Al-induced efflux of organic acid anions is poorly associated with internal organic acid metabolism in triticale roots

The secretion of organic acid anions from roots has been identified as a mechanism of resistance to Al. However, the process leading to the secretion of organic acid anions is poorly understood. The effect of Al on organic acid metabolism was investigated in two lines of triticale (xTriticosecale Wittmark) differing in Al-induced secretion of malate and citrate and in Al resistance. The site of Al-induced secretion of citrate and malate from a resistant line was localized to the root apices (terminal 5 mm). The levels of citrate (root apices and mature root segments) and malate (mature segments only) in roots increased during exposure to Al, but similar changes were observed in both triticale genotypes. The in vitro activities of four enzymes involved in malate and citrate metabolism (citrate synthase, phosphoenolpyruvate carboxylase, malate dehydrogenase, and NADP-isocitrate dehydrogenase) were similar for sensitive and resistant lines in both root apices and mature root segments. The response of these enzymes to pH did not differ between tolerant and sensitive lines or in the presence and absence of Al. Moreover, cytoplasmic and vacuolar pH were not affected by exposure to Al in either line. Together, these results indicate that the Al-dependent efflux of organic acid anions from the roots of triticale is not regulated by their internal levels in the roots or by the capacity of the root cells to synthesize malate and citrate.     

Aluminum-induced secretion of both citrate and malate in rye

Aluminum (Al)-resistant mechanisms responsible for Al-induced secretion of organic acids are poorly understood. In this study, we characterized the Al-induced secretion of both citrate and malate from rye (Secale cereale L. cv. King). Secretion of organic acids increased with increasing concentration (10, 30 and 50 M) and duration of Al treatments. Neither phosphorous (P) deficiency up to 15 days nor addition of 50M lanthanum, 50 M lead, 10 M cadmium, or 200 M manganese caused secretion of organic acids, suggesting that this secretion was a specific response to Al stress. Aluminum activated citrate synthase, the main enzyme for the synthesis of citrate, but its activation occurred only in the root tip. The elongation of roots of an Al-sensitive cultivar of wheat (Tritium aestivum L. cv. Scout 66) was not inhibited by 50 M Al in the presence of externally applied 50 M citrate or 400 M malate. The secretion of citrate and malate from intact rye roots exposed to 50 M Al corresponded to 31.3 ± 1.7 M and 11.5 ± 2.5 M, respectively, in the rhizosphere based on an assumption of a 2 mm thick unstirred layer around root tips. This result indicated that Al-resistance in rye was achieved by the Al-induced synthesis of citrate in root apices followed by Al-induced specific secretion of citrate from root tips.     

A comparative study on the aluminium- and copper-induced organic acid exudation from wheat roots

It is well established that aluminium (Al) and some heavy metals can elicit organic acid exudation from a range of species. In the present research we found that copper (Cu) can also induce organic acid exudation from the roots of wheat, rye, triticale, maize and soybean. Using intact wheat plants, we made a comparative study of Al- and Cu- induced organic acid exudation. In 5-day-old wheat seedlings, severe Cu stress (40 µ M CuCl₂) mainly induced the exudation of malate and citrate, and Al-tolerant genotypes could release significantly greater amounts of malate than Al-sensitive genotypes. The time course of the exudation of malate and citrate from the roots of 5-day-old seedlings of wheat (cv. Atlas) in 200 µ M AlCl₃ was similar to that in 40 µ M CuCl₂. In older wheat plants (15-day-old), moderate Cu stress (12 µ M CuCl₂) induced the exudation of large amounts of citrate and addition of Al or La sharply reduced Cu-induced citrate exudation, while Cu or La did not affect Al-induced malate efflux. When half of the root system of Atlas wheat was immersed in Al- or Cu-containing solution and the remaining half in Al- or Cu-free solution, organic acids were only exuded into the solution containing Al or Cu. This suggests that no long distance signal transport is involved in organic acid exudation induced by Al or Cu, and that direct contact of Al or Cu with plant roots is a prerequisite for the induction of organic acid exudation. The anion-channel inhibitor niflumic acid (NIF) significantly stimulated the exudation of both citrate and malate from 5-day-old wheat seedlings under severe Al or Cu stress. Our results suggest that Cu-induced organic acid efflux may be a common response, which may play a role in alleviating Cu toxicity in plants.     

Secretion of malate and citrate from roots is related to high Al-resistance in Lespedeza bicolor

Lespedeza bicolor (Lespedeza bicolor Turcz. cv. Jiangxi) is a leguminous shrub that is well adapted to acid infertile soils. However, the mechanisms of aluminum resistance in this species have not been established. This study aimed to assess the possible resistance mechanisms of this plant to Al. An Al-sensitive species of Lespedeza, sericea lespedeza [Lespedeza cuneata (Dum.-Cours.) G. Don cv. Zhejiang], was used as a reference. The roots of L. bicolor secreted both malate and citrate after exposure to Al, but roots of L. cuneata did not. The secretion of organic acids from L. bicolor was specific to Al; neither 15-day P starvation nor 50 μM lanthanum induced the secretion of these organic acid anions. Secretion of organic acid anions in L. bicolor was detected after 3–6 h exposure to Al, and the amount increased significantly after 6 h exposure, suggesting that this plant shows a pattern II-type organic acid secretion. This is supported by the finding that the secretion was significantly inhibited by a protein-synthesis inhibitor, cycloheximide. Two kinds of anion-channel inhibitors had different effects on Al-induced secretion of organic acids: 9-anthracene carboxylic acid completely inhibited secretion, phenylglyoxal had no effect. Root elongation in L. bicolor was more severely inhibited by Al in the presence of 9-anthracene carboxylic acid. All these results indicated that the secretion of malate and citrate is a specialized response to Al stress in L. bicolor roots, which might be one of the Al-resistance mechanisms in this species.     

Possible involvement of protein phosphorylation in aluminum-responsive malate efflux from wheat root apex

In many plants, efflux of organic anions from roots has been proposed as one of the major Al resistance mechanisms. However it remains unknown how plants regulate efflux of organic anions in response to Al. In this study, the regulatory mechanisms of Al-responsive malate efflux in wheat (Triticum aestivum) were characterized focusing on the role of protein phosphorylation. Al-resistant wheat (cv Atlas) initiated malate efflux at 5 min after addition of Al, and this response was sensitive to temperature. K-252a, a broad range inhibitor of protein kinases, effectively blocked the Al-induced malate efflux accompanied with an increased accumulation of Al and intensified Al-induced root growth inhibition. A transient activation of a 48-kD protein kinase and an irreversible repression of a 42-kD protein kinase were observed preceding the initiation of malate efflux, and these changes were canceled by K-252a. Malate efflux was accompanied with a rapid decrease in the contents of organic anions in the root apex, such as citrate, succinate, and malate but with no change in the contents of inorganic anions such as chloride, nitrate, and phosphate. These results suggest that protein phosphorylation is involved in the Al-responsive malate efflux in the wheat root apex and that the organic anion-specific channel might be a terminal target that responds to Al signaling mediated by phosphorylation.     

Differential Al resistance and citrate secretion in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

While barley ( Hordeum vulgare L.) is the most sensitive species to Al toxicity among small-grain crops, variation in Al resistance between cultivars does exist. We examined the mechanism responsible for differential Al resistance in 21 barley varieties. Citrate was secreted from the roots in response to Al stress. A positive correlation between citrate secretion and Al resistance [(root elongation with Al)/(root elongation without Al)] and a negative correlation between citrate secretion and Al content of root apices, were obtained, suggesting that citrate secretion from the root apices plays an important role in excluding Al and thereby detoxifying Al. The Al-induced secretion of citrate was characterized using an Al-resistant variety (Sigurdkorn) and an Al-sensitive variety (Kearney). In Sigurdkorn, Al-induced secretion of citrate occurred within 20 min, and the secretion did not increase with increasing external Al concentration. The Al-induced citrate secretion ceased at low temperature (6 degrees C) and was inhibited by anion-channel inhibitors. Internal citrate content of root apices was increased by Al exposure in Sigurdkorn, but was not affected in Kearney. The activity of citrate synthase was unaffected by Al in both Al-resistant and Al-sensitive varieties. The secretion rate of organic acid anions from barley was the lowest among wheat, rye and triticale.     

Recent progress in the research of external Al detoxification in higher plants: a minireview

Aluminum (Al) is highly toxic to plant growth. The toxicity is characterized by rapid inhibition of root elongation. However, some plant species and cultivars have evolved some mechanisms for detoxifying Al both internally and externally. In this review, the recent progress made in the research of external detoxification of Al is described. Accumulating evidence has shown that organic acids play an important role in the detoxification of Al. Some plant species and cultivars respond to Al by secreting citrate, malate or oxalate from the roots. Recently, the anion channel of malate and citrate in the plasma membrane has been characterized and a gene encoding the malate channel has been cloned. The metabolism of organic acids seems to be poorly correlated with the Al-induced secretion of organic acid anions. A number of QTLs (quantitative trait loci) for Al resistance have been identified in rice, Arabidopsis, and other species. Transgenic plants with enhanced resistance to Al have also been reported, but introduction of multiple genes may be required to gain high Al resistance in future.     

The role of phosphorus in aluminium-induced citrate and malate exudation from rape (Brassica napus)

Exudation of organic anions is believed to be a common tolerance mechanism for both aluminium toxicity and phosphorus deficiency. Nevertheless, which of these stresses that actually elicit the exudation of organic anions from rape ( Brassica napus L) remains unknown, and the combined effects of Al toxicity and P deficiency on rape have not been reported before. Therefore, in the current study, Brassica napus var. Natane nourin plants grown with or without 0.25 m M P were exposed to 0 or 50 µ M AlCl₃ and several parameters related to the exudation of organic anions from the roots were investigated. Eight days of P deficiency resulted in a significant growth reduction, but P deficiency alone did not induce exudation of organic anions. In contrast, Al strongly induced organic acid exudation, while simultaneously inhibiting root growth. Increased in-vitro activity of citrate synthase (CS, EC 4.1.3.7), malate dehydrogenase (MDH, EC 1.1.1.37) and phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), together with reduced root respiration, indicated that the Al-induced accumulation and subsequent exudation of citrate and malate were associated with both increased biosynthesis and reduced metabolism of citric and malic acid. Phosphorus-sufficient plants showed more pronounced aluminium-induced accumulation and exudation of organic anions than P-deficient plants. A divided root chamber experiment showed the necessity of direct contact between Al and roots to elicit exudation of organic anions. Prolonged exposure (10 days) to Al resulted in a decrease in the net exudation of citrate and malate, and the rate of decrease was much more rapid in P-deficient plants than in P-sufficient plants. It is concluded that P nutrition affects the level of Al-induced synthesis and exudation of organic anions. However, the mechanism needs further investigation.     

Comparative studies on the effect of a protein-synthesis inhibitor on aluminium-induced secretion of organic acids from Fagopyrum esculentum Moench and Cassia tora L. roots

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 microM, with or without exposure to 100 microm Al, but the secretion of citrate was completely inhibited by 30 microM CHM in C. tora. A time-course experiment showed that even prolonged exposure to 20 microM 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 microM 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 microM CHM. A 3-h pulse with 20 microM 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 microM 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 microM 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.     

Effect of phosphorus deficiency on aluminium-induced citrate exudation in soybean (Glycine max)

Aluminium (Al) toxicity or phosphorus (P) deficiency can induce exudation of organic acids from the roots of some plants, which is believed to be a tolerance mechanism against Al toxicity or P deficiency. In the present study, the effect of P deficiency on Al-induced citrate exudation was investigated in three soybean varieties differing in low-P tolerance. P starvation alone failed to induce secretion of organic acids from all three soybean varieties. However, P deficiency altered Al-induced citrate exudation over time, showing a complex interaction. Short × term P starvation (4 days) produced up to 50% increase in Al-induced citrate secretion, while longer-term (10 days) starvation reduced Al-induced citrate secretion to trace amounts. However, after a further 1 day in complete nutrient solution for recovery, Al-induced citrate exudation from the recovered roots was approximately 6 times higher than that from the continuously P-starved plants, but still approximately 3.6 times lower than that from the P-sufficient control. With increasing P or Al supply, Al-induced citrate exudation increased, while Al accumulation in soybean roots decreased in parallel with the decrease of P supply. The photosynthetic rate, stomatal conductance and transpiration were decreased by P deficiency, whereas the intracellular CO₂ concentration was increased. These findings indicate that P nutrition has a significant effect on Al-induced citrate exudation and Al accumulation in soybean root apices.     

Aluminum tolerance genes on the short arm of chromosome 3R are linked to organic acid release in triticale

Triticale, a hybrid between wheat and rye, shows a high degree of Al tolerance that is inherited from rye, but the mechanisms of high Al tolerance in both rye and triticale are unknown. We found that the short arm of chromosome 3R carries genes necessary for Al tolerance in triticale (x Triticosecale Wittmark cv Currency). Detailed comparative studies with a 3DS.3RL translocation line (ST22) and a non-substitution line (ST2) were conducted. Root elongation was similarly inhibited by Al in ST2 and ST22 during the first 12 h of Al treatment, but more strongly in ST22 than in ST2 at 18 h and thereafter. The root inhibition induced by other metals (Cu, Cd, and La) was similar between ST2 and ST22, suggesting that the action of the genes for Al tolerance on the short arm of triticale chromosome 3R is highly specific to Al. A 2-fold larger amount of malate and citrate was released from the roots of ST2 than from ST22 at 12 and 18 h after Al treatment, respectively. The marked lag phase in the inhibition of root elongation and the release of organic acids implies that the expression of genes on the short arm of triticale chromosome 3R is induced by Al, and that these genes are necessary for the release of organic acids.     

Possible Involvement of Al-Induced Electrical Signals in Al Tolerance in Wheat

The relationship between Al-induced depolarization of root-cell transmembrane electrical potentials (Em) and Al tolerance in wheat (Triticum aestivum L.) was investigated. Al exposure induced depolarizations of Em in the Al-tolerant wheat cultivars Atlas and ET3, but not in the Al-sensitive wheat cultivars Scout and ES3. The depolarizations of Em occured in root cap cells and as far back as 10 mm from the root tip. The depolarization was specific to Al3+; no depolarization was observed when roots were exposed to the rhizotoxic trivalent cation La3+. The Al-induced depolarization occurred in the presence of anion-channel antagonists that blocked the release of malate, indicating that the depolarization is not due to the electrogenic efflux of malate2-. K+-induced depolarizations in the root cap were of the same magnitude as Al-induced depolarizations, but did not trigger malate release, indicating that Al-induced depolarization of root cap cell membrane potentials is probably linked to, but is not sufficient to trigger, malate release.     

Nitric oxide protects sour pummelo (Citrus grandis) seedlings against aluminum-induced inhibition of growth and photosynthesis

Limited data are available on the amelioration of nitric oxide (NO) on aluminum (Al)-toxicity. Sour pummelo (Citrus grandis) seedlings were irrigated for 18 weeks with nutrient solution containing 0 and 1.2 mM AlCl₃·6H₂O × 0 and 10 μM sodium nitroprusside (SNP, an NO donor). Under Al stress, SNP increased root phosphorus (P) and Al, but decreased shoot Al. Al decreased photosynthesis, maximum quantum yield of primary photochemistry (F_v/F_m) and total performance index (PI_tot,sbs), but increased inactivation of oxygen-evolving complex (OEC), K-band and relative variable fluorescence at I-steps (V_I). SNP alleviated Al-induced changes for all these parameters. SNP stimulated Al-induced secretion of malate and citrate by excised roots from Al-treated seedlings, while Al did not increase their contents in roots. Antioxidant system in leaves and roots was up- and down-regulated by Al, respectively. SNP prevented Al-induced accumulation of malondialdehyde (MDA) in roots and leaves. In conclusion, SNP alleviates Al-induced inhibition of growth and impairment of the whole photosynthetic electron transport chain. This occurs through increasing Al-immobilization and P level in roots and Al-induced secretion of malate and citrate from roots, and decreasing Al accumulation in shoots. Thus, the decrease of photosynthesis is prevented. Increased P level and Al-immobilization in roots through SNP may be effected through enhanced secretion of malate and citrate.     

Influence of aluminum and phosphorus on growth and xylem sap composition in Melastoma malabathricum L.

Melastoma (Melastoma malabathricum L.) is an aluminum-accumulating woody plant that accumulates more than 10 000 mg kg^–1 of aluminum (Al) in mature leaves. The influence of Al and phosphorus (P) applications on plant growth and xylem sap was examined in the present study in order to elucidate the interaction between Al-induced growth enhancement and P nutrition, and to determine the form of Al for translocation from roots to shoots. Although the Al application significantly increased the growth of Melastomaseedlings with the high P pre-treatment, and P concentrations in the leaves and Pi concentrations in the xylem sap regardless of the P pre-treatment, we could not come to the conclusion that a primary cause of the Al-induced growth enhancement in Melastoma is the stimulation of P uptake. The degree of Al-induced growth enhancement corresponded not with the P concentrations but with the Al concentrations in the plant tissue, suggesting that the Al-induced growth enhancement in Melastoma is primarily caused by Al itself in the plant tissue rather than by the stimulation of P uptake. Through the analysis of organic acids and Al in the xylem sap and plant tissue, the form of Al for translocation from roots to shoots was shown to be an Al-citrate complex that was transformed into Al-oxalate complex for Al storage in the leaves. In addition, the xylem sap of Melastoma seedlings grown in the absence of Al contained higher concentrations of malate. In the presence of Al, however, higher concentrations of citrate were found, indicating that Melastoma changes its organic acid metabolism in the presence or absence of Al; more specifically, it increases the synthesis of citrate.     

Secretion of citrate from roots in response to aluminum and low phosphorus stresses in Stylosanthes

Excess aluminum (Al) ions and phosphorus (P) deficiency are the key factors that limit plant growth in acid soils. Secretion of organic acids (OA) from roots has been proposed as an Al-resistance mechanism. Nonetheless, the correlation between Al resistance and this mechanism has not been tested beyond a very small number of Al-resistant and Al-sensitive genotypes. To elucidate the mechanisms responsible for plant adaptability to acid soils, we studied the secretion of OA from roots of Stylosanthes in response to high-Al and low-P stresses using six different genotypes. Relative root inhibition by 50?µM Al ranged from 25–71% and differed significantly among six Stylosanthes genotypes. Al treatment induced the secretion of citrate from the roots of Stylosanthes seedling in a dose- and time-dependent manner. Moreover, the secretion rate was significantly higher in the Al-resistant genotype. On the other hand, inhibition of Al-induced citrate secretion by phenylisothiocyanate or 9-anthracenecarboxylic acid resulted in an increase in Al content in Stylosanthes root apices. P deficiency also induced citrate secretion from Stylosanthes seedling roots. Furthermore, citrate secretion was much more robust with exposure to both excess-Al and P-deficiency stresses than under either stress alone. Unlike Al-induced citrate secretion, which was rapid, low-P-induced secretion was a slow process, with significant increases in secretion only becoming evident after 6 d of treatment with free phosphate. The lag between treatment with Al and citrate secretion was approximately 4 h. These results suggest that the secretion of citrate is a mechanism for resistance to both excess-Al and low-P stresses in Stylosanthes.     

Characterization of aluminium-induced citrate secretion in aluminium-tolerant soybean (Glycine max) plants

Recently, we showed that secretion of citrate in an aluminium (Al) tolerant cultivar soybean (Glycine max) (cv. Suzunari) is a specific response to Al stress [Yang et al. (2000) Physiol Plant 110: 72–77]. Here we investigated the intrinsic mechanisms behind the secretion of citrate induced by Al. The amount of citrate secreted during the 24‐h Al treatment period increased with increasing concentration of Al (0–70 μM). We analysed citrate secretion basically under 3 conditions: (1) by varying light‐exposure, (2) with intact or excised shoots and (3) by using a divided chamber technique. Further, the content of organic acids in the tissue and the activity of enzymes involved in organic acid metabolism were analysed and evaluated. The results indicate that high rate of citrate secretion in soybean requires a 4‐h induction period. Al had a continuous effect on the citrate secretion when Al was removed from the treatment solution. Citrate secretion increased steadily under exposure to continuous light. However, when the shoots were excised the citrate secretion rate dropped to 3–6 times that of their control counterparts. Results of root manipulation experiments revealed that citrate secretion required the direct contact of Al. In other words, only the Al‐treated root portions secreted citrate. All these observations suggest that the shoots play a role in Al‐induced citrate secretion. Although shoots may not supply citrate for the secretion upon Al treatment, it seems that they may provide the carbon source and/or energy for citrate synthesis in the root. On the other hand, the root organic acid content (1‐cm apex) indicated that malate might contribute to citrate secretion by keeping the balance between citrate synthesis and release in the root apices. Quantification of enzymes involved in organic acid metabolism showed only a 16% increase in citrate synthase activity upon Al treatments (6 h) with no differences in other enzymes. Hence, we could not rule out completely the potential contribution of citrate from shoots and the results are discussed in the light of shoots contributing either energy or citrate itself for enhanced citrate secretion in the Al‐tolerant plant roots.     

Salicylic acid-induced aluminum tolerance by modulation of citrate efflux from roots of<Emphasis Type="Italic"> Cassia tora</Emphasis> L.

Aluminum-induced exudation of organic acids from roots has been proposed as a mechanism for Al tolerance in plants. To better understand the regulatory process leading to efflux of organic acids, the possible involvement of salicylic acid (SA) in regulating Al-induced citrate release in Cassia tora L. was identified. The response of citrate efflux to exogenous SA was concentration-dependent. Application of SA at 5 microM in solution containing 20 microM Al increased citrate efflux to levels 1.76-fold higher than in controls (20 microM Al alone). However, inhibition of citrate release was observed when SA concentrations increased to more than 20 microM. Increased citrate efflux due to the SA treatment was associated with decreased inhibition of root growth and Al content in root tips, suggesting that exogenous SA could confer Al tolerance by increasing citrate efflux. We also examined citrate synthase activities (EC 4.1.3.7) and citrate concentrations in root tips exposed to Al and/or SA. However, both citrate synthase activities and citrate accumulation remained unaffected. These results indicate that SA-promotion of Al-induced citrate efflux is not correlated with increase in citrate production. Total endogenous SA concentrations were measured in root tips and the SA concentrations were significantly enhanced by Al at levels of 10-50 microM.     

Aluminium triggers malate-independent potassium release via ion channels from the root apex in wheat

The regulatory mechanisms for the aluminium (Al)-induced efflux of K(+) and malate from the root apex of Al-resistant wheat ( Triticum aestivum L. cv. Atlas) were characterized. Treatment with 20 mM tetraethylammonium (TEA) chloride, a K(+)-channel inhibitor, blocked the Al-induced K(+) efflux by 65%, but blocked the Al-induced malate efflux only slightly. Lanthanum (La(3+)) or ytterbium (Yb(3+)) strongly inhibited the K(+) efflux, but slightly increased malate efflux. These lanthanides applied together with Al did not affect the Al-induced malate efflux, but reduced the Al-induced K(+) efflux by 57% for La(3+) and by 35% for Yb(3+). By contrast, pretreatment with 50 microM niflumic acid, an anion-channel inhibitor, strongly suppressed the Al-induced malate efflux, but did not affect the Al-induced K(+) efflux. The efflux of K(+) uncoupled with that of malate resulted in the alkalization of intracellular pH in the root apex, suggesting that the release of K(+) coupled with malate plays an important role in stabilizing intracellular pH. Copper (Cu(2+)) induced the release of K(+) via a TEA-insensitive pathway without the release of malate in both Al-resistant and Al-sensitive (cultivar Scout) wheat. Simultaneous application of Al and Cu(2+) to the root apices resulted in TEA-sensitive K(+) efflux in Atlas but not in Scout, suggesting that Al competes with Cu(2+) for K(+) efflux. Taken together, these results suggest that Al-induced K(+) efflux is mediated by both TEA- and lanthanide-sensitive K(+) channels, although this induction is not a prerequisite for the induction of the release of malate.     

The role of organic acids in the short- and long-term aluminum tolerance in maize seedlings (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

Aluminum (Al) affects numerous physiological processes in plants. However, Al tolerance mechanisms mediated by increased synthesis of organic acids (OAs) have been outlined recently. In this study, we examined the role of OAs in the short (1–8 h) and long-term (4 days) Al tolerance in maize seedlings. Exposure to Al stress for 4 days results in a rapid inhibition of root growth. Al induced morphological changes in the maize roots, especially at a higher solution of Al concentration (1,000 μM Al). The increase in Al accumulation in roots, including strongly elevated levels of Al accumulated in root cell walls suggests that Al tolerance in maize is mediated in part by higher accumulation of Al in the roots. The enhanced citrate exudation, which was only observed at 1,000 μM Al may lead to detoxification of Al by formation of OA–Al complexes in the root apoplast. This mechanism has been suggested to play a significant role in Al resistance response in maize. The short-term responses underlying internal detoxification via OA-chelators were also investigated. Succinate, malate, citrate and total root OA contents decreased markedly, 2 h after the Al exposure. At 4 and 8 h time points, OA contents increased or remained unchanged, except for that of malate which decreased. The level of OAs in shoots, on the other hand, showed alterations that were less pronounced in response to Al. Specifically, the citrate and total OA concentrations significantly increased at 4 h, but showed a pronounced decrease at the 8 h time point. Based on our findings, we propose that multiple responses, including Al exclusion by Al accumulation in root cells and citrate efflux, may contribute towards higher Al resistance in maize. The rapid OA changes in responses to short-term Al treatment may not be responsible for Al tolerance. However, increased OA synthesis observed in this study may be involved in diminishing the stress triggered by Al. The molecular aspects underlying Al resistance mechanism via Al-induced expression of the enzymes catalyzing OA synthesis and metabolism remain to be elucidated.