic and organic solutes The Na+ concentrations in leaves and roots with 25–100 mM NaCl increased four- to seven-fold and three- to six-fold, respectively, above control plants (Table 3). In addition, ClTable 1. Dry weights of leaves, stems, petioles, roots and shoot and shoot/ root ratio in Jatropha curcas seedlings exposed to NaCl treatment for 15 days. leaf DW
stem DW
treatment (mM NaCl)
g per plant
0 25 50 75 100
0.806a 0.599b 0.462c 0.448c 0.406d
6.53a 4.07b 3.71b 2.45c 2.17c
petiole DW
1.0a 0.694b 0.692b 0.407c 0.312d
root DW
0.889a 0.559b 0.526b 0.385c 0.372c
shoot DW
8.33a 5.361b 4.866b 3.305c 2.888c
shoot/ root
9.37a 9.59a 9.25a 8.58b 7.76c
Values represent means of four replicates. Means followed by the same letter did not differ significantly according to Tukey’s test (P = 0.05)
concentrations strongly increased with 25–100 mM NaCl, reaching values seven- to 14-fold higher in leaves and ten- to 12-fold higher in roots, when compared to control plants (Table 3). In contrast, K+ content fell in leaves at all NaCl concentrations; between 25 and 100 mM NaCl, the reduction was 61% to 87% (Table 3). In roots there was a reduction of 22% to 35% with 50 to 100 mM NaCl. The K+/Na+ ratios progressively fell in leaves as in roots, at increasing NaCl concentrations (Table 3). Regarding organic solute accumulation, it is important to mention that leaf free amino acid content increased 46% with 50 mM NaCl and 92% with 100 mM NaCl when compared to control plants (Table 4). Total soluble sugars slightly increased with NaCl, with a maximum of 16% at 100 mM. In contrast, proline content was stimulated in the presence of salt by 55% (50 mM) and 150% (100 mM) (Table 4). The glycine-betaine content was also enhanced in this interval to16% and 46% (Table 4). Effect of salt stress on transport and selectivity of ions in shoot and root The Na+ transport to shoots was more affected with 50 and 100 mM NaCl (Fig 1a). In parallel, Cl- transport increased with 75 and 100 mM NaCl (Fig. 1b), while K+ transport fell with 50 mM to 100 mM (Fig. 1c). In roots, Na+ and Cl- transport fell slightly only in the 50 mM (to Na+) and 50 and 100 mM (to Cl-) NaCl concentrations (Fig. 2a, b), while K+ transport fell significantly with increasing NaCl concentration (Fig. 2c). In addition, the selectivity (K-Na) in both shoot and root was progressively enhanced with increasing NaCl concentration (Fig. 3a, b). Physiological responses analysis (PRA) The physiological responses to salt stress suggest physiological adjustment in this species, as shown in the radial plot (Fig. 4). While some physiological variables fell with increasing salt stress (negative PRA), others increased (positive PRA). The most important information in the PRA is the modular value, which represents the magnitude of the response. According to this response, there are three PRA groups: low (PRA < 0.5), moderate (0.5 < PRA < 0.8) and high (PRA > 0.8). The highest PRA values were found in relation to concentrations of Na+ and Cl- in leaves and roots, concentration of K+ in leaves, K+/
Table 2. Relative water content (RWC), electrolyte leakage (EL) and osmotic adjustment (OA) in leaves of Jatropha curcas seedlings exposed to NaCl treatment for 15 days. treatment (mM NaCl)
RWC %
EL % MD
OA
0 25 50 75 100
68.11b 71.19b 72.46b 74.9b 93.67a
10.48d 11.36d 16.57c 18.34b 21.38a
0.37d 0.64c 0.85b 1.21a
Values represent means of four replicates. Means followed by the same letter did not differ significantly according to Tukey’s test (P = 0.05)
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Table 3. Content of Na+, Cl, K+ and K+/Na+ ratio in leaves and roots of Jatropha curcas seedlings exposed to NaCl treatment for 15 days. treatment mM NaCl 0 25 50 75 100
leaf Cl
leaf Na+
leaf K+
root Na+
mmolkg1 DW 376.81e 1710.14d 2202.89c 2376.81a 2492.75a
70d 525c 770b 956.67a 980a
1435.9a 547.01b 329.79c 256.41d 188.03e
leaf K+/Na+
mmolkg1 DW
20.51a 1.04b 0.43c 0.27d 0.19e
289.86e 898.55d 1101.45c 1391.3b 1681.16a
root Cl
root K+ root K+/Na+
116.67c 1166.67b 1376.67a 1423.33a 1458.33a
1606.84a 1589.74a 1247.86b 1162.39b 1042.73c
13.77a 1.36b 0.91c 0.82d 0.72e
Values represent means of four replicates. Means followed by the same letter did not differ significantly according to Tukey’s test (P = 0.05)
Table 4. Content of total free amino acids (TFAA), total soluble sugars (TSS), proline (Pro) and glycine-betaine (GB) in leaves of Jatropha curcas seedlings exposed to NaCl treatment for 15 days. TFAA
treatment (mM NaCl)
(mmolkg
0 z25 50 75 100
164.697d 199.875c 241.449b 267.033b 317.668a
1
TSS
Pro
GB
204.48b 217.72a 229.04a 231.59a 238.93a
0.89d 1.11c 1.39b 1.50b 2.24a
127.15c 138.62b 147.70b 184.51a 186.10a
a
DW)
b
Values represent means of four replicates. Means followed by the same letter did not differ significantly according to Tukey’s test (P = 0.05)
Na+ ratios in leaves and roots, transport of K+ and selectivity (K–Na) in roots. c
DISCUSSION Jatropha curcas display physiological changes in response to salinity The results of this study suggest that salt stress-induced changes in J. curcas contribute to its capacity to adjust to a saline environment. In general, the ability of a plant to acclimate to constraining environmental conditions is genetically determined (Donohue et al. 2005; Miner et al. 2005). It has been documented that, while some species exhibit substantial capacity for acclimation, others show only a modest plastic response to such changes (Gonzalez & Gianoli 2004; Valladares et al. 2007). The NaCl treatments induced significant alterations in physiological indicators associated with growth, accumulation, transport and selectivity of ions, and osmotic adjustment in J. curcas. For example, there was a large reduction in growth of all organs studied in mild salinity conditions (25 to 50 mM NaCl). These results corroborate previous studies of our group that demonstrated that J. curcas is a species sensitive to mild or moderate salt stress (Silva et al. 2010c; Rodrigues et al. 2012). This response may be strongly associated with ionic disturbance due to excess Na+ and Cl- uptake in plant tissues under NaCl treatment (Ashraf & Harris 2004). The high uptake of salt ions indicates that J. curcas does not have Na+ and Cl- redistribution and efficient exclusion mechanisms to reduce excess accumulation of salt ions in shoots under salinity (Silva et al. 2009a, 2010c, 2011). This response 1026
Fig 1. Shoot uptake rates of Na+ (a), Cl (b) and K+ (c) in Jatropha curcas seedlings exposed to NaCl treatment for 15 days. Values represent means of four replicates. Means followed by the same letter did not differ significantly according to Tukey’s test (P = 0.05).
varies among and within species, i.e. some genotypes have efficient exclusion mechanisms for Na+ and Cl- to minimise deleterious effects (Esta~ n et al. 2005), while others have salt-includer characteristics, similar to J. curcas (Martinez-Rodriguez et al. 2008; : Silva et al. 2009a). In addition, some plants have preferential Cl- accumulation, such as Citrus (Garcıa-Sanchez et al. 2002; Prior et al. 2007) and cashew (Ferreira-Silva et al. 2008; Silveira et al. 2012). In this context, our group found that J. curcas has high uptake of Na+ and Cl- ions, which are the most important solutes for osmotic adjustment under salinity (Silva et al. 2009b). Hence, J. curcas can be characterised as a salt includer species, especially for Na+ and Cl-, even at low NaCl levels (Silva et al. 2009a, 2010c). This response is frequently found in some
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a
b
Physiological adjustment to salt stress in J. curcas
a
b
c
Fig 3. (a) Selectivity index for K+ in relation to Na+ in the shoot (SK,Na) and (b) selectivity index for K+ in relation to Na+ in roots (SK,Na) of Jatropha curcas seedlings exposed to NaCl treatment for 15 days. Values represent means of four replicates. Means followed by the same letter did not differ significantly according to Tukey’s test (P = 0.05).
Fig 2. Root uptake rates of Na+ (a), Cl (b) and K+ (c) in Jatropha curcas seedlings exposed to NaCl treatment for 15 days. Values represent means of four replicates. Means followed by the same letter did not differ significantly according to Tukey’s test (P = 0.05).
glycophytes (Munns 2005; Ottow et al. 2005) and halophytes (Vasquez et al. 2006; Silveira et al. 2009). On the other hand, K+ content in leaves and roots may fall due to high Na+ accumulation in these tissues through antagonism between the two ions (Carillo et al. 2011). Regarding K+/Na+ ratios in plant tissues, it has widely been reported that elevated K+ in the external medium exerts a beneficial effect on plants exposed to high levels of Na+, restricting salt toxicity. Moreover, maintenance of a high cytosolic K+/ Na+ ratio is key to salt tolerance. Under optimal conditions, the cytosolic K+ content is about 150 mM with a negligible Na+ content (Carden et al. 2003). However, excess Na+ accumulation in the cytosol is associated with salt-induced K+ efflux, and the cytosolic K+/Na+ ratio falls dramatically under saline conditions (Shabala et al. 2003). This salinity-induced K+ loss from cells is a result of NaCl-induced membrane depolarisation, leading to activation of depolarisation-activated outwardrectifying K+ channels (Cuin et al. 2008). Several authors have suggested that adequate K+ levels might alleviate the toxic effects of Na+ in plant tissues (Cakmak 2005; Chen et al. 2007; Wu et al. 2009). It is also widely accepted that adequate K+/Na+ ratios in plant tissues (generally above 1.0) in the presence of NaCl salinity are essential for K+–Na+ homeostasis and salinity tolerance (Apse & Blumwald 2007; Britto
et al. 2010). Nevertheless, this assumption is based on very little experimental evidence. Others have suggested the use of K+/ Na+ ratios in leaves as a good indicator for the selection of genotypes with salt tolerance (Flowers 2004; Munns & Tester 2008). Our data show that only in the 25 mM NaCl treatment was there an adequate K+/Na+ ratio in both leaves and roots. All other salt treatments had values of K+/Na+ below 1.0 in both organs, suggesting that J. curcas presents an ionic imbalance and low tolerance under moderate to severe salt stress. This response reinforces previous results obtained by our group that demonstrate that this species has high retention of Na+ even under low NaCl concentrations. Moreover is important to mention that the ionic disturbances in leaves were more severe than in roots, as indicated by higher K+/Na+ ratios. This response may be associated with higher transport of K+ for the root alleviating the toxic effects of Na+. Although the toxic effects of salinity on shoots and roots of J. curcas seedlings were examined in this study, the leaf RWC increased with increasing NaCl concentrations. This response indicates maintenance of the water status of the leaves where the excess accumulated ions contributed to water retention in the tissues. In parallel, the osmotic potential decreased (data not shown). A reduction of Ψs of woody plants can be related to increased Na+ and Cl- accumulation (Ottow et al. 2005; Silveira et al. 2009). Taken together, these responses contribute to a mechanism for osmotic adjustment under salinity. Physiological response analysis and its importance for J. curcas under salt stress The physiological responses of J. curcas to salt stress revealed a low PRA to shoot/root ratio, RWC, concentration of K+ in
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Physiological adjustment to salt stress in J. curcas
Fig 4. Overall representation of physiological response analysis (PRA) in Jatropha curcas seedlings exposed to NaCl treatment as a radial plot. Data refer to the following variables: Leaf DW, stem DW, petiole DW, root DW, shoot DW, shoot/root ratio, RWC, electrolyte leakage, osmotic adjustment, leaf Na+ content, root Na+ content, leaf Cl content, root Cl content, leaf K+ content, root K+ content, leaf K+/Na+ ratio, root K+/Na+ ratio, total free amino acids, total soluble sugars, proline, glycinebetaine, shoot Na+ transport rate, shoot Cl transport rate, shoot K+ transport rate, root Na+ transport rate, root Cl transport rate, root K+ transport rate, shoot selectivity for K–Na and root selectivity for K–Na.
root, amino acids, soluble sugars, glycine-betaine and transport of Na+ and Cl- to shoots and roots. Moderate values of PRA were found for stem, petiole, root and shoot DW, EL, OA, proline content, transport of K+ to shoots and selectivity (K-Na) for the shoot. The highest PRA was found for concentrations of Na+ and Cl- in the leaves and roots, concentration of K+ in leaves, K+/Na+ ratio in leaves and roots, transport of K+ and selectivity (K-Na) for roots (Fig. 4). According to these responses, our data support the idea that accumulation of Na+ and Cl- in leaves and roots is evidence that J. curcas can adjust osmotically to salt stress. Silva et al. (2009b) demonstrated that Na+ and Cl- were the major solutes that contribute to osmotic adjustment of species under salinity conditions. Recently, Rodrigues et al. (2014) demonstrated that R. communis, a species in the same family, has efficient osmotic adjustment to salt stress through extension of Na+ and Cluptake. These results are in concordance with those obtained here, showing that both species present similar mechanisms of salinity tolerance. Although organic solutes, such as amino acids, soluble sugars and glycine-betaine did not reach high values of PRA, their accumulation is associated with maintenance of a good leaf REFERENCES Apse M.P., Blumwald E. (2007) Na+ transport in plants. FEBS Letters, 581, 2247–2254. Ashraf M., Foolad M.R. (2007) Roles of glycinebetaine and proline in improving plant abiotic stress resistance. Environmental and Experimental Botany, 59, 206–216. Ashraf M., Harris P.J.C. (2004) Potential biochemical indicators of salinity tolerance in plants. Plant Science, 166, 3–16.
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water status, which suggests that these osmo-solutes may be involved in the mechanism of osmotic adjustment, as previously shown (Silva et al. 2009b). In fact, they are known to contribute to osmotic adjustment of plants under salt stress conditions (Silveira et al. 2009). Here, our data support the occurrence of osmotic adjustment (see OA values in Table 2) and the involvement of these organic solutes in this mechanism. In summary, we have demonstrated that J. curcas presents changes in key physiological processes that allow it to adjust to a saline environment. These responses are related to accumulation of Na+ and Cl in leaves and roots, K+/Na+ homeostasis, transport of K+ and selectivity (K-Na) for roots and accumulation of organic solutes contributing to osmotic adjustment. ACKNOWLEDGEMENTS We thank the Conselho Nacional de Desenvolvimento Cientıfico e Tecnol ogico (CNPq) and Fundacß~ao Cearense de Apoio ao Desenvolvimento Cientıfico e Tecnol ogico (FUNCAP) for financial support, and the Tamandua Farm, Patos, Paraıba, Brazil, for supplying J. curcas seeds.
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