Genome-Wide Identification and Characterization of the AREB/ABF/ABI5 Subfamily Members from Solanum tuberosum

Abstract

Abscisic acid (ABA) plays crucial roles in plant development and adaption to environmental stresses. The ABA-responsive element binding protein/ABRE-binding factor and ABA INSENSITIVE 5 (AREB/ABF/ABI5) gene subfamily members, which belong to the basic domain/leucine zipper (bZIP) transcription factors family, participate in the ABA-mediated signaling pathway by regulating the expression of their target genes. However, information about potato (Solanum tuberosum) AREB/ABF/ABI5 subfamily members remains scarce. Here, seven putative AREB/ABF/ABI5 members were identified in the potato genome. Sequences alignment revealed that these members shared high protein sequence similarity, especially in the bZIP region, indicating that they might possess overlapping roles in regulating gene expression. Subcellular localization analysis illustrated that all seven AREB/ABF/ABI5 members were localized in the nucleus. Transactivation activity assays in yeast demonstrated that these AREB/ABF/ABI5 members possessed distinct transcriptional activity. Electrophoretic mobility shift assays (EMSA) confirmed that all of these AREB/ABF/ABI5 members could have an affinity to ABRE in vitro. The expression patterns of these AREB/ABF/ABI5 genes showed that they were in response to ABA or osmotic stresses in varying degrees. Moreover, most AREB/ABF/ABI5 genes were induced during stolon swelling. Overall, these results provide the first comprehensive identification of the potato AREB/ABF/ABI5 subfamily and would facilitate further functional characterization of these subfamily members in future work.

Keywords: AREB/ABF; abscisic acid; gene regulation; osmotic stress; potato; transcription factor.

Figure 1

Sequence analysis and chromosomal localization of potato AREB/ABF/ABI5 members. (a) Neighbor-joining tree of potato and Arabidopsis AREB/ABF/ABI5 proteins and the gene structure of each potato AREB/ABF/ABI5 member. The sizes (bp) of exons are as indicated. (b) The seven identified AREB/ABF/ABI5 members were mapped on the four chromosomes.

Figure 1

Sequence analysis and chromosomal localization of potato AREB/ABF/ABI5 members. (a) Neighbor-joining tree of potato and Arabidopsis AREB/ABF/ABI5 proteins and the gene structure of each potato AREB/ABF/ABI5 member. The sizes (bp) of exons are as indicated. (b) The seven identified AREB/ABF/ABI5 members were mapped on the four chromosomes.

Figure 2

Multiple sequence alignments of potato AREB/ABF/ABI5 members. Identical and conserved residues are shaded in blue and light blue, respectively. The positions of the C1 to C4 conserved domains and of the basic region are indicated by lines above the protein sequences. Potential phosphoresidues (R-X-X-S/T) corresponding to the characterized phosphorylation sites in Arabidopsis AREB1 are indicated by red frames. The positions of the conserved Leu residues in the Leu zipper domain are indicated by a black triangle. Protein sequence alignments were performed by Jalview 2.10 (http://www.jalview.org/).

Figure 3

Subcellular localization analysis of potato AREB/ABF/ABI5 members. The GFP alone and GFP-fusion proteins were transiently expressed in N. benthamiana leaves via agroinfiltration and the images of epidermal cells were taken at 60 hpi by CLSM. Green color indicates that GFP alone is localized in both the cytoplasm and nucleus, AREB/ABF/ABI5 members are mainly localized in the nucleus. Bars = 20 μm.

Figure 4

Transcriptional activation analysis of potato AREB/ABF/ABI5 members in yeast. The recombinant vectors were transferred into yeast strain AH109. The transcriptional activation ability of these proteins was analyzed by growing on SD/-Trp and SD/-Trp-His-Ade plates. The yeast strain AH109 harboring the pGBKT7 vector was used as a negative control.

Figure 5

Electrophoretic mobility shift assay (EMSA). The oligonucleotide (ABRE) containing the Em1a element (GGACACGTGGCG) was employed as a FAM-labeled probe in a mobility shift assay. In each assay, 1 μg of purified recombinant full-length or truncated AREB/ABF/ABI5 was used. The ‘+’ and ‘−’ indicate the presence and absence of the indicated probe or protein. Lanes 3 and 4, and 5 and 6 represent a 50-fold and 100-fold molar excess of the specific unlabeled competitor (ABRE) or unlabeled mutated oligonucleotide (mABRE) competitor. Sequences of oligonucleotides are as follows: ABRE, aattccGGACACGTGGCGtaagct; mABRE, aattccGGACctacaGCCtaagct. Shifted bands are indicated by an arrowhead.

Figure 6

Expression profiles of potato AREB/ABF/ABI5 members under treatments of ABA, dehydration, and high salinity. The relative expression levels are normalized to ef1α. ABA, dehydration, and high salinity represent the in vitro plants treated with 5 μM ABA, water deficit, and 125 mM NaCl. The numbers 0, 6, and 24 indicate the time (hour) after treatments. Error bars represent the standard deviation (SD) of three biological replicates. * and ** represents significant differences in p values < 0.5 and < 0.01, respectively.

Figure 7

Expression profiles of potato AREB/ABF/ABI5 members in distinct stages of stolon swelling. (a) Respective image showing distinct stages in the swelling process, ranging from no swelling (Stage 1) to prominent subapical swelling (Stage 4). (b–h) Graphs show the expression patterns of the potato AREB/ABF/ABI5 members in the four different stages. The Y-axis is the relative expression levels after normalization with ef1α. The numbers 1, 2, 3, and 4 indicate the four stages shown in (a). Error bars represent the standard deviation (SD) of three biological replicates.