GsMATE encoding a multidrug and toxic compound extrusion transporter enhances aluminum tolerance in Arabidopsis thaliana

Abstract

Background: Multidrug and toxic compound extrusion (MATE) transporters, which exist widely in plants, function as crucial regulators in plant resistance to aluminum (Al) toxicity by inducing citrate efflux. However, the functions of most MATE family members in soybean (Glycine soja) remain to be elucidated.

Results: Expression pattern analysis showed that GsMATE was constitutively expressed in different soybean organs, with the highest level in root compared with those in stem, leaf and cotyledon. In addition, Al stress induced expression of GsMATE in soybean. Temporal analysis indicated that GsMATE expression was greatly enhanced by increasing concentrations of aluminum [Al³⁺] after short exposure, reaching the high levels detected in the BW69 (Al-resistant) and the JW81 (Al-sensitive) lines of Glycine soja of wild soybean at 6 h and 8 h, respectively. Furthermore, transient GsMATE expression in Arabidopsis protoplasts showed that GsMATE protein localized to the plasma membrane. Overexpression of GsMATE on an Arabidopsis columbia-0 (Col-0) background resulted in increased Al tolerance in transgenic plants. Analysis of hematoxylin staining showed that the roots of GsMATE transgenic lines were stained less intensely than those of the wild-type exposured to the same AlCl₃ concentrations. Therefore, GsMATE enhanced the resistance of transgenic plants to Al toxicity by reducing Al accumulation in Arabidopsis roots.

Conclusions: In summary, our results indicate that GsMATE is responsive to aluminum stress and may participate in the regulation of sensitivity to Al toxicity in Arabidopsis. In addition, the GsMATE protein is an Al-induced citrate transporter of the MATE family and exerts an essential role in Al tolerance in Glycine soja.

Keywords: Al tolerance; Arabidopsis thaliana; Glycine soja; GsMATE.

Fig. 1

Homology analysis of GsMATE and other transmembrane proteins. All the available amino acid sequences and the accession numbers of MATE proteins were obtained from the NCBI databases (https://www.ncbi.nlm.nih.gov/). The MATE transporters have been characterized and their functions were identified except for GsMATE and the eight soybean MATE proteins [54]. The homology tree was produced using DNAMAN alignments. The accession numbers of the MATE proteins were shown in parentheses

Fig. 2

Tissue expression pattern of GsMATE. a Tissue expression pattern of GsMATE. b Expression of GsMATE in root sections. Tissue samples were obtained from the young BW69 and JW81 seedlings. Two days after germination, 20 seedlings were transferred to solutions of 0 μM and 50 μM AlCl₃ (pH 4.3, 0.5 mM CaCl₂). Samples were obtained from seedling roots (sections 0–2, 2–4, and > 4 cm) after 6 h of AlCl₃ treatment. Data were represented as the mean ± SE of three biological replicates; the same letter on each column set indicates no significant difference and different letters indicated a statistically significant difference according to analysis of variance (t-test, p = 0.05). BW69, JW81: two lines of Glycine soja. Data were measured using Image J software, analyzed using SPSS20 and plotted using EXCEL2000

Fig. 3

Pattern of GsMATE expression in response to acidic aluminum exposure. a Pattern of GsMATE expression under acidic aluminum exposure. b Temporal expression pattern of GsMATE under acidic aluminum exposure. Two days after germination, the seedlings were transferred to solutions of 0, 25, 50, 75, and 100 μM AlCl₃ (pH 4.3, 0.5 mM CaCl₂). After 6 h, root tip samples (6 cm long) were obtained from the seedlings for the analysis of the GsMATE expression patterns. To analyze the temporal expression pattern of GsMATE, seedlings (2 days after germination) were cultured in a solution of 0.5 mM CaCl₂ (pH 4.3) for 24 h, and then transferred to the solution of 50 μM AlCl₃ (pH 4.3, 0.5 mM CaCl₂). Root tip samples (6 cm long) were obtained from the seedlings after the treatments of 2, 4, 6, 8, 12, and 24 h. Data were represented as the mean ± SE of three biological replicates; the same letter on each column set indicates no significant difference and different letters indicate a statistically significant difference according to analysis of variance (t-test, p = 0.05). BW69, JW81: two lines of Glycine soja. Data were analyzed using SPSS20, and plotted using EXCEL2000

Fig. 4

Analysis of GsMATE protein localization. a Localization of GsMATE-GFP fusion protein or GFP alone in Arabidopsis protoplasts; b and e Corresponding bright-field images; c and f. Merged images; d pYL322-d1-eGFP vector. The GsMATE-GFP fusion vector was constructed by cloning the full coding sequence of GsMATE (without TAA) into the BamHI and KpnI sites of pYL322-d1-eGFP vector. Arabidopsis protoplasts were transformed using the heat-shock method. After 24 h, GFP or GsMATE-GFP fusion protein expression (driven by the CaMV 35S promoter) was visualized by fluorescence microscopy

Fig. 5

GsMATE increases Al tolerance of transgenic Arabidopsis lines. a Al tolerance phenotypes of GsMATE in transgenic lines. b Statistical analysis of relative root length. WT: wild-type of Arabidopsis (Col-0); OX1/OX2: GsMATE overexpression transgenic lines. Two days after germination, seedlings were transferred to culture medium containing 0, 50, 100, and 150 μM AlCl₃ (pH 4.3, 0.5 mM CaCl₂). After 7 days in culture, images of the phenotypes of the GsMATE transgenic lines were recorded for statistical analysis. Data were represented as the mean ± SE of three biological replicates (t-test, p = 0.05). Data were measured using Image J software, analyzed using SPSS20, and plotted using EXCEL2000