The ALMT gene was the first Al³⁺ tolerance gene identified in plants (Sasaki et al., 2004; Delhaize et al., 2007; Meyer et al., 2010). Membrane protein ALMTs possess 5–7 predicted transmembrane domains and a UPF0005 domain with unknown function (Delhaize et al., 2007). BoALMT1 (KF322104) cloned from cabbage contains an open reading frame of 1,497 bp, encoding a polypeptide of 498 amino acids. BLAST analysis revealed that the sequence of BoALMT1 was a 99% match to BnALMT1 from rape, 73% match to AtALMT1 from Arabidopsis, and 33% match to TaALMT1 from wheat. The HMMTOP transmembrane topology prediction server was used to predict the localization of helical transmembrane segments and the analysis indicated that BoALMT1 contained 5 predicted transmembrane domains (Figure 1A). Analysis of BoALMT1 and other reported ALMTs in plants indicated that BoALMT1 was most closely clustered with the BnALMT1 from Brassica napus (Figure 1B).

We performed real-time reverse transcription (RT)-PCR analysis to measure the expression of BoALMT1 in the roots and shoots, and found that BoALMT1 expression was primarily localized to the roots (Figure 2A). Al treatment enhanced its expression in all tissues (Figure 2A). Cabbage plants were exposed to a variety of trivalent cations, and the expression of BoALMT1 was not induced by lanthanum (La), cadmium (Cd), zinc (Zn), or copper (Cu), but was severely induced by aluminum (Figure 2B). A dose-response experiment and a time-course experiment indicated that increasing the external Al concentration and treatment time did not further increase the BoALMT1 transcript level (Figures 2C,D).

The subcellular localization of BoALMT1 was determined via localization of the GFP::BoALMT1 protein transiently expressed in onion epidermal cells (Figure 3). The GFP::BoALMT1 green fluorescence was only observed at the outer layer of the cell (Figures 3a,b), and the cells expressing GFP showed green fluorescence in the whole cell (Figures 3e,f). We induced plasmolysis by the addition of 0.8 M mannitol to distinguish localization in the plasma membrane and observed that the fluorescence of GFP::BoALMT1 was exclusively located in the plasma membrane in the plasmolysis cells (Figures 3c,d). These localization results were similar to those of some ALMTs identified in other species [TaALMT1 (Yamaguchi et al., 2005), BnALMT1 (Ligaba et al., 2006), ZmALMT1 (Piñeros et al., 2008), ZmALMT2 (Ligaba et al., 2008), and GmALMT1 (Liang et al., 2013)].

To investigate whether the secretion of malate was induced by Al treatment, we characterized malate exudation from cabbage roots. Cabbage roots secreted a low level of malate under normal conditions. After 3 h treatment with 50 μM Al, malate exudation was remarkably induced (Figure 4).

By treated the Arabidopsis mutant with Al stress, Degenhardt et al. (1998) observed that the pH of the root surface increased, while Bose et al. (2010) further confirmed Al stress correlated with lower H⁺ influx. So in our study, we used the non-invasive Scanning Ion-selective Electrode Technique (SIET) system to measure H⁺ fluxes crossing the surface of Xenopus oocytes with or without the per-injected malate (Figure 5A). We noticed that the H⁺ flux had no difference in the control oocytes under the absence or the present of Al. Furthermore, compared with the control cells, BoALMT1-expressing oocytes also secreted similar amount of H⁺ without pretreated with malate. However, when the malate was fed, the BoALMT1-expressing oocytes secreted more H⁺ compared with the control oocytes under the absence or the present of Al condition (Figure 5A). To further elucidate BoALMT1 served as a malate efflux transporter, we fed the control and the BoALMT1-expressing oocytes with ¹⁴C-labeled malate and then measured the efflux of radioactively labeled malate (Figure 5B). The BoALMT1-expressing oocytes excreted more labeled malate than the control cells. These results indicated that BoALMT1 was a malate efflux transporter and enhanced the H⁺ efflux according to malate secretion in Xenopus oocytes.

Al-activated membrane transporters, which mediate organic acid release from the root apex, are the primary physiological mechanism of plant Al tolerance (Kochian et al., 2004). Plant ALMTs that have been implicated in malate transport and Al tolerance are TaALMT1 in wheat (Sasaki et al., 2004), AtALMT1 in Arabidopsis (Hoekenga et al., 2006), BnAMLT1 and BnALMT2 in oilseed rape (Ligaba et al., 2006), GmALMT1 in soybean (Liang et al., 2013), and MsALMT1 in M. sativa (Chen et al., 2013).

In this study, to investigate whether the overexpression of BoALMT1 enhances malate exudation and Al tolerance, we induced expression of BoALMT1 driven by the CaMV 35S promoter in Arabidopsis plants. Successful introduction of BoALMT1 in two transgenic lines, but not the control line, was confirmed by RT-PCR (Figure 6A). Root malate exudation was then measured in the plants expressing BoALMT1 and demonstrating increased Al tolerance (Figure 6B). Plants expressing BoALMT1 showed a remarkable increase in root malate exudation rates in the presence of Al, but no difference was observed in the absence of Al. When grown in in agar plates without Al, the transgenic plants expressing BoALMT1 showed root growth similar to that of wild-type (Figure 6C). When grown in agar plates with 400 μM AlCl₃, root elongation of plants expressing BoALMT1 showed less root growth inhibition than that of the plants without expression (Figures 6C,D). To further determine the effect on H⁺ flow caused by overexpressing BoALMT1 in Arabidopsis, we performed SIET to detect the H⁺ flux at the root DEZ with 0 or 50 μM Al (pH = 4.5). Under low pH condition, the pattern of H⁺ influx exhibited no statistic difference between WT lines and BoALMT1 transgenic lines. However, treated with 50 μM Al, the H⁺ influx was inhibited in the WT lines, while the H⁺ was secreted from the roots BoALMT1 transgenic lines (Figure 6E).

Cabbage (B. oleracea cv. Zhonggan-11) was seeded at 25°C on moist filter paper in the dark for 2 days. The seedlings were then moved to a complete nutrient solution (Ligaba et al., 2006). After 5 days of culture, the uniform seedlings were moved to a new plastic pot wetted with 0.5 mM CaCl₂ (pH 4.5) solution and pre-incubated for ~24 h. To measure the spatial expression patterns of BoALMT1 in root tips (0–1 cm), after 6 h of 50 μM Al exposure, the roots and shoots were separately collected and subjected to qRT-PCR analysis. To test the specificity of Al-induced BoALMT1 gene expression, we exposed seedlings in a 0.5 mM CaCl₂ solution (pH 4.5) containing 50 μM AlCl₃, 25 μM CdCl₂, 10 μM LaCl₃, 0.5 μM CuCl₂, or 2.0 μM ZnCl₂ for 6 h. To investigate the dose effects of Al on BoALMT1 expression, the seedlings were exposed to a 0.5 mM CaCl₂ solution (pH 4.5) containing 0, 10, 50, or 100 μM AlCl₃ for 6 h. To analyze time-course effects of Al toxicity on BoALMT1 expression, the seedlings were exposed to a 0.5 mM CaCl₂ solution (pH 4.5) containing 50 μM AlCl₃ for 0, 2, 4, and 6 h.

To clone BoALMT1, RNA was isolated from cabbage seedlings roots treated with Al. To identify cabbage BoALMT1, we performed a BLAST search with the known AtALMT1 and BnALMT1 sequences on the NCBI website (http://www.ncbi.nlm.nih.gov/). For further amplification, two expressed sequence tags (ESTs) (DK499842 and DY012377) were selected. The two nucleotide sequences were combined to generate a full-length cDNA. The full-length cDNA of BoALMT1 was amplified with sense primer 5′-ATGGAGAAAGTGAGAGAGATAGTGAG-3′ and anti-sense primer 5′-TCAAATCTGAAGTATACGAACACCC-3′, and then constructed into the pMD18-T vector (Takara, Japan). HMMTOP was used for transmembrane protein prediction analysis. Multiple amino acid alignment was conducted by using ClustalX and MEGA4.1 software.

BoALMT1 expression was evaluated using quantitative real-time RT-PCR techniques. Primers for qPCR were designed using Primer 3.0. The first-strand cDNA synthesis was performed by using the Primescript reverse transcriptase (Takara, Japan). We performed real-time PCR with a SYBR Premix Ex Taq™ (perfect real time) kit (Takara, Japan) and using the Applied Biosystems 7500 Real-Time PCR System (ABI) using a relative standard curve method with the following primers: BoALMT1, 5′-AGAGAAGGAAGGAGGGTAGGAGAA-3′ (forward) and 5′-GAAGACAACAACGACGGTCA-3′ (reverse); Actin (LOC106327159), 5′-TAACAGGGAGAAGATGACTCAGATCA-3′ (forward) and 5′-AAGATCAAGACGAAGGATAGCATGAG-3′ (reverse). Quantitative PCR was performed with conditions of 95°C for 3 min, and then 40 cycles of 95°C for 10 s, 60°C for 30 s, and 72°C for 30 s. Expression data were normalized to the expression level of Actin by the ΔΔCt method.

The subcellular localization of BoALMT1 was determined in onion (Allium cepa) epidermal cells. We constructed a vector as 35S:BoALMT1::GFP. The coding region of BoALMT1 was subcloned into the expression vector pCAMBIA1302 using primers: 5′-CATGCCATGGTAATGGAGAAACTGAGAGAGATAGTG-3′ (forward) and 5′-GGACTAGTAATCTGAAGTATACGAACACCC-3′ (reverse). We transferred the chimera by particle bombardment. The gold particles (1 μm, 1.5 mg) were coated with 5 μg of plasmid DNA in a solution of 2.5 M CaCl₂ and 0.1 M spermidine (Sigma). We bombarded the epidermal onion peels at a helium pressure of 25–30 Mpa (Bio-rad, U.S.), and then incubated the tissue in MS medium at room temperature in the dark for 24 h. Confocal laser scanning microscopy (Leica DMI 6000B-CS, Germany) with a 488 nm excitation wavelength was used to detect the GFP fluorescence. We induced cell plasmolysis by adding 0.8 M mannitol for 3–5 min.

We cloned the coding regions (cDNA) of BoALMT1 into the MCS of a pCS107 vector. According to the manufacturer's (Ambion) recommendations, we synthesized the cRNA from 1 μg of AscI-linearized plasmid DNA template. We harvested stage V–VI Xenopus laevis oocytes as described previously (Golding, 1992; Hoekenga et al., 2006). We injected 50 nl RNase-free water containing 15 ng of cRNA encoding BoALMT1 or 50 nl RNase-free water into oocytes using a micro-injector and then incubated the injected oocytes at 18°C for 2 d in oocyte culture medium, OCM; 1L OCM contains 600 ml L-15 (Sigma L4386), 400 mg BSA (Sigma A4919), 5 ml Penicillin-Streptomycin (Gibco 15140-122), and 400 ml H₂O). Before flux measurements of H⁺, we preloaded malate in the Xenopus oocytes by injection of 50 nl of 0.1 M sodium malate or water. Two hours after preloading, the H⁺ fluxes were measured 30 μm away from X. laevis oocytes in a solution of 2 mM KCl, 96 mM NaCl, 1 mM MgCl₂, 0.3 mM MES, 1.8 mM CaCl₂ with or without 0.1 mM AlCl₃ and with the pH 4.5. Net H⁺ fluxes were measured using SIET (Xuyue Science and Technology Co., Ltd., Beijing, China) under steady conditions for 8–10 min to insure that no fluctuation was present. We used the OCM bath solution (pH = 4.5) to perform the ¹⁴C-labeled malate experiment as our previous study (Wu et al., 2014).

The coding region (cDNA) of BoALMT1 was amplified with primers (5′-GCTCTAGAATGGAGAAACTGAGAGAGATAGTG-3′ and 5′-CGCCCCGGGTCAAATCTGAAGTATACGAACACCC-3′) and was cloned into pBI121. We transformed the construct into Arabidopsis using Agrobacterium tumefaciens via the floral dip method (Clough and Bent, 1998). We used RT-PCR to measure the expression level of BoALMT1 in the transgenic plants. Root malate release and Al tolerance were analyzed in two independent homozygous transgenic T3 lines as follows. Arabidopsis seeds, stratified at 4°C for 3 days, were surface-sterilized and sown onto solid MS medium for 4 days. After germination, we removed uniform seedlings to 0.5 mM CaCl₂-agar plates containing 0 or 400 μM AlCl₃ (pH = 4.5). The seedlings were kept on agar plates for 2 days, and then the roots were scanned and the primary root length was measured by the Image J program (Liu et al., 2009). For malate exudation assays, two transgenic Arabidopsis and wild-type lines were surface sterilized and germinated on solid MS medium for 1 week. Next, we transferred the seedlings to a 25 ml solution with 0.5 mM CaCl₂ (pH 4.5) and without Al for 24 h. After this 24 h pre-incubation step, we then transfered the plants to 25 ml exudation medium (pH 4.5) with or without Al (50 μM AlCl₃). We collected the sample for malate assay by capillary electrophoresis, as described by Hoekenga et al. (2006). We measured the fluxes of H⁺ by using the non-invasive Scanning Ion-selective Electrode Technique (SIET) (Xuyue Science and Technology Co., Ltd., Beijing, China) as described by Bose et al. (2010). The 4- to 5-day-old wild type and BoALMT1 expressing Arabidopsis seedlings were equilibrated in a solution (0.1 mM CaCl2, 0.1 mM KCl, 0.3 mM MES, pH 4.5) with or without 50 mM Al for 5–10 min. H⁺ fluxes were measured 200 mm from the root tip for 6–10 min. The H⁺ fluxes were calculated by the JCal V3.1 (a free MS Excel spreadsheet, youngerusa.com or ifluxes.com). The H⁺ flux assay was replicated independently 4–6 times and the data were averaged.

All the statistical analysis was performed by one-way ANOVA and the t-test to determine the significance at the P < 0.05 level.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.