Inhibition of nitric oxide synthase (NOS) underlies aluminum-induced inhibition of root elongation in Hibiscus moscheutos

Authors

  • Qiu-Ying Tian,

    1. Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, the Chinese Academy of Sciences, Beijing 100093, People's Republic of China
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  • Dong-Hua Sun,

    1. Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, the Chinese Academy of Sciences, Beijing 100093, People's Republic of China
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  • Min-Gui Zhao,

    1. Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, the Chinese Academy of Sciences, Beijing 100093, People's Republic of China
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  • Wen-Hao Zhang

    1. Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, the Chinese Academy of Sciences, Beijing 100093, People's Republic of China
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Author for correspondence: Wen-Hao Zhang Tel: +86 10 6283 6697 Fax: +86 10 6259 2430 Email: whzhang@ibcas.ac.cn

Summary

  • • Aluminum (Al) is toxic to plants when solubilized into Al3+ in acidic soils, and becomes a major factor limiting plant growth. However, the primary cause for Al toxicity remains unknown.
  • • Nitric oxide (NO) is an important signaling molecule modulating numerous physiological processes in plants. Here, we investigated the role of NO in Al toxicity to Hibiscus moscheutos.
  • • Exposure of H. moscheutos to Al3+ led to a rapid inhibition of root elongation, and the inhibitory effect was alleviated by NO donor sodium nitroprusside (SNP). NO scavenger and inhibitors of NO synthase (NOS) and nitrate reductase had a similar inhibitory effect on root elongation. The inhibition of root elongation by these treatments was ameliorated by SNP.
  • • Aluminum inhibited activity of NOS and reduced endogenous NO concentrations. The alleviation of inhibition of root elongation induced by Al, NO scavenger and NOS inhibitor was correlated with endogenous NO concentrations in root apical cells, suggesting that reduction of endogenous NO concentrations resulting from inhibition of NOS activity could underpin Al-induced arrest of root elongation in H. moscheutos.

Introduction

Aluminum (Al) is the most abundant metal and the third most abundant chemical element in the earth's crust. Al becomes a major factor limiting crop growth and yield when solubilized in acid soils to phytotoxic species Al3+ (Kochian, 1995). Inhibition of root elongation is one of the earliest and most distinct symptoms of Al toxicity, which can be observed within hours or even minutes of exposure of roots to toxic Al3+ (Ryan et al., 1993; Jones et al., 1995; Zhang & Rengel, 1999). Al-induced inhibition of root elongation requires root apex to be exposed to Al (Ryan et al., 1993; Sivaguru & Horst, 1998; Kollmeier et al., 2000), suggesting that the root apex is a critical site of perception and expression of Al toxicity and resistance. Although it has been shown that Al can alter a myriad of physiological processes, ranging from distorting cytoskeleton to disrupting cytosolic Ca2+-dependent signaling cascades, the primary mechanism underlying the Al phytotoxicity remains largely unknown (Delhaize & Ryan, 1995; Horst, 1995; Kochian, 1995; Matsumoto, 2000; Barcelo & Poschenrieder, 2002; Rengel & Zhang, 2003).

Nitric oxide (NO) has emerged as an important signaling molecule that modulates numerous physiological processes in plants (Lamattina et al., 2003; Neill et al., 2003; Crawford & Guo, 2005), including regulation of root growth and development. For instance, NO promotes root growth and mediates IAA-induced adventitious rooting in maize (Gouvêa et al., 1997; Pagnussat et al., 2003). NO is also implicated in lateral root formation by modulating the expression of cell cycle regulatory genes in tomato (Correa-Aragunde et al., 2004, 2006). Furthermore, responses and adaptations of plants to several abiotic stresses have been shown to be associated with NO. This includes drought (Mata & Lamattina, 2001), salt (Zhao et al., 2004; Zhang et al., 2006a), heat (Neill et al., 2003) and heavy metal stresses (Hsu & Kao, 2004; Rodríguez-Serrano et al., 2006).

There are several potential pathways for generating endogenous NO in plants and it seems likely that the contribution of each pathway to the overall NO production is dependent upon species, developmental stages and environment in which plants are grown (reviewed by Neill et al., 2003). Nitric oxide synthase (NOS) and nitrate reductase (NR) are two key enzymes for NO synthesis in plants (Neill et al., 2003). In mammals, NO production is mainly mediated by NOS, which catalyzes the conversion of l-arginine to l-citrulline and NO (Furchgott, 1995). Mammalian NOS inhibitors inhibit NO production in response to various stimuli in plants (Lamattina et al., 2003; Neill et al., 2003), suggesting that an arginine-dependent NOS activity may also occur in plants. A gene (NOS1) has recently been isolated from Arabidopsis, which encodes a protein with sequence similarity to a protein that mediates NO synthesis in the snail Helix pomatia (Guo et al., 2003). The NOS1-mediated in vivo NO synthesis is involved in hormonal signaling (Guo et al., 2003), stomatal movement (Guo et al., 2003), flowering (He et al., 2004), pathogen defense (Zeidler et al., 2004), senescence (Guo & Crawford, 2005) and oxidative stress (Zhao et al., 2007). In addition to NOS-mediated NO generation, biosynthesis of NO in plants can also occur through NR-mediated reduction of nitrite under certain conditions (Klepper, 1990; Yamasaki et al., 1999). There is evidence to show that NR-mediated NO production is involved in pathogen attack (Molodo et al., 2005) and ABA-induced stomatal closure (Desikan et al., 2002; Bright et al., 2006).

The involvement of NO in responses of plants to abiotic stress is often inferred from results of NO donor and scavenger studies as well as from monitoring changes in endogenous NO concentrations under stressed conditions (Kopyra & Gwozdz, 2003; Zhao et al., 2004; Wang & Yang, 2005; Zhang et al., 2006b). In this context, it has been shown that NO donors mitigate and NO scavengers exaggerate syndromes associated with Al toxicity (Wang & Yang, 2005), cadmium toxicity (Kopyra & Gwozdz, 2003; Hsu & Kao, 2004) and salt stress (Zhao et al., 2004; Zhang et al., 2006a). The ameliorating effect of NO on plants suffering from abiotic stresses has been attributed to up-regulation of ATPases (Zhao et al., 2004; Zhang et al., 2006b) and protecting plants from oxidative stress (Kopyra & Gwozdz, 2003; Hsu & Kao, 2004; Zhao et al., 2004; Wang & Yang, 2005; Yu et al., 2005; Rodríguez-Serrano et al., 2006). For instance, Wang & Yang (2005) reported that exogenous NO recovers Al-induced inhibition of root elongation in Cassia tora by acting as an antioxidant to protect the plant against Al-induced oxidative stress. However, the authors did not explore whether the Al-induced inhibition of root elongation is associated with alterations of endogenous NO concentrations in root apical cells, which are critical sites of Al toxicity. Moreover, the origin of NO responsible for potential changes in endogenous NO concentrations in response to toxic Al3+ also remains unknown. To elucidate the role of NO in Al toxicity to Hibiscus moscheutos, an herbaceous perennial horticultural plant, we investigated effects of Al on endogenous NO concentrations, activities of NOS and NR and compared the effects with those of known NOS inhibitors, NO donors and NO scavengers.

Materials and Methods

Plant materials

Hibiscus moscheutos L. seeds were sterilized in 5% (v/v) sodium hypochlorite solution for 5 min, and then rinsed three times with deionized water. Seeds germinated on filter paper were grown in aerated 1/4 Hoagland's solution in a glasshouse under conditions of a 14 h light : 10 h dark cycle and a temperature of 23°C. The seedlings were incubated in solutions containing different chemicals with basal composition of 0.5 mm CaCl2, pH 4.5. During the pretreatment culture, the nutrient solution was changed every 2 d. Six seedlings were transferred to 250 ml glass chambers containing 0.5 mm CaCl2 (pH 4.5), and AlCl3 or sodium nitroprusside (SNP) was added to chosen concentrations. Root elongation was measured after 24 h incubation in the presence of various chemicals.

Measurements of root elongation

Four-day-old seedlings were incubated in control solution (0.5 mm CaCl2, pH 4.5) or treatment solutions containing the following chemicals (µm): 100 AlCl3, 100 SNP, 100 butylated hydroxyanisole (BHA); or 50 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) for 24 h. In order to avoid chelating Al3+ with SNP, we pretreated the seedlings with either AlCl3 or SNP for 12 h and thereafter incubated the seedlings in solutions containing either SNP or AlCl3 for another 12 h. To test whether the Al-induced inhibition of root elongation is related to endogenous NO, the effects of a NO scavenger (cPTIO), a NOS inhibitor Nù-nitro-L-arginine (L-NNA) and a NR inhibitor (tungstate) on root elongation were also investigated. All experiments were repeated at least three times.

To examine the short-term effect of AlCl3 and SNP on root elongation, H. moscheutos seedlings were incubated in 1/4 Hoagland's solution for 2 d and then transferred into Petri dishes with solutions containing either AlCl3 or SNP for varying time periods (1, 2, 4, 6, 8, 12, 24 h) in the basal solution of 0.5 mm CaCl2 (pH 4.5). Elongation of the primary root was measured after exposing the roots to different chemicals for varying time periods under an Olympus microscope. Values were given as means ± SE of at least eight independent measurements.

Determination of NO content

Nitric oxide was visualized using the specific NO fluorescent probe 4,5-diaminofluorescein diacetate (DAF-2DA), according to the method described by Correa-Aragunde et al. (2004). Briefly, 4-d-old H. moscheutos seedlings were incubated with 20 µm DAF-2DA in 20 mm Hepes-NaOH (pH 7.5) for 2 h. Thereafter, the roots were washed three times for 15 min with the Hepes-NaOH buffer. Seedlings were then incubated for another 20 min in the presence of various compounds (as indicated in appropriate figure legends) before visualization using a laser confocal scanning microscope (LSM 510; Zeiss, Oberkochen, Germany). The intracellular fluorescence of the root apices was excited with a 488 nm argon-krypton laser and emission signals at 515 nm were collected. Images were processed and analyzed using the Zeiss LSM 510 software.

Determination of NOS activity

Nitric oxide synthase activity was determined as described by Guo et al. (2003). Briefly, c. 1 g of roots, together with 50 mg of polyvinylpolypyrrolidone, were ground with liquid N2 and then resuspended in extraction buffer (50 mm Tris-HCl (pH 7.4), 1 mm EDTA, 320 mm sucrose, 1 mm DTT, 1 µm leupeptin, 1 µm pepstatin, 1 mm PMSF). After centrifuging at 10 000 g for 30 min at 4°C, the supernatant was used for NOS determination. NOS activity was determined by the citrulline assay using the NOS assay kit (Cayman Chemical, Ann Arbor, MI, USA). The reaction mixture (50 µl) contained 25 mm Tris-HCl (pH 7.4), 3 µm tetrahydrobiopterin (BH4), 1 µm FAD, 1 µm FMN, 1 mmβ-NADPH, 0.6 mm CaCl2, 0.1 µm camodulin, 0.3 µm (1 µCi) [3H] arginine (Amersham Biosciences, Piscataway, NJ, USA), and 10 µl enzyme extract. After incubation for 30 min at 37°C, the reaction was stopped by adding 400 µl stop buffer (50 mm Hepes (pH 5.5), 5 mm EDTA). A 100 µl resin slur was added to the reaction mixture, and the resin was removed by centrifuge. Flowthrough (400 µl) was added to 5 ml of scintillation liquid and radioactivity was counted (LS 6000; Beckman, Fullerton, CA, USA). The protein content in the supernatant was determined according to the method of Bradford (1976) with BSA as a standard.

Measurement of nitrate reductase activity

The activity of NR was assayed following the method of Scheible et al. (1997) with minor modifications. Approximately 2 g of root samples were homogenized using a chilled mortar and pestle in 500 µl of extraction buffer containing 100 mm HEPES-KOH (pH 7.5), 1 mm EDTA, 10% (v/v) glycerol, 5 mm DTT, 0.1% Triton X-100, 0.5 mm phenylmethylsulfonyl (PMSF), 20 µm FAD, 25 µm leupeptin, 5 µm Na2MoO4, and 1% polyvinylpyrolidone (PVP). Contents were centrifuged at 13 000 g for 20 min at 4°C. The activity of nitrate reductase was measured immediately by mixing one volume (200 µl) of extract with two volumes of prewarmed (25°C) assay buffer (100 mm HEPES-KOH (pH 7.5), 5 mm KNO3, 0.25 mm NADH). The reaction was started by addition of assay buffer and incubated at 30°C for 30 min and then stopped by adding 0.1 m Zn-acetate. The nitrite produced was measured colormetrically at 540 nm by adding 1 ml 1% (w/v) sulfanilamide in 3 m HCl plus 1 ml 0.02% (v/v) N-(1-naphthyl)-ethylenediamine in bidistilled water.

Results

NO donor alleviated Al-induced inhibition of root elongation

To examine the sensitivity of H. moscheutos to Al3+, the effect of AlCl3 on root elongation of H. moscheutos was investigated. Root elongation was reduced after 1 h exposure of H. moscheutos to 100 µm AlCl3 (pH 4.5) (Fig. 1a, inset), and the Al-induced inhibition of root growth became more distinct with time, such that root elongation was reduced from 10.2 ± 0.3 mm to 2.5 ± 0.2 mm after 24 h incubation in AlCl3 solution (Fig. 1a). The reduction in root elongation was recovered when roots were incubated in the solution containing both Al3+ and 100 µm NO-releasing compound, SNP (Fig. 1a). These results suggest that root elongation of H. moscheutos is sensitive to Al3+ and that the NO donor attenuates the Al3+-induced inhibition of root elongation. To decipher the nature of the SNP-induced alleviation of Al-dependent root elongation, the sensitivity of root elongation to SNP alone was also evaluated. Figure 1(b) shows that SNP at concentrations lower than 10 µm had no effect on root elongation, while a concentration-dependent inhibition of root elongation was observed when SNP concentration was > 50 µm. A SNP concentration of 100 µm, which inhibited root elongation by approx. 70%, was used to investigate the role of NO in Al toxicity throughout the study.

Figure 1.

Effect of aluminum (Al) and sodium nitroprusside (SNP) on root elongation of Hibiscus moscheutos. (a) Kinetics of root elongation in response to 100 µm AlCl3 and 100 µm SNP individually, and AlCl3 and SNP simultaneously. The inset shows the effect of the treatments on root elongation during the first 6 h. The control solution contained 0.5 mm CaCl2, pH 4.5. Data are means ± SE of more than eight roots measured. (b) Effect of varying concentrations of SNP on root elongation. Roots were incubated in the solutions containing different concentrations of SNP and elongation was measured after 24 h incubation. Data are presented as relative root elongation compared with control values (without SNP). All values are means ± SE of 12 roots.

The alleviation of Al-induced inhibition of root elongation by SNP could result from chelation of Al3+ by SNP, thus rendering Al3+ nontoxic. To rule out this possibility, we examined the effect of pretreatment of roots with Al3+ and SNP individually on root elongation and compared this with the effect of treatment of roots with Al and SNP simultaneously on root elongation. As shown in Fig. 2, a similar effect of SNP on the Al-induced inhibition of root elongation was observed regardless of whether SNP and Al were present together or separately. Therefore, these findings discount the possibility that the effect of SNP on root elongation in the presence of Al is the result of complexing toxic Al3+ by SNP in the incubation solution. A previous study by Wang & Yang (2005) suggested that NO may act as an antioxidant to ameliorate the Al-induced inhibition of root elongation in Cassia tora. To test whether a similar explanation may account for the observed effect of SNP on the Al-induced root elongation in H. moscheutos, the effect of BHA, a lipophilic antioxidant, on the Al-dependent inhibition of root elongation was also investigated. In contrast to SNP, the Al-induced inhibition of root elongation was not recovered by 100 µm BHA (Fig. 2).

Figure 2.

The effect of sodium nitroprusside (SNP) and butylated hydroxyanisole (BHA) on aluminum (Al)-induced inhibition of root elongation in Hibiscus moscheutos by incubating the roots in solutions supplemented with 100 µm AlCl3, 100 µm AlCl3 and 100 µm SNP or BHA together for 24 h, or incubating the roots in 100 µm AlCl3 for 12 h followed by another 12 h incubation in 100 µm SNP, or incubating the roots in 100 µm SNP for 12 h followed by another 12 h incubation in 100 µm AlCl3. Root elongation was expressed relative to root elongation in the control solution of 0.5 mm CaCl2, pH 4.5. Data are means ± SE of > 12 roots.

Effect of cPTIO, SNP and Al on root elongation

The alleviating effect of SNP on Al3+-induced inhibition of root elongation suggests that inhibition of root elongation by Al may result from reductions of endogenous NO concentrations in roots of H. moscheutos. To test this hypothesis, the effects of NO scavenger (cPTIO) on root elongation both in the absence and in the presence of SNP were evaluated and compared with that of Al3+. Like Al3+ and SNP, cPTIO also inhibited root elongation of H. moscheutos (Fig. 3). For instance, roots exposed to 50 µm cPTIO for 24 h inhibited root elongation by 27%. By comparison, root elongation was inhibited by 71 and 72% when treated with Al3+ and SNP, respectively. The Al- and cPTIO-induced inhibition of root elongation was markedly recovered by SNP (Fig. 3). On the other hand, cPTIO markedly recovered SNP-induced root elongation (Fig. 3). This result suggests that maintaining a critical concentration of endogenous NO is essential for root elongation. Further, there was an additive inhibitory effect of Al3+ and cPTIO on root elongation (Fig. 3), and SNP was no longer capable of recovering the reduction of root elongation caused by the combined effect of Al3+ and cPTIO (Fig. 3). However, SNP can recover some of the reductions of root elongation caused by exposure to Al and cPTIO for 6 h (Fig. 3), suggesting that longer treatments of H. moscheutos with Al and cPTIO simultaneously have detrimental impacts on root physiology.

Figure 3.

Effect of aluminum (Al), sodium nitroprusside (SNP) and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) alone and their combinations on root elongation of Hibiscus moscheutos. Roots were incubated in solutions containing either 100 µm AlCl3, 100 µm SNP and 50 µm cPTIO or containing two or three of the above chemicals for 24 h (hatched bars) or 6 h (open bars). The elongations are expressed relative to root elongation in control solutions of 0.5 mm CaCl2, pH 4.5. The values shown are means ± SE of > 12 roots.

Effect of NOS and NR inhibitors on root elongation

The ameliorating effect of SNP on Al-induced inhibition of root elongation suggests that a reduction of NO concentration may be a trigger to elicit inhibition of root elongation by Al. In plants, two biosynthesis pathways that are catalyzed by NR and NOS are responsible for endogenous NO production (Neill et al., 2003). To confirm the protective effect of SNP on root growth suffered from Al stress being related to endogenous NO production, we studied the effect of the NOS inhibitor, L-NNA, and the NR inhibitor, tungstate, on root elongation of H. moscheutos in both the absence and presence of Al and SNP. As shown in Fig. 4, root elongation of H. moscheutos was markedly reduced by treatments with L-NNA and tungstate. Similar to the Al-induced inhibition of root elongation, inhibition of root elongation by L-NNA and tungstate was substantially recovered by SNP (Fig. 4). Further, a synergetic effect of L-NNA and Al3+ on root elongation was observed (Fig. 4). By contrast, there was no effect of tungstate on root elongation treated with Al3+ (Fig. 4). These results suggest that changes in endogenous NO are likely to be involved in Al-induced inhibition of root elongation, and that both NOS- and NR-mediated NO production plays an important role in the process.

Figure 4.

Effect of a nitric oxide synthase (NOS) inhibitor (L-NNA; Nù-nitro-L-arginine) on root elongation of Hibiscus moscheutos in the absence and presence of aluminum (Al) and sodium nitroprusside (SNP) (a), and the effect of a nitrate reductase (NR) inhibitor (tungstate) on root elongation in the absence and presence of SNP and Al (b). 4-d-old H. moscheutos seedlings were incubated in solutions supplemented with 100 µm L-NNA or 100 µm tungstate alone or together with 100 µm AlCl3 and/or 100 µm SNP for 24 h. Elongation was then measured and normalized relative to root elongation in control solutions. All values are means ± SE (n = 12–18).

Effect of Al on activities of NOS and NR

The observations that root elongation displayed similar sensitivity to Al, cPTIO and two inhibitors of NO biosynthesis prompted us to examine the effect of Al on activities of NOS and NR in H. moscheutos. Treatments of H. moscheutos plants with Al significantly inhibited NOS activity, while NR activity was only marginally inhibited by Al3+ (Fig. 5).

Figure 5.

Effect of 100 µm AlCl3 on activities of nitric oxide synthase (NOS) (a) and nitrate reductase (NR) (b) in roots of Hibiscus moscheutos. To induce NR activity, 4-d-old H. moscheutos seedlings were precultured in 0.5 mm Ca(NO3)2 rather than 0.5 mm CaCl2 for 24 h and then exposed to 100 µm AlCl3. The control solutions contained either 0.5 mm Ca(NO3)2 or 0.5 mm CaCl2, pH 4.5, for determination of NR or NOS activity, respectively. The roots were collected for assaying activities of NOS and NR after 4 h exposure to the treatment solutions. Data are mean values ± SE of three independent experiments.

Effect of Al on endogenous NO concentrations

To confirm whether inhibition of root elongation by Al3+ is related to endogenous NO concentrations, these concentrations in root apices were labeled with a fluorescent probe 4,5-diaminofluorescein diacetate (DAF-2DA) and imaged by confocal microscopy. The DAF-2DA-dependent fluorescence was enhanced and reduced in response to the NO donor and scavenger, respectively (Fig. 6), indicating that DAF-2A-dependent fluorescence is associated with endogenous NO concentration. The fluorescence intensity remained relatively constant with time when measured in the control solution (Fig. 6). A decrease in endogenous NO concentration in root apices was found upon exposure of the roots to Al3+, as demonstrated by 34% reductions of fluorescence intensity following 20 min exposure to Al3+ (Fig. 6). The NOS inhibitor, L-NNA, markedly reduced the fluorescence intensity, while the NR inhibitor, tungstate, only induced a marginal and insignificant reduction in the fluorescence intensity (Fig. 6), suggesting that NOS-mediated NO production is a major source for endogenous NO in H. moscheutos. The reductions in DAF-2DA fluorescent intensity resulting from treatments with Al3+ and cPTIO were recovered by SNP (Fig. 6). Therefore, these results reveal that a reduction of endogenous NO concentration in the root apical cells resulting from inhibiting activities of NOS could be a critical event in Al3+ phytotoxicity to H. moscheutos.

Figure 6.

Effect of AlCl3, Nù-nitro-L-arginine (L-NNA), tungsate (Na2WO4), 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) and sodium nitroprusside (SNP) on endogenous nitric oxide (NO) concentration in Hibiscus moscheutos roots. Roots of 4-d-old H. moscheutos were loaded with 20 µm 4,5-diaminofluorescence (DAF-2DA) in a solution containing 20 mm Hepes-KOH, pH 7.5. Representative images showing endogenous NO concentrations in root apical cells treated with varying chemicals (control, 100 µm AlCl3, 100 µm SNP, 10 mm cPTIO, 10 mm L-NNA and 10 mm tungstate) for 20 min detected by confocal laser scanning microscopy are given in (a). Mean DAF-2DA fluorescence densities relative to the fluorescence densities measured in control solutions determined from images shown in (a) are given in (b). Data are means ± SE from measurements of at least four roots for each treatment. Bar, 200 µm (a).

Discussion

Numerous physiological processes are altered when plants suffer from the Al toxicity that commonly occurs in acidic soils, but the mechanism underlying this toxicity remains largely unknown (Matsumoto, 2000; Rengel & Zhang, 2003). There has been increasing evidence to show that NO serves as an important signaling molecule that modulates physiological processes such as plant growth and development, stomatal movement and responses to biotic and abiotic stresses (Lamattina et al., 2003; Neill et al., 2003; Crawford & Guo, 2005). In the present study, we found that exogenous NO in the form of the NO-releasing compound, SNP, can markedly ameliorate the Al-induced inhibition of root elongation (Figs 1, 2). Moreover, SNP was also capable of recovering root elongation inhibited by the NO scavenger cPTIO (Fig. 3). These results indicate that maintenance of homeostat NO concentrations in root cells is critical for root elongation, and that alterations of endogenous NO concentrations in root apical cells of H. moscheutos are closely associated with the Al-induced inhibition of root elongation. The observations that treatments with Al and cPTIO reduced endogenous NO concentrations in the root apical cells and that this effect was reversed by SNP (Fig. 6) are in line with this proposition. As NOS and NR are two key enzymes responsible for endogenous NO biosynthesis in plants (Neill et al., 2003; Crawford, 2006), we then tested sensitivity of NOS and NR to Al, and found that Al reduced activity of NOS, but not of NR (Fig. 5). Although we cannot exclude the possibility that the chemical agents used in the present study may not specifically target NO, these results collectively point to the fact that a reduction of endogenous NO concentration in root apical cells of H. moscheutos could be an important event in triggering Al toxicity in plants.

The SNP-induced recovery of root elongation treated by Al is unlikely to result from chelation of toxic Al3+ by SNP, as pretreatments of roots by SNP and Al had an identical effect on root elongation to the treatments with Al and SNP simultaneously (Fig. 2). These findings are consistent with those reported by Wang & Yang (2005), in which they found that SNP can ameliorate Al-induced inhibition of root elongation in Cassia tora. However, the ameliorating effect of SNP on the Al-induced reduction of root elongation was greater in H. moscheutos than in the roots of C. tora (Wang & Yang, 2005). For instance, 400 µm SNP recovered approx. 25% of 10 µm AlCl3-induced inhibition of root elongation in C. tora (Wang & Yang, 2005), while SNP at 100 µm recovered the Al-induced reduction of root elongation by 50% in H. moscheutos (Figs 1, 2). Moreover, the sensitivity of root elongation of H. moscheutos to SNP in the absence of Al also differed from that of root elongation of C. tora (Wang & Yang, 2005). For example, root elongation in C. tora is insensitive to SNP up to 800 µm (Wang & Yang, 2005). By contrast, we found that SNP at concentrations < 10 µm had no effect on root elongation, but SNP markedly inhibited root elongation in H. moscheutos at concentrations > 50 µm (Fig. 1b). A similar inhibitory effect of SNP on elongation of primary roots and hypocotyls has been reported (Beligni & Lamattina, 2000; Correa-Aragunde et al., 2004). The mechanism underlying the inhibitory effect of SNP at relatively high concentrations on root elongation is unclear. There are several potential possibilities to account for this inhibitory effect. For example, formation of cytotoxic peroxynitrite (ONOO) in the presence of excessive NO will alter physiological processes related to root elongation (Beligni & Lamattina, 1999). Furthermore, SNP at 100 µm up-regulates the genes for ACC synthase and ACC oxidase (Parani et al., 2004), the two enzymes responsible for ethylene biosynthesis. As root elongation is often inhibited by ethylene (Pierik et al., 2006), it is expected that the SNP-induced ethylene biosynthesis may contribute to the root elongation observed in the present study.

Root elongation of H. moscheutos was inhibited almost equally by exposure to the NO donor, SNP, and to Al3+ (Fig. 3). However, when exposed to the two chemicals together, the inhibitory effect on root elongation became less than the treatment with the two chemicals individually (Fig. 3). As SNP has been widely used as a compound to release the NO molecule (Furchgott, 1995), these results indicate that Al may disrupt NO homeostasis, leading to the endogenous NO concentration being lower than required for root elongation. This argument is consistent with the observation that SNP can recover NO scavenger-induced root elongation (Fig. 3). Another interesting observation in the present study is that Al and the NO scavenger (cPTIO) had a synergic effect on inhibition of root elongation (Fig. 3), and SNP was unable to recover the root elongation reduced by treatments with the two agents together for 24 h (Fig. 2). Because cPTIO functions to scavenge the endogenous NO, the inhibition of root elongation by cPTIO indicates that root elongation is closely related to endogenous NO concentration. The additive inhibitory effect of Al and cPTIO on root elongation may suggest that Al and cPTIO can reduce endogenous NO concentration through different mechanisms, resulting in a greater reduction of the endogenous NO concentrations that cannot readily be recovered by treatment with SNP. However, the reduction in root elongation caused by cPTIO and Al can be recovered to some extent by SNP when the roots are exposed to cPTIO and Al for 6 h (Fig. 3). This highlights that exposure of H. moscheutos to cPTIO and Al together for an extended time period may have detrimental impacts that may not be directly related to the endogenous NO concentration.

To unravel the mechanism underlying the ameliorating effect of SNP on the Al-induced inhibition of root elongation, we tested the effect of L-NNA and tungstate, which are potent inhibitors of NOS and NR, on root elongation of H. moscheutos in both the absence and presence of SNP. Similar to treatments with Al3+, both L-NNA and tungstate reduced root elongation and the inhibitory effect was substantially reversed by SNP (Fig. 4), suggesting that the reduction of root elongation by L-NNA and NR may result from alterations of NOS- and NR-mediated endogenous NO production in H. moscheutos. The similar effect of Al, L-NNA and tungstate on H. moscheutos in terms of inhibition of root elongation and the ameliorating effect by SNP prompted us to propose that the Al-induced inhibition of root elongation may occur through inhibiting activities of NOS and/or NR, thus leading to a reduction of the endogenous NO concentration. To test this hypothesis, we investigated the effect of Al on activities of NOS and NR, and found that Al significantly reduced activities of NOS, but had no effect on activities of NR (Fig. 5). We then further examined the responses of the endogenous NO concentration to Al and SNP by NO-specific fluorescent probe and confocal microscopy. The observations that Al rapidly reduced the endogenous NO concentration and SNP recovered the Al-induced reduction of NO concentration in the root apical cells provide direct evidence in support of alteration of the endogenous NO concentration resulting from inhibiting NOS activity being associated with the Al toxicity syndrome in H. moscheutos. There was an increase and decrease in the NO-dependent fluorescence by approx. 15 and 30% by equimolar SNP and Al, respectively (Fig. 6). However the two treatments led to almost identical inhibition of root elongation (Fig. 3). These suggest either that root elongation is more sensitive to a reduction of the endogenous NO concentration or that another mechanism in addition to NO may be involved in Al-induced root elongation.

The Al-induced reduction in the endogenous NO concentration in the root apical cells occurred earlier (e.g. 20 min exposure to Al) than the Al-induced reduction in root elongation (cf. Figs 1a, 6). Therefore, the reduction in the endogenous NO concentration is likely to be a cause rather than a consequence of Al toxicity. By contrast, a reduction of the endogenous NO concentration in pea roots after 15 d exposure to Cd (Rodríguez-Serrano et al., 2006) may reflect the consequence associated with Cd toxicity.

The observations that tungstate-induced inhibition of root elongation was ameliorated by SNP (Fig. 4) and that the endogenous NO concentration was insensitive to tungstate (Fig. 6) are not in line with the inhibition being related to modulation of endogenous NO concentration. The inhibition of root elongation was measured after 24 h treatments of roots with tungstate, and the changes in the endogenous NO concentrations were determined after 20 min exposure to tungstate. Therefore, there is a possibility that reduction of the endogenous NO concentration requires extended periods of exposure to tungstate. Alternatively, another unknown mechanism may account for the discrepancies between the effect of tungstate on root elongation and on endogenous NO concentration.

There have been several reports on the involvement of NO in response of plants to heavy metals (Kopyra & Gwozdz, 2003; Wang & Yang, 2005; Yu et al., 2005; Rodríguez-Serrano et al., 2006). However, most of these studies have emphasized the antioxidative effect of exogenous NO against the stress using NO donors and scavengers, and have paid little attention to the involvement of endogenous NO concentrations in response to the abiotic stresses. In this context, the ameliorating effect of SNP on the Al-dependent inhibition of root elongation in C. tora has been accounted for by the fact that NO functions as an antioxidant to counteract the Al-induced oxidative stress (Wang & Yang, 2005). However, we found that the Al-induced inhibition of root elongation was not alleviated by the lipophilic antioxidant BHA (Fig. 2). An identical effect of BHA on the Al-dependent inhibition of pea roots has been reported by Yamamoto et al. (2001). It has been shown that the Al-induced lipid peroxidation is an early symptom rather than the cause of the Al-induced inhibition of root elongation in pea (Yamamoto et al., 2001). Therefore, our results indicate that the effect of SNP on the Al-induced inhibition of root elongation in H. moscheuto cannot be exclusively accounted for by the antioxidative effect of NO on Al-induced oxidative stress. The improved Al tolerance by the NO donor seems unlikely to result from external Al tolerance, as treatment with SNP has no effect on Al-dependent exudation of organic anions (Wang & Yang, 2005). Therefore, it would be interesting to compare responses of the endogenous NO concentrations in root apical cells to Al using the plant species or genotypes with contrasting tolerance to Al in future studies.

In summary, we demonstrated that endogenous NO concentration in root apical cells of H. moscheuto were rapidly reduced by exposure to toxic Al3+ and that this reduction may result from inhibiting NOS activity. As NO has been shown to modulate the expression of a large number of genes at the transcriptional level (Parani et al., 2004), it is envisaged that a reduction of the endogenous NO concentration in root apical cells by Al will activate and/or deactivate numerous genes that are directly or indirectly involved in the regulation of cell elongation, leading to the observed inhibition of root elongation.

Acknowledgements

This work was supported by the Chinese Academy of Sciences through its Hundred Talent programme. We thank the two anonymous reviewers for their constructive suggestions.

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