Genome-wide analysis of MATE transporters and molecular characterization of aluminum resistance in Populus

Abstract

Ionic aluminum (Al) in acidic soils, comprising approximately 50% of arable land globally, is highly toxic to most plant species. Populus grow naturally in acidic soils and tolerate high concentrations of Al. Multidrug and toxic compound extrusion (MATE) family genes in plants are involved in responses to Al tolerance. To date, however, the functional roles of the MATE genes in Populus remain unclear. In the present study, 71 putative MATE transporters were predicted in the genome of Populus trichocarpa. The chromosome distribution, phylogenetic relationships, and expression level analysis revealed that four candidate MATE genes belonging to subgroup IIIc might contribute to high Al tolerance in poplar. Further, the expression levels of two subgroup IIIc members, PtrMATE1 and PtrMATE2, were induced by Al stress. Transient expression in onion epidermal cells showed that PtrMATE1 was localized to the plasma membrane. Overexpression of PtrMATE1 increased Al-induced secretion of citrate from the root apex of transgenic plants. Al-induced inhibition of root growths were alleviated in both PtrMATE1 overexpression lines in Populus and in Arabidopsis compared with wild-type plants. In addition, PtrMATE1 expression was induced at 12 h after exposure to Al stress whereas PtrMATE2 expression was induced at 24 h, indicating that these proteins coordinately function in response to Al stress in poplar. Taken together, these results provide important insights into the molecular mechanisms involved in Al tolerance in poplar.

Keywords: Aluminum stress; MATE transporter; Populus; citrate exudation; root apex.

Fig. 1.

Chromosomal location of Populus MATE genes. A total of 71 MATE genes are mapped to the 18 linkage groups (LG). Schematic view of chromosome reorganization through recent whole genome duplication in Populus is shown. Segmentally duplicated homologous blocks are indicated with the same color. The scale represents megabases (Mb). The LG numbers are indicated at the top of each bar.

Fig. 2.

The phylogenetic tree of MATE proteins from P. trichocarpa and other plant species. The phylogenetic tree was constructed using MEGA 6.0 with the Maximum Likelihood (ML) method. Bootstrap values in percentages (1000 replicates) are indicated on the nodes. Different subgroups are highlighted using different colors and marked with arcs outside the cycle tree.

Fig. 3.

The gene structures and conserved motifs of PtrMATE family members. (A) Exons and introns of PtrMATE genes are plotted using green boxes and black lines, respectively. The blue boxes indicate upstream/downstream sequences. (B) Protein motifs of the PtrMATE family. The motifs of Populus MATE proteins are shown as colored boxes, each motif is represented as a number in the colored box. The genes are listed according to the order of subfamily I to III from the phylogenetic tree and different subfamilies are highlighted with lines.

Fig. 4.

Expression analysis of subgroup III MATE genes using qRT-PCR. (A) Expression of four PtrMATE members in poplar roots under 500 μM Al³⁺ for 6 h and 24 h. (B) Relative quantities of four members in roots, stems, young leaves, and mature leaves are illustrated. Expression of PtrMATE1 in wild-type roots was arbitrarily fixed at one. The results are shown as the mean expression ± standard deviation (SD) of three independent experiments. Poplar ubiquitin (UBQ) expression was used as a control and gene-specific primers were used for qRT-PCR analysis of PtrMATE genes. Student’s t-test, *P<0.05, **P<0.01.

Fig. 5.

Subcellular localization of PtrMATE1. GFP alone (A–C) or fusion protein PtrMATE1::GFP (D–F) were transiently expressed in onion epidermal cells, respectively. The images were acquired before (D–F) and after (G–I) plasmolysing the cells with 0.1 M sucrose. The overlay images of the brightfield and fluorescence images are shown (C, F, I). Scale bar, 100 μm (C, F) or 50 μm (I). NU, nucleus; PM, plasma membrane; CW, cell wall.

Fig. 6.

Citrate release and PtrMATE1 expression in poplar roots in response to Al and La treatments. (A) Induction of citrate secretion from the root apices of poplar in response to Al treatment. (B) Time course of PtrMATE1 expression in the roots of poplar in response to Al treatment. (C) Induction of citrate secretion from root apices of poplar in response to La treatment. (D) PtrMATE1 expression in the roots of poplar in response to 12 h La treatment. Excised root apices, 10 mm in length, were placed in the solution containing 0 μM or 500 μM Al or La, respectively. Data are presented as the means ± standard deviation (SD) of three independent experiments. Poplar ubiquitin expression was used as a control. The results are shown as the mean expression ± standard deviation (SD) of three independent experiments. Student’s t-test, *P<0.05, **P<0.01.

Fig. 7.

GUS activity in roots of transgenic PtrMATE1p::GUS plants. The PtrMATE1 gene promoter-driven GUS expression vector was introduced into P. tomentosa Carr. Transgenic plant seedlings were treated with 500 μM Al³⁺ for 0 h (A) and 12 h (B), respectively. GUS staining was observed in the mature roots of transgenic poplar. Scale bars, 1 mm. (C) Quantitative GUS activity in the roots of transgenic plants. Student’s t-test, *P<0.05. Results are shown as mean expression ± standard deviation (SD) of three independent experiments.

Fig. 8.

Overexpression of PtrMATE1 in transgenic poplar confers citrate efflux and Al tolerance. (A) Root growth of wild-type and transgenic plants overexpressing PtrMATE1 in nutrient solution containing 500 μM Al³⁺ for 60 h. All results represent the means ±standard deviation (SD) of three independent experiments. (B) Induction of citrate secretion from root apices of wild-type and transgenic poplar in response to 500 μM Al³⁺ treatment for 12 h. Data are represented as the means ± standard deviation (SD) of three independent experiments. (C) Callose accumulation in the root tips following Al treatment. Poplar seedlings were exposed to a nutrient solution containing 0 M (-Al³⁺) or 500 μM AlCl₃ (+Al³⁺) for 12 h. Seedlings were subsequently fixed, stained with 0.1% aniline blue at pH 9.0, and observed using fluorescence microscopy. The fluorescence images indicate callose accumulation. WT-Al³⁺, wild-type plants in a solution without Al³⁺; WT+Al³⁺, wild-type plants in a solution containing 500 μM Al³⁺; 35S:PtrMATE1-Al³⁺, transgenic plants in a solution without Al³⁺; 35S:PtrMATE1+Al³⁺, transgenic plants in a solution containing 500 μM Al³⁺. (D) Relative expression levels of PtrMATE1, PtrMATE2, PtrDTX27, and PtrART1 in roots of wild-type and transgenic 35S:PtrMATE1 plants under 500 μM Al³⁺ treatment for 0 h and 12 h. Student’s t-test, **P<0.01.

Fig. 9.

Hypothetical model for PtrMATE transporter-mediated citrate secretion in response to Al stress in roots of Populus. (A) Root growth of wild-type plants in nutrient solution containing 500 μM Al³⁺ for 60 h. (B) Expression levels of PtrMATE1 and PtrMATE2 in poplar roots during 48 h under Al treatment. Expression of the poplar ubiquitin (UBQ) gene was used as a control. All results are shown as the mean expression ± standard deviation (SD) of three independent experiments. (C) Hypothetical model for Al-induced citrate secretion from roots of Populus. There are at least three pathways involved in the process. Pathway 1: the receptor (R) on the plasma membrane binds Al³⁺ and activates PtrMATE1 to transport citrate immediately out of the cell through the plasma membrane. Pathway 2: PtrMATE1 expression is induced by Al³⁺ and subsequently PtrMATE1 transports citrate out of the cell. Pathway 3: PtrMATE2 expression is induced by Al³⁺ at 24 h after treatment and subsequently PtrMATE2 transports citrate in cooperation with PtrMATE1.