Auxin enhances aluminium-induced citrate exudation through upregulation of <em>GmMATE</em> and activation of the plasma membrane H<sup>+</sup>-ATPase in soybean roots
INTRODUCTION
Aluminium (Al) is the most abundant metal in the earth’s crust (Tesfaye et al., 2001). In acidic soils (pHwater < 5·5), Al is released into the soil solution, becoming toxic to plants and limiting to crop production (Rengel and Zhang, 2003). The Al-activated release of organic acid anions from roots has been characterized as one of the most important Al-resistance mechanisms in plants (Ma, 2000), including the efflux of malate from wheat roots (Ryan et al., 1995), oxalate from buckwheat (Zheng et al., 1998) and citrate from soybean (Silva et al., 2001), rice bean (Yang et al., 2007) and broad bean (Chen et al., 2012). Exudation of organic acid anions decreases Al uptake by chelating Al to form stable and non-toxic complexes in the rhizosphere. Exudation of malate and citrate anions is mediated by membrane-localized anion transporters belonging to two families, ALMT1 (Al-activated malate transporter) and MATE (multidrug and toxic compound extrusion) (Sasaki et al., 2004; Magalhaes et al., 2007; Eticha et al., 2010; Liu et al., 2011; Wu et al., 2012). Several genes encoding ALMT1 and MATE protein families have been identified in roots of Arabidopsis (Liu et al., 2009), sorghum (Magalhaes et al., 2007), rice (Yokosho et al., 2011) and soybean (Wu et al., 2012).
The plasma membrane H-ATPase, as the most abundant plasma membrane protein, belongs to a large superfamily of pumps termed P-type ATPases (Rengel et al., 2015). Using the chemical energy of ATP, the H-ATPase extrudes protons from cells to create an electrochemical gradient across the plasma membrane that is necessary to activate secondary transporters and plays an important role in physiological functions such as nutrient uptake (Palmgren, 2001), stomatal opening (Kinoshita and Shimazaki, 1999) and polar auxin transport and cell growth (Rober-Kleber et al., 2003). In high cytosolic pH (7–7·5), citric acid dissociates to citrate and H, which indicates that the release of citric acid by plant roots is attributed to citrate export via citrate transporter (MATE) and proton export via the plasma membrane H-ATPase (Yan et al., 2002; Yu et al., 2016). Consistently, several studies have shown that the plasma membrane H-ATPase is also involved in regulating citrate exudation in some plant species under P deficiency or Al toxicity (Ohno et al., 2003; Shen et al., 2005; Yan et al., 2002). For example, citrate exudation from cluster roots of white lupin (Lupinus albus) under stress of P deficiency was dependent on the plasma membrane H-ATPase (Yan et al., 2002; Tomasi et al., 2009). In soybean and broad bean roots, an increase in citrate exudation accompanied by an elevation of plasma membrane H-ATPase activity was observed in Al-resistant but not Al-sensitive cultivars (Shen et al., 2005; Chen et al., 2013; Guo et al., 2013). Furthermore, the upregulation of plasma membrane H-ATPase and interaction with the 14-3-3 protein are involved in regulating Al-induced citrate exudation in Al-resistant soybean and broad bean roots (Shen et al., 2005; Chen et al., 2013, 2015; Guo et al., 2013). However, it is still poorly understood how plants modulate the activity of the plasma membrane H-ATPase in response to Al stress.
Auxin, an important growth regulator in plants, modulates not only plant growth and development, but also has a crucial function in stress responses. Auxin concentration and distribution have been shown to be altered upon exposure to abiotic stresses such as Cd (Xu et al., 2010), salt (Albacete et al., 2008) and low P (Shen et al., 2006). External application of IAA can alleviate heavy metal toxicity in Arabidopsis, wheat and eggplant (Yang et al., 2011; Agami and Mohamed, 2013; Zhu et al., 2013b; Singh and Prasad, 2015). For example, treatment with 100 µm IAA alleviated the toxic effects of 80 µm Cu in roots, as reflected in greater root length and root hair formation in sunflower (Ouzounidou and Ilias, 2005). Exogenous auxin application alleviated Cd toxicity by decreasing Cd-induced chlorosis and Cd accumulation in Arabidopsis roots and shoots (Zhu et al. 2013b). Auxin is also considered to be involved in mediating the P-starved effect because external application of IAA in P-sufficient plants resulted in proteoid root formation in L.albus (Gilbert et al., 2000). Furthermore, P starvation induced an increase in endogenous IAA concentration and exogenous application of IAA stimulated both activity of plasma membrane H-ATPase and P uptake in soybean roots (Shen et al., 2006).
Under Al stress, the Al-induced auxin accumulation in the root–apex transition zone was crucial for Al-induced root growth inhibition (Zhu et al., 2013a; Yang et al., 2014). Primary root elongation was less inhibited by Al in the Arabidopsis auxin polar transport mutants pin2 (PIN-FORMED) and aux1 (AUXIN RESISTANT 1) than wild-type plants (Sun et al., 2010). Consistently, Ruíz-Herrera (2013) found that auxin signalling is involved in Al-induced primary root growth inhibition, but promoted lateral root formation and maturation. In addition, transgenic rice overexpressing OsPIN2 showed greater Al resistance than wild-type plants (Wu et al., 2014). Also, Al-induced endogenous IAA accumulation correlated significantly with malate exudation, and exogenous treatment with 10 μm IAA or 30 μm naphthylphthalamc acid (its efflux transport inhibitor) enhanced or decreased malate efflux and Al concentration in wheat roots (Yang et al., 2011), thus demonstrating a possible role of IAA in alleviating Al toxicity. So far, there is no direct evidence regarding the mechanisms by which IAA enhances Al-activated organic acid anion exudation from roots.
In this study we provide evidence regarding whether and how IAA regulates Al-activated citrate exudation from soybean roots. The results show that the concentration of IAA was increased by Al and that external application of IAA decreased Al concentration in roots. Furthermore, exogenous IAA increased Al-induced citrate exudation through upregulation of GmMATE and activation of the plasma membrane H-ATPase.
MATERIALS AND METHODS
Plant culture and growth conditions
Seeds of soybean (Glycine max ‘Tamba kuro’), considered an Al-resistant cultivar (Guo et al., 2013; Wu et al., 2013), were soaked in deionized water for 12 h in the dark at 25 °C followed by surface-sterilization with 1 % (v/v) sodium hypochlorite. Then, the seeds were placed on a filter paper moistened with half-strength Hoagland solution for germination in the dark at 25 °C. Seedlings with roots approximately 1–2 cm long were transferred onto a floating mesh in a polypropylene pot with half-strength Hoagland solution (5 L) and grown under glasshouse conditions at 30/25 °C day/night temperatures with 12 h of light. The nutrient solution was renewed every other day.
Measurement of endogenous IAA
Two-week-old seedlings were pre-treated with 0·5 mm CaCl2 (pH 4·2) at 25 °C under constant light (100 μmol m2 s1) in a tissue culture room for 12 h. The seedlings were then transferred into 0·5 mm CaCl2 solution containing AlCl3 at 0, 25, 50 or 200 μm (pH 4·2) for the 24-h treatment. After treatment, root apices (0–2 cm) were excised, weighed and immediately frozen in liquid nitrogen. Free IAA concentration in roots was measured by HPLC according to Sun et al. (2014).
Effect of IAA on plant growth under Al stress
To analyse the effects of IAA on plant growth, uniform 5-day-old seedlings (without lateral roots) with the tap root about 1 cm long were transferred onto a mesh floating in a polypropylene pot with half-strength Hoagland solution (1 L) with 50 μm AlCl3 plus 0, 0·5, 1, 25, 50 μm IAA or 50 μm IAA + 10 μm TIBA (2,3,5-triiodobenzoic acid, an IAA polar transport inhibitor) for 8 d. Seedlings grown in half-strength Hoagland solution with 0 or 50 μm IAA were used as controls. The pH of the treatment solutions was adjusted to 4·2 by the addition of 1 m HCl and the nutrient solution was renewed every other day. After 8-d treatments, shoot and root fresh weights were determined.
Measurement of Al concentration in roots and citrate concentration in root exudates
Two-week-old seedlings were pre-treated with 0·5 mm CaCl2 solution (pH 4·2) at 25 °C overnight under constant light (as described above). The roots were thoroughly rinsed with deionized water and gently blotted; the seedlings were transferred into 0·5 mm CaCl2 solution with the following treatments: control (CK, 0·5 mm CaCl2), Al25 (25 μm AlCl3), Al25IAA50 (25 μm AlCl3 + 50 μm IAA), Al50 (50 μm AlCl3), Al50IAA50 (50 μm AlCl3 + 50 μm IAA), Al200 (200 μm AlCl3), Al200IAA50 (200 μm AlCl3 + 50 μm IAA) or Al50IAA50TIBA10 (50 μm AlCl3 + 50 μm IAA + 10 μm TIBA). The pH was adjusted to 4·2 with 1 m HCl. After treatment for 24 h, the root apices (0–2 cm) were excised, weighed, immediately frozen in liquid nitrogen and stored at −80 °C for later RNA and plasma membrane extraction. The root exudates were collected and concentrated for citrate analysis by HPLC (Chen et al., 2012).
For measurement of Al concentration in soybean roots, the excised roots were thoroughly rinsed with deionized water, gently blotted, weighed, transferred to borosilicate tubes and ashed at 550 °C for 12 h. The resulting ash was dissolved overnight in 1 mL concentrated HNO3 and then diluted to 50 mL with deionized water. Aluminium was analysed by inductively coupled plasma atomic emission spectroscopy (ICP-AES, model PS-1000, Leeman Labs, Lowell, MA, USA).
Isolation of plasma membrane proteins and measurement of plasma membrane H-ATPase activity
Two-week-old seedlings were treated as described above. After treatments, the root apices (0–2 cm) were excised, weighed, immediately frozen in liquid nitrogen and stored at −80 °C for isolation of the plasma membrane proteins. The plasma membrane proteins were isolated and the activity of the plasma membrane H-ATPase was assessed as we described elsewhere (Chen et al., 2015).
Membrane potential
The membrane potential was detected by a fluorescent probe, bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC4, a voltage-sensitive fluorescent dye; Sigma-Aldrich) as previously described (Sun et al., 2012). After treatments, a stock solution of DiBAC4 (2 mm in DMSO) was added to excised root tips (0–2 cm) for 10 min at 25 °C. The final concentration of DiBAC4 was 2 μm. Subsequently, the root tips were thoroughly rinsed with deionized water three times, mounted on glass slides and examined with a Nikon A1 confocal fluorescence system. The membrane potential dye was excited at 488 nm with an argon laser; fluorescence emission was filtered at 530 nm and fluorescence intensity was captured through a 20× objective. DiBAC4 enters depolarized cells and exhibits fluorescence upon binding to endomembranes or proteins, whereas a return of plasma membrane to the resting potential or hyperpolarization causes extrusion of the dye and a decrease in fluorescence (Sivaguru et al., 2005). The experiments were conducted three times, and one representative set of results is presented.
Real-time reverse transcription–PCR analysis
The excised 0–2 cm root tips were used to isolate total RNA using Trizol reagent, and the first-strand cDNA was synthesized as we described previously (Chen et al., 2011). Gene expression analyses were performed by adding 1 μL of 10-fold-diluted cDNA in an ABI Plus (StepOne, Applied Biosystems) 96-well reverse transcription PCR system with ABI SybrGreen PCR Master Mix following the manufacturer’s instructions. The sequences of the gene-specific primer pairs for GmMATE (GLYMA13G27300) and the plasma membrane H-ATPase gene ({"type":"entrez-nucleotide","attrs":{"text":"AF091303","term_id":"3644031","term_text":"AF091303"}}AF091303) were designed as follows: 5′-AGTAAGCGTAGCCACAGAA-3′ (sense primer)/5′-CTGAGATAGAGCCAAGGTC-3′ (antisense primer) and 5′-TGAGTGGGAAGGCTTGGGATAA-3′ (sense primer)/5′-AAGGTTGGTTGTTTCAGGTGGC-3′ (antisense primer), respectively. The 18s rRNA gene ({"type":"entrez-nucleotide","attrs":{"text":"M16859","term_id":"343347","term_text":"M16859"}}M16859) was used as a reference gene with 5′-CCCGTCTCAGATTGGTGTCATT-3′ (sense primer)/5′-ATAGCGAGCAAGTCGGTGGATT-3′ (antisense primer). The PCR was performed for 40 cycles, and triplicates were performed on three independent experiments.
Western blot analysis
After treatments, plasma membrane proteins were extracted, and an aliquot of 60 μg of plasma membrane proteins was separated by SDS–PAGE (10 %, w/w) for western analysis. The separated proteins were transferred to polyvinylidene difluoride (PVDF) membranes with a semi-dry transfer system. The membranes were first treated with rabbit antibodies raised against the C-terminal domain of the Arabidopsis plasma membrane H-ATPase (AHA3) or with rabbit antibodies raised against the specifically phosphorylated Thr-951 in the Vicia faba plasma membrane H-ATPase VHA2. The phosphorylation-specific antibody for VHA2, which has been shown to also detect the phosphorylation levels of the plasma membrane H-ATPase in soybean, was produced against a 20-amino-acid peptide (N′-ESVVKLKGLDIDTIQQHYT (p)V-C′) with a phosphorylated modification according to the amino acidic sequence of Vicia faba VHA2 (Chen et al., 2013).
Statistical analysis
Experiments contained from three to 12 replicates, and the data are expressed as means and standard errors. The SPSS 12·0 for Windows (SPSS, Chicago, IL, USA) software package was used to conduct the Least Significant Difference (LSD) test to determine statistical significance at P ≤ 0·05.
Plant culture and growth conditions
Seeds of soybean (Glycine max ‘Tamba kuro’), considered an Al-resistant cultivar (Guo et al., 2013; Wu et al., 2013), were soaked in deionized water for 12 h in the dark at 25 °C followed by surface-sterilization with 1 % (v/v) sodium hypochlorite. Then, the seeds were placed on a filter paper moistened with half-strength Hoagland solution for germination in the dark at 25 °C. Seedlings with roots approximately 1–2 cm long were transferred onto a floating mesh in a polypropylene pot with half-strength Hoagland solution (5 L) and grown under glasshouse conditions at 30/25 °C day/night temperatures with 12 h of light. The nutrient solution was renewed every other day.
Measurement of endogenous IAA
Two-week-old seedlings were pre-treated with 0·5 mm CaCl2 (pH 4·2) at 25 °C under constant light (100 μmol m2 s1) in a tissue culture room for 12 h. The seedlings were then transferred into 0·5 mm CaCl2 solution containing AlCl3 at 0, 25, 50 or 200 μm (pH 4·2) for the 24-h treatment. After treatment, root apices (0–2 cm) were excised, weighed and immediately frozen in liquid nitrogen. Free IAA concentration in roots was measured by HPLC according to Sun et al. (2014).
Effect of IAA on plant growth under Al stress
To analyse the effects of IAA on plant growth, uniform 5-day-old seedlings (without lateral roots) with the tap root about 1 cm long were transferred onto a mesh floating in a polypropylene pot with half-strength Hoagland solution (1 L) with 50 μm AlCl3 plus 0, 0·5, 1, 25, 50 μm IAA or 50 μm IAA + 10 μm TIBA (2,3,5-triiodobenzoic acid, an IAA polar transport inhibitor) for 8 d. Seedlings grown in half-strength Hoagland solution with 0 or 50 μm IAA were used as controls. The pH of the treatment solutions was adjusted to 4·2 by the addition of 1 m HCl and the nutrient solution was renewed every other day. After 8-d treatments, shoot and root fresh weights were determined.
Measurement of Al concentration in roots and citrate concentration in root exudates
Two-week-old seedlings were pre-treated with 0·5 mm CaCl2 solution (pH 4·2) at 25 °C overnight under constant light (as described above). The roots were thoroughly rinsed with deionized water and gently blotted; the seedlings were transferred into 0·5 mm CaCl2 solution with the following treatments: control (CK, 0·5 mm CaCl2), Al25 (25 μm AlCl3), Al25IAA50 (25 μm AlCl3 + 50 μm IAA), Al50 (50 μm AlCl3), Al50IAA50 (50 μm AlCl3 + 50 μm IAA), Al200 (200 μm AlCl3), Al200IAA50 (200 μm AlCl3 + 50 μm IAA) or Al50IAA50TIBA10 (50 μm AlCl3 + 50 μm IAA + 10 μm TIBA). The pH was adjusted to 4·2 with 1 m HCl. After treatment for 24 h, the root apices (0–2 cm) were excised, weighed, immediately frozen in liquid nitrogen and stored at −80 °C for later RNA and plasma membrane extraction. The root exudates were collected and concentrated for citrate analysis by HPLC (Chen et al., 2012).
For measurement of Al concentration in soybean roots, the excised roots were thoroughly rinsed with deionized water, gently blotted, weighed, transferred to borosilicate tubes and ashed at 550 °C for 12 h. The resulting ash was dissolved overnight in 1 mL concentrated HNO3 and then diluted to 50 mL with deionized water. Aluminium was analysed by inductively coupled plasma atomic emission spectroscopy (ICP-AES, model PS-1000, Leeman Labs, Lowell, MA, USA).
Isolation of plasma membrane proteins and measurement of plasma membrane H-ATPase activity
Two-week-old seedlings were treated as described above. After treatments, the root apices (0–2 cm) were excised, weighed, immediately frozen in liquid nitrogen and stored at −80 °C for isolation of the plasma membrane proteins. The plasma membrane proteins were isolated and the activity of the plasma membrane H-ATPase was assessed as we described elsewhere (Chen et al., 2015).
Membrane potential
The membrane potential was detected by a fluorescent probe, bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC4, a voltage-sensitive fluorescent dye; Sigma-Aldrich) as previously described (Sun et al., 2012). After treatments, a stock solution of DiBAC4 (2 mm in DMSO) was added to excised root tips (0–2 cm) for 10 min at 25 °C. The final concentration of DiBAC4 was 2 μm. Subsequently, the root tips were thoroughly rinsed with deionized water three times, mounted on glass slides and examined with a Nikon A1 confocal fluorescence system. The membrane potential dye was excited at 488 nm with an argon laser; fluorescence emission was filtered at 530 nm and fluorescence intensity was captured through a 20× objective. DiBAC4 enters depolarized cells and exhibits fluorescence upon binding to endomembranes or proteins, whereas a return of plasma membrane to the resting potential or hyperpolarization causes extrusion of the dye and a decrease in fluorescence (Sivaguru et al., 2005). The experiments were conducted three times, and one representative set of results is presented.
Real-time reverse transcription–PCR analysis
The excised 0–2 cm root tips were used to isolate total RNA using Trizol reagent, and the first-strand cDNA was synthesized as we described previously (Chen et al., 2011). Gene expression analyses were performed by adding 1 μL of 10-fold-diluted cDNA in an ABI Plus (StepOne, Applied Biosystems) 96-well reverse transcription PCR system with ABI SybrGreen PCR Master Mix following the manufacturer’s instructions. The sequences of the gene-specific primer pairs for GmMATE (GLYMA13G27300) and the plasma membrane H-ATPase gene ({"type":"entrez-nucleotide","attrs":{"text":"AF091303","term_id":"3644031","term_text":"AF091303"}}AF091303) were designed as follows: 5′-AGTAAGCGTAGCCACAGAA-3′ (sense primer)/5′-CTGAGATAGAGCCAAGGTC-3′ (antisense primer) and 5′-TGAGTGGGAAGGCTTGGGATAA-3′ (sense primer)/5′-AAGGTTGGTTGTTTCAGGTGGC-3′ (antisense primer), respectively. The 18s rRNA gene ({"type":"entrez-nucleotide","attrs":{"text":"M16859","term_id":"343347","term_text":"M16859"}}M16859) was used as a reference gene with 5′-CCCGTCTCAGATTGGTGTCATT-3′ (sense primer)/5′-ATAGCGAGCAAGTCGGTGGATT-3′ (antisense primer). The PCR was performed for 40 cycles, and triplicates were performed on three independent experiments.
Western blot analysis
After treatments, plasma membrane proteins were extracted, and an aliquot of 60 μg of plasma membrane proteins was separated by SDS–PAGE (10 %, w/w) for western analysis. The separated proteins were transferred to polyvinylidene difluoride (PVDF) membranes with a semi-dry transfer system. The membranes were first treated with rabbit antibodies raised against the C-terminal domain of the Arabidopsis plasma membrane H-ATPase (AHA3) or with rabbit antibodies raised against the specifically phosphorylated Thr-951 in the Vicia faba plasma membrane H-ATPase VHA2. The phosphorylation-specific antibody for VHA2, which has been shown to also detect the phosphorylation levels of the plasma membrane H-ATPase in soybean, was produced against a 20-amino-acid peptide (N′-ESVVKLKGLDIDTIQQHYT (p)V-C′) with a phosphorylated modification according to the amino acidic sequence of Vicia faba VHA2 (Chen et al., 2013).
Statistical analysis
Experiments contained from three to 12 replicates, and the data are expressed as means and standard errors. The SPSS 12·0 for Windows (SPSS, Chicago, IL, USA) software package was used to conduct the Least Significant Difference (LSD) test to determine statistical significance at P ≤ 0·05.
RESULTS
Aluminium exposure induces auxin accumulation in soybean roots
Because endogenous auxin accumulation is involved in plant responses to Al stress, we first analysed the changes in auxin concentration in soybean roots. Compared with the control treatment, the concentration of IAA was increased by 14 and 28 % after the roots were treated with 25 and 50 μm Al, respectively, but decreased to the control level at 200 μm Al (Fig. 1).
Effect of Al on IAA accumulation in soybean root tips. Two-week-old seedlings were treated with 0, 25, 50 or 200 μm Al in 0·5 mm CaCl2 for 24 h. Values are mean ± s.e. (n = 6). Means with different letters are significantly different (P≤0·05).
External application of IAA increased plant growth under Al stress
Having ascertained that auxin accumulation in soybean root tips was induced by Al, we then analysed the effect of IAA on soybean growth under Al stress. As shown in Fig. 2, the 50 μm Al treatment decreased shoot and root fresh weight as well as the root/shoot ratio, but these parameters were significantly restored by the application of up to 50 μm IAA in the Al treatment solution (Fig. 2A–C). These results suggested that external application of IAA increased plant growth under Al stress.
Effect of external application of IAA on soybean shoot fresh weight (A), root fresh weight (B) and shoot/root ratio (C) under Al stress. Five-day-old seedlings were treated by 50 μm AlCl3 plus 0, 0·5, 1, 25, 50 μm IAA or 50 μm IAA+10 μm TIBA for 8 days. Seedlings grown in half-strength Hoagland solution with 0 or 50 μm IAA or 50 μm IAA plus 50 μm AlCl3 were used as controls. Values are means ± s.e. (n = 12).
IAA decreased Al accumulation in soybean root tips
Increasing the Al concentration in the treatment solution from 25 to 200 μm resulted in a significant increase in Al concentration in root tips (from 3·7 to 6·9 mg g dry weight) (Fig. 3). Inclusion of 50 μm IAA in the treatment solutions containing 25, 50 or 200 μm Al decreased Al concentration in root tips by 18, 29 and 31 %, respectively (Fig. 3).
Effect of IAA on Al concentration in soybean root tips (0–2 cm). Two-week-old seedlings were treated with 0, 25, 50 or 200 μm Al with 0 or 50 μm IAA in 0·5 mm CaCl2 for 24 h. Subscripts represent concentrations in μm. Values are mean ± s.e. (n = 6). Means with different letters are significantly different (P ≤ 0·05).
Activation of plasma membrane H-ATPase by IAA was involved in the regulation of Al-induced citrate exudation
Aluminium-induced organic acid exudation from plant root tips is an Al-exclusion mechanism that can reduce Al accumulation in plant roots. We investigated the involvement of IAA in Al-induced citrate exudation under Al stress in soybean roots. No citrate exudation was detected in the control (0 μm Al) treatment (Fig. 4A). When the Al concentration was increased to 25 or 50 μm, citrate exudation increased progressively and significantly, but was lower at 200 than 25 or 50 μm Al (Fig. 4A). Compared with the Al-only treatments, citrate exudation was significantly increased after 50 μm IAA was added to 25, 50 and 200 μm Al treatment solutions (by 2·5-, 1·7- and 6·7-fold, respectively) (Fig. 4A).
Effect of IAA on citrate exudation (A), plasma membrane H-ATPase activity (B) and membrane potential (C, D) in soybean root tips (0–2 cm) under Al stress. Two-week-old seedlings were treated with 0, 25, 50 or 200 μm Al with 0 or 50 μm IAA and with or without 10 μm TIBA or 100 μm vanadate (VA) in 0·5 mm CaCl2 for 24 h. Subscripts represent concentrations in μm. In A, B and D, values are means ± s.e. (n = 6). In C and D, membrane potential was measured using the fluorescent dye DiBAC4. Decreased fluorescence indicates hyperpolarization of the plasma membrane. Fluorescence intensity was quantified using ImageJ (http://rsb.info.nih.gov/ij/). Means with different letters are significantly different (P ≤ 0·05).
It has been shown that citrate exudation from roots is accompanied by activation of the plasma membrane H-ATPase in soybean under Al stress (Shen et al., 2005; Guo et al., 2013); therefore, the activity of this enzyme was also determined. The IAA-only treatment significantly increased plasma membrane H-ATPase activity in soybean root tips compared with the control (0 μm IAA added) (Fig. 4B). Similarly, exposing soybean plants to 25 and 50 μm Al increased (up to 2-fold) the plasma membrane H-ATPase activity in the root tips, whereas at 200 µm Al the activity of this enzyme decreased to the control level (Fig. 4B). Adding exogenous IAA at 50 µM to the Al-containing treatment solutions resulted in a significant increase in the plasma membrane H-ATPase activity at 50 μm Al (74 % increase) and 200 μm Al (45 % increase) compared with the Al-only treatment (Fig. 4B). Adding TIBA (an IAA polar transport inhibitor) or vanadate (a plasma membrane H-ATPase inhibitor) to the treatment solution containing 50 μm Al plus 50 μm IAA resulted in a significant decrease in the plasma membrane H-ATPase activity (Fig. 4B) as well as citrate exudation (Fig. 4A). These results indicated that the plasma membrane H-ATPase was involved in IAA-dependent enhancement of Al-induced citrate exudation in soybean root tips.
DiBAC4(3), a fluorescent probe, is usually used for monitoring changes in membrane potential. When the membrane becomes hyperpolarized, the dye transports across the cell membrane and fluorescence decreases (Sun et al., 2012). In this study, the changes in membrane potential were monitored to investigate the action of Al and IAA using DiBAC4(3). The results showed that soybean roots exhibited relatively weak fluorescence after 24 h of exposure to Al compared with the control treatment (Fig. 4C). After application of IAA into the Al treatment solution, the relative fluorescence intensity was significantly decreased (about 68 % relative to the control and 38 % relative to the Al-only treatment) (Fig. 4D). However, TIBA supplied to the Al- and IAA-containing treatment solution increased the relative fluorescence intensity to the control level. These results suggested that both Al and IAA promoted hyperpolarization (Fig. 4C, D) by enhancing the plasma membrane H-ATPase (Fig. 4B) under Al stress.
IAA enhanced GmMATE but not plasma membrane H-ATPase gene expression under Al stress
To understand how IAA modulates the Al-induced citrate efflux and the plasma membrane H-ATPase activity, we explored the mRNA abundance of GmMATE and the plasma membrane H-ATPase gene. Exposure of soybean to 25 or 200 μm Al for 24 h resulted in a similar increase in the mRNA abundance of GmMATE in the root tips, and exposure to 50 μm Al increased it even further (Fig. 5A). For all three Al treatments, adding 50 μm IAA increased the expression of GmMATE significantly (1·6-, 1·7- and 1·5-fold for 25, 50 and 200 μm Al, respectively). However, compared with the Al + IAA treatment, application of TIBA reduced the expression of GmMATE by 62 % (Fig. 5A).
Real-time reverse transcription–PCR analysis of transcription (mRNA abundance) of GmMATE (A) and the plasma membrane H-ATPase gene (B) in soybean root tips (0–2 cm). Two-week-old seedlings were treated with 25, 50 or 200 μm Al with 0 or 50 μm IAA and with or without 10 μm TIBA in 0·5 mm CaCl2 for 24 h. Subscripts represent concentrations in μm. Values are mean ± s.e. (n = 6). Means with different letters are significantly different (P ≤ 0·05).
The expression of the plasma membrane H-ATPase gene was not influenced by an addition of 50 μm IAA, but was increased by 76, 95 and 59 % after roots were treated with 25, 50 or 200 μm Al, respectively (Fig. 5B). However, neither IAA nor IAA + TIBA addition to the Al-containing treatment solutions changed the expression of the plasma membrane H-ATPase gene. These results indicated that IAA enhanced Al-induced citrate exudation through increasing the expression of MATE but not the plasma membrane H-ATPase gene in the soybean root tips.
IAA enhanced phosphorylation of the plasma membrane H-ATPase under Al stress
To assess whether translational and/or post-translational regulation was involved in IAA-dependent enhancement of the plasma membrane H-ATPase activity, we analysed protein biosynthesis and phosphorylation. The total amount of plasma membrane H-ATPase protein in the root tips was increased by 35, 26 and 13 % after soybean plants were treated with Al, Al + IAA and Al + IAA + TIBA, respectively (Fig. 6A). Phosphorylation of the plasma membrane H-ATPase increased by 10 % after treatment with 50 μm Al and 34 % after Al + IAA. However, addition of TIBA to the Al + IAA treatment decreased phosphorylation to the control level (Fig. 6A).
Translation and phosphorylation of plasma membrane H-ATPase isolated from soybean root tips (0–2 cm). (A) Western blot analysis for detection of protein biosynthesis (bottom) and phosphorylation (top) of plasma membrane H-ATPase. (B) Coomassie brilliant blue staining of membrane proteins. Two-week-old seedlings were treated with 0 (control, CK), 50 μm Al (A50), 50 μm Al + 50 μm IAA (A50I50) or 50 μm Al + 50 μm IAA+10 μm TIBA (A50I50T10) in 0·5 mm CaCl2 for 24 h. Relative-intensity band quantification of western blots was performed using ImageJ (http://rsb.info.nih.gov/ij/). The experiment was conducted three times, and one representative set of results is shown.
Aluminium exposure induces auxin accumulation in soybean roots
Because endogenous auxin accumulation is involved in plant responses to Al stress, we first analysed the changes in auxin concentration in soybean roots. Compared with the control treatment, the concentration of IAA was increased by 14 and 28 % after the roots were treated with 25 and 50 μm Al, respectively, but decreased to the control level at 200 μm Al (Fig. 1).
Effect of Al on IAA accumulation in soybean root tips. Two-week-old seedlings were treated with 0, 25, 50 or 200 μm Al in 0·5 mm CaCl2 for 24 h. Values are mean ± s.e. (n = 6). Means with different letters are significantly different (P≤0·05).
External application of IAA increased plant growth under Al stress
Having ascertained that auxin accumulation in soybean root tips was induced by Al, we then analysed the effect of IAA on soybean growth under Al stress. As shown in Fig. 2, the 50 μm Al treatment decreased shoot and root fresh weight as well as the root/shoot ratio, but these parameters were significantly restored by the application of up to 50 μm IAA in the Al treatment solution (Fig. 2A–C). These results suggested that external application of IAA increased plant growth under Al stress.
Effect of external application of IAA on soybean shoot fresh weight (A), root fresh weight (B) and shoot/root ratio (C) under Al stress. Five-day-old seedlings were treated by 50 μm AlCl3 plus 0, 0·5, 1, 25, 50 μm IAA or 50 μm IAA+10 μm TIBA for 8 days. Seedlings grown in half-strength Hoagland solution with 0 or 50 μm IAA or 50 μm IAA plus 50 μm AlCl3 were used as controls. Values are means ± s.e. (n = 12).
IAA decreased Al accumulation in soybean root tips
Increasing the Al concentration in the treatment solution from 25 to 200 μm resulted in a significant increase in Al concentration in root tips (from 3·7 to 6·9 mg g dry weight) (Fig. 3). Inclusion of 50 μm IAA in the treatment solutions containing 25, 50 or 200 μm Al decreased Al concentration in root tips by 18, 29 and 31 %, respectively (Fig. 3).
Effect of IAA on Al concentration in soybean root tips (0–2 cm). Two-week-old seedlings were treated with 0, 25, 50 or 200 μm Al with 0 or 50 μm IAA in 0·5 mm CaCl2 for 24 h. Subscripts represent concentrations in μm. Values are mean ± s.e. (n = 6). Means with different letters are significantly different (P ≤ 0·05).
Activation of plasma membrane H-ATPase by IAA was involved in the regulation of Al-induced citrate exudation
Aluminium-induced organic acid exudation from plant root tips is an Al-exclusion mechanism that can reduce Al accumulation in plant roots. We investigated the involvement of IAA in Al-induced citrate exudation under Al stress in soybean roots. No citrate exudation was detected in the control (0 μm Al) treatment (Fig. 4A). When the Al concentration was increased to 25 or 50 μm, citrate exudation increased progressively and significantly, but was lower at 200 than 25 or 50 μm Al (Fig. 4A). Compared with the Al-only treatments, citrate exudation was significantly increased after 50 μm IAA was added to 25, 50 and 200 μm Al treatment solutions (by 2·5-, 1·7- and 6·7-fold, respectively) (Fig. 4A).
Effect of IAA on citrate exudation (A), plasma membrane H-ATPase activity (B) and membrane potential (C, D) in soybean root tips (0–2 cm) under Al stress. Two-week-old seedlings were treated with 0, 25, 50 or 200 μm Al with 0 or 50 μm IAA and with or without 10 μm TIBA or 100 μm vanadate (VA) in 0·5 mm CaCl2 for 24 h. Subscripts represent concentrations in μm. In A, B and D, values are means ± s.e. (n = 6). In C and D, membrane potential was measured using the fluorescent dye DiBAC4. Decreased fluorescence indicates hyperpolarization of the plasma membrane. Fluorescence intensity was quantified using ImageJ (http://rsb.info.nih.gov/ij/). Means with different letters are significantly different (P ≤ 0·05).
It has been shown that citrate exudation from roots is accompanied by activation of the plasma membrane H-ATPase in soybean under Al stress (Shen et al., 2005; Guo et al., 2013); therefore, the activity of this enzyme was also determined. The IAA-only treatment significantly increased plasma membrane H-ATPase activity in soybean root tips compared with the control (0 μm IAA added) (Fig. 4B). Similarly, exposing soybean plants to 25 and 50 μm Al increased (up to 2-fold) the plasma membrane H-ATPase activity in the root tips, whereas at 200 µm Al the activity of this enzyme decreased to the control level (Fig. 4B). Adding exogenous IAA at 50 µM to the Al-containing treatment solutions resulted in a significant increase in the plasma membrane H-ATPase activity at 50 μm Al (74 % increase) and 200 μm Al (45 % increase) compared with the Al-only treatment (Fig. 4B). Adding TIBA (an IAA polar transport inhibitor) or vanadate (a plasma membrane H-ATPase inhibitor) to the treatment solution containing 50 μm Al plus 50 μm IAA resulted in a significant decrease in the plasma membrane H-ATPase activity (Fig. 4B) as well as citrate exudation (Fig. 4A). These results indicated that the plasma membrane H-ATPase was involved in IAA-dependent enhancement of Al-induced citrate exudation in soybean root tips.
DiBAC4(3), a fluorescent probe, is usually used for monitoring changes in membrane potential. When the membrane becomes hyperpolarized, the dye transports across the cell membrane and fluorescence decreases (Sun et al., 2012). In this study, the changes in membrane potential were monitored to investigate the action of Al and IAA using DiBAC4(3). The results showed that soybean roots exhibited relatively weak fluorescence after 24 h of exposure to Al compared with the control treatment (Fig. 4C). After application of IAA into the Al treatment solution, the relative fluorescence intensity was significantly decreased (about 68 % relative to the control and 38 % relative to the Al-only treatment) (Fig. 4D). However, TIBA supplied to the Al- and IAA-containing treatment solution increased the relative fluorescence intensity to the control level. These results suggested that both Al and IAA promoted hyperpolarization (Fig. 4C, D) by enhancing the plasma membrane H-ATPase (Fig. 4B) under Al stress.
IAA enhanced GmMATE but not plasma membrane H-ATPase gene expression under Al stress
To understand how IAA modulates the Al-induced citrate efflux and the plasma membrane H-ATPase activity, we explored the mRNA abundance of GmMATE and the plasma membrane H-ATPase gene. Exposure of soybean to 25 or 200 μm Al for 24 h resulted in a similar increase in the mRNA abundance of GmMATE in the root tips, and exposure to 50 μm Al increased it even further (Fig. 5A). For all three Al treatments, adding 50 μm IAA increased the expression of GmMATE significantly (1·6-, 1·7- and 1·5-fold for 25, 50 and 200 μm Al, respectively). However, compared with the Al + IAA treatment, application of TIBA reduced the expression of GmMATE by 62 % (Fig. 5A).
Real-time reverse transcription–PCR analysis of transcription (mRNA abundance) of GmMATE (A) and the plasma membrane H-ATPase gene (B) in soybean root tips (0–2 cm). Two-week-old seedlings were treated with 25, 50 or 200 μm Al with 0 or 50 μm IAA and with or without 10 μm TIBA in 0·5 mm CaCl2 for 24 h. Subscripts represent concentrations in μm. Values are mean ± s.e. (n = 6). Means with different letters are significantly different (P ≤ 0·05).
The expression of the plasma membrane H-ATPase gene was not influenced by an addition of 50 μm IAA, but was increased by 76, 95 and 59 % after roots were treated with 25, 50 or 200 μm Al, respectively (Fig. 5B). However, neither IAA nor IAA + TIBA addition to the Al-containing treatment solutions changed the expression of the plasma membrane H-ATPase gene. These results indicated that IAA enhanced Al-induced citrate exudation through increasing the expression of MATE but not the plasma membrane H-ATPase gene in the soybean root tips.
IAA enhanced phosphorylation of the plasma membrane H-ATPase under Al stress
To assess whether translational and/or post-translational regulation was involved in IAA-dependent enhancement of the plasma membrane H-ATPase activity, we analysed protein biosynthesis and phosphorylation. The total amount of plasma membrane H-ATPase protein in the root tips was increased by 35, 26 and 13 % after soybean plants were treated with Al, Al + IAA and Al + IAA + TIBA, respectively (Fig. 6A). Phosphorylation of the plasma membrane H-ATPase increased by 10 % after treatment with 50 μm Al and 34 % after Al + IAA. However, addition of TIBA to the Al + IAA treatment decreased phosphorylation to the control level (Fig. 6A).
Translation and phosphorylation of plasma membrane H-ATPase isolated from soybean root tips (0–2 cm). (A) Western blot analysis for detection of protein biosynthesis (bottom) and phosphorylation (top) of plasma membrane H-ATPase. (B) Coomassie brilliant blue staining of membrane proteins. Two-week-old seedlings were treated with 0 (control, CK), 50 μm Al (A50), 50 μm Al + 50 μm IAA (A50I50) or 50 μm Al + 50 μm IAA+10 μm TIBA (A50I50T10) in 0·5 mm CaCl2 for 24 h. Relative-intensity band quantification of western blots was performed using ImageJ (http://rsb.info.nih.gov/ij/). The experiment was conducted three times, and one representative set of results is shown.
DISCUSSION
As one of the most widely accepted mechanisms of plant responses to Al, organic acid anion exudation has been validated by cloning of the relevant transporter genes, such as ALMT1 and MATE (Sasaki et al., 2004; Magalhaes et al., 2007; Eticha et al., 2010; Liu et al., 2011). In the present study, we found significantly increased abundance of GmMATE transcripts after a 24-h exposure to 25, 50 or 200 μm Al (Fig. 5A). Moreover, addition of IAA to the Al-containing treatment solutions significantly enhanced the mRNA abundance of GmMATE compared with Al-treated roots. These results suggested that upregulation of GmMATE is involved in IAA-enhanced citrate release under Al toxicity.
Acidification of the cell wall through activation of the plasma membrane H-ATPase is an important part of the growth-promoting effect of auxin (Hager et al., 1991; Frias et al., 1996), although the exact mechanisms mediating the effect of auxin on the plasma membrane H-ATPase are unclear (Fuglsang et al., 2007). Auxin polar transport and auxin gradient formation were demonstrated to be dependent on the activity of the plasma membrane H-ATPase (Hohm et al., 2014). In Arabidopsis, the pin2 mutant exhibited much reduced plasma membrane H-ATPase activity, auxin transport, proton secretion and root elongation under alkaline stress (Xu et al., 2012). In soybean roots, an increase in endogenous IAA was observed under low P, and an exogenous application of 10 µm IAA stimulated the activity of plasma membrane H-ATPase and enhanced P uptake (Shen et al., 2006).
Plasma membrane H-ATPase is the pivotal enzyme for creating an electrochemical H gradient and generating the proton motive force across the plasma membrane that is necessary to activate citrate transporters under Al stress and low-P conditions (Rengel et al., 2015; Yu et al., 2016). For example, the plasma membrane H-ATPase is involved in citrate exudation from cluster roots of Lupinus albus under P deficiency (e.g. in cluster roots; Yan et al., 2002) and from soybean (Shen et al., 2005) and Vicia faba roots (Chen et al., 2012) of Al-resistant but not Al-sensitive cultivars. In the present study, 50 µm IAA added to Al-containing solution enhanced both Al-induced citrate exudation and Al-mediated increase in the plasma membrane H-ATPase activity (Fig. 4B, C); this enhancement was repressed by the addition of the plasma membrane H-ATPase inhibitor vanadate or the auxin polar transport inhibitor TIBA (Fig. 4). The results of using DiBAC4 dye also suggested that IAA strengthened hyperpolarization of the plasma membrane caused by Al; such a hyperpolarization effect (Fig. 4C, D) of Al or IAA was underpinned by increased activity of H-ATPase caused by Al or IAA (Fig. 4B).
Aluminium caused depolarization of the plasma membrane, suggesting decreased H-ATPase activity, in many experimental systems, including wheat, barley, maize and squash (for references see Rengel and Zhang, 2003), tobacco (Sivaguru et al., 2005) and Arabidopsis (Illes et al., 2006), but both depolarization and hyperpolarization of the plasma membrane due to Al exposure may occur in Arabidopsis depending on the root part and the degree of Al sensitivity (Bose et al., 2010a, b). Similarly, in wheat root tips, Al exposure caused depolarization in Al-sensitive and hyperpolarization in Al-resistant genotypes (Ahn et al., 2004). In the apoplastic space (pH ∼5·5), IAA is protonated as IAAH (pKa 4·85); in contrast, higher cytosolic pH (7–7·5) promoted the dissociation of IAAH to deprotonated IAA (IAA) and H. Like citric acid, efflux of IAAH across the membrane must be accomplished by at least two structurally separated plasma membrane transport processes: PIN-FORMED (PIN) protein family of auxin transporters mediated IAA (IAA) efflux and plasma membrane H-ATPase-mediated H efflux. In the present study, hyperpolarization of the root-cell plasma membrane was caused by Al, and this effect was further enhanced by exposure to Al + IAA (Fig. 4C, D); these results were consistent with those obtained with Al and IAA individually and in combination, enhancing plasma membrane H-ATPase activity (Fig. 4B). Therefore, it is speculated that hyperpolarization of the membrane through activation of the plasma membrane H-ATPase is necessary to activate auxin and citrate transport across the membrane.
The activity of plasma membrane H-ATPase can be modulated at transcriptional, translational and post-translational levels (Portillo, 2000). For example, external IAA treatment enhanced the mRNA abundance and protein biosynthesis of the plasma membrane H-ATPase in maize (Hager et al., 1991; Frias et al., 1996). Aluminium toxicity stress induced gene expression and protein biosynthesis of the plasma membrane H-ATPase in Al-resistant cultivars of soybean and broad bean (Shen et al., 2005; Chen et al., 2013). Phosphorylation at the penultimate Thr residue of the plasma membrane H-ATPase is a common example of post-translational modification altering the activity of this enzyme. In Arabidopsis, IAA activates the plasma membrane H-ATPase by enhancing phosphorylation and the interaction with 14-3-3 protein during hypocotyl elongation under P-deficiency conditions (Takahashi et al., 2012). The penultimate Thr-directed phosphorylation of the plasma membrane H-ATPase is also involved in citrate exudation from soybean (Shen et al., 2005) and Vicia faba roots (Chen et al., 2013) under Al toxicity. Furthermore, the post-translational modification of the plasma membrane H-ATPase, rather than transcriptional and translational modification, was involved in Mg enhancement of Al-induced citrate exudation in Vicia faba roots (Chen et al., 2015). The results presented here showed that IAA did not induce changes in transcription (Fig. 5A) and translation (Fig. 6A) of the plasma membrane H-ATPase under Al stress, whereas phosphorylation of the penultimate Thr-oriented residue in the plasma membrane H-ATPase was increased in the presence of IAA under Al stress (Fig. 6A).
Conclusions
The results of this study provide evidence that Al induced IAA accumulation in soybean root tips, and that external application of IAA reduced Al uptake in soybean root tips. An IAA-dependent upregulation of GmMATE expression and phosphorylation of the plasma membrane H-ATPase were involved in enhancing activity of the plasma membrane H-ATPase, resulting in increased citrate exudation from soybean roots under Al stress.
Abstract
Background and Aims Aluminium (Al) toxicity is a limiting factor for plant growth and crop production in acidic soils. Citrate exudation and activation of the plasma membrane H-ATPase are involved in soybean responses to Al stress. Auxin has crucial functions in plant growth and stress responses. However, little is known about possible interactions between auxin and citrate exudation under Al stress. In this study, we elucidated the regulatory roles of IAA in Al-induced citrate exudation in soybean roots.
Methods We measured IAA content, Al concentration, citrate exudation, plasma membrane H-ATPase activity, expression of the relevant genes and phosphorylation of the plasma membrane H-ATPase by integrating physiological characterization and molecular analysis using hydroponically grown soybean.
Key Results The concentration of IAA was increased by 25 and 50 μm Al, but decreased to the control level at 200 μm Al. External addition of 50 μm IAA to the root medium containing 25, 50 or 200 μm Al decreased root Al concentration and stimulated Al-induced citrate exudation and the plasma membrane H-ATPase activity. Reverse transcription–PCR analysis showed that exogenous IAA enhanced the expression of citrate exudation transporter (GmMATE) but not the plasma membrane H-ATPase gene. The western blot results suggested that IAA enhanced phosphorylation of the plasma membrane H-ATPase under Al stress.
Conclusions Auxin enhanced Al-induced citrate exudation through upregulation of GmMATE and an increase in phosphorylation of the plasma membrane H-ATPase in soybean roots.
ACKNOWLEDGEMENTS
This work was supported by the National Natural Science Foundation of China (No. 31360340), the National Basic Research Program of China (No. 2014CB138701) and the Talent Training Program in Yunnan Province (KKSY201326062). Z.R. was supported by Australian Research Council (DP160104434).