Exogenously Applied 24-Epibrassinolide (EBL) Ameliorates Detrimental Effects of Salinity by Reducing K⁺ Efflux via Depolarization-Activated K⁺ Channels

Introduction

Salinity is a serious and growing problem for agricultural production in many countries that affects almost 20% of the total cultivated and half of the irrigated land of the world (Pitman and Läuchli 2002, Figueroa and Lunn 2016). Physiological and genetic complexity of salinity tolerance (Zhu 2000, Flowers 2004, Munns and Tester 2008) has significantly handicapped the progress in plant breeding for this trait (Shabala 2013).

Another viable and widely explored option to minimize detrimental effects of salinity on plant growth and performance is by exogenous application of nutrients (e.g. silicon), hormones [e.g. brassinosteroids (BRs)] and various growth regulators (proline, glycinebetaine, ascorbic acid, salicyclic acid or polyamines). These chemicals can either be added directly to the growth medium or applied by pre-soaking seeds or as foliar sprays, and were reported to enhance salt tolerance in different plant species (Hoque et al. 2007, Arafa et al. 2009, Ashraf et al. 2010, Shi and Chan, 2014). However, most of these studies have reported beneficial effects of such treatment without exploring in-depth the underlying physiological and/or molecular mechanisms. Because of that, the credibility of some of these reports is sometimes questioned by the research community.

Brassinolide is the most bioactive form of the growth-promoting plant steroids termed BRs regulating a wide range of physiological processes during the plant life cycle, from seed development and cell division to flowering and senescence (Bishop and Koncz 2002, Gruszka 2013). Recent reports indicated that BRs positively modulate plant metabolic response to environmental biotic and abiotic stresses, enhancing plant tolerance to salinity, drought, high temperatures, oxidative stress, pathogens, herbicides and pesticides (reviewed by Vriet et al. 2012). 24-Epibrassinolide (EBL) is a natural BR occurring in Vicia faba (Ikekawa et al. 1988). Numerous research studies have reported that exogenous application of EBL ameliorates the inhibition of growth and development induced by salt stress in a broad range of plant species (Derevyanchuk et al. 2015, Khalid and Aftab 2016, Zheng et al. 2016). Most of these studies attributed the positive effects of EBL to the increment in activities of antioxidant enzymes, soluble proteins and hormones (Agami 2013, Sadeghi and Shekafandeh 2014), but failed to link these mostly phenomenological observations with underlying cellular mechanisms.

The cytosolic K⁺/Na⁺ ratio is widely accepted as one of the key features conferring salinity stress tolerance in plants (Maathuis and Amtmann 1999, Shabala and Cuin 2008, Anschütz et al. 2014, Shabala et al. 2016), and this trait is often considered as a potential screening tool for plant breeders (Shannon 1997, Poustini and Siosemardeh, 2004). This ratio can be regulated by either active exclusion of Na⁺ from the cytosol [either into the vacuole (Blumwald et al. 2000, Maathuis et al. 2014) or into the apoplast (Zhu 2001)], or improved K⁺ retention in the cytosol (Carden et al. 2003).

Depolarization-activated outward-rectifying K⁺ channels and reactive oxygen species (ROS)-activated non-selective cation channels (NSCCs) are believed to be the major pathways that mediate K⁺ efflux under salinity stress (Demidchik et al. 2014, Shabala and Pottosin 2014). Literature reports revealed that exogenous application of EBL decreased Na⁺ accumulation and increased K⁺ content in the above-ground parts of various species grown under saline conditions (Ding et al. 2012, Sadeghi and Shekafandeh 2014, Mei et al. 2015), implying beneficial effects of EBL on prevention of K⁺ loss under NaCl stress. However, the specific ionic and molecular mechanisms by which EBL modulates ion transport processes and controls intracellular ionic homeostasis in salt stress-exposed plants remains largely unexplored.

The aim of this study was to investigate downstream targets of EBL and the impact of exogenous EBL application on kinetics of net ion fluxes and tissue ionic relations in salt-stressed plants. The reported data shows clear evidence that exogenously applied EBL ameliorates detrimental effects of salinity by reduced K⁺ efflux via gated outward-rectifying K⁺ channels (GORKs), most probably as a result of EBL-induced activation of H⁺-ATPase activity.

Results

Effects of different concentrations of NaCl and EBL on seed germination and root growth in barley

As shown in Fig. 1, seeds treated with salinity showed a significant reduction in root length and germination percentage, and an increase in the root hair density with increasing concentration from 150 mM (T₂) to 300 mM (T₃) compared with control (T₁), in the absence of EBL treatment. To investigate the effects of EBL on alleviating salt stress, the root length, root hair length, root hair density and seed germination percentage were measured under 0.1, 0.25 and 0.5 mg l^–1 EBL only (T₁₀ to T₁₂) or their combination with 150 mM (T₄ to T₆) and 300 mM NaCl (T₇ to T₉). In comparison with those of control, seeds treated with EBL only did not show any significant difference in any of the above characteristics, while compared with 150 mM NaCl, addition of 0.25 and 0.5 mg l^–1 EBL markedly increased the root hair length (Fig. 1B). EBL application (0.1, 0.25 and 0.5 mg l^–1) decreased the 150 mM NaCl-induced increase of root hair density (Fig. 1C). Moreover, 150 mM NaCl-induced reduction in germination was not influenced by addition of 0.1 and 0.25 mg l^–1 EBL, but was decreased by 0.5 mg l^–1 EBL (Fig. 1D). Under 300 mM NaCl, addition of 0.1 mg l^–1 EBL showed no effects on root growth, while 0.25 and 0.5 mg l^–1 EBL treatments significantly inhibited the growth of root hairs, with a minor effect on root hair length and root hair density (Fig. 1A–C). In addition, inhibition of germination induced by 300 mM NaCl was alleviated by addition of 0.1 and 0.5 mg l^–1 EBL. The beneficial effects of EBL became apparent with the addition of 0.25 mg l^–1 EBL under 150 mM NaCl conditions. Therefore, we selected 0.25 mg l^–1 EBL + 150 mM NaCl for further experimentation.

Fig. 1

Root length (A), root hair length (B), root hair density (C) and germination percentage (D) of barley seedlings under different treatments. T₁, distilled water as control; T₂, 150 mM NaCl; T₃, 300 mM NaCl; T₄, 0.1 mg l^–1 EBL and 150 mM NaCl; T₅, 0.25 mg l^–1 EBL and 150 mM NaCl; T₆, 0.5 mg l^–1 EBL and 150 mM NaCl; T₇, 0.1 mg l^–1 EBL and 300 mM NaCl; T₈, 0.25 mg l^–1 EBL and 300 mM NaCl; T₉, 0.5 mg l^–1 EBL and 300 mM NaCl; T₁₀, 0.1 mg l^–1 EBL; T₁₁, 0.25 mg l^–1 EBL; T₁₂, 0.5 mg l^–1L EBL. Data are the mean ± SE (n = 8–10). Data labeled with different lower case letters represent significant differences at P < 0.05.

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EBL alleviates salt-induced inhibition in shoot length, shoot weight and relative water content of barley plants

To further confirm the beneficial effect of exogenous EBL treatment on salinity stress, hydroponically grown barley seedlings were treated with 150 mM NaCl, 0.25 mg l^–1 EBL or their combination for 7 d. Plants grown under 150 mM NaCl exhibited a significant reduction in shoot length, and shoot fresh and dry weights, of 32.4, 45.3 and 28%, respectively (Fig. 2A–C). Exogenous application of EBL (0.25 mg l^–1) reduced the adverse effects of salinity on shoot agronomical characteristics (length, fresh weight and dry weight; Fig. 2A–C) but caused no significant (at P < 0.05) effects on either root length or biomass. In addition, relative water content (RWC) was reduced significantly under saline conditions, while the addition of EBL effectively restored RWC under salt stress to nearly the same level as the control (Fig. 2D).

Fig. 2

Effect of EBL and NaCl on (A) shoot and root lengths, (B) shoot and root dry weight, (C) shoot and root fresh weight and (D) shoot relative water content (RWC) of barley seedlings. After 3 d in the dark, barley seedlings were treated with 0.25 mg l^–1 EBL, 150 mM NaCl or their combination, and grown under light conditions for another 7 d, and then samples were collected for measurement. Data are the mean ± SE (n = 10). Data labeled with different lower case letters represent significant differences at P < 0.05.

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EBL reduces salt-induced increase of osmolality and Na⁺ content, and enhances K⁺ contents in shoot and root

Seedlings subjected to 150 mM NaCl exhibited higher leaf sap osmolality and much lower K⁺ content both in the shoot and in the root (∼45% and 10% of control, respectively), while with the addition of EBL beneficial effects of EBL on these parameters were observed, with lower leaf sap osmolality and higher K⁺ content (Fig. 3A–C). Salt treatment caused a significant increase in Na⁺ content in both shoots and roots, whereas EBL pre-treatment lowered Na⁺ accumulation in these tissues by 46% and 18%, respectively (Fig. 3D, E).

Fig. 3

Effect of EBL and NaCl on (A) leaf sap osmolality, (B) shoot K⁺ content, (C) root K⁺ content (D) shoot Na⁺ content and (E) root Na⁺ content of barley seedlings. After 3 d in the dark, barley seedlings were treated with 0.25 mg l^–1 EBL, 150 mM NaCl or their combination, and grown under light conditions for another 7 d, and then samples were collected for measurement. Data are the mean ± SE (n = 4). Data labeled with different lower case letters represent significant differences at P < 0.05.

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Effect of EBL on the K⁺ and H⁺ flux in barley root

Transient K⁺ and H⁺ flux responses were measured upon addition of 0.25 mg l^–1 EBL in both elongation (∼2 mm) and mature (∼10 mm) barley root zones. Addition of EBL stimulated root K⁺ uptake in both the zones, with a more pronounced effect in the elongation zone (Fig. 4A). Thus, the elongation zone was used in further electrophysiological experiments. EBL also enhanced net H⁺ efflux in the elongation zone, while no significant EBL effect on H⁺ fluxes was reported for the mature zone (Fig. 4B). The above stimulation of net H⁺ efflux by EBL was suppressed in roots treated with sodium orthovanadate (Supplementary Fig. S1), suggesting involvement of H⁺-ATPase in this process.

Fig. 4

Transient net K⁺ (A) and H⁺ flux (B) kinetics measured from the mature zone (∼10 mm) and elongation zone (∼2 mm) in response to 0.25 mg l^–1 EBL in barley roots. Values are the mean ± SE (n = 6–8). The sign convention for all MIFE measurements is ‘efflux negative’.

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Pre-treatment with EBL decreases NaCl-induced K⁺ efflux

To determine the effects of EBL on NaCl-induced K⁺ and H⁺ efflux and the optimal duration of EBL treatment, barley roots were pre-treated with EBL for either 1 or 24 h before measurement. The EBL treatment significantly reduced NaCl-induced K⁺ efflux, and 24 h pre-treatment was more effective than 1 h pre-treatment (Fig. 5A), indicating that this effect may be time dependent, implying the cytosolic mode of action. EBL treatment also progressively shifted steady-state H⁺ fluxes towards net H⁺ efflux, even in the absence of salt (zero to 4 min time interval in Fig. 5B), also consistent with the cytosolic mode of EBL action.

Fig. 5

Transient net K⁺ (A) and H⁺ (B) flux kinetics measured from the elongation zone (∼2 mm) in response to 150 mM NaCl in Gairdner barley roots without treatment, with 24 h or 1 h pre-treatment with 0.25 mg l^–1 EBL. Values are the mean ± SE (n = 6–8).

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EBL pre-treatment did not influence the root response to oxidative stress

To test the effect of EBL on the root response to oxidative stress, net K⁺ and Ca²⁺ fluxes were measured in response to H₂O₂ and Cu²⁺/ascorbate (Cu/A; a hydroxyl radical-generating agent) treatment from the elongation zone of barley roots under control (without EBL treatment) and with 1 h EBL pre-treatment. Both H₂O₂ and Cu/A induced massive K⁺ efflux from the roots; yet, no beneficial effect of 1 h EBL pre-treatment was observed (Fig. 6A, C). Similarly, EBL pre-treatment did not affect H₂O₂-induced net Ca²⁺ uptake (Fig. 6B).

Fig. 6

Transient net K⁺ (A) and Ca²⁺ (B) fluxes in response to 10 mM H₂O₂ and net K⁺ flux response to 0.3 mM Cu/A (C) measured from the elongation zone (∼2 mm) in barley roots. Roots were without treatment (control) or pre-treated with 0.25 mg l^–1 EBL for 1 h. Values are the mean ± SE (n = 6–8).

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EBL pre-treatment decreased NaCl-induced K⁺ efflux via depolarization-activated GORK channels

Net K⁺ and H⁺ fluxes were measured in two Arabidopsis mutants (i) the gork1-1 mutant that lacks functional depolarization-activated KOR channels in the root epidermal cells and (ii) the akt1 mutant that lacks a K⁺ uptake channel. NaCl caused a massive K⁺ loss in the elongation zone of Arabidopsis roots, in the sequence akt1 > wild type (WT) > gork1-1 (Fig. 7A). Pre-treatment with EBL reduced the NaCl-induced K⁺ efflux by 50- to 100-fold in the WT and akt1 (where the GORK channel is functional) but only <10-fold in the gork mutant (Fig. 7A). These results indicated that the GORK channel is one of the downstream targets of EBL.

Fig. 7

Effects of EBL pre-treatment on the transient net K⁺ (A) and H⁺ (B) flux kinetics of 5-day-old Arabidopsis wild-type (WT) and potassium channel mutants (akt1 and gork1-1) roots in response to 150 mM NaCl measured from the elongation zone (∼500 µm from the root tip). Before measurement, Arabidopsis roots were treated without (circles) or with 0.25 mg l^–1 EBL (diamonds) for 1 h. Values are the mean ± SE (n = 10). The insert in (A) shows steady-state K⁺ flux values in control and EBL-pre-treated roots prior to stress onset.

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EBL pre-treatment shifted net H⁺ fluxes towards efflux, where gork1-1 showed a lower efflux in comparison with the WT and akt1 (Fig. 7B). This EBL-induced H⁺ efflux implied an increased H⁺ pumping.

Discussion

Epibrassinolides decrease NaCl-induced K⁺ loss from barley shoot and root

Salinity tolerance is a complex trait made up of various subtraits; among these, K⁺ retention has received increasing attention in recent years (Shabala et al. 2006, Jayakannan et al. 2013). As high cytosolic K⁺ levels are essential for the activity of a plethora of enzymes, the ability to maintain cytosolic K⁺ in plant cells under high Na⁺ is crucial to tolerate salinity (Flowers and Dalmond 1992, Maathuis and Amtmann 1999). Indeed, higher salt tolerance resulting from efficient K⁺ preservation has been reported in many plant species (reviewed in Shabala and Cuin 2008, Wang et al. 2013, Houmani and Francisco 2016, Shabala et al. 2016, Zhao et al. 2016). The results presented here showed that NaCl treatment induced a significant K⁺ loss both in shoots (by approximately 55%) and in roots (by approximately 85%) (Fig. 3B, C). The massive decrease in cytosolic K⁺ may also activate caspase-like proteases and endonucleases, resulting in programmed cell death (PCD) (Shabala 2009, Demidchik et al. 2014). The presence of EBL in the growth medium prevented NaCl-induced K⁺ leak from shoots and roots (Fig. 3B, C). This was further supported by a MIFE (microelectrode ion flux estimation) experiment where EBL pre-treatment significantly decreased NaCl-induced K⁺ efflux in the elongation zone of barley roots (Fig. 5A). Therefore, the positive effect of EBL may be related to the better control of K⁺ retention under saline conditions.

GORK channels are downstream targets of EBL

NaCl stress-induced K⁺ efflux from roots can be mediated by two major pathways: (i) via depolarization-activated outwardly-rectifying K⁺ (GORK in Arabidopsis) channels (Cuin et al. 2008) and (ii) via ROS-activated non-selective cation channels (NSCCs) (Demidchik et al. 2003). Here to test the hypothesis that GORK channels are the downstream targets of EBL, the net K⁺ and H⁺ fluxes were measured using two Arabidopsis mutants, namely gork1-1 (lacking a functional GORK channel) and akt1 (lacking a functional K⁺ uptake channel). Comparison of K⁺ efflux induced by NaCl between the WT, gork1-1 and akt1 (Fig. 7A) suggested that EBL did not reduce the K⁺ flux in the gork-1mutant, while it induced 50- and 100-fold decreases of K⁺ loss from the WT and akt1 under salt treatment. Hence, these results provide strong evidence that EBL decreases K⁺ efflux under salinity conditions, improving plant tolerance through controlling depolarization-activated GORK channels. In this experiment, the gork1-1 mutant still lost some K⁺ which can be due to two possible reasons: (i) not all GORK transporters were eliminated (e.g. knock-down rather than knock-out) or (ii) K⁺ may be lost through a population of NSCCs (K⁺ permeable but showing only weak voltage dependence).

Epibrassinolide pre-treatment enhances the H⁺ efflux during salt stress

EBL pre-treatment progressively shifted steady-state H⁺ fluxes towards net H⁺ efflux during the first 4 min after salt treatment in barley roots (Fig. 5B) and shifted H⁺ influx towards efflux in Arabidopsis roots (Fig. 7B). Given the fact that the above shift was sensitive to vanadate (Supplementary Fig. S1), a known inhibitor of the plasma membrane-based H⁺-ATPase, these results suggest the EBL is likely to enhance H⁺-ATPase pump activity under salinity stress. In root cells, membrane potential (MP) is maintained predominantly by the activity of the plasma membrane H⁺-ATPase (Falhof et al. 2016). A significant membrane depolarization could be induced by NaCl treatment, which will activate depolarization-activated GORK channels and result in K⁺ leak (Jayakannan et al. 2013). Thus, we suggest that better K⁺ retention (and overall beneficial effects of EBL) is achieved via EBL-induced activation of the H⁺-ATPase. This conclusion is further supported by analysis of the effect of EBL on steady-state K⁺ fluxes in barley (Fig. 5) and Arabidopsis (Fig. 7) in the absence of salt stress (e.g. prior to NaCl treatment). A positive shift of about 350 nmol m^–2 s^–1 was observed in barley roots following 24 h of EBL treatment (Fig. 5). A similar shift was observed in all three Arabidopsis genotypes (by 110 ± 15 nmol m^–2 s^–1 in WT Columbia; by 185 ± 17 nmol m^–2 s^–1 in akt1; but by only by 38 ± 4 nmol m^–2 s^–1 in gork1-1; see insert in Fig. 7A). These data are fully consistent with the idea of EBL activating the H⁺-ATPase pump (even in the absence of salt) and shifting the MP towards more negative values, reducing K⁺ efflux via GORK channels. Additional support comes from the literature showing that BRs were found to enhance both vacuolar and plasma membrane H⁺-ATPase activity in BR-induced cell elongation and cell wall expansion through binding to brassinosteroid insensitive1 (BRI1) (Friedrichsen and Chory 2001, Witthöft and Harter 2011).

Epibrassinolide pre-treatment decreases the net Na⁺ uptake and root-to-shoot Na⁺ transport

High Na⁺ concentrations in the cytoplasm result in deleterious effects on cell biology, such as photosynthetic activity and membrane integrity (Nieves-Cordones et al. 2016). Thus, the ability to maintain low Na⁺ contents in shoots is a key factor under salinity stress for glycophytes (Tester and Davenport 2003). Compared with those grown in NaCl, barley seedlings grown in solutions with EBL and NaCl simultaneously showed much lower Na⁺ contents in both shoots and roots, especially in shoots (by nearly 50%) (Fig. 3D, E). As the shoot and root dry weight of barley seedlings showed no difference between NaCl treatment and NaCl + EBL treatment (Fig. 2B), this suggests that EBL reduces the net Na⁺ uptake by roots. This is one of the main mechanisms by which glycophytes minimize Na⁺ accumulation in shoots (Tester and Davenport 2003). In addition, the ratio between shoot Na⁺ content and total Na⁺ content was reduced from 68% in EBL-untreated seedlings to 55% in EBL-treated seedlings under salt stress (Fig. 3D, E), implying a decrease of long-distance transport of Na⁺ into shoots, due to reduced xylem Na⁺ loading in roots and/or increased Na⁺ retrieval from shoots during long-term salt stress (Jayakannan et al. 2013). Another hypothesis is that EBL-induced enhancement of H⁺ pumping may ‘fuel’ SOS₁ and thus exclude more Na⁺ from uptake. Indeed, upon salinity exposure, EBL-treated barley seedlings showed observable improvement in the shoot length and fresh weight, indicating reduced Na⁺ toxicity. Reduced Na⁺ content in the leaf apoplast is also essential for normal stomata operation to support continued water uptake under salinity conditions, and can explain the higher RWC in the leaves of EBL-treated plants (Fig. 2A, C, D).

Epibrassinolide did not improve the antioxidant capacity of barley roots

Salinity enhances the production of ROS and causes oxidative damage to proteins, DNA and lipids (Miller et al. 2010). Some researches have shown that application of EBL protects cells from oxidative stress and improves plant salt tolerance through enhancing the activities of antioxidant enzymes, such as catalase, peroxidase and superoxide dismutase (Carange et al. 2011, Fariduddin et al. 2014, Guerrero et al. 2015). However, in the present study, the net K⁺ and Ca²⁺ fluxes in response to H₂O₂ and net K⁺ flux to Cu/A did not show any difference between control and EBL-treated roots (Fig. 6). As most previous studies focused on the changes of antioxidant activity in shoots (Fariduddin et al. 2014, Guerrero et al. 2015, Khalid and Aftab 2016), here we show evidence that EBL-induced alleviation of salt stress in barley roots was not related to the improved antioxidant capability. It should be noted, however, that in order to rule out the involvement of EBL-induced modulation of antioxidant activity in roots completely, longer salinity treatments (e.g. at least 24 h) should be tested.

In summary, EBL treatment enhances the salinity tolerance of barley seedlings, reflected in the improvement of shoot length, shoot fresh weight and RWC in leaves under salinity stress. The whole-plant experiments and MIFE analysis showed that EBL treatment increases K⁺ contents in both shoots and roots under salt conditions, and 1 and 24 h EBL pre-treatment significantly decreases NaCl-induced K⁺ efflux, implying that the beneficial effects of EBL are related to better K⁺ retention. K⁺ loss is regulated by GORK channels and NSCCs, and the results from Arabidopsis mutants (gork1-1 and akt1) provide direct evidence that depolarization-activated GORK channels are the downstream target of EBL. EBL-induced H⁺ shift from influx to efflux, and the sensitivity of this shift to vanadate, a known inhibitor of the plasma membrane-based H⁺-ATPase, suggests that enhanced H⁺-ATPase activity maintains the MP at more negative values to decrease K⁺ leak from GORK channels. On the other hand, a lower Na⁺ content in shoots and roots of EBL-treated seedlings during long-term salt stress also contributes to the better K⁺ retention, as Na⁺ entry will lead to depolarization of the plasma membrane which will further activate depolarization-activated GORK channels.

Materials and Methods

Seed treatments with 24-epibrassinolide

Seeds of the salt-sensitive barley variety (Hordeum vulgare cv. Franklin; Australian Winter Cereal collection) were surface sterilized with 1% (v/v) sodium hypochlorite (commercial bleach) for 10 min and thoroughly rinsed with distilled water. Seeds were germinated in a dark growth cabinet at 24°C for 3 d in Petri dishes containing 0, 150 and 300 mM NaCl solutions and 0, 0.1, 0.25 and 0.5 mg l^–1EBL. All these treatments were also applied in combinations of EBL and NaCl; thus 12 different treatments were designed as: T₁ (Control), T₂ (150 mM NaCl), T₃ (300 mM NaCl), T₄ (150 mM NaCl + 0.1 mg l^–1 EBL), T₅ (150 mM NaCl + 0.25 mg l^–1 EBL), T₆ (150 mM NaCl + 0.5 mg l^–1 EBL), T₇ (300 mM NaCl + 0.1 mg l^–1 EBL), T₈ (300 mM NaCl + 0.25 mg l^–1 EBL), T₉ (300 mM NaCl + 0.5 mg l^–1 EBL), T₁₀ (0.1 mg l^–1 EBL), T₁₁ (0.25 mg l^–1 EBL) and T₁₂ (0.5 mg l^–1 EBL). To make EBL solution, 2 mg of 24-epibrassinolide (Sigma-E 1641) was first dissolved in 2 ml of ethanol and then stock solution was made with distilled water. Further different concentrations of EBL were made by diluting a volume of stock solution with distilled water. Each experiment had three replications with 20 seeds in each Petri dish. After 3 d, their root lengths, root hair length, root hair density and germination percentage were recorded.

Hydroponic experiments

Barley seeds were sterilized as above and germinated in an aerated hydroponic culture unit comprising a 1.5 liter plastic container over which seeds were suspended on a plastic grid and completely immersed in the nutrient solution (modified Rygol–Johnson solution; see Shabala et al. 2010 for details). Aeration was provided by two aquarium air pumps via flexible plastic tubing. Seeds were germinated in a dark growth cabinet at 24°C for 3 d. After that, lights (16/8 h light/dark cycle at 23°C with an irradiance of 150 µmol m⁻² s⁻¹) were turned on and seedlings were treated with different treatments: T₁ (Control), T₂ (150 mM NaCl), T₃ (0.25 mg l^–1 EBL+ 150 mM NaCl) and T₄ (0.25 mg l^–1 EBL). Plant roots were protected from direct light exposure. The plants were grown for another 7 d.

Biomass measurement

Plants were harvested at the age of 10 d. Each plant was separated into roots and shoots, and excess water was removed with paper towels. Root and shoot fresh weights were measured immediately by a Mettler BB2440 Delta Range balance (Mettler-Toledo) and their lengths were also recorded. Roots and shoots were then dried at 65°C in a Unitherm Dryer to constant weight and weighed again. The RWCs were then calculated.

Determination of Na⁺ and K⁺ content in plant samples

Dry barley roots and shoots were ground and passed through a 2 mm mesh sieve. A 0.1 g weighed sample was digested in 10 ml of 98% H₂SO₄ and 3 ml of 30% H₂O₂ for 5 h essentially as described by Skoog et al. (2000). The Na⁺ and K⁺ contents were determined by using a flame photometer (PFP7, Jenway, Bibby Scientific Ltd.).

Leaf sap osmolality

One day prior to harvest for biomass, four segments (2 cm) from flag leaf blades for each treatment were sampled and immediately preserved at –20°C. Flag leaf blade sap was extracted using the freeze–thaw method (Cuin et al. 2009) and its osmolality was determined using a vapor pressure osmometer (Vapro; Wescor Inc.).

Plant material and growth conditions for MIFE experiments

Barley seeds were surface sterilized and grown in the dark in an aerated hydroponic solution for 3 d as described above. Barley roots of about 60–80 mm long were pre-treated with 0.25 mg l^–1 EBL for 0, 1 and 24 h (as required) and placed into the bathing solution containing 0.5 mM KCl, 0.1 mM CaCl₂, 4 mM MES and 2 mM Tris (pH 6), still in the presence of EBL.

For experiments with Arabidopsis, seeds of Arabidopsis thaliana wild-type Columbia, gork1-1 and akt1 mutants were grown at 22°C and 16/8 h day/night regime (fluorescent lighting 100 mmol m^–2 s^–1 irradiance) in sterile conditions in vertically oriented 90 mm Petri dishes containing 0.35% Phytagel (Sigma) with half-strength Murashige and Skoog medium (Duchefa) and 1% (w/v) sucrose (see Demidchik et al. 2002 for more details). Seeds were surface sterilized with commercial bleach plus 0.01% Triton and sown on the surface of the Phytogel. The dishes were sealed with Parafilm and placed on edge vertically, so roots grew down the Phytagel surface without penetrating it. Six- to eight-day-old roots were used for K⁺ and H⁺ flux measurement in the elongation zone (∼0.5 mm).

Ion flux measurements

Net fluxes of H⁺, K⁺ and Ca²⁺ were measured non-invasively using ion-selective vibrating microelectrodes (University of Tasmania) as described previously (Shabala et al. 1997, Shabala et al. 2003). Electrodes with Nernst slope responses of <50 mV per decade were discarded. Electrodes were mounted on a manually operated 3D-micromanipulator (MMT-5; Narishige) and their tips were aligned and positioned 40 µm away from the root surface. During the measurements, a computer-controlled stepper motor moved electrodes in a slow (10 s cycle, 40 µm amplitude) square-wave between the two positions, close to (40 µm) and away from (80 µm) the root surface. CHART software (Shabala et al. 1997, Newman 2001) recorded the potential difference between the two positions and converted it to electrochemical potential difference using the calibrated Nernst slope of the electrode. Net ion fluxes were calculated using the MIFEFLUX software for cylindrical diffusion geometry (Newman 2001).

Ion flux measuring protocols for barley roots

One hour prior to measurement, a seedling was taken from the growth cabinet and its root was placed in a 6 ml Perspex measuring chamber with 3 ml of bathing solution (control solution: 0.5 mM KCl and 0.1 mM CaCl₂, 4 mM MES, 2 mM Tris, pH 6). The root was centered within the chamber and fixed horizontally by immobilizing the root using moveable cross-bars within the chamber. Transient recordings of the flux kinetics of K⁺, H⁺ and Ca²⁺ were measured for 5 min before and 30 min after treatment application in all the experiments. The data for the first minute after changing the solution were removed from the analysis to allow the establishment of the diffusion gradients and meet non-stirred layer conditions (Shabala and Hariadi 2005).

Transient K⁺ and H⁺ flux responses were measured upon addition of 0.25 mg l^–1 EBL in elongation (∼2 mm) and mature (∼10 mm) zones of roots of 3-day-old barley seedlings. For the salinity stress responses, roots were pre-treated with 0.25 mg l^–1 EBL for 0, 1 and 24 h in bathing solution, prior to addition of 150 mm NaCl. After the specified pre-treatment duration, net ion fluxes were measured in the same pre-treatment solution for 5 min to ensure steady initial values, and then salinity treatment (150 mM NaCl was applied as double stock made up in 3 ml of the bath solution) was given. For the responses to oxidative stress, barley roots were pre-treated with 0.25 mg l^–1 EBL for 1 h in bathing solution. After the specified pre-treatment duration, net ion fluxes were measured in the same pre-treatment solution for 5 min to ensure steady initial values, then responses to 10 mM H₂O₂ and 0.3 mM Cu/A (a hydroxyl radical-generating mix; Demidchik et al. 2003) were measured from barley roots.

Ion flux measuring protocols for Arabidopsis seedlings

Arabidopsis seedlings were taken out from the Petri dish where they had been growing on half-strengh Murashige and Skoog medium, and their roots were immersed in 0.25 mg l^–1 EBL for 1 h. After this, the root was immobilized in a horizontal position on a microscope slide using Parafilm, placed in a chamber with 3 mo of basal salt medium (0.5 mM KCl and 0.1 mM CaCl₂) solution, and left to equilibrate for 40 min. Then net K⁺ and H⁺ fluxes were measured for 5–10 min, followed by NaCl stress.

Statistical analysis

Statistical significance of mean and SE values was determined using the standard least significant difference test at P ≤ 0.05. All the statistical analyses were performed using SAS 9.4 for Windows. Data were subjected to analysis of variance (ANOVA), and mean values were compared by Duncan’s multiple range test (P < 0.05).

Supplementary data

Supplementary data are available at PCP online.

Acknowledgement

This work was supported by the Australian Research Council grant to Sergey Shabala and Australia Endeavour fellowship to Nazila Azhar.

Disclosures

The authors have no conflicts of interest to declare.

Abbreviations

Abbreviations
 
• BR

  brassinosteroid

 
• Cu/A

  Cu²⁺/ascorbate

 
• EBL

  24-epibrassinolide

 
• GORK

  gated outward-rectifying K⁺ channel

 
• MIFE

  microelectrode ion flux estimation

 
• MP

  membrane potential

 
• NSCC

  non-selective cation channel

 
• ROS

  reactive oxygen species

 
• RWC

  relative water content

 
• WT

  wild-type

References