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Front Plant Sci
2017 Jan 01;8:2156. doi: 10.3389/fpls.2017.02156.
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BoALMT1, an Al-Induced Malate Transporter in Cabbage, Enhances Aluminum Tolerance in Arabidopsis thaliana.
Zhang L
,
Wu XX
,
Wang J
,
Qi C
,
Wang X
,
Wang G
,
Li M
,
Li X
,
Guo YD
.
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Aluminum (Al) is present in approximately 50% of the arable land worldwide and is regarded as the main limiting factor of crop yield on acidic soil. Al-induced root malate efflux plays an important role in the Al tolerance of plants. Here, the aluminum induced malate transporter BoALMT1 (KF322104) was cloned from cabbage (Brassica oleracea). BoALMT1 showed higher expression in roots than in shoots. The expression of BoALMT1 was specifically induced by Al treatment, but not the trivalent cations lanthanum (La), cadmium (Cd), zinc (Zn), or copper (Cu). Subcellular localization studies were performed in onion epidermal cells and revealed that BoALMT1 was localized at the plasma membrane. Scanning Ion-selective Electrode Technique was used to analyze H+ flux. Xenopus oocytes and Arabidopsis thaliana expressing BoALMT1 excreted more H+ under Al treatment. Overexpressing BoALMT1 in transgenic Arabidopsis resulted in enhanced Al tolerance and increased malate secretion. The results suggested that BoALMT1 functions as an Al-resistant gene and encodes a malate transporter. Expressing BoALMT1 in Xenopus oocytes or A. thaliana indicated that BoALMT1 could increase malate secretion and H+ efflux to resist Al tolerance.
Figure 1. Amino acid sequence (A) and phylogenetic (B) analysis of Brassica oleracea BoALMT1. (A) Multiple sequence alignment of cabbage BoALMT1, maize ZmALMT1, wheat TaALMT1, Arabidopsis AtALMT1, rape BnALMT1, rye ScALMT1, and Barley HvALMT1. Identical amino acids and similar amino acids were indicated by dark shading and light shading, respectively. Lines depict the 5 predicted transmembrane domains in BoALMT1 as predicted by HMMTOP. (B) Phylogenetic relationship of BoALMT1 and other known Al-activated malate transporters (ALMT). The amino acid sequences were aligned by ClustalW.
Figure 2. Analysis of BoALMT1 expression in cabbage seedlings by quantitative real-time PCR. (A) Tissue-specific expression of BoALMT1. Seedlings were exposed to a 0.5 mM CaCl2 solution (pH 4.5) containing 50 μM AlCl3 for 6 h. Expression of BoALMT1 gene in the root tip, rest of root (RoR) and shoots were determined. (B) Effect of Al, La, Cd, Cu, and Zn on BoALMT1 expression. Seedlings were exposed to a 0.5 mM CaCl2 solution (pH 4.5) containing 50 μM AlCl3, 25 μM Cd, 10 μM La, 0.5 μM Cu, or 2.0 μM Zn. (C) Time-dependent expression of BoALMT1. Cabbage seedlings were exposed to a solution containing 50 μM AlCl3 for different time. (D) Dose-response expression analysis of BoALMT1 gene in cabbage roots. The roots were exposed to a 0.5 mM CaCl2 solution (pH 4.5) containing 0, 10, 50, and 100 μM AlCl 3 for 6 h. Actin expression was used as an internal control. Bars represent means ± SD of three replicates and independent experiments were performed at least three times. Different letters above the columns indicate significant differences (P < 0.05) between treatments.
Figure 3. Cellular localization of the BoALMT1 protein by transient expression of the GFP::BoALMT1 fusion protein in epidermal onion cells. (a–d) The plasma membrane localization of BoALMT1 in onion epidermal cells before (a,b) and after cell plasmolysis with 0.8 M mannitol (c,d). (e–h) GFP protein in onion epidermal cells before (e,f) and after cell plasmolysis with 0.8M manitol (g,h). PM, CW, and TM labels denote the plasma membrane cell wall and tonoplast membrane localization, respectively. White bars = 100 μm.
Figure 4. Time course of malate secretion after exposure of 50 μM Al in cabbage roots. Data are means ± SD (n = 5).
Figure 5. Characterization of BoALMT1 in Xenopus oocytes. (A) Mean values of H+ fluxes in the absence or presence of 50 μM Al with pretreated malate or water. Each value represents the average of at least 3 different cells, the error bars represent SD (n = 4–6). (B) Malate efflux transport activity. Control and BoALMT1-expressing oocytes injected with 14C-labeled malate were kept in OCM solution. The radioactivity in the bathing solution was measured at the indicated time points; values are expressed as a percentage of the total radioactivity injected. Data was given as means ± SD (n = 3). Different letters above the columns indicate significant differences (P < 0.05) between treatments.
Figure 6. Expression of BoALMT1 in transgenic Arabidopsis plants results in enhanced citrate release and Al tolerance. (A)
BoALMT1 expression in the two transgenic lines (A1 and A2) and a control line (WT). (B) Root malate exudation in the absence and presence of 50 μM AlCl3. Experiments were repeated at least three times (n = 100). (C) Root growth of representative plants from two independent transgenic lines grown in agar medium in the absence or presence of Al for 2 days. (D) Relative root growth of the plants subjected to 400 μM Al for 2 d. Each bar represents the mean of three replicates ± SD (n = 4). (E) Comparison H+ flux at the DEZ of 4- to 5-day-old Arabidopsis thaliana seedlings in the presence and absence of Al. Data are given as means ± SD (n = 3–5). Different letters above the columns indicate significant differences (P < 0.05) between treatments.
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