Al-induced Citrate Exudation Research Articles

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.

Auxin enhances aluminium-induced citrate exudation through upregulation of GmMATE and activation of the plasma membrane H+-ATPase in soybean roots.

Background and Aims Aluminium (Al) toxicity is a limiting factor for plant growth and crop production in acidic soils. Citrate exudation and activation of the plasma membrane H+-ATPase are involved in soybean responses to Al stress. Auxin has crucial functions in plant growth and stress responses. However, little is known about possible interactions between auxin and citrate exudation under Al stress. In this study, we elucidated the regulatory roles of IAA in Al-induced citrate exudation in soybean roots. Methods We measured IAA content, Al concentration, citrate exudation, plasma membrane H+-ATPase activity, expression of the relevant genes and phosphorylation of the plasma membrane H+-ATPase by integrating physiological characterization and molecular analysis using hydroponically grown soybean. Key Results The concentration of IAA was increased by 25 and 50μm Al, but decreased to the control level at 200μm Al. External addition of 50μm IAA to the root medium containing 25, 50 or 200μm Al decreased root Al concentration and stimulated Al-induced citrate exudation and the plasma membrane H+-ATPase activity. Reverse transcription-PCR analysis showed that exogenous IAA enhanced the expression of citrate exudation transporter (GmMATE) but not the plasma membrane H+-ATPase gene. The western blot results suggested that IAA enhanced phosphorylation of the plasma membrane H+-ATPase under Al stress. Conclusions Auxin enhanced Al-induced citrate exudation through upregulation of GmMATE and an increase in phosphorylation of the plasma membrane H+-ATPase in soybean roots.

Phosphorylation and Interaction with the 14-3-3 Protein of the Plasma Membrane H+-ATPase are Involved in the Regulation of Magnesium-Mediated Increases in Aluminum-Induced Citrate Exudation in Broad Bean (Vicia faba. L).

Several studies have shown that external application of micromolar magnesium (Mg) can increase the resistance of legumes to aluminum (Al) stress by enhancing Al-induced citrate exudation. However, the exact mechanism underlying this regulation remains unknown. In this study, the physiological and molecular mechanisms by which Mg enhances Al-induced citrate exudation to alleviate Al toxicity were investigated in broad bean. Micromolar concentrations of Mg that alleviated Al toxicity paralleled the stimulation of Al-induced citrate exudation and increased the activity of the plasma membrane (PM) H(+)-ATPase. Northern blot analysis shows that a putative MATE-like gene (multidrug and toxic compound extrusion) was induced after treatment with Al for 4, 8 and 12 h, whereas the mRNA abundance of the MATE-like gene showed no significant difference between Al plus Mg and Al-only treatments during the entire treatment period. Real-time reverse transcription-PCR (RT-PCR) and Western blot analyses suggest that the transcription and translation of the PM H(+)-ATPase were induced by Al but not by Mg. In contrast, immunoprecipitation suggests that Mg enhanced the phosphorylation levels of VHA2 and its interaction with the vf14-3-3b protein under Al stress. Taken together, our results suggest that micromolar concentrations of Mg can alleviate the Al rhizotoxicity by increasing PM H(+)-ATPase activity and Al-induced citrate exudation in YD roots. This enhancement is likely to be attributable to Al-induced increases in the expression of the MATE-like gene and vha2 and Mg-induced changes in the phosphorylation levels of VHA2, thus changing its interaction with the vf14-3-3b protein.

Citrate exudation induced by aluminum is independent of plasma membrane H+-ATPase activity and coupled with potassium efflux from cluster roots of phosphorus-deficient white lupin

Exudation of organic acid anions is one of the mechanisms responsible for aluminum (Al) tolerance. The plasma membrane (PM) H+-ATPase is involved in the exudation of organic acid anions. However, the relationship between organic acid exudation and PM H+-ATPase under Al toxicity remains unclear. This study aims to investigate the correlation among Al-induced citrate exudation, PM H+-ATPase activity and counterions for citrate release from cluster roots of phosphorus-deficient (−P) white lupin. Al and various pharmacological agents were applied to incubate the cluster roots of P-deficient white lupin; the citrate exudation rate, PM H+-ATPase activity and ion exudation of cluster roots were examined. Citrate exudation from cluster roots of P-deficient white lupin was induced by 50 μM Al treatment within 1.5 h, but no extra increase was found when the duration of Al treatment increased. The PM H+-ATPase activity of cluster roots was insensitive to Al treatment, irrespective of Al concentration and duration of Al treatment. Al treatment increased K+ efflux but not H+ efflux from cluster roots. After application of pharmacological agents to P-deficient cluster roots under Al treatment or not, vanadate decreased H+ efflux and increased K+ efflux, but had no inhibitory effect on citrate exudation; fusicoccin increased H+ efflux and citrate exudation, but decreased K+ efflux; tetraethylammonium (TEA) chloride, a K+-channel inhibitor, inhibited K+ efflux and increased H+ efflux. These results indicate that citrate exudation induced by combined treatment with P-deficiency and Al is independent of PM H+-ATPase activity, and is coupled with K+ efflux, which may compensate H+ efflux for keeping the charge balance for Al-induced citrate exudation from cluster roots of P-deficient white lupin.

Salicylic acid involved in the process of aluminum induced citrate exudation in Glycine max L.

Background and aims Aluminum (Al)-induced citrate exudation has been identified as an important Al-resistance mechanism in soybean. Salicylic acid (SA) is a crucial signal molecule modulating numerous environment stresses in plants. This study was to investigate the role of SA in Al-induced citrate exudation from soybean roots.

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).

Citrate exudation from white lupin induced by phosphorus deficiency differs from that induced by aluminum

Both phosphorus (P) deficiency and aluminum (Al) toxicity induce root exudation of carboxylates, but the relationship between these two effects is not fully understood. Here, carboxylate exudation induced by Al in Lupinus albus (white lupin) was characterized and compared with that induced by P deficiency. Aluminum treatments were applied to whole root systems or selected root zones of plants with limited (1 microM) or sufficient (50 microM) P supply. Aluminum stimulated citrate efflux after 1-2 h; this response was not mimicked by a similar trivalent cation, La(3+). P deficiency triggered citrate release from mature cluster roots, whereas Al stimulated citrate exudation from the 5- to 10-mm subapical root zones of lateral roots and from mature and senescent cluster roots. Al-induced citrate exudation was inhibited by P limitation at the seedling stage, but was stimulated at later growth stages. Citrate exudation was sensitive to anion-channel blockers. Al treatments did not affect primary root elongation, but inhibited the elongation of lateral roots. The data demonstrate differential patterns of citrate exudation in L. albus, depending on root zone, developmental stage, P nutritional status and Al stress. These findings are discussed in terms of possible functions and underlying mechanisms.

Combined Effect of Short-Term Water Deficit Stress and Aluminum Toxicity on Citrate Secretion from Soybean Roots

In order to investigate the combined effect of drought stress and aluminum (Al) toxicity on citrate exudation in soybean, hydroponic cultivation with addition of Al and PEG-6000 was conducted to simulate Al-toxic dry soil. Results showed that 2-day exposure of soybeans to 5.5% (w/v) PEG-6000 or 100 µM AlCl3 mainly hindered root growth, while combined exposure to PEG and Al (PEG/Al) reduced both root and shoot growth. Photosynthetic rate of first trifoliolate for the Al-tolerant genotype PI 416937 (PI) was not affected by imposition of 5.5% PEG/100 µM AlCl3 (38–40 h), whereas photosynthetic rate for the Al-sensitive genotype YC was significantly reduced. Based on root fresh weight, Al-induced citrate exudation in the roots of soybean genotypes recovered from pre-treatment with 5.5 or 9% PEG was not altered, but was increased in the plants pretreated with 7% PEG without recovery. After 2 days of recovery from 2-day combined exposure to 5.5% PEG/100 µM AlCl3, the Al-tolerant PI exuded more citrate than its control, but the Al-sensitive YC exuded significantly less citrate than its corresponding control. Split root experiment revealed that Al-induced citrate exudation in one half of the root system was significantly reduced by exposing the other half of the root system to 0.5 mM CaCl2 solution containing 9% PEG or 9% PEG/50 µM AlCl3. However, organic acid secretion was not observed in the half of the root system in the Ca solution when the other part of the root system was exposed to the Ca solution containing 50 µM AlCl3, 9% PEG or 9% PEG/50 µM AlCl3. This suggests that no Al- or drought-induced signals such as ABA are involved in the citrate secretion in soybean.

Citrate Secretion Induced by Aluminum Stress May Not be a Key Mechanism Responsible for Differential Aluminum Tolerance of Some Soybean Genotypes

Eighteen soybean genotypes differing in aluminum (Al) tolerance were used to investigate genotypic differences in Al-induced citrate exudation and its role in Al tolerance. Aluminum accumulation and localization in soybean roots were examined by analysis of total Al and hematoxylin staining. Soybean genotypes exhibited a wide range of Al tolerance. Based on relative root elongation, several Al-tolerant genotypes from Brazil such as B1, B10, and B15 were more tolerant than the Al-tolerant PI 416937 (PI) and Perry. All soybeans exuded citrate in response to Al stress, and some Al-sensitive genotypes secreted more citrate than tolerant ones, showing no correlation between the Al tolerance and Al-induced citrate exudation. Further study found that both copper (Cu) and cadmium (Cd) stimulated citrate and malate exudation in soybean, indicating that organic acid secretion is not specifically induced by Al. Aluminum concentrations were significantly higher in 2–3 and 3–4 cm of segments than that in 0–1 and 1–2 cm segments under 15 μM AlCl3. Both the root mature zone and apex were heavily stained by hematoxylin after exposure to 10, 15, or 20 μM AlCl3 (24 h), whereas root elongation zone was not stained. After exposure to 50 μM AlCl3 for 20 min, the Al-tolerant PI was less stained by hematoxylin than the Al-sensitive Young, suggesting that Al accumulation in root apices seem to be an immediate response to Al stress, and related to differential Al sensitivity. Present results suggest that citrate secretion induced by Al stress may not be a key mechanism responsible for the differential Al tolerance of some soybean genotypes and other mechanism(s) conferring Al exclusion should exist and operate immediately after exposure to Al stress.

Aluminum regulation of citrate metabolism for Al-induced citrate efflux in the roots of Cassia tora L.

Aluminum-induced exudation of organic acid from plant roots has been proposed to be an important mechanism for Al tolerance. In search for the physiological basis of citrate efflux, the activities of several enzymes involved in citrate metabolism and internal citrate accumulation in the root tips of Cassia tora L. were examined. Results indicated that aluminum at 10–50 μM progressively stimulated activities of citrate synthase (CS, EC 4.1.3.7). A time-course experiment revealed that the increase in CS activities occurred within 3 h after the Al exposure, while the substantial citrate exudation began 6 h after Al treatment, suggesting that citrate exudation could be due to the increased CS capacity. By contrast, aconitase (Aco, EC 4.2.1.3) activities in Al-treated root tips were always suppressed compared with controls, indicating that aconitase was possibly involved in the aluminum-induced citrate efflux. The increase in CS activity and decrease in aconitase activity with Al treatments were associated with the enhanced citrate accumulation in root tips. We also examined NADP-isocitrate dehydrogenase (NADP-ICDH, EC 1.1.1.42), malate dehydrogenase (MDH, EC 1.1.1.37) and phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) in root tips exposed to Al. However, none of the enzymes showed significant alterations in activities. These results suggested that NADP-ICDH, MDH and PEPCase were not directly involved in the Al-induced citrate exudation in the roots of C. tora.

Citrate Secretion Induced by Aluminum Stress May Not be a Key Mechanism Responsible for Differential Aluminum Tolerance of Some Soybean Genotypes

Abstract Eighteen soybean genotypes differing in aluminum (Al) tolerance were used to investigate genotypic differences in Al-induced citrate exudation and its role in Al tolerance. Aluminum accumulation and localization in soybean roots were examined by analysis of total Al and hematoxylin staining. Soybean genotypes exhibited a wide range of Al tolerance. Based on relative root elongation, several Al-tolerant genotypes from Brazil such as B1, B10, and B15 were more tolerant than the Al-tolerant PI 416937 (PI) and Perry. All soybeans exuded citrate in response to Al stress, and some Al-sensitive genotypes secreted more citrate than tolerant ones, showing no correlation between the Al tolerance and Al-induced citrate exudation. Further study found that both copper (Cu) and cadmium (Cd) stimulated citrate and malate exudation in soybean, indicating that organic acid secretion is not specifically induced by Al. Aluminum concentrations were significantly higher in 2–3 and 3–4 cm of segments than that in 0–1 and 1–2 cm segments under 15 μM AlCl3. Both the root mature zone and apex were heavily stained by hematoxylin after exposure to 10, 15, or 20 μM AlCl3 (24 h), whereas root elongation zone was not stained. After exposure to 50 μM AlCl3 for 20 min, the Al-tolerant PI was less stained by hematoxylin than the Al-sensitive Young, suggesting that Al accumulation in root apices seem to be an immediate response to Al stress, and related to differential Al sensitivity. Present results suggest that citrate secretion induced by Al stress may not be a key mechanism responsible for the differential Al tolerance of some soybean genotypes and other mechanism(s) conferring Al exclusion should exist and operate immediately after exposure to Al stress.

Probing the role of calmodulin in Al toxicity in maize

The role of calmodulin on Al toxicity was studied in two maize ( Zea mays L.) inbred lines, Cat 100-6 (Al-tolerant) and S 1587-17 (Al-sensitive). Increasing levels of Al induced the release of malate at similar rate by roots of both genotypes, while the exudation of citrate, a stronger Al-binding compound, was 3.5 times higher in Cat 100-6 seedlings exposed to 16.2×10 −6 Al 3+ activity. The calmodulin inhibitor trifluoperazine significantly reduced the root growth in both genotypes, mimicking the main effect of Al. However, when Cat 100-6 and S 1587-17 seedlings were challenged with Al in conjunction with trifluoperazine, no further reduction in root growth or any other effect of Al toxicity was observed. The rate of Al-induced citrate exudation by both genotypes was not affected by treatment with trifluoperazine or calmidazolium, another calmodulin inhibitor. The Al 3+ interaction with cytoplasmic CaM was estimated using models for the binding of Al 3+ and Mg 2+ with CaM and physiological concentrations of citrate, CaM, InsP 3, ATP, ADP, Al 3+ and Mg 2+. In this simulation, Al 3+ associated with citrate and InsP 3, but not with CaM. We conclude that calmodulin is not relevant to the physiological processes leading to the Al tolerance in maize, nor is it a primary target for Al toxicity.