Al-induced Citrate Secretion Research Articles

Plasma membrane proteomic analysis by TMT-PRM provides insight into mechanisms of aluminum resistance in tamba black soybean roots tips.

Aluminum (Al) toxicity in acid soil is a worldwide agricultural problem that inhibits crop growth and productivity. However, the signal pathways associated with Al tolerance in plants remain largely unclear. In this study, tandem mass tag (TMT)-based quantitative proteomic methods were used to identify the differentially expressed plasma membrane (PM) proteins in Tamba black soybean (TBS) root tips under Al stress. Data are available via ProteomeXchange with identifier PXD017160. In addition, parallel reaction monitoring (PRM) was used to verify the protein quantitative data. The results showed that 907 PM proteins were identified in Al-treated plants. Among them, compared to untreated plants, 90 proteins were differentially expressed (DEPs) with 46 up-regulated and 44 down-regulated (fold change > 1.3 or < 0.77, p < 0.05). Functional enrichment based on GO, KEGG and protein domain revealed that the DEPs were associated with membrane trafficking and transporters, modifying cell wall composition, defense response and signal transduction. In conclusion, our results highlight the involvement of GmMATE13, GmMATE75, GmMATE87 and H+-ATPase in Al-induced citrate secretion in PM of TBS roots, and ABC transporters and Ca2+ have been implicated in internal detoxification and signaling of Al, respectively. Importantly, our data provides six receptor-like protein kinases (RLKs) as candidate proteins for further investigating Al signal transmembrane mechanisms.

Functional characterization of three MATE genes in relation to aluminum-induced citrate efflux from soybean root

Citrate transporters responsible for Aluminum (Al)-induced citrate efflux have not been identified in soybean. Three soybean multi-drug and toxic compound extrusion (MATE) family genes were characterized by transcriptional expression, subcellular localization and overexpression experiments. GmMATE75, GmMATE79 and GmMATE87 are localized to plasma membrane. Their over-expression respectively resulted in more citrate efflux and less Al contents in soybean hairy roots, alleviated root elongation inhibition in Arabidopsis and partially restored root growth in atmate mutant under Al stress. Al increased their transcriptional expression at either the root apex or the base zone. Cu2+, Cd2+, La3+ increased the expression of GmMATE79 and GmMATE87. Ten day of –Fe culture increased the expression of GmMATE75 and GmMATE79. Al treatment extended β-glucuronidase (GUS) staining from central cylinder to cortical and epidermis cells for pGmMATE75::GUS or pGmMATE79::GUS soybean hairy roots transformation. But GUS staining restricted within central cylinder for pGmMATE87::GUS transformation with or without Al treatment. GmMATE75, GmMATE79 and GmMATE87 are plasma-membrane-localized citrate transporters and have capabilities to increase citrate efflux. They played different role in Al-induced citrate secretion from soybean because their different tissue localization and expression patterns.

Interactions between hydrogen sulphide and nitric oxide regulate two soybean citrate transporters during the alleviation of aluminium toxicity.

Hydrogen sulphide (H2 S) is emerging as an important signalling molecule involved in plant resistance to various stresses. However, the underlying mechanism of H2 S in aluminium (Al) resistance and the crosstalk between H2 S and nitric oxide (NO) in Al stress signalling remain elusive. Citrate secretion is a wide-spread strategy for plants against Al toxicity. Here, two citrate transporter genes, GmMATE13 and GmMATE47, were identified and characterized in soybean. Functional analysis in Xenopus oocytes and transgenic Arabidopsis showed that GmMATE13 and GmMATE47 mediated citrate exudation and enhanced Al resistance. Al treatment triggered H2 S generation and citrate exudation in soybean roots. Pretreatment with an H2 S donor significantly elevated Al-induced citrate exudation, reduced Al accumulation in root tips, and alleviated Al-induced inhibition of root elongation, whereas application of an H2 S scavenger elicited the opposite effect. Furthermore, H2 S and NO mediated Al-induced GmMATE expression and plasma membrane (PM) H+ -ATPase activity and expression. Further investigation showed that NO induced H2 S production by regulating the key enzymes involved in biosynthesis and degradation of H2 S. These findings indicate that H2 S acts downstream of NO in mediating Al-induced citrate secretion through the upregulation of PM H+ -ATPase-coupled citrate transporter cotransport systems, thereby conferring plant resistance to Al toxicity.

Nitric oxide mediates aluminum-induced citrate secretion through regulating the metabolism and transport of citrate in soybean roots

Citrate secretion is a kind of typical strategy for plant against aluminum (Al) toxicity. However, the signaling process in Al-activated citrate secretion needs to be clarified. Physiological and biochemical methods as well as gene expression analysis were employed to examine the regulatory roles of nitric oxide (NO) in Al-activated citrate secretion in soybean roots. Application of NO donor alleviated root growth inhibition and decreased Al content in Al-treated root apices. Al-induced NO production and citrate secretion were further elevated by NO donor, but inhibited by NO scavenger. Inhibition of citrate synthase (CS) or plasma membrane (PM) H+-ATPase activity significantly decreased Al-induced secretion of citrate, but inhibition of aconitase (ACO) activity enhanced citrate secretion under Al stress. Furthermore, NO mediated Al-stimulated CS and PM H+-ATPase activities, but decreased ACO activity under Al stress. Further investigation showed that NO modulated Al-activated transcriptional expression of CS and PM H+-ATPase as well as GmMATE. Overexpression of GmMATE in soybean hairy roots caused an enhanced Al-induced citrate efflux and Al resistance. Our findings suggest that NO-dependent up-regulation of citrate synthesis and activation of PM H+-ATPase-coupled MATE transporter co-transport system participates in Al-activated citrate exudation, thus conferring plant resistance to Al toxicity.

Transcription factor WRKY22 promotes aluminum tolerance via activation of OsFRDL4 expression and enhancement of citrate secretion in rice (Oryza sativa).

Whilst WRKY transcription factors are known to be involved in diverse plant responses to biotic stresses, their involvement in abiotic stress tolerance is poorly understood. OsFRDL4, encoding a citrate transporter, has been reported to be regulated by ALUMINUM (Al) RESISTANCE TRANSCRIPTION FACTOR 1 (ART1) in rice, but whether it is also regulated by other transcription factors is unknown. We define the role of OsWRKY22 in response to Al stress in rice by using mutation and transgenic complementation assays, and characterize the regulation of OsFRDL4 by OsWRKY22 via yeas one-hybrid, electrophoretic mobility shift assay and ChIP-quantitative PCR. We demonstrate that loss of OsWRKY22 function conferred by the oswrky22 T-DNA insertion allele causes enhanced sensitivity to Al stress, and a reduction in Al-induced citrate secretion. We next show that OsWRKY22 is localized in the nucleus, functions as a transcriptional activator and is able to bind to the promoter of OsFRDL4 via W-box elements. Finally, we find that both OsFRDL4 expression and Al-induced citrate secretion are significantly lower in art1 oswrky22 double mutants than in the respective single mutants. We conclude that OsWRKY22 promotes Al-induced increases in OsFRDL4 expression, thus enhancing Al-induced citrate secretion and Al tolerance in rice.

Two citrate transporters coordinately regulate citrate secretion from rice bean root tip under aluminum stress.

Aluminum (Al)-induced organic acid secretion from the root apex is an important Al resistance mechanism. However, it remains unclear how plants fine-tune root organic acid secretion which can contribute significantly to the loss of fixed carbon from the plant. Here, we demonstrate that Al-induced citrate secretion from the rice bean root apex is biphasic, consisting of an early phase with low secretion and a later phase of large citrate secretion. We isolated and characterized VuMATE2 as a possible second citrate transporter in rice bean functioning in tandem with VuMATE1, which we previously identified. The time-dependent kinetics of VuMATE2 expression correlates well with the kinetics of early phase root citrate secretion. Ectopic expression of VuMATE2 in Arabidopsis resulted in increased root citrate secretion and Al resistance. Electrophysiological analysis of Xenopus oocytes expressing VuMATE2 indicated VuMATE2 mediates anion efflux. However, the expression regulation of VuMATE2 differs from VuMATE1. While a protein translation inhibitor suppressed Al-induced VuMATE1 expression, it releases VuMATE2 expression. Yeast one-hybrid assays demonstrated that a previously identified transcription factor, VuSTOP1, interacts with the VuMATE2 promoter at a GGGAGG cis-acting motif. Thus, we demonstrate that plants adapt to Al toxicity by fine-tuning root citrate secretion with two separate root citrate transport systems.

Sulfur-Mediated-Alleviation of Aluminum-Toxicity in Citrus grandis Seedlings.

Limited data are available on the sulfur (S)-mediated-alleviation of aluminum (Al)-toxicity in higher plants. Citrus grandis seedlings were irrigated for 18 weeks with 0.5 mM MgSO4 or 0.5 mM MgSO4 + 0.5 mM Na2SO4, and 0 (−Al) or 1 mM AlCl3·6H2O (+Al, Al-toxicity). Under Al-toxicity, S decreased the level of Al in leaves; increased the relative water content (RWC) of roots and leaves, the contents of phosphorus (P), calcium (Ca) and magnesium (Mg) per plant, the dry weights (DW) of roots and shoots, the ratios of root DW/shoot DW, and the Al-induced secretion of citrate from root; and alleviated the Al-induced inhibition of photosynthesis via mitigating the Al-induced decrease of electron transport capacity resulting from the impaired photosynthetic electron transport chain. In addition to decreasing the Al-stimulated H2O2 production, the S-induced upregulation of both S metabolism-related enzymes and antioxidant enzymes also contributed to the S-mediated-alleviation of oxidative damage in Al-treated roots and leaves. Decreased transport of Al from roots to shoots and relatively little accumulation of Al in leaves, and increased leaf and root RWC and P, Ca, and Mg contents per plant might also play a role in the S-mediated-alleviation of Al-toxicity.

Genome-wide analysis of MATE transporters and molecular characterization of aluminum resistance in Populus.

Ionic aluminum (Al) in acidic soils, comprising approximately 50% of arable land globally, is highly toxic to most plant species. Populus grow naturally in acidic soils and tolerate high concentrations of Al. Multidrug and toxic compound extrusion (MATE) family genes in plants are involved in responses to Al tolerance. To date, however, the functional roles of the MATE genes in Populus remain unclear. In the present study, 71 putative MATE transporters were predicted in the genome of Populus trichocarpa. The chromosome distribution, phylogenetic relationships, and expression level analysis revealed that four candidate MATE genes belonging to subgroup IIIc might contribute to high Al tolerance in poplar. Further, the expression levels of two subgroup IIIc members, PtrMATE1 and PtrMATE2, were induced by Al stress. Transient expression in onion epidermal cells showed that PtrMATE1 was localized to the plasma membrane. Overexpression of PtrMATE1 increased Al-induced secretion of citrate from the root apex of transgenic plants. Al-induced inhibition of root growths were alleviated in both PtrMATE1 overexpression lines in Populus and in Arabidopsis compared with wild-type plants. In addition, PtrMATE1 expression was induced at 12 h after exposure to Al stress whereas PtrMATE2 expression was induced at 24 h, indicating that these proteins coordinately function in response to Al stress in poplar. Taken together, these results provide important insights into the molecular mechanisms involved in Al tolerance in poplar.

Two MATE Transporters with Different Subcellular Localization are Involved in Al Tolerance in Buckwheat.

Buckwheat (Fagopyrum esculentum) shows high tolerance to aluminum (Al) toxicity, but the molecular mechanisms responsible for this high Al tolerance are still poorly understood. Here, we investigated the involvement of two MATE (multi-drug and toxic compound extrusion) genes in Al tolerance. Both FeMATE1 and FeMATE2 showed efflux transport activity for citrate, but not for oxalate when expressed in Xenopus oocytes. A transient assay with buckwheat leaf protoplasts using green fluorescent protein (GFP) fusion showed that FeMATE1 was mainly localized to the plasma membrane, whereas FeMATE2 was localized to the trans-Golgi and Golgi. The expression of FeMATE1 was induced by Al only in the roots, but that of FeMATE2 was up-regulated in both the roots and leaves. Furthermore, the expression of both genes only responded to Al toxicity, but not to other stresses including low pH, cadmium (Cd) and lanthanum (La). Heterologous expression of FeMATE1 or FeMATE2 in the Arabidopsis mutant atmate partially rescued its Al tolerance. Expression of FeMATE1 also partially recovered the Al-induced secretion of citrate in the transgenic lines, whereas expression of FeMATE2 did not complement the citrate secretion. Further physiological analysis showed that buckwheat roots also secreted citrate in addition to oxalate in response to Al in a dose-responsive manner. Taken together, our results indicate that FeMATE1 is involved in the Al-activated citrate secretion in the roots, while FeMATE2 is probably responsible for transporting citrate into the Golgi system for the internal detoxification of Al in the roots and leaves of buckwheat.

Retrotransposon-Mediated Aluminum Tolerance through Enhanced Expression of the Citrate Transporter OsFRDL4.

High aluminum (Al) tolerance of rice (Oryza sativa) is controlled by multiple tolerance genes, but the regulatory mechanisms underlying the differential expression of these genes are poorly understood. Here, we investigated the factors regulating the expression of OsFRDL4, a gene encoding a citrate efflux transporter involved in Al-induced citrate secretion from the roots. Analysis with chromosome segment substitution lines derived from cv Nipponbare (high OsFRDL4 expression) and cv Kasalath (low OsFRDL4 expression) revealed that the differential expression of OsFRDL4 is responsible for the quantitative trait locus for Al tolerance detected previously on chromosome 1. Comparison of the OsFRDL4 gene structure in cv Nipponbare and cv Kasalath showed that there was no difference in the position of the transcriptional start site, but a 1.2-kb insertion showing high similarity to the solo long terminal repeat of the retrotransposon was found in the promoter region of OsFRDL4 in cv Nipponbare. This insertion showed higher promoter activity and contained nine cis-acting elements for ALUMINUM RESISTANCE TRANSCRIPTION FACTOR1 (ART1). However, this insertion did not alter the spatial expression or cellular localization of OsFRDL4. Furthermore, this insertion was found in most japonica varieties but was largely absent from indica varieties or wild rice species. These results indicate that the 1.2-kb insertion in the OsFRDL4 promoter region in japonica subspecies is responsible for their higher expression level of OsFRDL4 due to the increased number of cis-acting elements of ART1. Our results also suggest that this insertion event happened at the initial stage of domestication of japonica subspecies.

Functional Analysis of a MATE Gene OsFRDL2 Revealed its Involvement in Al-Induced Secretion of Citrate, but a Lower Contribution to Al Tolerance in Rice.

The multidrug and toxic compound extrusion (MATE) transporters represent a large transporter family in plants, but the role of most genes in this family has not been examined. We functionally characterized a MATE family member, OsFRDL2, in rice (Oryza sativa). OsFRDL2 showed an efflux transport activity for citrate when it was expressed in both Xenopus oocytes and cultured tobacco cells. OsFRDL2 was mainly expressed in the roots and its expression was not induced by iron (Fe) deficiency, but it was rapidly up-regulated by aluminum (Al). Furthermore, the expression of OsFRDL2 was regulated by ART1, a C2H2-type zinc-finger transcription factor for Al tolerance. OsFRDL2 protein was localized at unidentified vesicles in the cytosol, but not co-localized with either mitochondria or peroxisomes when expressed in both onion epidermal cells and cultured tobacco cells. Knockout of OsFRDL2 decreased Al-induced secretion of citrate from the roots, but did not affect the internal citrate concentration. The Al-induced inhibition of root elongation was similar between the OsFRDL2 knockout line and its wild-type rice. Knockout of OsFRDL2 did not affect the translocation of Fe from the roots to the shoots. A double mutant between osfrdl2 and osfrdl4 or osfrdl1 did not further decrease the Al-induced citrate secretion and Fe translocation compared with the single mutant. Collectively, our results indicate that although OsFRDL2 is involved in the Al-induced secretion of citrate, its contribution to high Al tolerance is relatively small in rice.

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.

Al-enhanced Expression and Interaction of 14-3-3 Protein and Plasma Membrane H+-ATPase is Related to Al-induced Citrate Secretion in an Al-resistant Black Soybean

The up-regulated expression of plasma membrane (PM) H+-ATPase is related to Al-enhanced citrate exudation in soybean. Moreover, the interaction with a 14-3-3 protein can activate PM H+-ATPase. This study investigated if the expression and interaction level of the 14-3-3 protein and PM H+-ATPase is associated with PM H+-ATPase activity as well as citrate secretion in Al-tolerant (RB) and Al-sensitive (SB) black soybeans, respectively, under 50 mM Al stress. The results showed that Al stress significantly induced transcription of the 14-3-3a gene in RB but not in SB roots. The Al-enhanced transcription of the PM H+-ATPase encoding gene (gha2) was higher in RB than in SB roots. The translation and phosphorylation of PM H+-ATPase, the interaction between the 14-3-3 protein and the phosphorylated PM H+-ATPase all showed an increasing pattern in RB but displayed a descending pattern in SB during the whole Al stress period. Consistently, H+ pump activity and citrate secretion were also higher in RB than in SB under Al stress. Furthermore, citrate secretion was correlated positively with PM H+-ATPase activity in RB and SB under Al stress. These results indicated that Al-enhanced expression of 14-3-3 protein and PM H+-ATPase and the interaction between 14-3-3 protein and phosphorylated PM H+-ATPase increased the activity of PM H+-ATPase and the H+ pump, thereby augmenting citrate secretion in RB. This might be an important molecular mechanism underlying the higher Al-tolerance in RB compared to SB.

Hydrogen sulfide alleviates aluminum toxicity in barley seedlings

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. 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. 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. 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.

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 1.2mM AlCl3·6H2O×0 and 10μM 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 (Fv/Fm) and total performance index (PItot,sbs), but increased inactivation of oxygen-evolving complex (OEC), K-band and relative variable fluorescence at I-steps (VI). 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.

Transcriptomic responses to aluminum stress in soybean roots

Aluminum (Al) toxicity is the primary limitation to crop production and plant growth in acid soils. Soybean has multiple mechanisms of Al resistance including the complexing and exclusion of Al in root apices by Al-induced citrate secretion. Microarray analysis is available for the identification of genes in soybean. In the present study, Affymetrix soybean genome array was used to identify the Al-induced differentially expressed genes in Al-resistant genotype Jiyu 70. With a cutoff of > 2.0-fold (p < 0.05) between non Al-treated and Al-treated root apices, 561 genes were upregulated and 78 genes were downregulated when roots were exposed to 30 μmol/L AlCl(3) for 4 h. Quantitative real-time PCR was used to test the microarray data. The analysis showed that nearly half of the Al-responsive genes were of unknown biological function. A higher proportion of genes related to transcription regulation and cell wall processes were observed in Al-induced up- and downregulated genes, respectively. Some genes homologous to the citrate transporter MATE family gene or C(2)H(2) family transcription factor gene, STOP1, were detected in our analysis. Some genes related to lignin deposition were upregulated, which might be related to Al-induced root elongation inhibition.

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).

Corrigendum to: Mitochondrial enzymes and citrate transporter contribute to the aluminium-induced citrate secretion from soybean (Glycine max) roots

The concentration of soluble aluminium (Al) in the soil solution increases at low pH and the prevalence of toxic Al3+ cations represent the main factor limiting plant growth on acid soils. Citrate secretion from roots is an important Al-tolerance mechanism in many species including soybean. We isolated mitochondria from the roots of an Al-resistant soybean (Glycine max L.) cv. Jiyu 70 to investigate the relationship between citrate metabolism and Al-induced citrate secretion. Spectrophotometric assays revealed that the activities of mitochondrial malate dehydrogenase and citrate synthase increased and aconitase decreased with increasing of Al concentration (0–50 µM) and duration of Al treatment (30 µM Al, 0.5–9 h). Al-induced citrate secretion was inhibited by the citrate synthase inhibitor suramin, and enhanced by the aconitase inhibitor fluorocitric acid. Mersalyl acid, an inhibitor of a citrate carrier located in mitochondria membrane, also suppressed Al-induced citrate secretion. Transcript level of the mitochondrial citrate synthase gene increased in soybean roots exposed to Al, whereas expression of aconitase showed no significant difference. Expression of Gm-AlCT, a gene showing homology to Al-activated citrate transporters was also induced after 4 h in Al treatment. The Al-dependent changes in activity and expression of these enzymes are consistent with them supporting the sustained release of citrate from soybean roots.

Mitochondrial enzymes and citrate transporter contribute to the aluminium-induced citrate secretion from soybean (Glycine max) roots

The concentration of soluble aluminium (Al) in the soil solution increases at low pH and the prevalence of toxic Al3+ cations represent the main factor limiting plant growth on acid soils. Citrate secretion from roots is an important Al-tolerance mechanism in many species including soybean. We isolated mitochondria from the roots of an Al-resistant soybean (Glycine max L.) cv. Jiyu 70 to investigate the relationship between citrate metabolism and Al-induced citrate secretion. Spectrophotometric assays revealed that the activities of mitochondrial malate dehydrogenase and citrate synthase increased and aconitase decreased with increasing of Al concentration (0–50 µM) and duration of Al treatment (30 µM Al, 0.5–9 h). Al-induced citrate secretion was inhibited by the citrate synthase inhibitor suramin, and enhanced by the aconitase inhibitor fluorocitric acid. Mersalyl acid, an inhibitor of a citrate carrier located in mitochondria membrane, also suppressed Al-induced citrate secretion. Transcript level of the mitochondrial citrate synthase gene increased in soybean roots exposed to Al, whereas expression of aconitase showed no significant difference. Expression of Gm-AlCT, a gene showing homology to Al-activated citrate transporters was also induced after 4 h in Al treatment. The Al-dependent changes in activity and expression of these enzymes are consistent with them supporting the sustained release of citrate from soybean roots.

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.