Role of the plasma membrane H⁺-ATPase in the regulation of organic acid exudation under aluminum toxicity and phosphorus deficiency

Abstract

Aluminum (Al) toxicity and phosphorus (P) deficiency are 2 major limiting factors for plant growth and crop production in acidic soils. Organic acids exuded from roots have been generally regarded as a major resistance mechanism to Al toxicity and P deficiency. The exudation of organic acids is mediated by membrane-localized OA transporters, such as ALMT (Al-activated malate transporter) and MATE (multidrug and toxic compound extrusion). Beside on up-regulation expression of organic acids transporter gene, transcriptional, translational and post-translational regulation of the plasma membrane H⁺-ATPase are also involved in organic acid release process under Al toxicity and P deficiency. This mini-review summarizes the current knowledge about this field of study on the role of the plasma membrane H⁺-ATPase in organic acid exudation under Al toxicity and P deficiency conditions.

After oxygen and silicon, aluminum (Al) is the third most abundant element and the most abundant metal in the Earth crust. Most Al combines efficiently with oxygen and silicon to form insoluble oxides and aluminosilicates, which are harmless to plants.¹ However, solubilisation of Al from insoluble oxides is enhanced in acidic soils (pH < 5.50), which covers over 50% of the world's potential arable lands.² Al toxicity has been shown to be capable of affecting the plasma membrane structure, reducing nutrient assimilation, inhibiting root growth, which lead to significant reductions in plant growth and development.^3,4 A further problem of acid soils is that phosphorous (P) is easily fixed by Al- and Fe-oxyhydroxides, and hence rendering it unavailable for root uptake.^5,6 Therefore, Al toxicity and P deficiency are 2 major limiting factors for crop production in acidic soils.

It is now clear that organic acids, such as citrate, malate and oxalate, exuded from roots can protect roots by detoxifying Al in the rhizosphere and benefit the P nutrition of plants. First, organic acids can alleviate Al toxicity by chelating Al, which effectively decreases the uptake of free Al. Second, the release of organic acids converts fixed P to a soluble form that is more readily absorbed by plants.⁷ For example, wheat, soybean and buckwheat reduce toxic levels of Al via exudation of malate, citrate and oxalic acid from root tips.^8-11 Similarly, purple lupin and white lupin improve P availability by stimulating citrate efflux from their roots.^7,12

The exudation of organic acids is mediated by membrane-localized organic acids transporters, which belong to 2 families, ALMT (Al-activated malate transporter) and MATE (multidrug and toxic compound extrusion). Several genes encoding ALMT1 and MATE-family proteins have been identified from the roots of wheat,¹³ Arabidopsis,¹⁴ sorghum,¹⁵ rice bean,¹⁶ soybean,^17,18 and alfalfa,^19,20 And the identification and function of organic transporter genes in plant resistance to Al stress have been widely reviewed.^21-23 Beside on Al-induced organic acids transporter gene's expression, plasma membrane H⁺-ATPase is also involved in Al-induced organic acid process. In this review, we summarize what is understood about this field of study on the role of the plasma membrane H⁺-ATPase in organic acid exudation under Al toxicity and P deficiency conditions.

The H⁺-ATPase is the most abundant protein in the plasma membrane. Using the chemical energy of ATP, the H⁺-ATPase extrudes protons from cells to create an electrochemical proton gradient across the plasma membrane.^24,25 And this electrochemical gradient plays an important role both in P-deficiency and Al-induced citrate exudation from plant roots. For examples, Gardner et al.^26-28 provided the first evidence that organic acid exudation from specialized proteoid roots (dense bottle-brush-like clusters of rootlets) of white lupin (Lupinus albus) to enhance P uptake by molubilization of poorly available soil P. Since then, there has been increased interest worldwide in the role of the plasma membrane H⁺-ATPase in the regulation of organic acids exudation from plant roots. In white lupin, Yan et al.¹² reported that P deficiency induced higher plasma membrane H⁺-ATPase activity and its concentration in active proteoid roots than in other roots. In greater purple lupin, Ligaba et al.⁷ also observed that P-deficiency enhanced plasma membrane H⁺-ATPase activity coupled with citrate exudation. While 1 mM vanadate inhibited plasma membrane H⁺-ATPase activity by 80%, citrate content in root exudates, but not malate, was decreased by 60%.⁷ Under Al stress conditions, Ahn et al.²⁹ showed that PM H⁺-ATPase activity and H⁺-transport rate were decreased and potential was depolarized in plasma membrane H⁺-ATPase vesicles from root tips of Al-sensitive wheat cultivar (ES8) but not of Al-tolerant ET8 after 2.6 μM Al treated for 4 h. This indicated that the Al-induced exudation of malate from wheat roots was associated with changes in plasma membrane surface potential and activation of H⁺-ATPase.²⁹ And then, Shen et al.⁹ provided the direct evidences linking organic acid exudation from roots to the activity of the plasma membrane H⁺-ATPase under Al stress. In Al-resistance soybean roots, while FC or VA significantly enhanced or inhibited activity of the plasma membrane H⁺-ATPase by approximately 85% or 53%, Al-induced citrate exudation also increased or decreased about 58% or 52%, respectively.⁹ In broad bean and Tamble balck soybean roots, the activity of plasma membrane H⁺-ATPase exhibited a significant linear correlation with the rates of citrate exudation both in Al-resistance and Al-sensitive cultivars.^30-33 Furthermore, activation of the plasma membrane H⁺-ATPase is also involved in micromollar concentrations of magnesium (Mg) enhancement of Al-induced citrate exudation in ricebean and broad bean roots.³¹ However, several studies also described the plasma membrane H⁺-ATPase activity was inhibited by Al. For instance, the proton transport activity was inhibited by 50% in the presence of 100 μM Al at pH 6.5 but the inhibition was only 10% at pH 7.5.³⁴ Furthermore, 50 and 70 μM Al inhibited the plasma membrane H⁺-ATPase activity in wheat and soybean roots, respectively.^9,35 Furthermore, other authors reported that the enhanced release of H⁺ and the activity of plasma membrane H⁺-ATPase did not coincide with increased exudation of carboxylate anions in tomato plant under Al stress.³⁶ and tomato, wheat and soybean under P deficiency conditions, respectively.^37,38 These different responses of the plasma membrane H⁺-ATPase to Al toxicity and P deficiency might be due to differences in the plant species, stress levels and experimental conditions.

The activity of the plasma membrane H⁺-ATPase can be modulated by various environmental stimuli at transcriptional, translational and post-translational levels.³⁹ In white lupin, higher plasma membrane H⁺-ATPase activity and its concentration coincidence with more abundance of the gene expression and protein synthesis of the plasma membrane H⁺-ATPase in active proteoid roots than in other roots.⁴⁰ Using plasma membrane vesicles isolated from roots grown in the presence or absence of Al, soybean.⁴¹ and broad bean.³⁰ roots exhibited an increased plasma membrane H⁺-ATPase activity that was correlated with changes in the corresponding protein expression.

Furthermore, transgenic Arabidopsis thaliana overexpressing the plasma membrane H⁺-ATPase exuded more citrate compared with wild-type plants.⁴¹ Phosphorylation at the penultimate Thr residue of the plasma membrane H⁺-ATPase is a common example of post-translational modification altering the activity of this enzyme.⁴² The association of 14-3-3 proteins with the phosphothreonine-containing sequence of the plasma membrane H⁺-ATPase maintains the phosphorylation state and results in its activation.^43,44 The 14-3-3 proteins, which are members of a highly conserved protein family with regulatory roles in all eukaryotes, interact with phosphorylated target proteins such as nitrate reductase, sucrose-phosphate synthase and plasma membrane H⁺-ATPase.⁴⁵ In broad bean and soybean roots, 50 μM Al increased the phosphorylation levels of the plasma membrane and its interaction with the 14-3-3 protein with a time dependent manner in Al-resistance cultivars but not in Al-sensitive cultivars.^9,30,32 Furthermore, while the phosphorylation and interaction with the 14-3-3 protein of the plasma membrane H⁺-ATPase were stimulated or inhibited by the addition of FC or 5′-monophosphate (5′-AMP), both the activity of this enzyme coupled with the Al-induced citrate exudation were significantly decreased or decreased in broad bean roots.³⁰ A more recent study also provided the evidence that post-translational modification of the plasma membrane H⁺-ATPase is involved in Mg activation of this enzyme activity and its related citrate exudation. In broad bean roots, Chen et al.³¹ found that the gene and protein expression of the plasma membrane H⁺-ATPase as well as the mRNA abundance of a putative MATE-like gene were induced by Al, but not Mg. Whereas, immunoprecipitation and western blot analyses showed that phosphorylation of the penultimate Thr-residue in VHA2 (Vicia faba plasma membrane H⁺-ATPase 2), as well as its in vivo association with the vf14-3-3b protein, both increased in the presence of Mg under Al stress.³¹

In conclusions, phosphorus deficiency and Al toxicity are 2 main contraints for plant grwoth and crop production in acidic soils (Fig. 1, left). Plasma membrane H⁺-ATPase plays important roles in organic acids exudation in plant response to Al toxicity and P deficiency stresses (Fig. 1, right). It is proposed that genes and proteins, including organic acid transporter and plasma membrane H⁺-ATPase are up-regulated by Al stress and/ or P deficiency. Phosphorylation levels of the plasma membrane H⁺-ATPase and its interaction with the 14-3-3 protein are enhanced, which results in the increase of the plasma membrane H⁺-ATPase activity and proton efflux across the cell membrane. The created electrochemical potential across the plasma membrane further promoted the activity of the organic acid transporters and a passive of organic anions efflux from the root tips. Organic acids exude from roots form a stable complex with Al and make P soluble for plant assimilation. Further work is needed to further characterize the regulatory mechanisms of the plasma membrane H⁺-ATPase on organic acids exudation, especially for their transcriptional, translational and post-translational regulation of this enzyme under Al toxicity and P deficiency.

Figure 1.

The potential role of the plasma membrane H⁺-ATPase in the regulation of organic acid exudation under Al stress and P deficiency stresses referring to references in this review.

Acknowledgments

We thank Dr. Frantisek Baluska for kindly inviting this mini-review.

Funding

This work was supported by the National Natural Science Foundation of China (No. 31360340) and Talent Training Program in Yunnan Province (KKSY201326062).