Altered cell wall properties are responsible for ... - Wiley Online Library

bs_bs_banner

Plant, Cell and Environment (2015) 38, 1382–1390

doi: 10.1111/pce.12490

Original Article

Altered cell wall properties are responsible for ammonium-reduced aluminium accumulation in rice roots Wei Wang1,2, Xue Qiang Zhao1, Rong Fu Chen1, Xiao Ying Dong1, Ping Lan1, Jian Feng Ma1,3 & Ren Fang Shen1 1

State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China, 2University of Chinese Academy of Sciences, Beijing 100049, China and 3Institute of Plant Science and Resources, Okayama University, Kurashiki 710-0046, Japan

ABSTRACT The phytotoxicity of aluminium (Al) ions can be alleviated by ammonium (NH4+) in rice and this effect has been attributed to the decreased Al accumulation in the roots. Here, the effects of different nitrogen forms on cell wall properties were compared in two rice cultivars differing in Al tolerance. An in vitro Al-binding assay revealed that neither NH4+ nor NO3− altered the Al-binding capacity of cell walls, which were extracted from plants not previously exposed to N sources. However, cell walls extracted from NH4+-supplied roots displayed lower Al-binding capacity than those from NO3−supplied roots when grown in non-buffered solutions. Fourier-transform infrared microspectroscopy analysis revealed that, compared with NO3−-supplied roots, NH4+supplied roots possessed fewer Al-binding groups (-OH and COO-) and lower contents of pectin and hemicellulose. However, when grown in pH-buffered solutions, these differences in the cell wall properties were not observed. Further analysis showed that the Al-binding capacity and properties of cell walls were also altered by pHs alone. Taken together, our results indicate that the NH4+-reduced Al accumulation was attributed to the altered cell wall properties triggered by pH decrease due to NH4+ uptake rather than direct competition for the cell wall binding sites between Al3+ and NH4+. Key-words: Al-binding capacity; nitrate; pH.

INTRODUCTION Acid soils account for around 35% of the world’s arable land and nearly 50% of potentially arable land (Uexküll & Mutert 1995; Ryan et al. 2011). The major factor limiting crop production on these acid soils is aluminium (Al) toxicity. At low concentrations, Al ion inhibits root elongation, resulting in root structural and functional damage (Kochian 1995; Ma et al. 2014). However, some plant species and cultivars have developed strategies for detoxifying Al internally and/or externally. The most-studied mechanism is the secretion of organic acid anions such as citrate, malate and oxalate from roots to chelate toxic Al ions in the rhizosphere (Delhaize et al. 1993; Ma et al. 1997, 2004a; Zhao et al. 2003). Recently, genes involved in the Al-induced secretion of malate and Correspondence: R. F. Shen. e-mail: [email protected] 1382

citrate have been identified in several plant species (Sasaki et al. 2004; Furukawa et al. 2007; Magalhaes et al. 2007). In addition, a number of other Al tolerance genes have also been identified especially in rice (Huang et al. 2009; Yamaji et al. 2009; Delhaize et al. 2012; Ma et al. 2014). Different from simple solution culture, in real acid soils, Al toxicity is affected by many other factors, such as nitrogen (N) forms. The predominant inorganic form of N in acid soils is ammonium (NH4+) due to the weak nitrification capacity and the application of ammonium fertilizers on these soils (McCain & Davies 1983; De Boer & Kowalchuk 2001; Guo et al. 2010; Schroder et al. 2011). Uptake of NH4+ by the roots is known to be accompanied with release of H+, resulting in pH decrease in the rhizosphere (Schubert & Yan 1997). In contrast, uptake of NO3− consumes H+, resulting in rhizosphere alkalization (Marschner & Römheld 1983; Moorby et al. 1985). Since Al solubility increases with decreasing soil pH, it is reasonable to speculate that the presence of NH4+ increases the sensitivity of plants to Al toxicity, whereas NO3− has opposite effect. Although these predicted effects have been observed in some species (Fleming 1983; Taylor & Foy 1985a,b,c; Tan et al. 1992), the opposite cases have also been found in others (Cumming 1990; Cumming & Weinstein 1990; Grauer & Horst 1990; Tsuji et al. 1994; Zhao et al. 2009; Chen et al. 2010). In rice (Oryza sativa), an alleviative effect of NH4+ on Al toxicity has been observed in both japonica and indica cultivars differing in Al sensitivity (Zhao et al. 2013). This effect of NH4+ is associated with decreased Al accumulation in the root tips (Zhao et al. 2009). Furthermore, a significant correlation between N preference and Al tolerance has recently been observed in 30 rice cultivars (Zhao et al. 2013). The alleviative effect of NH4+ on Al toxicity has been partly attributed to a direct competition for root binding sites between Al3+ and NH4+ in lespedeza (Zhao & Shen 2013), but the exact mechanism is still unclear. The cell wall is the primary Al-binding site in root cells (Kochian et al. 2005; Horst et al. 2010). In monocots and dicots, almost 80–90% of Al is accumulated in the cell walls (Clarkson 1967; Ma et al. 2004b; Yang et al. 2008, 2011; Rangel et al. 2009). Al binding to cell walls decreases their extensibility, thereby causing inhibition of root elongation (Ma et al. 2004b). Using haematoxylin staining method, a previous study reported that NH4+-supplied roots showed © 2014 John Wiley & Sons Ltd

Ammonium affects Al binding of root cell wall light intensity of staining, suggesting that NH4+ decreased Al binding to the cell wall (Zhao et al. 2009). In the present study, we further compared Al-binding capacity in the cell wall of the roots exposed to NH4+ and NO3− in two rice cultivars differing in Al tolerance. We also investigated the effects of different N forms on cell wall properties, including functional group numbers, pectin and hemicellulose contents, and surface charge. In roots of both Al-sensitive and Al-tolerant rice cultivars, we found that the NH4+-reduced accumulation of Al is a consequence of altered cell wall properties, which was triggered by pH decrease due to NH4+ uptake, rather than direct competition for the cell wall binding sites between Al3+ and NH4+.

MATERIALS AND METHODS Plant materials and growth conditions Two cultivars (Wuyunjing7 and Yangdao6) of rice (O. sativa) were used in this study. Wuyunjing7 (WYJ7) is an Al-tolerant cultivar (japonica), while Yangdao6 (YD6) is an Al-sensitive cultivar (indica) (Zhao et al. 2009). Seeds were surfacesterilized in a 1% (v/v) sodium hypochlorite solution for 30 min, followed by washing thoroughly with deionized water, and by soaking in deionized water overnight. The seeds were germinated in an incubator at 30 °C for 24 h and then transferred to a nylon net floating on a 0.5 mm CaCl2 (pH 4.5) solution in a 6.5 L container. The solution was renewed every 2 d. All experiments were conducted in a growth chamber with a 14 h/30 °C day and a 10 h/25 °C night regime under a relative humidity of 65%. After 3 d preculture, seedlings were used for the following experiments. The root elongation was monitored in all treatments by measuring the root length before and after the treatments with a ruler. Root elongations of all treatment, except for Al treatment, were higher than 1 cm/24 h (Supporting Information Table S1).

Cell wall preparation Seedlings (200 plants per treatment) were exposed to a 0.5 mm CaCl2 solution (pH 4.5) buffered with or without 5 mm Homopiperazine-1,4-bis(2-ethanesulfonic acid) (i.e. Homo-pipes) in the presence of 0, 1 mm NH4Cl or 1 mm NaNO3. To investigate the effect of Al on cell wall composition, the roots were exposed to the same solution containing 50 μm AlCl3. To investigate the effects of pH on cell wall composition, the roots were exposed to a 0.5 mm CaCl2 solution buffered with or without 5 mm Homo-pipes at different pHs (3.5, 4.5 and 5.5) in the absence of N. After 24 h, root tips (0–1 cm from the tip) were excised and subjected to cell wall extraction according to Zhong & Lauchli (1993). Briefly, the roots were ground with a mortar and pestle in liquid N, and transferred to a 50 mL centrifuge tube containing 15 mL icecold 75% ethanol. After re-suspended by vortexing, the mixtures were kept in an ice-water bath for 20 min and then centrifuged at 1200 g for 10 min at 4 °C. The pellets were washed for 20 min each with acetone, methanol: chloroform

1383

(1:1) and methanol, respectively. Pellets (i.e. cell wall materials) were freeze-dried and stored at −20 °C until use.

Al adsorption kinetics in cell wall Since the adsorption of Al into cell wall materials may cause pH change of static adsorption solution, peristaltic pump was used to drive the adsorption solution passed through the column which was filled with cell wall materials. Briefly, the root cell wall materials (10 mg), extracted as described above, were placed in a 3 mL column equipped with a filter at the bottom. The cell walls were washed with 0.5 mm CaCl2 (pH 4.5) with a flow speed of 0.2 mL min−1 for 2 h (wetting and balance), followed by passage of adsorption solution (10 μm AlCl3 in 0.5 mm CaCl2 at pH 4.5) through the column at the same flow speed. These elutes were collected at 20 min intervals. Al in the adsorption solution was determined with pyrocatechol violet (Kerven et al. 1989; Zheng et al. 2004). The amount of Al adsorbed by the cell walls was calculated based on the difference of Al concentration in the solution before and after passing through the column. To investigate direct competition between NH4+ and Al in cell walls, cell walls of roots without N pretreatment were prepared as described above. The root cell walls were either incubated with 10 μm AlCl3 in 0.5 mm CaCl2 (pH 4.5) with or without 1 mm NH4Cl or NaNO3 or first incubated with or without 1 mm NH4Cl or NaNO3 for 24 h, followed by 10 μm AlCl3 in 0.5 mm CaCl2 (pH 4.5) solution. For Al binding at different pHs, cell walls extracted from roots pretreated at pH 3.5, 4.5 and 5.5 were subjected to a 0.5 mm CaCl2 solution (pH 4.5) containing 10 μm AlCl3. Other procedures were the same as described above.

Effect of NH4+ and other monovalent cations on Al-binding capacity in isolated intact root cell walls To examine the effect of NH4+ and other monovalent cations (i.e. Na+ and K+) on Al binding to the isolated intact root cell walls, roots of 3-day-old seedlings without N pretreatment were boiled in methanol for 5 min and then washed three times with fresh methanol (Ma et al. 2004b). The roots were then exposed to a 0.5 mm CaCl2 (pH 4.5) solution containing 0, 1 or 5 mm of NH4Cl, NaNO3, KCl or NaCl with or without 50 μm AlCl3. After 24 h, root tips (1 cm) were excised and subjected to extraction with 2 m HCl for 24 h with occasional shaking. The concentrations of Al in the solution were determined using a graphite furnace atomic absorption spectrophotometer (Varian GTA 110Z, Palo Alto, CA, USA).

Determination of cell wall polysaccharide content Fifteen milligrams of cell wall materials prepared as above were subjected to polysaccharide extraction. The pectin fraction was extracted twice with boiling water for 1 h each and

© 2014 John Wiley & Sons Ltd, Plant, Cell and Environment, 38, 1382–1390

1384

W. Wang et al.

the supernatants were pooled. The pellets were subsequently subjected to triple extractions with 4% KOH containing 0.1% NaBH4 at 25 °C for a total of 24 h, followed by similar extraction with 24% KOH containing 0.1% NaBH4. The pooled supernatants from 4% and 24% KOH extractions contained the hemicellulose fraction. The content of uronic acid in each cell wall fraction was determined according to the meta-hydroxydiphenyl method (Blumenkrantz & Asboe-Hansen 1973).

Fourier-transform infrared (FT-IR) microspectroscopic analysis Spectra of the root cell walls were obtained using a Nicolet 6700 spectrometer equipped with a Model 300 photoacoustic component (METC,Waltham, MA, USA). Each recorded spectrum was the average of 32 scans, with a spectral resolution of 2 cm−1 from 400 to 4000 cm−1. The speed of the moving mirror was 0.32 cm s−1, and a background spectrum was recorded before analysis. Spectra were analysed and fitted using OriginPro (version 8.5) software (OriginLab Corporation, Northampton, MA, USA) equipped with a peak-fitting module.

Zeta (ζ)-potential measurement

Figure 1. Effect of different N forms on cell wall Al binding in two rice cultivars. Rice cultivars ‘WYJ7’ (Al-tolerant) and ‘YD6’ (Al-sensitive) were exposed to 0 (control), 1 mm NH4Cl or 1 mm NaNO3 in 0.5 mm CaCl2 at pH 4.5. After 24 h, root tips (0–1 cm) were excised and subjected to cell wall extraction. An Al-binding assay was performed by incubating cell wall materials in a solution of 10 μm AlCl3 in 0.5 mm CaCl2 (pH 4.5) for 700 min. Data are means ± SE (n = 4). Different letters show significant differences (P < 0.05, Duncan’s multiple range test) among treatments.

Al competition for cell wall binding sites

To investigate the surface charge properties of cell walls at different pHs, 5 mg of cell wall materials, extracted from roots pretreated in 0.5 mm CaCl2 without N at pH 4.5, was suspended in a 0.5 mm CaCl2 solution at pH 4.5. The mixture was subsequently vortexed for several minutes and balanced on a reciprocating shaker at 200 r.p.m. and 25 °C for 24 h.The mixture was then filtered with 100 μm cell strainers, followed by centrifugation at 3000 g for 5 min. After the supernatant was discarded, the pellet was re-suspended in 0.5 mm CaCl2 at pH 3.5, 4.0, 4.5, 5.0 or 5.5 for 24 h. Final pH values were measured and re-adjusted to initial values. ζ-potential was measured with a NanoBrook Zeta Plus ζ-potential analyser (Brookhaven Instruments, Holtsville, NY, USA).

RESULTS Effects of NH4+ and NO3− treatments on Al binding to cell walls To investigate the effects of different N forms on Al-binding capacity in the cell walls in rice, the cell walls were extracted from root tips of two cultivars, Al-sensitive YD6 and Al-tolerant WYJ7, which had been exposed to NH4+, NO3− or no N (control) in a non-buffered solution. The pH was decreased to 3.56 from initial 4.50 in the presence of NH4+ (Supporting Information Table S1), while increased to 5.43 in the presence of NO3−. In the absence of N, the solution pH was changed from 4.50 to 4.72. An Al-binding assay revealed that the cell walls isolated from NH4+-treated roots of either cultivar had a lower Al-binding capacity, whereas those from NO3−-treated roots had a higher Al-binding capacity compared with the control (no N pretreatment; Fig. 1). Within a given treatment, Al-binding capacity of the cell wall was much higher in YD6 than in WYJ7 (Fig. 1).

To investigate competition for cell wall binding sites, we extracted cell walls from roots not pretreated with NH4+ or NO3−. After the cell walls were incubated with or without of NH4+ and NO3−, the Al-binding capacity was compared. Activity of Al in the different treatment solutions was similar (Supporting Information Table S2). Coexistence of Al and NH4+ or NO3− did not affect the cell wall Al-binding capacity in either cultivar (Fig. 2a). The Al-binding capacity was also not altered when the cell wall was first incubated by 1 mm NH4+ before Al treatment. These results indicated that neither NH4+ (NH4Cl) nor Na+ (NaNO3) competed with Al3+ for the binding sites of cell wall. Again, the cell walls of Al-tolerant rice cultivar WYJ7 displayed a lower Al-binding capacity than those of Al-sensitive YD6, at either N forms (Fig. 2a,b). Because Al is usually bound only to outer root cells (Ma et al. 2004b), the use of extracted cell walls may overestimate their Al-binding capacity. We therefore tested Al-binding capacity using isolated intact cell walls, which were prepared by boiling the whole roots in methanol. Al-binding capacity of isolated intact root cell wall was not altered by the presence of NH4+ or NO3− at low (1 mm) or high (5 mm) concentrations in either cultivar (Fig. 3). Presence of K+ and Na+ also did not affect the cell wall Al-binding capacity (Fig. 3). Consistently, a genotypic difference in Al-binding capacity was also observed in the isolated intact root cell walls; the Al-tolerant cultivar always exhibited lower Al-binding capacity than the Al-sensitive one (Fig. 3).

Effects of N forms on cell wall functional groups The effects of different N forms on cell wall functional groups involved in Al binding were examined by FT-IR


Ammonium affects Al binding of root cell wall

(a)

(b) +

Control

a 30

b

b

NO3

a

–

40

a

b

20

a Amount of AI adsorbed (mmol·g–1 cell wall)

NH4

40

Amount of AI adsorbed (mmol·g–1 cell wall)

1385

10

30

b

b

a

a

b

20

10

0

0 WYJ7

YD6

WYJ7

YD6

Figure 2. Competition assay for extracted cell wall binding sites. Cell walls were extracted from root tips (0–1 cm) of two rice cultivars (WYJ7, Al-tolerant and YD6, Al-sensitive) without N pretreatment. (a) Extracted cell walls were incubated with 10 μm AlCl3 in 0.5 mm CaCl2 (pH 4.5) in the absence or presence of 1 mm NH4Cl or NaNO3 for different durations. (b) Extracted cell walls were first incubated with or without 1 mm NH4Cl or NaNO3 for 24 h, followed by incubation in 0.5 mm CaCl2 (pH 4.5) containing 10 μm AlCl3 for 700 min. Data are means ± SE (n = 4). Different letters show significant differences (P < 0.05, Duncan’s multiple range test) among treatments.

microspectroscopy. Because strong hydrogen-bonded -OH bands in the range of 4000–2995 cm−1 were commonly observed in all spectra (Oh et al. 2005), the peak band at 3345 cm−1 was generally assigned to -OH (Synytsya et al. 2003). The quantity of these functional groups could be semiquantitatively reflected by the photoacoustic intensity, which is generated by converting the absorbed modulated

radiation to heat (Zhang et al. 2009). In both cultivars, the photoacoustic intensity of -OH bands was significantly lower in cell walls isolated from NH4+-treated roots than those from NO3−-treated roots (Fig. 4a). Photoacoustic intensities of the symmetric -COOH band situated at 1529 cm−1 and C-C and C-OH bands at 1051 cm−1, corresponding to pectin, hemicellulose and cellulose, respectively (Kacˇuráková et al. 2000; Toole et al. 2004; Mimmo et al. 2005). These bands, corresponding to pectin and hemicellulose, were also lower in NH4+-treated root cell walls than NO3−-treated ones in both cultivars (Fig. 4b). Bands corresponding to -OH, -COOH, C-C and C-OH were more intense in YD6 than those in WYJ7 (Fig. 4).

Effect of N forms on cell wall composition

Figure 3. Effect of NH4+ and other monovalent cations on Al binding to isolated intact cell walls in two rice cultivars. Isolated intact cell walls were prepared by boiling the roots in methanol. For the binding assay, the fixed roots were incubated with different solutions at pH 4.5 for 24 h and then the root tips (0–1 cm) were excised for analysis. Error bars indicate standard errors of three replicates. Different letters indicate significant differences (P < 0.05, Duncan’s multiple range test) among treatments.

To examine the effect of different N forms on cell wall composition, the cell walls were extracted from roots grown in a non pH-buffered or pH-buffered solution containing NH4+ or NO3− in the presence or absence of Al. Since the root elongations after all treatments were close to 1 cm, the cell wall materials extracted from the root tips were comparable between different treatments (Supporting Information Table S1). In a non-pH-buffered solution without Al, pH in the solution changed from 4.5 to 3.56 in the presence of NH4+ (Supporting Information Table S1), while increased to 5.43 in the presence of NO3−. Under such condition, NH4+ treatment resulted in lower pectin (Fig. 5a,b) and hemicellulose (Fig. 5c,d) contents in both cultivars compared with NO3− treatment. Exposure to Al significantly increased polysaccharide contents in both cultivars (Fig. 5b,d). Treatment with NO3− and Al increased pectin content in WYJ7 and hemicellulose content in YD6 (Fig. 5b,d), but no


1386

W. Wang et al.

difference in pectin and hemicellulose contents was observed between Al alone and Al and NH4+ treatments in either cultivar (Fig. 5b,d). In most treatments, YD6 exhibited higher pectin and hemicellulose contents than WYJ7 (Fig. 5). On the other hand, when roots were exposed to a pH-buffered solution, in which pH was hardly changed during treatment (Supporting Information Table S1), the differences in polysaccharide content between different N forms disappeared (Fig. 6). However, Al-induced increase of polysaccharide and genotypic differences in polysaccharide content were still observed (Fig. 6b,d).

(a)

(b)

(c)

(d)

(a)

Figure 5. Uronic acid content of cell wall fractions extracted

Photoacoustic intensity

from root tips of two rice cultivars grown in a non pH-buffered solution. Three-day-old seedlings of rice cultivars ‘WYJ7’ and ‘YD6’ were exposed to 0 (control), 1 mm NH4+ or 1 mm NO3− in a 0.5 mm CaCl2 solution without (a, c) or with 50 μm Al (b, d) at pH 4.5 for 24 h. Root apices (0–1 cm) were excised, and cell wall polysaccharides were fractionated into pectin (a, b) and hemicellulose (c, d) portions for uronic acid content measurement. Error bars indicate standard errors of three replicates. All data were subjected to a three-way analysis of variance (anova) after testing the normality and homogeneity, C: cultivars (i.e. WYJ7 and YD6); T, treatment (i.e. -Al or +Al ); N, nitrogen treatment(i.e. NH4+, none and NO3−); **P < 0.01. No significant difference between C × T, N × C, T × C and C × T × N interaction effects on pectin and HC contents. Boxes with different letters show significant differences (P < 0.05, Duncan’s multiple range test) among treatments.

Effect of pH on cell wall composition

Wavenumber (cm–1)

To confirm that alteration of cell wall composition observed above results from different pH due to uptake of ammonium, the roots were exposed to different pHs in the absence of N. The content of both pectin and hemicellulose was lower at pH 3.5 than at pH 5.5 in both cultivars (Fig. 7a,b). Consistent with these results, the Al-binding capacity of the cell wall was also lower at pH 3.5 than at pH 5.5 in either cultivar (Fig. 7c). The Al-sensitive cultivar showed a higher Al binding of the cell wall than the Al-tolerant cultivar at either pH (Fig. 7c).

Photoacoustic intensity

(b)

Effects of pH on ζ-potential of root cell walls The effect of different pH on ζ-potential was investigated. In both rice cultivars, the cell wall ζ-potential became more negative at higher pH (Fig. 8). At each pH level, the ζ-potential was more negative in cell walls of YD6 than in WYJ7 (Fig. 8).

DISCUSSION

Wavenumber (cm–1)

Figure 4. Fourier-transform infrared (FT-IR) microspectroscopy of cell walls. The two rice cultivars ‘WYJ7’ and ‘YD6’ were treated with 0, 1 mm NH4Cl or 1 mm NaNO3 in 0.5 mm CaCl2 at pH 4.5 for 24 h. Cell walls were then extracted for scanning of -OH (a) and -COO− (b) by FT-IR microspectroscopy.

The ammonium-alleviated Al toxicity in rice has been attributed to the decreased Al accumulation in roots (Zhao et al. 2009). In the present study, we found that this effect of ammonium is associated with alterations of root cell wall properties.


Ammonium affects Al binding of root cell wall

NH4+ does not compete with Al3+ for cell wall binding sites The ammonium-induced decrease of Al accumulation in the roots was suggested to be direct competition between NH4+ and Al for the binding sites (Chen et al. 2010). To test this, we performed a competition experiment using extracted and isolated intact cell wall. However, either coexistence of ammonium and Al or pretreatment with ammonium did not affect the Al binding of the root cell walls (Figs 2 and 3), indicating that the direct competition between NH4+ and Al3+ for cell wall binding sites did not occur. This result was different from results observed in dicots. In root cell walls of Lespedeza bicolor and soybean, NH4+ was able to compete with Al3+ (Klotz & Horst 1988; Chen et al. 2010). This inconsistency may be attributed to cell wall components among plant species. It is well known that the compositions of cell wall in graminaceous monocots (i.e. cereals), belongs to typeII, which was different from those of dicots and nongraminaceous monocot species with type-I cell walls (Jarvis et al. 1988; Carpita 1996). Type-II cell walls are characterized by less pectin material and high hemicellulose glucuronoarabinoxylans that cross-link with cellulose. Type-I cell walls, in contrast, are enriched in pectin and the high hemicellulose xyloglucan (Carpita & Gibeaut 1993; Fleischer et al.

(a)

1387

(a)

(b)

(c)

(b)

Figure 7. Effects of pH on uronic acid content and Al binding of

(c)

(d)

Figure 6. Uronic acid content of cell wall fractions extracted from root tips of two cultivars grown in a pH-buffered solution. Three-day-old seedlings of cultivars ‘WYJ7’ and ‘YD6’ were exposed to 0 (control), 1 mm NH4+ or 1 mm NO3− in 0.5 mm CaCl2 solution (pH 4.5) buffered with 5 mm Homo-pipes in the absence (a, c) or presence of 50 μm Al (b, d) for 24 h. Root apices (0–1 cm) were excised, and cell wall polysaccharides were fractionated into pectin (a, b) and hemicellulose (c, d) portions for uronic acid content measurement. Error bars indicate standard errors of three replicates. The statistics analysis was performed as described in Fig. 5. C, cultivars (i.e. WYJ7 and YD6); T, treatment (i.e. -Al or +Al); N, nitrogen forms (i.e. NH4+, none and NO3−); ns, non-significant; **P < 0.01. No significant difference between C × T, N × C, T × C and C × T × N interaction effects on pectin and HC contents. Boxes with different letters show significant differences (P < 0.05, Duncan’s multiple range test) among treatments.

cell wall fractions extracted from root tips of two rice cultivars. Three-day-old seedlings of cultivars ‘WYJ7’ and ‘YD6’ were exposed to pH 3.5, 4.5 and 5.5 in a 0.5 mm CaCl2 solution buffered with 5 mm Homo-pipes in the absence Al for 24 h. Root apices (0–1 cm) were excised, and cell wall polysaccharides were fractionated into pectin (a) and hemicellulose (b) portions for uronic acid content measurement. An Al-binding assay was also performed by incubating cell wall materials in a 0.5 mM CaCl2 (pH 4.5) containing 10 μm AlCl3 for 700 min (c). Data are means ± SE (n-4) for Al-binding capacity. The statistics analysis was performed as described in Fig. 5. C, cultivars (i.e. WYJ7 and YD6); pH, pH treatment (i.e. 3.5, 4.5 or 5.5); **P < 0.01. No significant difference between pH × C interaction effects on pectin and HC contents. Boxes with different letters show significant differences (P < 0.05, Duncan’s multiple range test) among treatments.

1999). It seems that NH4+ and Al3+ may interact differently in monocots and dicots because of the different cell wall compositions.

Cell wall properties are altered by different N forms Pectin and hemicellulose were decreased when plants were grown in the presence of NH4+, but increased with NO3− (Fig. 5a,c). Negative charged functional groups (e.g.


1388

W. Wang et al. that the altered cell wall composition was attributed to pH changes caused by uptake of the different N forms.

Cell wall electrical charge is also altered by pH

Figure 8. Cell wall zeta (ζ)-potential of two rice cultivars differing in Al tolerance. Extracted cell walls were incubated with 0.5 mm CaCl2 for 24 h at different pH values, followed by measurement of ζ-potential using a NanoBrook ZetaPlus analyser. The line was drawn by best fit according to the function of Boltzmann. All data were subjected to a two-way analysis of variance (anova) after testing the normality and homogeneity, C, cultivars (i.e. WYJ7 and YD6); pH, pH treatment (i.e. 3.5, 4.0, 4.5 or 5.5); **P < 0.01. No significant difference between pH × C interaction effects on ζ-potential. Least significant difference (LSD; one way anova, P < 0.05) is 2.51 mv.

COO- and -OH) in these polysaccharides possess a particularly high capacity for binding positively charged Al3+ (Blamey et al. 1990; Chang et al. 1999; Yang et al. 2011; Zhu et al. 2012). FT-IR analysis revealed that cultivation with NH4+ decreased, while that with NO3− increased the number of -OH and COO- functional groups in the cell walls (Fig. 4). These changes in the cell wall functional groups were consistent with the content of pectin and hemicellulose (Fig. 5a,c). These results suggested that ammonium-reduced aluminium accumulation in the cell wall might be caused by the alteration of cell wall composition as well as functional groups. These observed changes in the cell wall properties may result from pH changes caused by uptake of the different N forms. Under our experimental conditions, the solution pH was drastically reduced in the presence of NH4+, from initial 4.5 to around 3.6, whereas in the presence of NO3−, it was significantly raised to around 5.2 (Supporting Information Table S1). These pH changes during the treatments may be a trigger for altering the cell wall properties (Fig. 7a,b). This is supported by that when roots were grown in a pH-buffered solution, the cell wall composition was not altered by different N forms (Fig. 6) and by that lowering pH alone also resulted in lower pectin and hemicellulose and less Al accumulation in the cell walls (Fig. 7c). Consistent with our results, Zhao et al. (2009) also found that the ammoniumalleviated effect of Al accumulation was hardly observed in a pH-buffered solution. It is known that pH level is crucial to the activities of expansin and pectinase, which play roles in cell wall extension (Cosgrove 1999, 2000). It was reported that degradation of cell wall matrix polymers such as xyloglucan and pectic polymers was enhanced at low pH (Jacobs & Ray 1975; Bates & Ray 1981).These results suggest

Changes of pH caused by uptake of different N forms may also affect the electrical charge of cell walls. We therefore compared ζ-potential between different pHs. ζ-potential represents the potential difference between a dispersion medium and the stationary layer of fluid attached to a dispersed particle (Wang et al. 2008). Therefore, it could reflect the charge on the cell wall. ζ-potential in both Al-sensitive and Al-tolerant cultivars increased with increasing pHs (Fig. 8). This result was consistent with the results measured by microelectrode (Shomer et al. 2003). This was also in agreement with our observation that cell wall surfaces were more negatively charged because of increased generation of -COO− and -O− functional groups in pectin and hemicellulose after treatment with NO3− (Fig. 4). The ζ-potentials in cell wall surface of WYJ7 are significantly lower than that of YD6 (Fig. 4), which might be caused by less pectin and hemicellulose in WYJ7 than YD6 (Figs 5–7).

Al-binding capacity of cell wall is probably responsible for the genotypic difference in Al tolerance Rice (O. sativa) is the most Al-tolerant species among small grain cereal crops (Ma et al. 2002). Generally, japonica varieties showed higher Al tolerance than indica varieties (Ma et al. 2002; Famoso et al. 2011; Zhao et al. 2013). Several genes involved in Al tolerance have recently been identified in rice (Huang et al. 2009, 2012; Xia et al. 2010, 2013; Yokosho et al. 2011; Chen et al. 2012). Some of these genes, such as OsFRDL4, are associated with genotypic differences in Al tolerance (Yokosho et al. 2011), whereas others, such as STAR1/2, are required for basic detoxification of Al (Huang et al. 2009). In the present study, the Al-tolerant cultivar WYJ7 (japonica) showed lower Al-binding capacity, lower pectin and hemicellulose contents, and a lower number of functional groups than the Al-sensitive cultivar YD6 (indica) (Figs 2–5 and 7). Since the differences in Al binding of cell wall were observed at different pHs (Fig. 7), it seems that the basic cell wall composition (pectin and hemicellulose) is responsible for the genotypic differences in Al binding, but the mechanisms underlying this genotypic difference in cell wall composition remain to be examined in future. On the other hand, Al notably increased the content of pectin and hemicellulose in either cultivar (Fig. 5). This phenomenon was also found in many other studies (Chang et al. 1999; Tabuchi & Matsumoto 2001; Tabuchi et al. 2004; Yang et al. 2008). One possible explanation was that Al binding to pectin and hemicellulose modifies the degradation of polysaccharide metabolism, which is a prerequisite for auxininduced cell elongation (Van et al. 1994), resulting in accumulation of Al-polysaccharide (Chang et al. 1999; Tabuchi & Matsumoto 2001; Tabuchi et al. 2004).


Ammonium affects Al binding of root cell wall In conclusion, our results show that NH4+-decreased Al accumulation was due to altered cell wall properties, rather than direct competition between NH4+ and Al3+. Furthermore, we found that these cell wall alterations were caused by pH decrease arising from uptake of NH4+.

ACKNOWLEDGMENTS This work was supported by the National Key Basic Research Program of China (No. 2014CB441000), ‘Strategic Priority Research Program’ of the Chinese Academy of Sciences (Nos. XDB15030302 and XDB15030202) and the Natural Science Foundation of China (No. 41025005).

REFERENCES Bates G.W. & Ray P.M. (1981) pH-dependent interactions between pea cell wall polymers possibly involved in wall deposition and growth. Plant Physiology 68, 158–164. Blamey F.P.C., Edmeades D.C. & Wheeler D.M. (1990) Role of root cationexchange capacity in differential aluminum tolerance of lotus species. Journal of Plant Nutrition 13, 729–744. Blumenkrantz N. & Asboe-Hansen G. (1973) New method for quantitative determination of uronic acids. Analytical Biochemistry 54, 484–489. Carpita N.C. (1996) Structure and biogenesis of the cell walls of grasses. Annual Review of Plant Physiology and Plant Molecular Biology 47, 445– 476. Carpita N.C. & Gibeaut D.M. (1993) Structural models of primary cell walls in flowering plants: consistency of molecular structure with the physical properties of the walls during growth. The Plant Journal 3, 1–30. Chang Y.C., Yamamoto Y. & Matsumoto H. (1999) Accumulation of aluminium in the cell wall pectin in cultured tobacco (Nicotiana tabacum L.) cells treated with a combination of aluminium and iron. Plant, Cell & Environment 22, 1009–1017. Chen Z.C., Zhao X.Q. & Shen R.F. (2010) The alleviating effect of ammonium on aluminum toxicity in Lespedeza bicolor results in decreased aluminuminduced malate secretion from roots compared with nitrate. Plant and Soil 337, 389–398. Chen Z.C., Yamaji N., Motoyama R., Nagamura Y. & Ma J.F. (2012) Up-regulation of a magnesium transporter gene OsMGT1 is required for conferring aluminum tolerance in rice. Plant Physiology 159, 1624–1633. Clarkson D. (1967) Interactions between aluminium and phosphorus on root surfaces and cell wall material. Plant and Soil 27, 347–356. Cosgrove D.J. (1999) Enzymes and other agents that enhance cell wall extensibility. Annual Review of Plant Physiology and Plant Molecular Biology 50, 391–417. Cosgrove D.J. (2000) Loosening of plant cell walls by expansins. Nature 407, 321–326. Cumming J.R. (1990) Nitrogen source effects on Al toxicity in nonmycorrhizal and mycorrhizal pitch pine (Pinus rigida) seedlings. II. Nitrate reduction and NO3− uptake. Canadian Journal of Botany 68, 2653–2659. Cumming J.R. & Weinstein L.H. (1990) Nitrogen source effects on Al toxicity in nonmycorrhizal and mycorrhizal pitch pine (Pinus rigida) seedlings. I. Growth and nutrition. Canadian Journal of Botany 68, 2644–2652. De Boer W. & Kowalchuk G.A. (2001) Nitrification in acid soils: microorganisms and mechanisms. Soil Biology and Biochemistry 33, 853–866. Delhaize E., Ryan P.R. & Randall P.J. (1993) Aluminum tolerance in wheat (Triticum aestivum L.) (II. aluminum-stimulated excretion of malic acid from root apices). Plant Physiology 103, 695–702. Delhaize E., Ma J.F. & Ryan P.R. (2012) Transcriptional regulation of aluminium tolerance genes. Trends in Plant Science 17, 341–348. Famoso A.N., Zhao K.Y., Clark R.T., Tung C.W., Wright M.H., Bustamante C. & McCouch S.R. (2011) Genetic architecture of aluminum tolerance in rice (Oryza sativa) determined through genome-wide association analysis and QTL mapping. PLoS Genetics 7, e1002221. Fleischer A., O’Neill M.A. & Ehwald R. (1999) The pore size of nongraminaceous plant cell walls is rapidly decreased by borate ester crosslinking of the pectic polysaccharide rhamnogalacturonan II. Plant Physiology 121, 829–838.

1389

Fleming A.L. (1983) Ammonium uptake by wheat varieties differing in Al tolerance. Agronomy Journal 75, 726–730. Furukawa J., Yamaji N., Wang H., Mitani N., Murata Y., Sato K. & Ma J.F. (2007) An aluminum-activated citrate transporter in barley. Plant & Cell Physiology 48, 1081–1091. Grauer U.E. & Horst W.J. (1990) Effect of pH and nitrogen source on aluminium tolerance of rye (Secale cereale L.) and yellow lupin (Lupinus luteus L. Plant and Soil 127, 13–21. Guo J.H., Liu X.J., Zhang Y., Shen J.L., Han W.X., Zhang W.F. & Zhang F.S. (2010) Significant acidification in major Chinese croplands. Science 327, 1008–1010. Horst W.J., Wang Y.X. & Eticha D. (2010) The role of the root apoplast in aluminium-induced inhibition of root elongation and in aluminium resistance of plants: a review. Annals of Botany 106, 185–197. Huang C.F., Yamaji N., Mitani N., Yano M., Nagamura Y. & Ma J.F. (2009) A bacterial-type ABC transporter is involved in aluminum tolerance in rice. The Plant Cell 21, 655–667. Huang C.F., Yamaji N., Chen Z.C. & Ma J.F. (2012) A tonoplast-localized half-size ABC transporter is required for internal detoxification of aluminum in rice. The Plant Journal 69, 857–867. Jacobs M. & Ray P.M. (1975) Promotion of xyloglucan metabolism by acid pH. Plant Physiology 56, 373–376. Jarvis M.C., Forsyth W. & Duncan H.J. (1988) A survey of the pectic content of nonlignified monocot cell walls. Plant Physiology 88, 309–314. Kacˇuráková M., Capek P., Sasinková V., Wellner N. & Ebringerová A. (2000) FT-IR study of plant cell wall model compounds: pectic polysaccharides and hemicelluloses. Carbohydrate Polymers 43, 195–203. Kerven G., Edwards D., Asher C., Hallman P. & Kokot S. (1989) Aluminum determination in soil solution. II. Short-term colorimetric procedures for the measurement of inorganic monomeric aluminum in the presence of organic acid ligands. Soil Research 27, 91–102. Klotz F. & Horst W.J. (1988) Effect of ammonium- and nitrate-nitrogen nutrition on aluminium tolerance of soybean (Glycine max L.). Plant and Soil 111, 59–65. Kochian L.V. (1995) Cellular mechanisms of aluminum toxicity and resistance in plants. Annual Review of Plant Physiology and Plant Molecular Biology 46, 237–260. Kochian L.V., Piñeros M.A. & Hoekenga O.A. (2005) The physiology, genetics and molecular biology of plant aluminum resistance and toxicity. Plant and Soil 274, 175–195. Ma J.F., Zheng S.J., Matsumoto H. & Hiradate S. (1997) Detoxifying aluminium with buckwheat. Nature 390, 569–570. Ma J.F., Shen R.F., Zhao Z.Q., Wissuwa M., Takeuchi Y., Ebitani T. & Yano M. (2002) Response of rice to Al stress and identification of quantitative trait loci for Al tolerance. Plant & Cell Physiology 43, 652–659. Ma J.F., Nagao S., Sato K., Ito H., Furukawa J. & Takeda K. (2004a) Molecular mapping of a gene responsible for Al-activated secretion of citrate in barley. Journal of Experimental Botany 55, 1335–1341. Ma J.F., Shen R.F., Nagao S. & Tanimoto E. (2004b) Aluminum targets elongating cells by reducing cell wall extensibility in wheat roots. Plant & Cell Physiology 45, 583–589. Ma J.F., Chen Z.C. & Shen R.F. (2014) Molecular mechanisms of Al tolerance in gramineous plants. Plant and Soil 381, 1–12. McCain S. & Davies M. (1983) The influence of background solution on root responses to aluminium in Holcus lanatus L. Plant and Soil 73, 425–430. Magalhaes J.V., Liu J.P., Guimaraes C.T., Lana U.G.P., Alves V.M.C., Wang Y.H. & Kochian L.V. (2007) A gene in the multidrug and toxic compound extrusion (MATE) family confers aluminum tolerance in sorghum. Nature Genetics 39, 1156–1161. Marschner H. & Römheld V. (1983) In vivo measurement of root-induced pH changes at the soil-root interface: effect of plant species and nitrogen source. Zeitschrift für Pflanzenphysiologie 111, 241–251. Mimmo T., Marzadori C., Montecchio D. & Gessa C. (2005) Characterisation of Ca- and Al-pectate gels by thermal analysis and FT-IR spectroscopy. Carbohydrate Research 340, 2510–2519. Moorby H., Nye P.H. & White R.E. (1985) The influence of nitrate nutrition on H+ efflux by young rape plants (Brassica napus cv. emerald). Plant and Soil 84, 403–415. Oh S.Y., Yoo D.I., Shin Y. & Seo G. (2005) FTIR analysis of cellulose treated with sodium hydroxide and carbon dioxide. Carbohydrate Research 340, 417–428. Rangel A.F., Rao I.M. & Horst W.J. (2009) Intracellular distribution and binding state of aluminum in root apices of two common bean (Phaseolus


1390 W. Wang et al. vulgaris) genotypes in relation to Al toxicity. Physiologia Plantarum 135, 162–173. Ryan P.R., Tyerman S.D., Sasaki T., Furuichi T., Yamamoto Y., Zhang W.H. & Delhaize E. (2011) The identification of aluminium-resistance genes provides opportunities for enhancing crop production on acid soils. Journal of Experimental Botany 62, 9–20. Sasaki T., Yamamoto Y., Ezaki B., Katsuhara M., Ahn S.J., Ryan P.R. & Matsumoto H. (2004) A wheat gene encoding an aluminum-activated malate transporter. The Plant Journal 37, 645–653. Schroder J.L., Zhang H.L., Girma K., Raun W.R., Penn C.J. & Payton M.E. (2011) Soil acidification from long-term use of nitrogen fertilizers on winter wheat. Soil Science Society of America Journal 75, 957–964. Schubert S. & Yan F. (1997) Nitrate and ammonium nutrition of plants: effects on acid/base balance and adaptation of root cell plasmalemma H+-ATPase. Zeitschrift für Pflanzenernährung und Bodenkunde 160, 275–281. Shomer I., Novacky A.J., Pike S.M., Yermiyahu U. & Kinraide T.B. (2003) Electrical potentials of plant cell walls in response to the ionic environment. Plant Physiology 133, 411–422. ˇ opíková J., Mateˇjka P. & Machovicˇ V. (2003) Fourier transform Synytsya A., C Raman and infrared spectroscopy of pectins. Carbohydrate Polymers 54, 97–106. Tabuchi A. & Matsumoto H. (2001) Changes in cell-wall properties of wheat (Triticum aestivum) roots during aluminum-induced growth inhibition. Physiologia Plantarum 112, 353–358. Tabuchi A., Kikui S. & Matsumoto H. (2004) Differential effects of aluminium on osmotic potential and sugar accumulation in the root cells of Al-resistant and Al-sensitive wheat. Physiologia Plantarum 120, 106–112. Tan K.Z., Keltjens W.G. & Findenegg G.R. (1992) Effect of nitrogen form on aluminum toxicity in sorghum genotypes. Journal of Plant Nutrition 15, 1383–1394. Taylor G.J. & Foy C.D. (1985a) Mechanisms of aluminum tolerance in Triticum aestivum (wheat). IV. The role of ammonium and nitrate nutrition. Canadian Journal of Botany 63, 2181–2186. Taylor G.J. & Foy C.D. (1985b) Mechanisms of aluminum tolerance in Triticum aestivum L. (wheat). I. Differential pH induced by winter cultivars in nutrient solutions. American Journal of Botany 72, 695–701. Taylor G.J. & Foy C.D. (1985c) Mechanisms of aluminum tolerance in Triticum aestivum L. (wheat). II. Differential pH induced by spring cultivars in nutrient solutions. American Journal of Botany 72, 702–706. Toole G.A., Kacˇuráková M., Smith A.C., Waldron K.W. & Wilson R.H. (2004) FT-IR study of the Chara corallina cell wall under deformation. Carbohydrate Research 339, 629–635. Tsuji M., Kuboi T. & Konishi S. (1994) Stimulatory effects of aluminum on the growth of cultured roots of tea. Soil Science and Plant Nutrition 40, 471–476. Uexküll H.R. & Mutert E. (1995) Global extent, development and economic impact of acid soils. Plant and Soil 171, 1–15. Van H.L., Kuraishi S. & Sakurai N. (1994) Aluminum-induced rapid root inhibition and changes in cell-wall components of squash seedlings. Plant Physiology 106, 971–976. Wang P., Zhou D.M., Kinraide T.B., Luo X.S., Li L.Z., Li D.D. & Zhang H.L. (2008) Cell membrane surface potential (ψ0) plays a dominant role in the phytotoxicity of copper and arsenate. Plant Physiology 148, 2134–2143. Xia J.X., Yamaji N., Kasai T. & Ma J.F. (2010) Plasma membrane-localized transporter for aluminum in rice. Proceedings of the National Academy of Sciences of the United States of America 107, 18381–18385.

Xia J.X., Yamaji N. & Ma J.F. (2013) A plasma membrane-localized small peptide is involved in rice Al tolerance. The Plant Journal 76, 345– 355. Yamaji N., Huang C.F., Nagao S., Yano M., Sato Y., Nagamura Y. & Ma J.F. (2009) A zinc finger transcription factor ART1 regulates multiple genes implicated in aluminum tolerance in rice. The Plant Cell 21, 3339–3349. Yang J.L., Li Y.Y., Zhang Y.J., Zhang S.S., Wu Y.R., Wu P. & Zheng S.J. (2008) Cell wall polysaccharides are specifically involved in the exclusion of aluminum from the rice root apex. Plant Physiology 146, 602–611. Yang J.L., Zhu X.F., Peng Y.X., Zheng C., Li G.X., Liu Y., . . . Zheng S.J. (2011) Cell wall hemicellulose contributes significantly to aluminum adsorption and root growth in Arabidopsis. Plant Physiology 155, 1885–1892. Yokosho K., Yamaji N. & Ma J.F. (2011) An Al-inducible MATE gene is involved in external detoxification of Al in rice. The Plant Journal 68, 1061– 1069. Zhang W.R., Lowe C. & Smith R. (2009) Depth profiling of coil coating using step-scan photoacoustic FTIR. Progress in Organic Coatings 65, 469–476. Zhao X.Q. & Shen R.F. (2013) Interactive regulation of nitrogen and aluminum in rice. Plant Signaling & Behavior 8, e24355. Zhao X.Q., Shen R.F. & Sun Q.B. (2009) Ammonium under solution culture alleviates aluminum toxicity in rice and reduces aluminum accumulation in roots compared with nitrate. Plant and Soil 315, 107–121. Zhao X.Q., Guo S.W., Shinmachi F., Sunairi M., Noguchi A., Hasegawa I. & Shen R.F. (2013) Aluminium tolerance in rice is antagonistic with nitrate preference and synergistic with ammonium preference. Annals of Botany 111, 69–77. Zhao Z.Q., Ma J.F., Sato K. & Takeda K. (2003) Differential Al resistance and citrate secretion in barley (Hordeum vulgare L.). Planta 217, 794–800. Zheng S.J., Lin X.Y., Yang J.L., Liu Q. & Tang C.X. (2004) The kinetics of aluminum adsorption and desorption by root cell walls of an aluminum resistant wheat (Triticum aestivum L.) cultivar. Plant and Soil 261, 85–90. Zhong H.L. & Lauchli A. (1993) Changes of cell wall composition and polymer size in primary roots of cotton seedlings under high salinity. Journal of Experimental Botany 44, 773–778. Zhu X.F., Shi Y.Z., Lei G.J., Fry S.C., Zhang B.C., Zhou Y.H., . . . Zheng S.J. (2012) XTH31, encoding an in vitro XEH/XET-active enzyme, regulates aluminum sensitivity by modulating in vivo XET action, cell wall xyloglucan content, and aluminum binding capacity in Arabidopsis. The Plant Cell 24, 4731–4747.

Received 17 January 2014; received in revised form 13 November 2014; accepted for publication 18 November 2014

SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Table S1. Changes of solution pH and root elongation after treatment of N or pH. Table S2. Ionic activities of Al species, H+, NH4+, Na+, K+ and Ca2+ in solutions used in the Al-binding assay shown in Figs 1–3.
