Genome-Wide Identification and Characterization of ABC Transporters in Nine Rosaceae Species Identifying MdABCG28 as a Possible Cytokinin Transporter linked to Dwarfing

Abstract

ATP-binding cassette (ABC) transporters constitute a large, diverse, and ubiquitous superfamily that is involved in a broad range of processes. The completion of genome sequencing provides an opportunity to understand the phylogenetic history of the ABC transporter superfamily among Rosaceae species. This study identified a total of 1323 ABC transporter genes from nine Rosaceae genomes: 191 from Malus domestica, 174 from Pyrus communis, 138 from Prunus persica, 118 from Prunus avium, 141 from Prunus dulcis, 122 from Fragaria vesca, 98 from Rubus occidentalis, 162 from Prunus mume, and 179 from Rosa chinensis. Their chemical characterization, phylogenetic analysis, chromosomal localization, gene structure, gene duplication, and tissue-specific expression were studied. Their subcellular localization, transmembrane structures, and protein motifs were predicted. All the ABC transporter genes were grouped into eight subfamilies on the basis of their phylogenetic relationships and structural features. Furthermore, cis-element and expression analysis of 10 potential phytohormone transporters in MdABCG subfamily genes were also performed. Loss of the W-box in the promoter region of MdABCG28 was found to reduce the gene expression level and was linked to the dwarfing phenotype in apple rootstocks. MdABCG28 overexpression promoted shoot growth of atabcg14 mutants in Arabidopsis.

Keywords: ABC transporter; Rosaceae; apple; bioinformatics; cytokinin transport; dwarfing.

Figure 1

Numbers of ATP-binding cassette (ABC) transporter family members for each classification in Rosaceae: (a) subfamily; (b) isoelectric point; (c) instability index; (d) grand average of hydropathicity; (e) transmembrane structure.

Figure 2

Phylogenetic trees, motif analysis, and gene structures of potential phytohormone transporters in ABCG subfamily genes of nine Rosaceae species. (a) The phylogenetic tree was constructed on the basis of the full-length sequences of known phytohormone transporter genes in Arabidopsis and homologous genes in nine Rosaceae species using the neighbor-joining (NJ) method and 1000 replications for bootstrap analysis in MEGA 6.0. Red stars indicate apple homologues. (b) Motif analyses. Twenty motifs are indicated with different colored boxes. Logos of each motif are shown in Table 1. All motifs were identified via online analysis with the package MEME Suite 5.0.2. (c) Gene structures. Exons are represented by yellow boxes. Black lines connecting two exons represent introns. Green boxes represent untranslated regions (UTRs). Gene structure maps were drawn with TBtools.

Figure 3

Expression profiles of 10 potential phytohormone transporters in MdABCG subfamily genes. (a) Tissue expression profiles obtained from ArrayExpress data displaying diverse expression levels of 10 potential phytohormone transporters in MdABCG subfamily genes in different tissues. Relative transcript levels based on ArrayExpress data are presented as heat maps from blue to red, reflecting relative signal values. (b) Cis-elements analysis. These cis-elements responded to environmental stress and phytohormone signal in the sense strands of 10 MdABCG subfamily gene promoters. The x-axis indicates the number of each cis-element. (c) Inducible expression profiles of 10 MdABCG subfamily genes under indole-3-acetic acid (IAA) (1 mM), indole-3-butyric acid (IBA) (1 mM), trans-zeatin (tZ) (1 mM), N⁶-(Δ²-isopentenyl) adenine (iP) (1 mM), or abscisic acid (ABA) (1 mM) in apple callus. The color scale represents the log2 transformed gene relative expression compared with controls.

Figure 4

Functional analysis of MdABCG28 and its variant promoter. (a) Relative expression of MdABCG28 in roots of apple rootstocks. (b) Transient GFP expression assay in Arabidopsis protoplasts testing the effects of the 17 bp deletion variant in the MdABCG28 promoter. The 2 kb promoter region of MdABCG28 was amplified and cloned into the pCambia1302 vector (GFP tag) by inserting it into the EcoRI/KpnI site. The M.9 construct contained a 17 bp deletion sequence (-GAACCGTCTTGACATGT-) in the region from 1186 to 1202 bp with a W-box (-TTGAC-) compared with the robusta construct. The A1 construct was generated on the basis of the M.9 construct with a 17 bp deletion sequence inserted back. The A2 construct was generated on the basis of robusta construct with W-box mutated (-GAACCGTCTTCACATGT-). The pCAMBIA1301 vector (GUS tag) was used as internal control. The panel shows the GFP expression levels relative to GUS (n = 10). (c) Cytokinin contents and (d) phenotypes of the atabcg14 mutant and its overexpressed lines of MdABCG28. The full-length cDNA of MdABCG28 was amplified and cloned into the pCambia1300 vector by inserting it into the EcoRI/KpnI site. The resulting recombinant vector was inserted into Agrobacterium tumefaciens LBA4404 cells to transform atabcg14 mutant plants. Homozygous T3 transgenic lines were used. Biological replicates were performed in triplicate. Error bars represent the standard error. Asterisks indicate p-values below 0.05, obtained using a two-tailed Student’s t-test. Different letters indicate p-values below 0.05, obtained using Duncan’s test.