Secretion Of Organic Acid Anions Research Articles

The LRR receptor-like kinase ALR1 is a plant aluminum ion sensor

Plant survival requires an ability to adapt to differing concentrations of nutrient and toxic soil ions, yet ion sensors and associated signaling pathways are mostly unknown. Aluminum (Al) ions are highly phytotoxic, and cause severe crop yield loss and forest decline on acidic soils which represent ∼30% of land areas worldwide. Here we found an Arabidopsis mutant hypersensitive to Al. The gene encoding a leucine-rich-repeat receptor-like kinase, was named Al Resistance1 (ALR1). Al ions binding to ALR1 cytoplasmic domain recruits BAK1 co-receptor kinase and promotes ALR1-dependent phosphorylation of the NADPH oxidase RbohD, thereby enhancing reactive oxygen species (ROS) generation. ROS in turn oxidatively modify the RAE1 F-box protein to inhibit RAE1-dependent proteolysis of the central regulator STOP1, thus activating organic acid anion secretion to detoxify Al. These findings establish ALR1 as an Al ion receptor that confers resistance through an integrated Al-triggered signaling pathway, providing novel insights into ion-sensing mechanisms in living organisms, and enabling future molecular breeding of acid-soil-tolerant crops and trees, with huge potential for enhancing both global food security and forest restoration.

Loss of LaMATE impairs isoflavonoid release from cluster roots of phosphorus-deficient white lupin.

White lupin (Lupinus albus L.) forms brush-like root structures called cluster roots under phosphorus-deficient conditions. Clusters secrete citrate and other organic compounds to mobilize sparingly soluble soil phosphates. In the context of aluminum toxicity tolerance mechanisms in other species, citrate is released via a subgroup of MATE/DTX proteins (multidrug and toxic compound extrusion/detoxification). White lupin contains 56 MATE/DTX genes. Many of these are closely related to gene orthologs with known substrates in other species. LaMATE is a marker gene for functional, mature clusters and is, together with its close homolog LaMATE3, a candidate for the citrate release. Both were highest expressed in mature clusters and when expressed in oocytes, induced inward-rectifying currents that were likely carried by endogenous channels. No citrate efflux was associated with LaMATE and LaMATE3 expression in oocytes. Furthermore, citrate secretion was largely unaffected in P-deficient composite mutant plants with genome-edited or RNAi-silenced LaMATE in roots. Moderately lower concentrations of citrate and malate in the root tissue and consequently less organic acid anion secretion and lower malate in the xylem sap were identified. Interestingly, however, less genistein was consistently found in mutant exudates, opening the possibility that LaMATE is involved in isoflavonoid release.

Interactive effects of pH and aluminum on the secretion of organic acid anions by roots and related metabolic factors in Citrus sinensis roots and leaves

Low pH and aluminum (Al)-toxicity often coexist in acidic soils. Citrus sinensis seedlings were treated with nutrient solution at a pH of 2.5, 3.0, 3.5 or 4.0 and an Al concentration of 0 or 1 mM for 18 weeks. Thereafter, malate, citrate, isocitrate, acid-metabolizing enzymes, and nonstructural carbohydrates in roots and leaves, and release of malate and citrate from roots were measured. Al concentration in roots and leaves increased under Al-toxicity, but it declined with elevating nutrient solution pH. Al-toxicity increased the levels of glucose, fructose, sucrose and total soluble sugars in leaves and roots at each given pH except for a similar sucrose level at pH 2.5–3.0, but it reduced or did not alter the levels of starch and total nonstructural carbohydrates (TNC) in leaves and roots with the exception that Al improved TNC level in roots at pH 4.0. Levels of nonstructural carbohydrates in roots and leaves rose with reducing pH with a few exceptions with or without Al-toxicity. A potential model for the possible role of root organic acid (OA) metabolism (anions) in C. sinensis Al-tolerance was proposed. With Al-toxicity, the elevated pH upregulated the OA metabolism, and increased the flow of carbon to OA metabolism, and the accumulation of malate and citrate in roots and subsequent release of them, thus reducing root and leaf Al and hence eliminating Al-toxicity. Without Al-toxicity, low pH stimulated the exudation of malate and citrate, an adaptive response of Citrus to low pH. The interactive effects of pH and pH on OA metabolism were different between roots and leaves.

Mechanisms and regulation of aluminum-induced secretion of organic acid anions from plant roots.

Aluminum (Al) is the most abundant metal element in the earth's crust. On acid soils, at pH 5.5 or lower, part of insoluble Al-containing minerals become solubilized into soil solution, with resultant highly toxic effects on plant growth and development. Nevertheless, some plants have developed Al-tolerance mechanisms that enable them to counteract this Al toxicity. One such well-documented mechanism is the Al-induced secretion of organic acid anions, including citrate, malate, and oxalate, from plant roots. Once secreted, these anions chelate external Al ions, thus protecting the secreting plant from Al toxicity. Genes encoding the citrate and malate transporters responsible for secretion have been identified and characterized, and accumulating evidence indicates that regulation of the expression of these transporter genes is critical for plant Al tolerance. In this review, we outline the recent history of research into plant Al-tolerance mechanisms, with special emphasis on the physiology of Al-induced secretion of organic acid anions from plant roots. In particular, we summarize the identification of genes encoding organic acid transporters and review current understanding of genes regulating organic acid secretion. We also discuss the possible signaling pathways regulating the expression of organic acid transporter genes.

Phosphorus application reduces aluminum toxicity in two Eucalyptus clones by increasing its accumulation in roots and decreasing its content in leaves.

Under acidic conditions, aluminum (Al) toxicity is an important factor limiting plant productivity; however, the application of phosphorus (P) might alleviate the toxic effects of Al. In this study, seedlings of two vegetatively propagated Eucalyptus clones, E. grandis × E. urophylla ‘G9’ and E. grandis × E. urophylla ‘DH32-29’were subjected to six treatments (two levels of Al stress and three levels of P). Under excessive Al stress, root Al content was higher, whereas shoot and leaf Al contents were lower with P application than those without P application. Further, Al accumulation was higher in the roots, but lower in the shoots and leaves of G9 than in those of DH32-29. The secretion of organic acids was higher under Al stress than under no Al stress. Further, under Al stress, the roots of G9 secreted more organic acids than those of DH32-29. With an increase in P supply, Al-induced secretion of organic acids from roots decreased. Under Al stress, some enzymes, including PEPC, CS, and IDH, played important roles in organic acid biosynthesis and degradation. Thus, our results indicate that P can reduce Al toxicity via the fixation of elemental Al in roots and restriction of its transport to stems and leaves, although P application cannot promote the secretion of organic acid anions. Further, the higher Al-resistance of G9 might be attributed to the higher Al accumulation in and organic acid anion secretion from roots and the lower levels of Al in leaves.

Physiological and Molecular Analysis of Aluminium-Induced Organic Acid Anion Secretion from Grain Amaranth (Amaranthus hypochondriacus L.) Roots.

Grain amaranth (Amaranthus hypochondriacus L.) is abundant in oxalate and can secrete oxalate under aluminium (Al) stress. However, the features of Al-induced secretion of organic acid anions (OA) and potential genes responsible for OA secretion are poorly understood. Here, Al-induced OA secretion in grain amaranth roots was characterized by ion charomatography and enzymology methods, and suppression subtractive hybridization (SSH) together with quantitative real-time PCR (qRT-PCR) was used to identify up-regulated genes that are potentially involved in OA secretion. The results showed that grain amaranth roots secrete both oxalate and citrate in response to Al stress. The secretion pattern, however, differs between oxalate and citrate. Neither lanthanum chloride (La) nor cadmium chloride (Cd) induced OA secretion. A total of 84 genes were identified as up-regulated by Al, in which six genes were considered as being potentially involved in OA secretion. The expression pattern of a gene belonging to multidrug and toxic compound extrusion (MATE) family, AhMATE1, was in close agreement with that of citrate secretion. The expression of a gene encoding tonoplast dicarboxylate transporter and four genes encoding ATP-binding cassette transporters was differentially regulated by Al stress, but the expression pattern was not correlated well with that of oxalate secretion. Our results not only reveal the secretion pattern of oxalate and citrate from grain amaranth roots under Al stress, but also provide some genetic information that will be useful for further characterization of genes involved in Al toxicity and tolerance mechanisms.

Erratum to: Possible involvement of heterotrimeric G-protein signaling in Al-induced secretion of organic acid anions in Arabidopsis and rye

Erratum to: Possible involvement of heterotrimeric G-protein signaling in Al-induced secretion of organic acid anions in Arabidopsis and rye

Possible involvement of heterotrimeric G-protein signaling in Al-induced secretion of organic acid anions in Arabidopsis and rye

Secretion of organic acid anions (OAs) from the roots in response to aluminum (Al) is a common mechanism for Al resistance in plants. Transporter genes involved in the Al-induced secretion of malate and citrate have been identified, but the signal cascades mediating the regulation of the secretion of OAs has not been understood. Here, we investigated the possible role of heterotrimeric G-proteins in the Al-induced secretion of OAs. Seedlings of rye and Arabidopsis thaliana were grown in an environment-controlled growth room. Organic acid anions in the exudates of roots were determined spectrophotometrically. Heterotrimeric G-proteins and GTPase activity were assayed using protein gel blot analyses with Gα antibody and spectrofluorometer, respectively. GPA1 gene expression level was determined by semi-quantitative RT-PCR. Immunoblotting revealed a heterotrimeric G-protein in the plasma membrane fraction of rye and Arabidopsis roots. Exposure of the rye roots to Al resulted in activation of GTPase. Al-induced secretion was inhibited by pertussis toxin, a heterotrimeric G-protein antagonist, but was stimulated by cholera toxin, a heterotrimeric G-protein agonist. Furthermore, Al-induced OA secretion was blocked in Gα- or Gβ-deficient mutants of Arabidopsis. Al induced GPA1 expression in Arabidopsis roots. Heterotrimeric G-proteins-coordinated transduction of Al-stress signals may be associated with the secretion of OAs from roots.

Brassica oleracea MATE encodes a citrate transporter and enhances aluminum tolerance in Arabidopsis thaliana.

The secretion of organic acid anions from roots is an important mechanism for plant aluminum (Al) tolerance. Here we report cloning and characterizing BoMATE (KF031944), a multidrug and toxic compound extrusion (MATE) family gene from cabbage (Brassica oleracea). The expression of BoMATE was more abundant in roots than in shoots, and it was highly induced by Al treatment. The (14)C-citrate efflux experiments in oocytes demonstrated that BoMATE is a citrate transporter. Electrophysiological analysis and SIET analysis of Xenopus oocytes expressing BoMATE indicated BoMATE is activated by Al. Transient expression of BoMATE in onion epidermal cells demonstrated that it localized to the plasma membrane. Compared with the wild-type Arabidopsis, the transgenic lines constitutively overexpressing BoMATE enhanced Al tolerance and increased citrate secretion. In addition, Arabidopsis transgenic lines had a lower K(+) efflux and higher H(+) efflux, in the presence of Al, than control wild type in the distal elongation zone (DEZ). This is the first direct evidence that MATE protein is involved in the K(+) and H(+) flux in response to Al treatment. Taken together, our results show that BoMATE is an Al-induced citrate transporter and enhances aluminum tolerance in Arabidopsis thaliana.

Molecular mechanisms of Al tolerance in gramineous plants

Aluminum (Al) toxicity has limited the productivity and expansion of cereal crops on acid soils; however, a number of plant species or cultivars have developed different strategies for detoxifying aluminum both internally and externally. This review focuses on recent progress on molecular mechanisms of Al tolerance in gramineous plants. A common mechanism in all gramineous plants is the secretion of organic acid anions (citrate and malate) from the roots. Genes belonging to ALMT (for Aluminum-activated malate transporter) and MATE (Multidrug and toxic compound extrusion) family involved in the secretion have been identified in several plant species; however, different plant species show different gene expression patterns including Al-induction, spatial and temporal expression, and tissue localization. Furthermore, the mechanisms regulating the gene expression also differ with plant species, which are achieved by increased tandem repeated element, increase of copy number, insertion of transposon, or alteration of cis-acting element. In addition to these common Al exclusion mechanisms, rice as a highly Al-tolerant species has developed a number of other mechanisms for detoxification of Al. A transcription factor for Al tolerance ART1 identified in rice regulates at least 30 genes implicated in internal and external detoxification of Al. These multiple genes may contribute to the high Al tolerance of rice. In the future, regulation mechanisms of Al-tolerance genes need to be further investigated.

Adaptation to acidic soil is achieved by increased numbers of cis‐acting elements regulating ALMT1 expression in Holcus lanatus

Yorkshire fog (Holcus lanatus), which belongs to the Poaceae family and is a close relative of the agronomic crop oat (Avena sativa), is a widely adaptable grass species that is able to grow on highly acidic soils with high levels of Al, but the mechanism underlying the high Al tolerance is unknown. Here, we characterized two accessions of H.lanatus collected from an acid plot (soil pH 3.6, HL-A) and a neutral plot (pH 7.1, HL-N) in terms of Al tolerance, organic acid anion secretion and related gene expression. In response to Al (pH 4.5), the HL-A roots secreted approximately twice as much malate as the HL-N roots, but there was no difference in citrate secretion. Cloning of the gene HlALMT1 responsible for malate secretion showed that the encoded amino acid sequence did not differ between two accessions, but the expression level in the outer cell layers of the HL-A roots was twice as high as in the HL-N roots. This difference was not due to the genomic copy number, but was due to the number of cis-acting elements for an Al-responsive transcription factor (HlART1) in the promoter region of HlALMT1, as demonstrated by both a yeast one-hybrid assay and a transient assay in tobacco protoplasts. Furthermore, introduction of HlALMT1 driven by the HL-A promoter into rice resulted in significantly more Al-induced malate secretion than introduction of HlALMT1 driven by the HL-N promoter. These findings indicate that the adaptation of H.lanatus to acidic soils may be achieved by increasing number of cis-acting elements for ART1 in the promoter region of the HlALMT1 gene, enhancing the expression of HlALMT1 and the secretion of malate.

Roles of organic acid anion secretion in aluminium tolerance of higher plants.

Approximately 30% of the world's total land area and over 50% of the world's potential arable lands are acidic. Furthermore, the acidity of the soils is gradually increasing as a result of the environmental problems including some farming practices and acid rain. At mildly acidic or neutral soils, aluminium(Al) occurs primarily as insoluble deposits and is essentially biologically inactive. However, in many acidic soils throughout the tropics and subtropics, Al toxicity is a major factor limiting crop productivity. The Al-induced secretion of organic acid (OA) anions, mainly citrate, oxalate, and malate, from roots is the best documented mechanism of Al tolerance in higher plants. Increasing evidence shows that the Al-induced secretion of OA anions may be related to the following several factors, including (a) anion channels or transporters, (b) internal concentrations of OA anions in plant tissues, (d) temperature, (e) root plasma membrane (PM) H+-ATPase, (f) magnesium (Mg), and (e) phosphorus (P). Genetically modified plants and cells with higher Al tolerance by overexpressing genes for the secretion and the biosynthesis of OA anions have been obtained. In addition, some aspects needed to be further studied are also discussed.

Mechanisms of aluminum-tolerance in two species of citrus: Secretion of organic acid anions and immobilization of aluminum by phosphorus in roots

‘Sour pummelo’ (Citrus grandis) and ‘Xuegan’ (C. sinensis) seedlings were irrigated for 18 weeks with nutrient solution containing 0 (−Al) and 1.2mM AlCl3·6H2O (+Al)×0, 50 and 200μM KH2PO4. C. sinensis was more tolerant to aluminum (Al) than C. grandis. Phosphorus (P) alleviated the toxic effects of Al on seedlings. Under Al stress, P increased root Al, but decreased shoot Al; C. sinensis accumulated more Al in roots and less Al in shoots than C. grandis. Al decreased root and shoot P. P content was higher in C. sinensis than in C. grandis seedlings. C. sinensis roots secreted more malate and citrate than C. grandis ones in response to Al. Al-induced-secretion of malate and citrate from +Al excised roots was higher than from −Al ones, while Al-preculture did not increase root malate and citrate. Al-induced-secretion of malate and citrate from +Al excised roots decreased with increasing P supply. To conclude, P can alleviate Al-toxicity through increasing immobilization of Al in roots and P level in seedlings rather than through increasing organic acid (OA) anion secretion. The higher Al-tolerance of C. sinensis may involve secretion of OA anions and precipitation of Al by P in roots.

The alleviating effect of ammonium on aluminum toxicity in Lespedeza bicolor results in decreased aluminum-induced malate secretion from roots compared with nitrate

NH 4 + and NO 3 − supply often affect aluminum (Al) toxicity in plants growing in acidic soils. Lespedeza bicolor is a leguminous shrub well adapted to acid infertile soils. Secretion of organic acid anions has been demonstrated to be the main mechanism responsible for its Al resistance. The objective of this study was to investigate how NH 4 + and NO 3 − supply affect Al toxicity in L. bicolor and whether Al-induced organic acid anion secretion is involved in this effect. Long-term (36 d) Al treatment increased L. bicolor root and shoot growth in a NH 4 + medium. NH 4 + treatment also decreased Al, Ca, Mg, Fe, and Mn contents in the roots of L. bicolor compared with NO 3 − treatment. After short-term (24 h) Al treatment, L. bicolor seedlings had higher absolute root elongation and relative root elongation, and lower Al contents in root tips with NH 4 + treatment than with NO 3 − treatment. In vitro Al adsorption kinetics experiment with isolated root cell wall showed that the accumulative amount of Al adsorbed in root cell walls was lower with NH 4 + treatment than with NO 3 − treatment. The roots of L. bicolor seedlings subjected to Al stress secreted less malate with NH 4 + treatment than with NO 3 − treatment, whereas there was no significant difference in the malate content of L. bicolor roots between the treatments after exposure to Al. Taken together, these results show that NH 4 + alleviated the toxicity of Al in L. bicolor in solution culture compared with NO 3 − , and this alleviating effect further resulted in decreased Al-induced malate secretion from L. bicolor roots.

Crop production on acidic soils: overcoming aluminium toxicity and phosphorus deficiency

Crop production on acidic soils: overcoming aluminium toxicity and phosphorus deficiency

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.

Differential Aluminum Resistance and Organic Acid Anions Secretion in Triticale

Triticale (X Triticosecale Wittmack), a hybrid of wheat and rye, shows a high degree of aluminum (Al) tolerance, but variation in Al resistance between cultivars does exist. The mechanisms responsible for differential Al resistance in 10 triticale cultivars were investigated in this study. Triticale roots secreted both malate and citrate in response to Al stress. The amount of organic acid anions secreted was correlated positively to the relative root elongation (an index for Al resistance) and negatively to the Al content in root apices under Al stress, suggesting that the secretion of malate and citrate seems to be involved in the exclusion of Al from root tip. The Al‐induced secretion of malate and citrate was characterized using an Al‐resistant cultivar (ZC 237) and an Al‐sensitive cultivar (OH 1621). Root elongation was significantly inhibited in both ZC 237 and OH 1621 after 24 h of exposure to 30, 50, or 100 µM Al but was more strongly in OH 1621 than in ZC 237 at all Al concentrations tested. A marked lag phase (3 h) between the addition of Al and the secretion of organic acid anions was observed in both triticale cultivars, and the secretion increased with increasing external Al concentration. The two anion‐channel inhibitors, phenylglyoxal and niflumic acid, significantly inhibited the secretion of malate and citrate in ZC 237, with the degree of the inhibition of niflumic acid greater than that of phenylglyoxal. The Al‐induced secretion of malate and citrate decreased to a very low level at low temperature (4°C) in both cultivars. These results indicate that Al‐induced malate and citrate secretion from roots play important roles in excluding Al and thereby detoxifying Al in triticale. The Al‐induced organic acid anions were inhibited by anion‐channel inhibitors and were dependent on temperature.

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.

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.