Aluminium (Al)-induced secretion of organic acids from plant roots is considered a mechanism of Al resistance, but the processes leading to the secretion of organic acids are still unknown. In the present study, a protein-synthesis inhibitor, cycloheximide (CHM), was used to investigate its effect on Al-induced organic acid secretion in a pattern I (rapid exudation of organic acids under Al stress) plant buckwheat (Fagopyrum esculentum Moench) and a pattern II (exudation of organic acids was delayed by several hours under Al stress) plant Cassia tora L. A dose–response experiment showed that the secretion of oxalate by buckwheat roots was not affected by CHM when added in the range from 0 to 50 µm, with or without exposure to 100 µm Al, but the secretion of citrate was completely inhibited by 30 µm CHM in C. tora. A time-course experiment showed that even prolonged exposure to 20 µm CHM did not affect oxalate secretion in buckwheat, but significantly inhibited citrate secretion in C. tora. However, citrate synthase (CS) activity in C. tora was not affected during 12 h exposure to 100 µm Al when compared with that in control roots, although CHM can inhibit CS activity effectively. These results indicated that CS activity was not related to Al-regulated citrate efflux in C. tora. The total protein was decreased by 14.0% and 32.3% in C. tora and buckwheat root tip, respectively, after 3-h treatment with 20 µm CHM. A 3-h pulse with 20 µm CHM completely inhibited citrate efflux in C. tora during the next 6-h exposure to Al, although a small amount of citrate was exuded after 9-h exposure. However, oxalate efflux in buckwheat was not influenced by a similar treatment. In buckwheat, a 3-h pulse with 100 µm Al maintained oxalate secretion at a high level during the next 9 h, with or without CHM treatment. Conversely, in C. tora a 6-h pulse with 100 µm Al induced significant secretion of citrate which was inhibited by the CHM. Taken together, these findings suggest that both de novo synthesis and activation of an anion channel are needed for Al-induced secretion of citrate in C. tora, but in buckwheat the plasma membrane protein responsible for oxalate secretion pre-exists.
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Aluminium (Al) toxicity is one of the most serious factors limiting crop production on acid soils (Kochian 1995). However, many native plants and local crop cultivars can grow well on such soils and there is scope to further increase Al resistance in more crop species by using biotechnology (Kochian et al. 2002).
The mechanisms involved in Al resistance in different plant species have been extensively studied over the last two decades (Matsumoto 2000). Two strategies for the detoxification of Al by plants have been suggested (Taylor 1991). One is the exclusion of Al from the cell cytosol (exclusion mechanism) and the other is the tolerance of Al that enters the plant (internal tolerance mechanism). Several possibilities have been proposed for each type of mechanism, but most of them are speculative. However, evidence is accumulating to show that organic acids play an important role in both internal and external detoxification of Al (Ma, Ryan & Delhaize 2001; Ryan, Delhaize & Jones 2001; Kochian, Hoekenga & Piñeros 2004). Two patterns of Al-induced secretion of organic acids have been characterized on the basis of the timing of secretion (Ma 2000). In pattern I no discernible delay is observed between the addition of Al and the onset of organic acid release. By contrast, in pattern II organic acid secretion is delayed for several hours after exposure to Al. Different processes are thought to be involved in the two secretion patterns. In pattern I, Al may activate a preexisting transporter (possibly an anion channel) in the plasma membrane to initiate organic anion efflux, and the induction of a novel protein is not required. On the other hand, in pattern II, exposure to Al induces the expression of genes and proteins involved in organic acid synthesis or transport which are necessary for efflux from root cells. However, there is not much direct experimental evidence to support this supposition.
Buckwheat is highly resistant to Al stress and Ma et al. (1997a) proposed that rapid activation of oxalate release from roots by Al treatment plays a key role in its tolerance mechanism. Furthermore, the amount of oxalate secreted represented only 15% of the total soluble oxalate in the roots, indicating that oxalate synthesis may not be required for Al-induced oxalate efflux in buckwheat (Zheng, Ma & Matsumoto 1998). Cassia tora L. has also been reported to be an Al-resistant species by means of the secretion of citrate from roots (Ma, Zheng & Matsumoto 1997b). In contrast to the pattern of rapid secretion of oxalate from buckwheat, the secretion of citrate from C. tora was very slow during the first few hours, but significantly enhanced thereafter. This implies that some inducible process leading to the secretion of citrate, such as the induction of enzymes required for citrate synthesis or de novo synthesis of transport proteins to facilitate organic acid efflux, is occurring in C. tora. In this study, we have investigated the different types of organic acid secretion responses of the two species to Al stress. A protein-synthesis inhibitor, cycloheximide, was applied before, together with or after exposure to Al stress in buckwheat and C. tora. Our results demonstrated for the first time that de novo protein synthesis is critical for Al-activated organic acid exudation in C. tora but not in buckwheat.
MATERIALS AND METHODS
Plant materials and growth environments
Seeds of buckwheat (Fagopyrum esculentum Moench cv Jiangxi) and Cassia tora L. were soaked in deionized water overnight, and then placed between two layers of filter paper moistened with deionized water in the dark at 25 °C for germination. After germination the seeds were placed onto a net tray, which was floated on a 0.5 mm CaCl2 solution at pH 4.5 in a plastic container. The solution was renewed daily. On day 5, seedlings of a similar size were transplanted into a 1 L plastic pot (12 seedlings per pot) containing aerated nutrient solution. One-fifth-strength of Hoagland solution was used, which contained the macronutrients KNO3 (1.0 mm), Ca(NO3)2 (1.0 mm), MgSO4 (0.4 mm), and (NH4)H2PO4 (0.2 mm), and the micronutrients NaFeEDTA (20 µm), H3BO3 (3 µm), MnCl2 (0.5 µm), CuSO4 (0.2 µm), ZnSO4 (0.4 µm), and (NH4)6Mo7O24 1 µm. The solution was adjusted to pH 4.5 with HCl and renewed every other day. The plants were grown in a greenhouse for 2 weeks, Two days before the treatments the pots were moved to a controlled-environment room with a 14-h/26 °C day and a 10-h/22 °C night regime, a light intensity of 150 µmol photons m−2 s−1 and a relative humidity of 65%. Each experiment was conducted at least twice independently.
Protein synthesis inhibitor and Al treatments
Before the treatments with protein synthesis inhibitor and Al stress, plant roots were rinsed with 0.5 mm CaCl2 solution, then soaked in an aerated CaCl2 solution at pH 4.5 overnight. Four experiments with different treatments were conducted, the basic medium of all treatments was 0.5 mm CaCl2, and the pH was adjusted to pH 4.5:
Five concentrations of cycloheximide (soluble in de-ionized water, Nacalai Tesque, Inc. Kyoto, Japan) (referred to as CHM hereafter), 0, 10, 20, 30 and 50 µm, either in the absence or in the presence of 100 µm Al (added as AlCl3). Root exudates were collected 3 or 6 h after the addition of Al and CHM.
Four treatments (0 or 100 µm Al, 20 µm CHM, or 100 µm Al +20 µm CHM) were used. Root exudates were collected at 3-h intervals for 12 h.
Roots were subjected to 20 µm CHM for 3 h. Then the roots were cleaned by quickly immersing them in a 0.5 mm CaCl2 solution. Then the seedlings were transferred to a 0.5 mm CaCl2 solution with or without 100 µm Al solution, and the root exudates were collected every 3 h for 9 h.
Roots were exposed to 100 µm Al (in 0.5 mm CaCl2) for either 3 or 6 h, and then Al exposure was terminated by immersing the roots in 0.5 mm CaCl2 without Al. Next the seedlings were transferred to a 0.5 mm CaCl2 solution, with or without 20 µm CHM, and the root exudates were collected every 3 h for 9 h.
Measurement of organic acids in root exudates
The root exudates were passed through a cation-exchange resin column (16 × 14 cm) filled with 5 g of Amberlite IR-120 resin (H+ form, Muromachi Chemical, Tokyo, Japan), followed by an anion-exchange resin column filled with 2 g Dowex 1 × 8 (100–200 mesh, format form). The organic acid absorbed on anion-exchange resin were eluted by 1 m HCl, and the eluate was concentrated to dryness using a rotary evaporator at 40 °C. The residue was re-dissolved in dilute HClO4 solution at pH 2.1; the concentration of organic acids was determined by HPLC equipped with an ion-exclusion column (Shodex RSpak KC-811, 300 × 8 mm) and a guard column (50 × 8 mm). The eluant was a dilute HClO4 solution at pH 2.1 with a flow rate of 0.8 mL min−1 at 40 °C. The peaks at 210 nm were recorded.
Measurement of enzyme activity
For the determination of citrate synthase (CS) activity, root apices (0–10 mm) were extracted in a buffer containing 100 mm Tris-HCl (pH 8.0), 5 mm MgCl2, 5 mm EDTA, 10% glycerol, 0.1% Triton X-100, and 0.5 mm phenylmethylsulphonyl fluoride (PMSF). Samples were centrifuged for 5 min (20 000 g at 4 °C), and the supernatant was used to assay CS. The activity of CS was assayed spectrophotometrically by monitoring the reduction of acetyl coenzyme A with 5,5′-dithio-bis-2-nitrobenzonic acid (DTNB) at 412 nm for 3 min (Srere 1967). The reaction mixture contained 100 mm Tris-HCl buffer (pH 8.0), 5 mm MgCl2, 100 mm DTNB, 0.3 mm acetyl coenzyme A, and 0.5 mm oxaloacetic acid. The reaction mixture without oxaloacetic acid was used for each sample as the blank.
Total protein extraction and quantitative analysis
In order to investigate the effectiveness of CHM on the inhibition of protein synthesis, 4-day-old seedlings were exposed to 0.5 mm CaCl2 solution (pH 4.5) containing 0 or 20 µm CHM for 3 h. Total protein extraction was carried out according to Martínez-García, Monte & Quail (1999). Root apices (0–10 mm) were cut and placed in 1.5 mL Eppendorf tubes containing the extraction buffer (125 mm Tris-HCl pH 8.8, 1% (w/v) SDS, 10% (v/v) glycerol, 50 mm Na2S2O5). Samples were centrifuged for 10 min (13 000 g at 20 °C). The protein concentration was assayed colorimetrically (Lowry et al. 1951).
A progressive reduction in Al-activated citrate exudation with exposure to increasing CHM concentrations was observed in C. tora, with 10 and 20 µm CHM eliciting inhibitions of 65% and 85%, respectively. CHM concentrations of 30 µm and above completely abolished Al-activated citrate exudation (Fig. 1a). Whereas Al-induced oxalate secretion was not inhibited in buckwheat, even when exposed to 50 µm CHM (Fig. 1b). In order to investigate whether the treatment time with CHM was too short, roots were exposed to 20 µm CHM together with 100 µm Al for up to 12 h. Even prolonged treatment with CHM did not affect oxalate secretion in buckwheat; exudation even slightly increased but the difference was not significant (Fig. 2b). In contrast, Al-dependent citrate exudation from C. tora was inhibited by nearly 90% over the whole treatment period (Fig. 2a).
A 3-h pulse of 20 µm CHM was sufficient to inhibit citrate secretion for the next 6 h in C. tora even after removal of the CHM treatment (Fig. 3a). However, oxalate secretion from buckwheat was not affected (Fig. 3b). It has been reported that oxalate exudation in buckwheat and citrate secretion in C. tora can be maintained after its induction by Al treatment (Ma et al. 1997b; Zheng et al. 1998). In the present study, oxalate exudation in buckwheat or citrate exudation in C. tora was initiated by 3 or 6 h pulses with 100 µm Al; the exudation was monitored during the subsequent 9 h, with or without addition of 20 µm CHM. Clearly, the Al-pulse treatment successfully induced oxalate and citrate exudation. After removal of Al, the exudation was significantly inhibited by CHM treatment in C. tora, but the pattern was not changed in buckwheat (Fig. 4a & b). In C. tora Al treatment did not affect the activity of CS over the 12 h exposure, when compared with the control (no Al), but CHM effectively inhibited CS activity (Fig. 5). Moreover, the total protein content was reduced by 32.3% and 14.0% in buckwheat and C. tora root tip after 3-h exposure of 20 µm CHM indicating that CHM was effectively inhibiting protein synthesis in both species(Fig. 6).
Two patterns of Al-induced organic acid exudation have been identified for a range of plant species (Ma et al. 2001). It had been suggested that the induction of gene expression was involved in pattern II species, but not in pattern I plants. However, there was no direct evidence to support this contention, until now. Gene expression would finally result in de novo protein synthesis. The nonspecific protein-synthesis inhibitor, CHM, was used to investigate the relationship between Al stress and organic acid secretion. The secretion of organic acid in buckwheat typically belongs to pattern I (Fig. 2b, Zheng et al. 1998), and that in C. tora to pattern II (Fig. 2a, Ma et al. 1997b). Here we demonstrated that the induction of oxalate secretion from buckwheat was rapid, and CHM did not affect the process (Figs 1b & 2b), implying that all the necessary metabolic ‘machinery’ was constitutively expressed in buckwheat roots, and that oxalate secretion was simply triggered by the Al stimulus. However, a lag phase was observed between the addition of Al and the start of citrate secretion in C. tora, and CHM inhibited the citrate efflux (Figs 1a & 2a), suggesting that intermediate steps occur between the perception of the stimulus and citrate exudation.
In a preliminary experiment, we found that CHM at 5 µm significantly inhibited the biosynthesis of plasma membrane iron reductase activity of red clover roots within 3 h, indicating that CHM was effective in inhibiting de novo protein synthesis (data not shown). As CHM might influence Al activity and ion speciation in the treatment solution, a pulse experiment was designed in the present study. In C. tora a 3-h pulse treatment with 20 µm CHM completely inhibited citrate exudation in the subsequent 6 h (Fig. 3a); there was a slight citrate efflux thereafter, but the amount was significantly lower when compared with that measured for roots not treated with CHM (Figs 2a & 3a). In buckwheat, a 3-h pulse with 20 µm CHM had no effect on oxalate exudation (Fig. 3b). Furthermore, a 3-h exposure with 20 µm CHM was sufficient to inhibit the de novo protein synthesis both in buckwheat and C. tora (Fig. 6). These results indicate that de novo protein synthesis accounted for organic anion efflux in C. tora, but that protein synthesis was not required for organic acid release in buckwheat. Therefore, our present results provide direct evidence that protein synthesis is needed in pattern II but not in pattern I species.
The effect of Al on organic acid metabolism has been examined in several studies, but there has been no consensus regarding the effect of Al on internal organic acid contents or CS activity. Al-induced increase in internal organic acids has been reported in several species, e.g. soybean (Yang et al. 2001), rye (Li, Ma & Matsumoto 2000) and maize (Piñeros et al. 2002). A significant accumulation of organic acid in root tips probably results from increased citrate synthesis. For example, CS activity increased by 30% in the root tips of rye exposed to 50 µm Al (Li et al. 2000). An increased CS activity was also observed in roots of soybean (Yang et al. 2001) and snapbean (Mugai, Agong & Matsumoto 2000) in response to Al stress. Recent work has suggested that genetic transformation of plants to overexpress enzymes involved in organic acid synthesis may lead to increased efflux of organic acids and Al resistance (de la Fuente et al. 1997; Tesfaye et al. 2001; Anoop et al. 2003). On the other hand, Ryan, Delhaize & Randall (1995) reported that the activities of malate dehydrogenase and phosphoenolpyruvate carboxylase in wheat roots were unaffected, even when rapid malate efflux occurred. Zhao et al. (2003) found that CS activity was not affected by Al in either resistant or sensitive barley varieties, although the internal citrate content was increased by Al exposure in the resistant cultivar. A recent study on a pair of triticale lines differing in Al resistance, compared root elongation and organic acid secretion with the internal content of organic acid, and with enzyme activity (Hayes & Ma 2003). The results showed that the metabolism of organic acids was poorly correlated with the Al-induced secretion of organic acids, indicating that the Al-dependent efflux of organic acid anions from the roots of triticale is not regulated by the root internal levels or by the capacity of root cells to synthesize malate and citrate. Thus, the mechanisms behind Al-induced organic acid exudation in Pattern II may differ among plant species. In the present study, CS activity was not affected by the Al treatment in C. tora (Fig. 5), and hence Al-induced efflux of citrate was not related to the CS activity. However, the question remains whether the inhibition by CHM of citrate efflux was due to the decrease of CS activity or to the inhibition of anion channel synthesis, because CHM can effectively inhibit CS activity (Fig. 5). Therefore, further studies are required to elucidate the relationship between Al-regulated citrate metabolism and enzymes involved in the process.
Although it remains a matter of debate whether organic acid synthesis was responsible for the enhanced organic acid efflux, several reports support the hypothesis that organic acid secretion in response to Al stress occurs through an anion channel in the plasma membrane (Ryan et al. 1997; Ma et al. 2001). However, it was not clear whether the anion channel pre-existed on the plasma membrane or how the opening and closing of the channel is controlled. As application of CHM, both before and during Al treatment, did not change the frequency of anion channel activation and conductance in patch-clamp studies on protoplasts isolated from maize root apices, Kollmeier et al. (2001) proposed that the biochemical machinery for transduction of the Al signal to channel activation was already established before addition of Al in that species. Piñeros & Kochian (2001) also demonstrated that Al itself directly activated the anion channel in isolated patches of membrane from maize root protoplasts.
In this study, a pulse of Al induced significant exudation of citrate after transfer to an Al-free solution in both species, but exudation was completely inhibited by the addition of 20 µm CHM only in C. tora (Fig. 4a). Considering the inhibition of CS activity by CHM and the poor association of the enzyme with Al-induced citrate efflux (Fig. 5), the enhancement of citrate efflux could have been caused by the de novo synthesis of anion channels. The data in Fig. 4a indicate that the turnover time of the protein(s) affected by CHM could have been ∼ 6 h. Furthermore, an anion channel inhibitor, anthracene-9-carboxylic acid, inhibited citrate exudation in C. tora (Chiba 1999), which is consistent with the involvement of an anion channel in the process. Thus, we hypothesized that the efflux of citrate in C. tora was mainly due to Al-induced de novo synthesis of anion channels. However, it cannot be ruled out that de novo synthesis of other proteins involved in the process is important. In buckwheat, a pulse of Al successfully induced oxalate exudation, and after removal of Al the exudation pattern was not changed by CHM treatment (Fig. 4b), indicating that once the exudation started, the anion channel could be functionally active for a relatively long time, and further de novo protein synthesis was not required. This strongly suggests that the protein functioning as an anion channel for oxalate release preexisted on the plasma membrane and therefore Al directly acted as a signal for opening of the channel in buckwheat.
It was noteworthy that the turnover rates of plasma membrane proteins that functioned as anion channels were different in buckwheat and C. tora L. Al-induced oxalate efflux was not affected (Figs 2b & 3b), implying that the turnover time of anion channels facilitating oxalate efflux in buckwheat was > 9 h. However the turnover time of anion channels in C. tora was at most 6 h (Fig. 3a). Ryan et al. (1995) reported that pretreatment of wheat roots with CHM at ∼ 35 µm for 15 min can effectively inhibit Al-activated malate efflux. This result indicated the requirement of de novo protein synthesis, although malate was rapidly stimulated after Al addition. Ryan et al. (1995) provided two reasons for this apparent contradiction. One was that the turnover time of anion channels was < 15 min, and the other was due to some direct effect of CHM on malate transport. Schmidt (1994) also reported that CHM can inhibit both FeEDTA and ferricyanide reduction activities effectively after 4 h treatment. However, the NADH-linked Fe chelate reduction activity was not sensitive to CHM, indicating that the redox chain involved in iron uptake was composed of multiple components (Schmidt & Bartels 1998). Ryan et al. (2001) suggested three possible mechanisms for Al-activated organic acid efflux. From the work presented in this paper it cannot be ruled out that the inhibition of organic acid efflux by CHM may be due to effects on intermediates, such as a possible receptor protein, involved in the processes of the Al-regulated organic acid efflux.
In conclusion, anion-channel activation by Al stress accounts for oxalate exudation in buckwheat, but de novo synthesis and activation of speculative anion channels would be required for citrate exudation in C. tora.
This study was financially supported by the program for New Century Excellent Talents in University (NCET), a Fund for Excellent Young Teachers from the Chinese Ministry of Education and grants from the Natural Science Foundation of China. We thank Dr Tony Miller from Rothamsted Research for checking the English text.