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.     

Citrate transporters play an important role in regulating aluminum-induced citrate secretion in Glycine max

To further understand the process of Al-induced citrate secretion from soybean roots, the effect of protein synthesis inhibitor, anion channel blockers, and citrate carrier inhibitors on Al-induced citrate exudation was investigated in Al-resistant soybean cultivar PI 416937. Citrate exudation from roots increased with the increase of Al concentration from 10 to 50 μM and initiated after 4 h of Al exposure. Protein synthesis inhibitor, cycloheximide (CHM; 25 μM) completely inhibited Al-induced citrate secretion during 12-h exposure, suggesting that novel protein synthesis was necessary in Al-induced citrate efflux. Also both anion channel blocker anthracene-9-carboxylic acid (A-9-C) and citrate carrier inhibitor mersalyl acid (Mersalyl) significantly reduced citrate secretion, suggesting that both anion channels in plasma membrane and citrate carriers in mitochondria membrane were the rate limiting factors of Al dependent citrate release. However, Al-induced citrate secretion was insensitive to anion channel blockers phenylglyoxal (PG), 4,4′-diisothiocyanostibene-2,2′-disulfonat (DIDS) and citrate carrier inhibitor pyridoxal 5′-P (PP).     

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.     

Molecular mapping of a gene responsible for Al-activated secretion of citrate in barley

Aluminium (Al) toxicity is an important limitation to barley (Hordeum vulgare L.) on acid soil. Al-resistant cultivars of barley detoxify Al externally by secreting citrate from the roots. To link the genetics and physiology of Al resistance in barley, genes controlling Al resistance and Al-activated secretion of citrate were mapped. An analysis of Al-induced root growth inhibition from 100 F2 seedlings derived from an Al-resistant cultivar (Murasakimochi) and an Al-sensitive cultivar (Morex) showed that a gene associated with Al resistance is localized on chromosome 4H, tightly linked to microsatellite marker Bmag353. Quantitative trait locus (QTL) analysis from 59 F4 seedlings derived from an F3 plant heterozygous at the region of Al resistance on chromosome 4H showed that a gene responsible for the Al-activated secretion of citrate was also tightly linked to microsatellite marker Bmag353. This QTL explained more than 50% of the phenotypic variation in citrate secretion in this population. These results indicate that the gene controlling Al resistance on barley chromosome 4H is identical to that for Al-activated secretion of citrate and that the secretion of citrate is one of the mechanisms of Al resistance in barley. The identification of the microsatellite marker associated with both Al resistance and citrate secretion provides a valuable tool for marker-assisted selection of Al-resistant lines.     

Overexpression of MsALMT1, from the Aluminum-Sensitive Medicago sativa, Enhances Malate Exudation and Aluminum Resistance in Tobacco

Aluminum (Al) toxicity is a major limiting factor for plant growth and crop production in acidic soils. Al-induced organic acid (OA) exudation plays an important role in plant Al resistance. The exudation of OAs is mediated by membrane-localized OA transporters. In our previous study, a gene encoding the Al-induced malate transporter (MsALMT1) was identified in the roots of the Al-sensitive plant Medicago sativa L. cv. Yumu no. 1 (YM1). To further validate the function of MsALMT1, transgenic plants that overexpressed MsALMT1 under the control of the CaMV 35S (35S) promoter were generated. This transgenic tobacco showed an enhanced capacity for malate efflux and better Al resistance than wild type (WT) plants after exposure to 30 μM Al for 24 h. The Al content in the transgenic plant roots decreased to 40–52 % of that in WT plant roots. These results demonstrate that MsALMT1 is an Al-resistant gene in YM1 and encodes a malate transporter, the overexpression of which effectively enhances the Al resistance of transgenic tobacco plants.     

Involvement of putrescine and nitric oxide in aluminum tolerance by modulating citrate secretion from roots of red kidney bean

Background and Aims

Polyamines and nitric oxide (NO) are two important molecules modulating numerous environment stresses in plants. This study was to investigate the roles of polyamines and NO in aluminum (Al) tolerance in red kidney bean.

Methods

The interaction between putrescine (Put) and NO under Al stress was examined. NO donor and scavenger were used to further examine the role of NO in Al-induced citrate secretion from roots by high performance liquid chromatography.

Results

Al stress caused increase of endogenous free Put, and exogenous Put alleviated Al-induced inhibition of root elongation and Al accumulation. In addition, Put induced NO production and nitrate reductase (NR) activity under Al stress. Al- and Put-induced NO production could be reversed by NR inhibitor. Furthermore, Al stress stimulated citrate secretion from roots, and this response was stimulated by NO donor, whereas NO scavenger inhibited Al-induced citrate secretion from roots. Concomitantly, NO donor reduced Al accumulation in root apexes, while NO scavenger further enhanced Al accumulation. Al-induced inhibition of root growth was significantly improved by exogenous citrate treatment.

Conclusions

Put and NO enhanced Al tolerance by modulating citrate secretion from roots, and NO may act downstream of Put in red kidney bean under Al stress.     

Aluminum resistance in maize cannot be solely explained by root organic acid exudation. A comparative physiological study

Root apical aluminum (Al) exclusion via Al-activated root citrate exudation is widely accepted as the main Al-resistance mechanism operating in maize (Zea mays) roots. Nonetheless, the correlation between Al resistance and this Al-exclusion mechanism has not been tested beyond a very small number of Al-resistant and Al-sensitive maize lines. In this study, we conducted a comparative study of the physiology of Al resistance using six different maize genotypes that capture the range of maize Al resistance and differ significantly in their genetic background (three Brazilian and three North American genotypes). In these maize lines, we were able to establish a clear correlation between root tip Al exclusion (based on root Al content) and Al resistance. Both Al-resistant genotypes and three of the four Al-sensitive lines exhibited a significant Al-activated citrate exudation, with no evidence for Al activation of root malate or phosphate release. There was a lack of correlation between differential Al resistance and root citrate exudation for the six maize genotypes; in fact, one of the Al-sensitive lines, Mo17, had the largest Al-activated citrate exudation of all of the maize lines. Our results indicate that although root organic acid release may play a role in maize Al resistance, it is clearly not the only or the main resistance mechanism operating in these maize roots. A number of other potential Al-resistance mechanisms were investigated, including release of other Al-chelating ligands, Al-induced alkalinization of rhizosphere pH, changes in internal levels of Al-chelating compounds in the root, and Al translocation to the shoot. However, we were unsuccessful in identifying additional Al-resistance mechanisms in maize. It is likely that a purely physiological approach may not be sufficient to identify these novel Al-resistance mechanisms in maize and this will require an interdisciplinary approach integrating genetic, molecular, and physiological investigations.     

Aluminium tolerance and high phosphorus efficiency helps Stylosanthes better adapt to low-P acid soils

Backgrond and Aims

Stylosanthes spp. (stylo) is one of the most important pasture legumes used in a wide range of agricultural systems on acid soils, where aluminium (Al) toxicity and phosphorus (P) deficiency are two major limiting factors for plant growth. However, physiological mechanisms of stylo adaptation to acid soils are not understood.

Methods

Twelve stylo genotypes were surveyed under field conditions, followed by sand and nutrient solution culture experiments to investigate possible physiological mechanisms of stylo adaptation to low-P acid soils.

Key Results

Stylo genotypes varied substantially in growth and P uptake in low P conditions in the field. Three genotypes contrasting in P efficiency were selected for experiments in nutrient solution and sand culture to examine their Al tolerance and ability to utilize different P sources, including Ca-P, K-P, Al-P, Fe-P and phytate-P. Among the three tested genotypes, the P-efficient genotype ‘TPRC2001-1’ had higher Al tolerance than the P-inefficient genotype ‘Fine-stem’ as indicated by relative tap root length and haematoxylin staining. The three genotypes differed in their ability to utilize different P sources. The P-efficient genotype, ‘TPRC2001-1’, had superior ability to utilize phytate-P.

Conclusions

The findings suggest that possible physiological mechanisms of stylo adaptation to low-P acid soils might involve superior ability of plant roots to tolerate Al toxicity and to utilize organic P and Al-P.Key words: Stylosanthes, phosphorus, P efficiency, organic P, Al toxicity, acid soil     

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.     

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.     

Aluminum resistance of cowpea as affected by phosphorus-deficiency stress

Plants growing in acid soils suffer both phosphorus (P) deficiency and aluminum (Al) toxicity stresses. Selection of genotypes for adaptation to either P deficiency or Al toxicity has sometimes been unsuccessful because these two soil factors often interact. Two experiments were conducted to evaluate eight cowpea genotypes for Al resistance and to study the combined effect of P deficiency and Al toxicity stress on growth, P uptake, and organic acid anion exudation of two genotypes of contrasting Al resistance selected from the first experiment. Relative root inhibition by 30 μM Al ranged from 14% to 60% and differed significantly among the genotypes. Al significantly induced callose formation, particularly in Al-sensitive genotypes. P accumulation was significantly reduced (28% and 95%) by Al application for both the Al-resistant and the Al-sensitive genotypes. Al supply significantly enhanced malate release of root apices of both genotypes. However, the exudation rate was significantly higher in the Al-resistant genotype. P deprivation induced an enhanced malate exudation in the presence of Al only in the Al-resistant genotype IT89KD-391. Citrate exudation rate of the root apices was lower than malate exudation by a factor of about 10, and was primarily enhanced by P deficiency in both genotypes. Al treatment further enhanced citrate exudation in P-sufficient, but not in P-deficient plants. The level of citrate exudation was consistently higher in the Al-resistant genotype IT89KD-391 particularly in presence of Al.It is concluded that the Al-resistant genotype is better adapted to acid Al-toxic and P-deficient soils than the Al-sensitive genotype since both malate and citrate exudation were more enhanced by combined Al and P-deficiency stresses.     

Aluminum-induced citric acid secretion is not the sole mechanism of Al-resistance in maize

Aluminum-induced citric acid (CA) root secretion is a widely accepted mechanism to explain Al-resistance in maize. Nonetheless, several aspects of this mechanism remain controversial. In this study, we used paclobutrazol (PBZ), a plant growth retardant, to gain new insights into the relationship between Δ⁵-sterol composition, membrane permeability, (PM) H⁺-ATPase activity and CA secretion in an Al-sensitive (UFVM-100) and Al-resistant (UFVM-200) maize genotypes challenged with Al. The Al-sensitive genotype displayed greater concentrations of Al in the root tips and greater inhibition of root elongation (RE), which was accompanied by greater electrolyte leakage and greater reduction in the Δ⁵-sterols content after Al treatment. CA secretion by roots increased in both genotypes after Al treatment but to a greater extent in the Al-resistant genotype. The (PM) H⁺-ATPase activity was down-regulated in the sensitive cultivar and up-regulated in its resistant counterpart upon Al treatment. A significant correlation between (PM) H⁺-ATPase activity and CA secretion was observed, but only in the Al-resistant genotype. Upon adding PBZ to the Al-treated plants, differences in the RE and Δ⁵-sterol composition between the maize genotypes were fully abolished, whereas genotypic differences in CA secretion and (PM) H⁺-ATPase activity were reduced but not completely eliminated. Taken together, this information suggests the existence of other processes or mechanisms operating in the Al resistance in these two maize genotypes.     

An aluminum-activated citrate transporter in barley

Soluble ionic aluminum (Al) inhibits root growth and reduces crop production on acid soils. Al-resistant cultivars of barley (Hordeum vulgare L.) detoxify Al by secreting citrate from the roots, but the responsible gene has not been identified yet. Here, we identified a gene (HvAACT1) responsible for the Al-activated citrate secretion by fine mapping combined with microarray analysis, using an Al-resistant cultivar, Murasakimochi, and an Al-sensitive cultivar, Morex. This gene belongs to the multidrug and toxic compound extrusion (MATE) family and was constitutively expressed mainly in the roots of the Al-resistant barley cultivar. Heterologous expression of HvAACT1 in Xenopus oocytes showed efflux activity for (14)C-labeled citrate, but not for malate. Two-electrode voltage clamp analysis also showed transport activity of citrate in the HvAACT1-expressing oocytes in the presence of Al. Overexpression of this gene in tobacco enhanced citrate secretion and Al resistance compared with the wild-type plants. Transiently expressed green fluorescent protein-tagged HvAACT1 was localized at the plasma membrane of the onion epidermal cells, and immunostaining showed that HvAACT1 was localized in the epidermal cells of the barley root tips. A good correlation was found between the expression of HvAACT1 and citrate secretion in 10 barley cultivars differing in Al resistance. Taken together, our results demonstrate that HvAACT1 is an Al-activated citrate transporter responsible for Al resistance in barley.     

A Second Mechanism for Aluminum Resistance in Wheat Relies on the Constitutive Efflux of Citrate from Roots

The first confirmed mechanism for aluminum (Al) resistance in plants is encoded by the wheat (Triticum aestivum) gene, TaALMT1, on chromosome 4DL. TaALMT1 controls the Al-activated efflux of malate from roots, and this mechanism is widespread among Al-resistant genotypes of diverse genetic origins. This study describes a second mechanism for Al resistance in wheat that relies on citrate efflux. Citrate efflux occurred constitutively from the roots of Brazilian cultivars Carazinho, Maringa, Toropi, and Trintecinco. Examination of two populations segregating for this trait showed that citrate efflux was controlled by a single locus. Whole-genome linkage mapping using an F₂ population derived from a cross between Carazinho (citrate efflux) and the cultivar EGA-Burke (no citrate efflux) identified a major locus on chromosome 4BL, Xce_c, which accounts for more than 50% of the phenotypic variation in citrate efflux. Mendelizing the quantitative variation in citrate efflux into qualitative data, the Xce_c locus was mapped within 6.3 cM of the microsatellite marker Xgwm495 locus. This linkage was validated in a second population of F_2:3 families derived from a cross between Carazinho and the cultivar Egret (no citrate efflux). We show that expression of an expressed sequence tag, belonging to the multidrug and toxin efflux (MATE) gene family, correlates with the citrate efflux phenotype. This study provides genetic and physiological evidence that citrate efflux is a second mechanism for Al resistance in wheat.     

Aluminium tolerance is achieved by exudation of citric acid from roots of soybean (Glycine max)

Fourteen soybean ( Glycine max [L.] Merr.) cultivars were analysed and found to differ considerably in aluminium (Al) resistance. The cultivars Suzunari (Al-resistant) and Shishio (Al-sensitive) were selected for further analysis of physiological mechanisms of Al-resistance. The relative root growth of Shishio was 48% compared to 76% for Suzunari in response to 15 μ M Al (24 h). Aluminium accumulation and Al-induced callose formation in root apices were 50 and 25% of that in Suzunari, respectively. Al inhibited both Suzunari and Shishio during the first 6 h of exposure. However, the root growth inhibition was further increased in Shishio but not in Suzunari, suggesting an Al-induced Al-resistant mechanism operating in Suzunari. Organic acid analysis in root exudates of both cultivars revealed that they specifically exuded citrate in response to Al. However, the citrate exudation rate was significantly higher in Suzunari during the 6 h/24 h Al treatment, which was 52/330 compared to Shishio's 26/118 (nmol [g root fresh weight]⁻¹ [6 h]⁻¹), respectively. This Al-induced citric acid exudation was found to be specific for Al, as several other metals failed to induce citrate exudation in both cultivars. Fourteen days of P deficiency did not elicit citrate excretion in both cultivars, while application of Al to P-deficient plants rapidly induced citrate exudation in both cultivars, confirming the specificity of the response of these soybean cultivars to Al. To our knowledge, this is the first report demonstrating an Al-exclusion mechanism in soybean cultivars, which is conferred by enhanced and specific Al-induced exudation of citrate.     

Hydrogen sulfide alleviates aluminum toxicity in barley seedlings

Aims

Aluminum (Al) toxicity is one of the major factors that limit plant growth. Low concentration of hydrogen sulfide (H₂S) has been proven to function in physiological responses to various stresses. The objective of this study is to investigate the possible role of H₂S in Al toxicity in barley (Hordeum vulgare L) seedlings.

Methods

Barley seedlings pre-treated with sodium hydrosulfide (NaHS), a H₂S donor, and subsequently exposed to Al treatment were studied for their effects on root elongation, Al accumulation in seedlings, Al-induced citrate secretion and oxidative stress, and plasma membrane (PM) H⁺-ATPase expression.

Results

Our results showed that H₂S had significant rescue effects on Al-induced inhibition of root elongation which was correlated well with the decrease of Al accumulation in seedlings. Meanwhile, Al-induced citrate secretion was also significantly enhanced by NaHS pretreatment. Al-induced oxidative stress as indicated by lipid peroxidation and reactive oxygen species burst was alleviated by H₂S through the activation of the antioxidant system. Moreover, Al-induced reduction in PM H⁺-ATPase expression was reversed by exogenous NaHS.

Conclusions

Altogether, our results suggest H₂S plays an ameliorative role in protecting plants against Al toxicity by inducing the activities of antioxidant enzymes, increasing citrate secretion and citrate transporter gene expression, and enhancing the expression of PM H⁺-ATPase.     

Effect of anion channel antagonists and La3+ on citrate release, Al content and Al resistance in maize roots

The correlation between organic acid anion release and Al content was examined in two maize (Zea mays L.) inbred lines, Cat 100-6 (Al-resistant) and S 1587-17 (Al-sensitive) treated with anion channel antagonists and La3+, a cation channel blocker. In the intact roots of Al-resistant maize, the Al-induced excretion of citrate was inhibited by the anion channel antagonists niflumic acid, anthracene-9-carboxylic and ethacrinic acid. Citrate release in excised root apices was reduced by 60% in the presence of 15 microM niflumic acid, while the Al content increased by 42%. Nevertheless, Cat 100-6 accumulated less Al than S 1587-17 when the rate of citrate release was similar in both lines, indicating that other mechanisms of Al-resistance are operating in Cat 100-6. The presence of 60 microM La3+ did not change the rate of citrate release, but the Al content in excised root apices of Al-resistant plants was reduced by 70%. These results suggest that the Al distributed uniformly in the roots does not contribute to citrate release and possibly the activity of anion channels is correlated with the free activities of extracellular Al3+ close to anion channels.