Mol. Cells 2017; 40(12): 966-975
Published online December 20, 2017
https://doi.org/10.14348/molcells.2017.0229
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: kim1312@gnu.ac.kr (WYK); jycha@gnu.ac.kr (JYC)
Excessive salt disrupts intracellular ion homeostasis and inhibits plant growth, which poses a serious threat to global food security. Plants have adapted various strategies to survive in unfavorable saline soil conditions. Here, we show that humic acid (HA) is a good soil amendment that can be used to help overcome salinity stress because it markedly reduces the adverse effects of salinity on
Keywords Arabidopsis, calcium, HKT1, humic acid, salt stress
One of the largest global concerns is climate change, which is bringing about rapid soil erosion in agricultural lands worldwide. One of the major abiotic stresses, salinity, causes soil degradation and inhibits nutrient absorption, consequently reducing crop yields (Ashraf and Foolad, 2007; Tester and Davenport, 2003). Plants must cope with both osmotic and ionic stress under high-salinity conditions. Osmotic stress reduces water uptake and cell expansion and delays lateral bud development (Munns and Tester, 2008). Ionic stress is induced when toxic ions such as Na+ accumulate at high levels in cells, specifically in leaves and shoots, leading to increased leaf mortality along with chlorosis and necrosis (Glenn et al., 1999; Yeo and Flowers, 1986). In addition, high Na+ concentrations interrupt the uptake of potassium (K+) and inhibit the activity of several enzymes (Murguia et al., 1995; Wu et al., 1996). High cytosolic K+/Na+ ratios in the shoot are critical for salt tolerance in glycophytes, which can only tolerate relatively low salt concentrations (Blumwald, 2000; Gorham et al., 1987; 1990; Hauser and Horie, 2010; Ren et al., 2005; Sunarpi et al., 2005; Yamaguchi and Blumwald, 2005).
When a Na+ ion enters the plant root, it can be selectively transported through three independent biological membranes: the plasma membrane in epidermal cells, the vacuolar membrane in root and shoot cells, and the plasma membrane in xylem parenchyma cells (Horie et al., 2012). Accumulated cytosolic Na+ can be removed by efflux systems such as Na+/H+ antiporters, which transport Na+ across the plasma membrane (Apse et al., 1999; Blumwald, 2000; Pardo et al., 2006), as well as the Salt-Overly Sensitive (SOS) pathway (Park et al., 2016). This pathway is composed of three SOSs, including the calcium binding protein SOS3, which senses salt-triggered increases in calcium levels and binds to the Ser/Thr protein kinase SOS2. The SOS2:SOS3 complex is translocated to the plasma membrane through the N-terminal myristoylation of SOS3, where it activates the Na+/H+ antiporter SOS1 by phosphorylating its C-terminus (Guo et al., 2001; 2004; Halfter et al., 2000; Ishitani et al., 2000; Liu and Zhu, 1998; Liu et al., 2000; Qiu et al., 2002). SOS3 possessing three potential Ca2+-binding sites that recognize the cytosolic calcium ion (Ca2+) signal elicited by salt stress (Ishitani et al., 2000; Liu and Zhu, 1998; Moncrief et al., 1990). Exogenous application of calcium (Ca2+) enhances salt tolerance in glycophytic plants (Läuchli, 1990), likely due to the SOS3-mediated activation of the SOS pathway at the epidermis.
Na+ that enters the root cell and is transported to leaf tissue must be compartmentalized in the vacuole to avoid the cytosolic accumulation of toxic Na+. This process is mediated by the vacuolar Na+/H+ antiporter, NHX, which moves Na+ into the vacuole in exchange for H+ (Blumwald et al., 2000). This process might also be regulated by the SOS signaling pathway (Qiu et al., 2004).
Na+ reabsorption occurs from the xylem stream to the surrounding tissues. This process, which reduces the net flow of Na+ into shoots (Läuchli, 1984; Lacan and Durand, 1996), is mediated by H+ influx carriers, particularly HIGH-AFFINITY POTASSIUM (K+) TRANSPORTER (HKT) family members (Horie et al., 2001). HKTs are involved in the retrieval of Na+ from the xylem to reduce its transport to/accumulation in the shoot in several plant species (Davenport et al., 2007; Mäser et al., 2002a; Ren et al., 2005; Sunarpi et al., 2005). Plant HKTs, which function as Na+ influx transporters, are divided into two subclasses based on protein sequence and ion selectivity (Mäser et al., 2002b). In rice, a Ser residue with high selectivity for Na+ over K+ at the first pore-loop domain is conserved in class 1 (HKT1) family members, while a Gly at the same position in class 2 (HKT2) members is permeable to both Na+ and K+ (Horie et al., 2001).
Humin, which is composed of humic and fulvic acids (commonly known as humic substances [HS]), is a complex supramolecular association of abiotically transformed bio-molecules that are released into soils after cell lysis (Orsi, 2014). Humin influences plant growth both directly and indirectly by functioning as a major source of organic compounds in soil (Sangeetha et al., 2006). These substances can improve soil properties such as aggregation, aeration, permeability, water holding capacity, micronutrient transport, and availability. Furthermore, the direct uptake of HS into plant tissues results in diverse biochemical outcomes (Arancon et al., 2006; Nardi et al., 2002; Selim et al., 2009; Tan, 2003). Humic acid (HA) improves plant development by regulating metabolic and signaling pathways by acting directly on certain targets in diverse physiological processes (Quaggiotti et al., 2004; Trevisan et al., 2010). The application of HA to plants increases cell membrane permeability, oxygen uptake, respiration, photosynthesis, phosphate uptake, and root elongation (Cacco and Dell Agnolla, 1984; Russo and Berlyn, 1990; Vaughan, 1974). HA treatment enhances the mobilization of toxic heavy metals, especially from abandoned mine tailings, indicating that HA could be utilized as a possible remedy to reduce further soil contamination (Wang and Mulligan, 2009). Moreover, HA has protective effects against high saline stress by inhibiting Na+ uptake in barley (Marketa et al., 2016), and it reduces yield losses in maize under salt stress (Masciandaro et al., 2002). However, recent extensive studies have failed to further explain the physiological and molecular mechanisms underlying how HA confers salt tolerance to plants.
In the current study, we investigated how HA increases salt tolerance in Arabidopsis seedlings. The salt-induced degradation of HKT1 was impaired by HA treatment, and HA increased the protein abundance of HKT1 in the root stele, which resulted in greater reabsorption of Na+ from xylem vessels into xylem parenchyma cells and, consequently, less translocation of Na+ to the shoot.
Five-day-old WT Arabidopsis seedlings were transferred to 1/2 MS medium containing 250 mM NaCl alone or supplemented with 86 or 860 mg L−1 HA (Sigma-Aldrich). The fresh weight of 15 plants was measured at 7 days after treatment, with three independent replications.
Five-day-old Arabidopsis seedlings (WT,
Five-day-old Arabidopsis seedlings treated with 100 mM NaCl with or without HA (860 mg L−1) for 14 h were stained with 5 μM CoroNa-Green AM (Invitrogen) for 3 h in the presence of pluronic acid (Sigma-Aldrich) at a final concentration of 0.02% in the dark (Leshem et al., 2006; Mazel et al., 2004; Meier et al., 2006). The stained roots were examined under a confocal microscope (Olympus FV1000) at excitation and emissions wavelengths of 488 nm and 516 nm, respectively. The cell walls and dead cells were stained with 1 μg ml−1 propidium iodide (Invitrogen).
Nine-day-old
Nine-day-old
Nine-day-old
Three-week-old WT plants were transferred to 1/2 MS plates containing 100 mM NaCl with or without 860 mg L−1 HA and grown for an additional 2 days. The plants were rinsed with deionized water and dried at 65°C for 2 days. The dry tissues were ground using a mortar and pestle, and 100 mg of dry tissue was extracted with 10 ml of HCIO4:H2O:H2SO4 (9:5:1, v/v/v) on a heating block with a gradual increase in temperature from 100°C to 320°C. After digestion, the samples were diluted to a final volume of 100 ml with deionized water and filtered through filter paper (Whatman No. 2). The Na+ ion content was analyzed using Inductively Coupled Plasma-Mass Spectroscopy (ICP-MS, Perkin Elmer Optima 2200 DV).
The protective effect of HA in plants under salt stress has primarily been demonstrated in cereals such as maize and wheat (Aydin et al., 2012; Khaled and Fawy, 2011). Very recently, we also reported that HA increases seed germination rates in Arabidopsis in a dose-dependent manner and that the application of 86 mg L−1 HA confers salt stress tolerance to this plant under excessive salt concentrations (250 mM NaCl) (Cha et al., 2017). In the current study, to determine whether the use of HA at concentrations greater than 86 mg L−1 would cause a dramatic increase in salt tolerance, we performed a salt tolerance assay in which WT Arabidopsis seedlings were grown on 1/2 MS agar plates under a fixed concentration of NaCl (250 mM) and various concentrations of HA (0, 86, and 860 mg L−1). In the absence of HA, the seedlings were nearly dead when grown on salt stress medium, with chlorosis observed in shoots. However, seedlings grown on 86 or 860 mg L−1 HA medium plus 250 mM NaCl had green shoots (Fig. 1A). We measured the fresh weight of plants, finding that increasing the concentration of HA in seedlings under salinity stress increased salt stress tolerance in a dose-dependent manner (Fig. 1B). In tomato, the application of HA to the soil increased plant growth up to a concentration of 1 g kg−1, but the effects were reduced at 2 g kg−1 (Türkmen et al., 2004). HA has a positive effect on various plants, but how HA promotes plant growth and salt tolerance has remained elusive.
HA possesses many ionizable sites that allow it to bind to and chelate various cations, including Na+ (Tunstall, 2005). Thus, we performed ICP-MS analysis to determine whether HA can bind to and chelate Na+ ions in agar medium. Interestingly, plants grown on medium containing either NaCl or HA plus NaCl absorbed similar amounts of Na+ ions, suggesting that HA does not inhibit the uptake of NaCl by plants in culture (
HA promotes lateral root formation by exhibiting auxin-like activity in maize, tomato, and Arabidopsis (Dobbss et al., 2007; Trevisan et al., 2010; Zandonadi et al., 2007). By contrast, salt stress reduces root growth, including primary and lateral root growth, although primary root growth is more severely affected than lateral root growth (Julkowska et al., 2014). In addition, we have previously reported that primary root growth of
Na+ ions that reach the xylem by passing through several barriers in the root under salinity stress are transported to the shoot. Altering specific Na+ transport processes in specific cell types can reduce Na+ accumulation in the shoot, which is quite harmful to higher plants (Møller et al., 2009). To examine how HA enhances salinity stress tolerance in the shoot under salt stress conditions, we monitored the long-distance transport of Na+ in the root after short-term salinity treatment (Fig. 4). Five-day-old WT seedlings were treated with 100 mM NaCl in the absence or presence of HA (860 mg L−1) for 14 h. We visualized the distribution of sodium using the fluorescent, sodium-specific dye CoroNa-Green AM. After salt treatment, the intensity of fluorescent staining in WT roots became stronger than in the non-treated control, as shown previously (Oh et al., 2010) (Fig. 4A). In WT, when HA was added to salt-containing medium, the intensity of fluorescent staining moved upward to the elongation zone, while the intensity in the root-tip zone was reduced (Fig. 4A). Like WT, the intensity of fluorescent staining in the root tip of
To investigate the possibility that HA enhances the unloading of Na+ to xylem parenchyma cells, we first examined whether HA affects HKT1 protein levels using
HA is a component of humus. This heterogeneous, relatively large, high molecular weight organic complex, which ranges in color from brown to black, is amorphous, hydrophilic, molecularly flexible, and composed of polyelectrolytic compounds. HS contains a large number of complex humate molecules. Humate can bind to positive metal cations such as Iron (Fe2+), copper (Cu2+), zinc (Zn2+), calcium (Ca2+), manganese (Mg2+), and magnesium (Mg2+) (Tunstall, 2005). Salinity (NaCl) stress induces Ca2+ influx; the elevated levels of cytosolic free Ca2+ serve as a second messenger (Tracy et al., 2008). To investigate the positive effects of HA on HKT1 stability/activity due to the Ca2+ chelating effect of humate on plants under salt stress conditions, we used various pharmacological agents to inhibit Ca2+ release and flux (Fig. 6). Nicotinamide inhibits cyclic ADP-ribose (cADPR), a potent Ca2+-releasing agent, while GdCl3 blocks stretch-activated cation channels, thereby functioning as a Ca2+ influx inhibitor, and U73122 inhibits phospholipase C, thus acting as an inhibitor of Ca2+ efflux (Dodd et al., 2007; Tracy et al., 2008). In the absence of salt, HKT1 protein abundance increased by nicotinamide, U73122, or GdCl3 treatment compared to the control condition (Fig. 6). In the presence of 50 mM nicotinamide or 1 mM GdCl3, HKT1 was stabilized against NaCl-induced degradation under salt stress conditions. However, treatment with 5 μM U73122 (to inhibit vacuolar calcium release) failed to restore HKT1 protein to normal levels in the presence of NaCl-induced degradation (Fig. 6). These results suggest that the increase in cytosolic Ca2+ levels plays a role in NaCl-mediated HKT1 protein destabilization upon salt stress.
In this study, we demonstrated that HA treatment improves plant growth and reduces plant sensitivity to salinity stress. HA can function as a growth regulator by regulating hormone levels, plant growth, and stress responses (Piccolo et al., 1992; Serenella et al., 2002). HA treatment reduces the toxicity of salt in strawberry, maize, and garden cress seedlings (Masciandaro et al., 2002; Pilanal and Kaplan, 2003; Türkmen et al., 2004). Here, we showed that HA application also increases plant growth and enhances salt stress tolerance in Arabidopsis (Figs. 1–3; Cha et al., 2017). Treatment with 1 g L−1 HA has a positive effect on plant growth under saline soil conditions (Türkmen et al., 2004), which is consistent with our observation that 860 mg L−1 HA caused a significant increase in seedling survival, even under saline conditions (Fig. 1). David et al. (1994) reported that HS promotes plant growth and mineral nutrient uptake due to improved root system development. In addition, HS influences protein synthesis in higher plants (Carletti et al., 2008). Na+ strongly accumulated in both shoots and roots after the addition of NaCl, which is consistent with the findings for various barley cultivars exposed to 150 mM NaCl (Kamboj et al., 2015). HKT transporters are thought to be intricately involved in Na+ uptake and salt toxicity in plants (Ali et al., 2012; Mäser et al., 2002a; 2002b; Uozumi et al., 2000; Xue et al., 2011). AtHKT1;1 localized to the plasma membrane of xylem parenchyma cells mediates the removal of Na+ from xylem vessels during salinity stress (Sunarpi et al., 2005). When
HA treatment improves ion uptake and mineral nutrition in plants (Trevisan et al., 2010). Asik et al. (2009) determined that both soil and foliar application of small amounts of HS increase nutrient uptake in wheat under salt stress conditions. Murat et al. (2011) reported that adding humus to the soil increases nutrient uptake in plants under 45 and 60 mM NaCl treatment. Indeed, the protective effect of HA on plants under salt stress has been demonstrated in many cereals, such as maize and wheat (Aydin et al., 2012; Khaled and Fawy, 2011). In the current study, we showed that HA treatment relieved the growth inhibition induced by NaCl via the stabilization of HKT1 protein (Fig. 5).
Higher calcium levels in soil protect the cell membrane from the negative effects of salinity (Busch, 1995). Kwon et al. (2009) demonstrated that the addition of 60 mM NaCl to growth medium increases Na+ uptake in plants, as expected, but supplemental Ca2+ reverses this effect. Ca2+ also reduces the translocation of Na+ to the shoot and retains this ion in the roots. Under particular conditions, HS can stimulate plant growth, including increased plant height and dry/fresh weight (Blanchet, 1958; Guminski, 1968). These findings are consistent with our hypothesis that influx of the secondary messenger Ca2+ and cytosolic Ca2+ participate in NaCl-mediated destabilization of HKT1 protein under salt stress (Fig. 6). Several studies have confirmed the hypothesis that HS has a direct effect on plant physiology, specifically concerning lateral root development (Canellas et al., 2002; Carletti et al., 2008; Zandonadi et al., 2007). More recently, the auxin-like activity of HS in promoting lateral root development was investigated in the model plant Arabidopsis using a combination of genetic and molecular approaches (Trevisan et al., 2009).
In conclusion, this study demonstrates that HA plays an important role in improving salt tolerance by regulating the sodium transporters HKT1 in post-transcriptional levels. It is difficult to monitor changes in protein abundance after HA treatment due to the complex network of signaling pathways. To the best of our knowledge, HKT1 is the first protein whose levels were found to change in Arabidopsis after exposure to HA under salt stress treatment. Further research is needed to elucidate the specific functions and regulatory mechanisms underlying the effects of HA on HKT transporters and its role in salinity tolerance.
Mol. Cells 2017; 40(12): 966-975
Published online December 31, 2017 https://doi.org/10.14348/molcells.2017.0229
Copyright © The Korean Society for Molecular and Cellular Biology.
Laila Khaleda1, Hee Jin Park2,3, Dae-Jin Yun3, Jong-Rok Jeon4, Min Gab Kim5, Joon-Yung Cha1,*, and Woe-Yeon Kim1,4,*
1Division of Applied Life Science (BK21Plus), Plant Molecular Biology and Biotechnology Research Center (PMBBRC), Research Institute of Life Sciences (RILS), Gyeongsang National University, Jinju 52828, Korea, 2Institute of Glocal Disease Control, Konkuk University, Seoul 05029, Korea, 3Department of Biomedical Science and Engineering, Konkuk University, Seoul 05029, Korea, 4Department of Agriculture Chemistry and Food Science & Technology, Institute of Agriculture and Life Science (IALS), Gyeongsang National University, Jinju 52828, Korea, 5College of Pharmacy and Research Institute of Pharmaceutical Science, PMBBRC, Gyeongsang National University, Jinju 52828, Korea
Correspondence to:*Correspondence: kim1312@gnu.ac.kr (WYK); jycha@gnu.ac.kr (JYC)
Excessive salt disrupts intracellular ion homeostasis and inhibits plant growth, which poses a serious threat to global food security. Plants have adapted various strategies to survive in unfavorable saline soil conditions. Here, we show that humic acid (HA) is a good soil amendment that can be used to help overcome salinity stress because it markedly reduces the adverse effects of salinity on
Keywords: Arabidopsis, calcium, HKT1, humic acid, salt stress
One of the largest global concerns is climate change, which is bringing about rapid soil erosion in agricultural lands worldwide. One of the major abiotic stresses, salinity, causes soil degradation and inhibits nutrient absorption, consequently reducing crop yields (Ashraf and Foolad, 2007; Tester and Davenport, 2003). Plants must cope with both osmotic and ionic stress under high-salinity conditions. Osmotic stress reduces water uptake and cell expansion and delays lateral bud development (Munns and Tester, 2008). Ionic stress is induced when toxic ions such as Na+ accumulate at high levels in cells, specifically in leaves and shoots, leading to increased leaf mortality along with chlorosis and necrosis (Glenn et al., 1999; Yeo and Flowers, 1986). In addition, high Na+ concentrations interrupt the uptake of potassium (K+) and inhibit the activity of several enzymes (Murguia et al., 1995; Wu et al., 1996). High cytosolic K+/Na+ ratios in the shoot are critical for salt tolerance in glycophytes, which can only tolerate relatively low salt concentrations (Blumwald, 2000; Gorham et al., 1987; 1990; Hauser and Horie, 2010; Ren et al., 2005; Sunarpi et al., 2005; Yamaguchi and Blumwald, 2005).
When a Na+ ion enters the plant root, it can be selectively transported through three independent biological membranes: the plasma membrane in epidermal cells, the vacuolar membrane in root and shoot cells, and the plasma membrane in xylem parenchyma cells (Horie et al., 2012). Accumulated cytosolic Na+ can be removed by efflux systems such as Na+/H+ antiporters, which transport Na+ across the plasma membrane (Apse et al., 1999; Blumwald, 2000; Pardo et al., 2006), as well as the Salt-Overly Sensitive (SOS) pathway (Park et al., 2016). This pathway is composed of three SOSs, including the calcium binding protein SOS3, which senses salt-triggered increases in calcium levels and binds to the Ser/Thr protein kinase SOS2. The SOS2:SOS3 complex is translocated to the plasma membrane through the N-terminal myristoylation of SOS3, where it activates the Na+/H+ antiporter SOS1 by phosphorylating its C-terminus (Guo et al., 2001; 2004; Halfter et al., 2000; Ishitani et al., 2000; Liu and Zhu, 1998; Liu et al., 2000; Qiu et al., 2002). SOS3 possessing three potential Ca2+-binding sites that recognize the cytosolic calcium ion (Ca2+) signal elicited by salt stress (Ishitani et al., 2000; Liu and Zhu, 1998; Moncrief et al., 1990). Exogenous application of calcium (Ca2+) enhances salt tolerance in glycophytic plants (Läuchli, 1990), likely due to the SOS3-mediated activation of the SOS pathway at the epidermis.
Na+ that enters the root cell and is transported to leaf tissue must be compartmentalized in the vacuole to avoid the cytosolic accumulation of toxic Na+. This process is mediated by the vacuolar Na+/H+ antiporter, NHX, which moves Na+ into the vacuole in exchange for H+ (Blumwald et al., 2000). This process might also be regulated by the SOS signaling pathway (Qiu et al., 2004).
Na+ reabsorption occurs from the xylem stream to the surrounding tissues. This process, which reduces the net flow of Na+ into shoots (Läuchli, 1984; Lacan and Durand, 1996), is mediated by H+ influx carriers, particularly HIGH-AFFINITY POTASSIUM (K+) TRANSPORTER (HKT) family members (Horie et al., 2001). HKTs are involved in the retrieval of Na+ from the xylem to reduce its transport to/accumulation in the shoot in several plant species (Davenport et al., 2007; Mäser et al., 2002a; Ren et al., 2005; Sunarpi et al., 2005). Plant HKTs, which function as Na+ influx transporters, are divided into two subclasses based on protein sequence and ion selectivity (Mäser et al., 2002b). In rice, a Ser residue with high selectivity for Na+ over K+ at the first pore-loop domain is conserved in class 1 (HKT1) family members, while a Gly at the same position in class 2 (HKT2) members is permeable to both Na+ and K+ (Horie et al., 2001).
Humin, which is composed of humic and fulvic acids (commonly known as humic substances [HS]), is a complex supramolecular association of abiotically transformed bio-molecules that are released into soils after cell lysis (Orsi, 2014). Humin influences plant growth both directly and indirectly by functioning as a major source of organic compounds in soil (Sangeetha et al., 2006). These substances can improve soil properties such as aggregation, aeration, permeability, water holding capacity, micronutrient transport, and availability. Furthermore, the direct uptake of HS into plant tissues results in diverse biochemical outcomes (Arancon et al., 2006; Nardi et al., 2002; Selim et al., 2009; Tan, 2003). Humic acid (HA) improves plant development by regulating metabolic and signaling pathways by acting directly on certain targets in diverse physiological processes (Quaggiotti et al., 2004; Trevisan et al., 2010). The application of HA to plants increases cell membrane permeability, oxygen uptake, respiration, photosynthesis, phosphate uptake, and root elongation (Cacco and Dell Agnolla, 1984; Russo and Berlyn, 1990; Vaughan, 1974). HA treatment enhances the mobilization of toxic heavy metals, especially from abandoned mine tailings, indicating that HA could be utilized as a possible remedy to reduce further soil contamination (Wang and Mulligan, 2009). Moreover, HA has protective effects against high saline stress by inhibiting Na+ uptake in barley (Marketa et al., 2016), and it reduces yield losses in maize under salt stress (Masciandaro et al., 2002). However, recent extensive studies have failed to further explain the physiological and molecular mechanisms underlying how HA confers salt tolerance to plants.
In the current study, we investigated how HA increases salt tolerance in Arabidopsis seedlings. The salt-induced degradation of HKT1 was impaired by HA treatment, and HA increased the protein abundance of HKT1 in the root stele, which resulted in greater reabsorption of Na+ from xylem vessels into xylem parenchyma cells and, consequently, less translocation of Na+ to the shoot.
Five-day-old WT Arabidopsis seedlings were transferred to 1/2 MS medium containing 250 mM NaCl alone or supplemented with 86 or 860 mg L−1 HA (Sigma-Aldrich). The fresh weight of 15 plants was measured at 7 days after treatment, with three independent replications.
Five-day-old Arabidopsis seedlings (WT,
Five-day-old Arabidopsis seedlings treated with 100 mM NaCl with or without HA (860 mg L−1) for 14 h were stained with 5 μM CoroNa-Green AM (Invitrogen) for 3 h in the presence of pluronic acid (Sigma-Aldrich) at a final concentration of 0.02% in the dark (Leshem et al., 2006; Mazel et al., 2004; Meier et al., 2006). The stained roots were examined under a confocal microscope (Olympus FV1000) at excitation and emissions wavelengths of 488 nm and 516 nm, respectively. The cell walls and dead cells were stained with 1 μg ml−1 propidium iodide (Invitrogen).
Nine-day-old
Nine-day-old
Nine-day-old
Three-week-old WT plants were transferred to 1/2 MS plates containing 100 mM NaCl with or without 860 mg L−1 HA and grown for an additional 2 days. The plants were rinsed with deionized water and dried at 65°C for 2 days. The dry tissues were ground using a mortar and pestle, and 100 mg of dry tissue was extracted with 10 ml of HCIO4:H2O:H2SO4 (9:5:1, v/v/v) on a heating block with a gradual increase in temperature from 100°C to 320°C. After digestion, the samples were diluted to a final volume of 100 ml with deionized water and filtered through filter paper (Whatman No. 2). The Na+ ion content was analyzed using Inductively Coupled Plasma-Mass Spectroscopy (ICP-MS, Perkin Elmer Optima 2200 DV).
The protective effect of HA in plants under salt stress has primarily been demonstrated in cereals such as maize and wheat (Aydin et al., 2012; Khaled and Fawy, 2011). Very recently, we also reported that HA increases seed germination rates in Arabidopsis in a dose-dependent manner and that the application of 86 mg L−1 HA confers salt stress tolerance to this plant under excessive salt concentrations (250 mM NaCl) (Cha et al., 2017). In the current study, to determine whether the use of HA at concentrations greater than 86 mg L−1 would cause a dramatic increase in salt tolerance, we performed a salt tolerance assay in which WT Arabidopsis seedlings were grown on 1/2 MS agar plates under a fixed concentration of NaCl (250 mM) and various concentrations of HA (0, 86, and 860 mg L−1). In the absence of HA, the seedlings were nearly dead when grown on salt stress medium, with chlorosis observed in shoots. However, seedlings grown on 86 or 860 mg L−1 HA medium plus 250 mM NaCl had green shoots (Fig. 1A). We measured the fresh weight of plants, finding that increasing the concentration of HA in seedlings under salinity stress increased salt stress tolerance in a dose-dependent manner (Fig. 1B). In tomato, the application of HA to the soil increased plant growth up to a concentration of 1 g kg−1, but the effects were reduced at 2 g kg−1 (Türkmen et al., 2004). HA has a positive effect on various plants, but how HA promotes plant growth and salt tolerance has remained elusive.
HA possesses many ionizable sites that allow it to bind to and chelate various cations, including Na+ (Tunstall, 2005). Thus, we performed ICP-MS analysis to determine whether HA can bind to and chelate Na+ ions in agar medium. Interestingly, plants grown on medium containing either NaCl or HA plus NaCl absorbed similar amounts of Na+ ions, suggesting that HA does not inhibit the uptake of NaCl by plants in culture (
HA promotes lateral root formation by exhibiting auxin-like activity in maize, tomato, and Arabidopsis (Dobbss et al., 2007; Trevisan et al., 2010; Zandonadi et al., 2007). By contrast, salt stress reduces root growth, including primary and lateral root growth, although primary root growth is more severely affected than lateral root growth (Julkowska et al., 2014). In addition, we have previously reported that primary root growth of
Na+ ions that reach the xylem by passing through several barriers in the root under salinity stress are transported to the shoot. Altering specific Na+ transport processes in specific cell types can reduce Na+ accumulation in the shoot, which is quite harmful to higher plants (Møller et al., 2009). To examine how HA enhances salinity stress tolerance in the shoot under salt stress conditions, we monitored the long-distance transport of Na+ in the root after short-term salinity treatment (Fig. 4). Five-day-old WT seedlings were treated with 100 mM NaCl in the absence or presence of HA (860 mg L−1) for 14 h. We visualized the distribution of sodium using the fluorescent, sodium-specific dye CoroNa-Green AM. After salt treatment, the intensity of fluorescent staining in WT roots became stronger than in the non-treated control, as shown previously (Oh et al., 2010) (Fig. 4A). In WT, when HA was added to salt-containing medium, the intensity of fluorescent staining moved upward to the elongation zone, while the intensity in the root-tip zone was reduced (Fig. 4A). Like WT, the intensity of fluorescent staining in the root tip of
To investigate the possibility that HA enhances the unloading of Na+ to xylem parenchyma cells, we first examined whether HA affects HKT1 protein levels using
HA is a component of humus. This heterogeneous, relatively large, high molecular weight organic complex, which ranges in color from brown to black, is amorphous, hydrophilic, molecularly flexible, and composed of polyelectrolytic compounds. HS contains a large number of complex humate molecules. Humate can bind to positive metal cations such as Iron (Fe2+), copper (Cu2+), zinc (Zn2+), calcium (Ca2+), manganese (Mg2+), and magnesium (Mg2+) (Tunstall, 2005). Salinity (NaCl) stress induces Ca2+ influx; the elevated levels of cytosolic free Ca2+ serve as a second messenger (Tracy et al., 2008). To investigate the positive effects of HA on HKT1 stability/activity due to the Ca2+ chelating effect of humate on plants under salt stress conditions, we used various pharmacological agents to inhibit Ca2+ release and flux (Fig. 6). Nicotinamide inhibits cyclic ADP-ribose (cADPR), a potent Ca2+-releasing agent, while GdCl3 blocks stretch-activated cation channels, thereby functioning as a Ca2+ influx inhibitor, and U73122 inhibits phospholipase C, thus acting as an inhibitor of Ca2+ efflux (Dodd et al., 2007; Tracy et al., 2008). In the absence of salt, HKT1 protein abundance increased by nicotinamide, U73122, or GdCl3 treatment compared to the control condition (Fig. 6). In the presence of 50 mM nicotinamide or 1 mM GdCl3, HKT1 was stabilized against NaCl-induced degradation under salt stress conditions. However, treatment with 5 μM U73122 (to inhibit vacuolar calcium release) failed to restore HKT1 protein to normal levels in the presence of NaCl-induced degradation (Fig. 6). These results suggest that the increase in cytosolic Ca2+ levels plays a role in NaCl-mediated HKT1 protein destabilization upon salt stress.
In this study, we demonstrated that HA treatment improves plant growth and reduces plant sensitivity to salinity stress. HA can function as a growth regulator by regulating hormone levels, plant growth, and stress responses (Piccolo et al., 1992; Serenella et al., 2002). HA treatment reduces the toxicity of salt in strawberry, maize, and garden cress seedlings (Masciandaro et al., 2002; Pilanal and Kaplan, 2003; Türkmen et al., 2004). Here, we showed that HA application also increases plant growth and enhances salt stress tolerance in Arabidopsis (Figs. 1–3; Cha et al., 2017). Treatment with 1 g L−1 HA has a positive effect on plant growth under saline soil conditions (Türkmen et al., 2004), which is consistent with our observation that 860 mg L−1 HA caused a significant increase in seedling survival, even under saline conditions (Fig. 1). David et al. (1994) reported that HS promotes plant growth and mineral nutrient uptake due to improved root system development. In addition, HS influences protein synthesis in higher plants (Carletti et al., 2008). Na+ strongly accumulated in both shoots and roots after the addition of NaCl, which is consistent with the findings for various barley cultivars exposed to 150 mM NaCl (Kamboj et al., 2015). HKT transporters are thought to be intricately involved in Na+ uptake and salt toxicity in plants (Ali et al., 2012; Mäser et al., 2002a; 2002b; Uozumi et al., 2000; Xue et al., 2011). AtHKT1;1 localized to the plasma membrane of xylem parenchyma cells mediates the removal of Na+ from xylem vessels during salinity stress (Sunarpi et al., 2005). When
HA treatment improves ion uptake and mineral nutrition in plants (Trevisan et al., 2010). Asik et al. (2009) determined that both soil and foliar application of small amounts of HS increase nutrient uptake in wheat under salt stress conditions. Murat et al. (2011) reported that adding humus to the soil increases nutrient uptake in plants under 45 and 60 mM NaCl treatment. Indeed, the protective effect of HA on plants under salt stress has been demonstrated in many cereals, such as maize and wheat (Aydin et al., 2012; Khaled and Fawy, 2011). In the current study, we showed that HA treatment relieved the growth inhibition induced by NaCl via the stabilization of HKT1 protein (Fig. 5).
Higher calcium levels in soil protect the cell membrane from the negative effects of salinity (Busch, 1995). Kwon et al. (2009) demonstrated that the addition of 60 mM NaCl to growth medium increases Na+ uptake in plants, as expected, but supplemental Ca2+ reverses this effect. Ca2+ also reduces the translocation of Na+ to the shoot and retains this ion in the roots. Under particular conditions, HS can stimulate plant growth, including increased plant height and dry/fresh weight (Blanchet, 1958; Guminski, 1968). These findings are consistent with our hypothesis that influx of the secondary messenger Ca2+ and cytosolic Ca2+ participate in NaCl-mediated destabilization of HKT1 protein under salt stress (Fig. 6). Several studies have confirmed the hypothesis that HS has a direct effect on plant physiology, specifically concerning lateral root development (Canellas et al., 2002; Carletti et al., 2008; Zandonadi et al., 2007). More recently, the auxin-like activity of HS in promoting lateral root development was investigated in the model plant Arabidopsis using a combination of genetic and molecular approaches (Trevisan et al., 2009).
In conclusion, this study demonstrates that HA plays an important role in improving salt tolerance by regulating the sodium transporters HKT1 in post-transcriptional levels. It is difficult to monitor changes in protein abundance after HA treatment due to the complex network of signaling pathways. To the best of our knowledge, HKT1 is the first protein whose levels were found to change in Arabidopsis after exposure to HA under salt stress treatment. Further research is needed to elucidate the specific functions and regulatory mechanisms underlying the effects of HA on HKT transporters and its role in salinity tolerance.
Jae Yong Ryu, Chung-Mo Park*, and Pil Joon Seo*
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