Sweetpotato bZIP Transcription Factor IbABF4 Confers Tolerance to Multiple Abiotic Stresses

Abstract

The abscisic acid (ABA)-responsive element binding factors (ABFs) play important regulatory roles in multiple abiotic stresses responses. However, information on the stress tolerance functions of ABF genes in sweetpotato (Ipomoea batatas [L.] Lam) remains limited. In the present study, we isolated and functionally characterized the sweetpotato IbABF4 gene, which encodes an abiotic stress-inducible basic leucine zipper (bZIP) transcription factor. Sequence analysis showed that the IbABF4 protein contains a typical bZIP domain and five conserved Ser/Thr kinase phosphorylation sites (RXXS/T). The IbABF4 gene was constitutively expressed in leaf, petiole, stem, and root, with the highest expression in storage root body. Expression of IbABF4 was induced by ABA and several environmental stresses including drought, salt, and heat shock. The IbABF4 protein localized to the nucleus, exhibited transcriptional activation activity, and showed binding to the cis-acting ABA-responsive element (ABRE) in vitro. Overexpression of IbABF4 in Arabidopsis thaliana not only increased ABA sensitivity but also enhanced drought and salt stress tolerance. Furthermore, transgenic sweetpotato plants (hereafter referred to as SA plants) overexpressing IbABF4, generated in this study, exhibited increased tolerance to drought, salt, and oxidative stresses on the whole plant level. This phenotype was associated with higher photosynthetic efficiency and lower malondialdehyde and hydrogen peroxide content. Levels of endogenous ABA content and ABA/stress-responsive gene expression were significantly upregulated in transgenic Arabidopsis and sweetpotato plants compared with wild-type plants under drought stress. Our results suggest that the expression of IbABF4 in Arabidopsis and sweetpotato enhances tolerance to multiple abiotic stresses through the ABA signaling pathway.

Keywords: IbABF4; abiotic stress; drought tolerance; salt tolerance; sweetpotato.

FIGURE 1

Multiple sequence alignment of the highly conserved bZIP domains and phylogenetic analysis of selected ABF-related proteins. (A) Multiple sequence alignment of the conserved bZIP domains of selected ABF-related proteins. Color-coding indicates sequence similarity, with black indicating the highest sequence similarity, pink indicating lower similarity, and blue indicating the least similarity. (B) Phylogenetic analysis of IbABF4 homologs in plants.

FIGURE 2

Expression profiles of IbABF4 in various tissues and under various abiotic stress conditions. (A) Tissue-specific expression pattern of IbABF4. L, leaf; P, petiole; S, stem; FR, fibrous root; PR, pencil root; SRp, proximal end of storage root; SRb, storage root body; SRd, distal end of storage root. (B) Expression pattern of IbABF4 in response to various abiotic stresses. Three-week-old sweetpotato plants grown in half-strength Hoagland nutrient solution were subjected to ABA (10 μM), dehydration (25% PEG8000), high salt (350 mM NaCl), and heat shock (47°C) treatments. Expression levels of genes were determined in the third fully expanded intact leaf (from the top) by qRT-PCR analysis. Expression levels of genes were normalized relative to the IbActin gene (internal control). Data represent mean ± standard deviation (SD) of three biological replicates.

FIGURE 3

Subcellular localization and transactivation assay of the IbABF4 protein. (A) Subcellular localization of IbABF4. (B) Vectors used in the yeast one-hybrid assay. (C) Transactivation activity assay of the IbABF4 protein. The pGAL4 and pBD empty vectors were used as positive and negative controls, respectively, in yeast one-hybrid assay. (D) Relative quantitative assay of β-galactosidase activity (expressed in Miller units). Data represent mean ± SD of three independent biological replicates. Asterisks indicate significant differences between pBD and other vectors at P < 0.01.

FIGURE 4

Binding of IbABF4 to the cis-acting ABRE in EMSA. (A) Oligonucleotide probes containing the ABRE repeat motif. The underlined letters indicate the ABRE core sequence. (B) Analysis of binding specificity of IbABF4. FP indicates free probe. EMSA was performed using ³²P-labeled ABRE probe with GST protein (lane 2) or GST-IbABF4. Triangles indicate increasing amounts of GST-IbABF4 protein (5 and 10 μg) used for DNA-binding analysis. (C) Competition analysis of unlabeled probe in EMSA. Triangles indicate increasing amounts of unlabeled probe. GST-IbABF4 protein was preincubated with 1-, 20-, 40-, and 80-fold molar excess of ABRE before the addition of probe.

FIGURE 5

Seed germination assay of transgenic Arabidopsis lines overexpressing IbABF4 under ABA, drought, and salt stresses. (A) Seed germination phenotype of transgenic and WT Col-0 seeds on 1/2 MS medium supplemented with or without (control) 0.6 μM ABA, 200 mM mannitol, or 125 mM NaCl. (B) Germination rate of transgenic and Col-0 seeds under ABA, drought, and salt stresses. A total of 100 seeds were used for the calculation of germination rate in each treatment. Data represent mean ± SD of three biological replicates. Asterisks indicate significant differences at P < 0.01.

FIGURE 6

Post-germination growth assay of transgenic Arabidopsis plants overexpressing IbABF4 under drought and salt stresses. (A) Growth of transgenic Arabidopsis plants on 1/2 MS medium supplemented with or without (control) 300 mM mannitol, or 125 mM NaCl. (B) Root length of Arabidopsis plants subjected to drought and salt stresses. (C) Fresh weight of Arabidopsis plants exposed to drought and salt stresses. A total of 40 seedlings were used in each treatment. Data represent mean ± SD of three biological replicates. Asterisks indicate significant differences at P < 0.01.

FIGURE 7

Overexpression of IbABF4 in Arabidopsis confers drought tolerance. (A) Drought tolerance phenotypes of 3-week-old transgenic Arabidopsis (OE9 and OE13) lines and Col-0 plants grown in soil without irrigation for 10 days, followed by recovery for 1 day. Contents of MDA (B), H₂O₂ (C) and ABA (D) in leaves of Col-0 and OE plants grown without irrigation for 3 and 6 days. (E) Transcript levels of stress-responsive marker genes in OE9, OE13, and Col-0 plants. The AtActin gene was used as an internal control. Data represent mean ± SD of three biological replicates. Asterisks indicate significant differences at P < 0.01.

FIGURE 8

Generation of SA plants overexpressing IbABF4. (A) Schematic representation of the constructs used for sweetpotato transformation. (B) PCR analysis of SA plants using GFP-specific primers. M, DNA size markers; P, positive control. (C) qRT-PCR analysis of SA plants using IbABF4-specific primers. The IbActin gene was used as an internal control. Data represent mean ± SD of three biological replicates.

FIGURE 9

Phenotypic and physiological characterization of SA plants under drought stress. (A) Visible damage in the leaves of sweetpotato plants after drought stress treatment for 17 days, followed by recovery for 2 days. (B) Photosynthetic efficiency (Fv/Fm) of PSII. Contents of MDA (C) and H₂O₂ (D) in leaves of 5-week-old WT and SA plants subjected to drought stress for 17 days, followed by recovery for 2 days. (E) ABA content of leaves of 5-week-old WT and SA plants subjected to drought stress for 3 and 6 days. (F) Transcript levels of stress-responsive marker genes in WT and SA plants determined by qRT-PCR. Samples of WT plants were collected prior to drought stress treatment, and transcript levels of genes were normalized relative to IbActin transcripts. Data represent mean ± SD of three biological replicates. Asterisks indicate significant differences between WT and SA plants at P < 0.01.

FIGURE 10

Phenotypic and physiological characterization of SA plants under salt stress. (A) Visible damage in the leaves of sweetpotato plants after 6 days of salt stress treatment. (B) PSII photosynthetic efficiency (Fv/Fm). Contents of MDA (C), H₂O₂ (D), and ABA (E) in leaves of 5-week-old WT and SA plants subjected to salt stress treatment for 6 days. Data represent mean ± SD of three biological replicates. Asterisks indicate significant differences between WT and SA plants at P < 0.01.

FIGURE 11

Oxidative stress tolerance of SA plants. (A) Visible damage in detached leaves of WT and SA plants treated with 2 μM MV. (B) Relative membrane permeability of detached leaves treated with 2 μM MV at 48 h. Data represent mean ± SD of three biological replicates. Asterisks indicate significant differences between WT and SA plants at P < 0.01. (C) Analysis of H₂O₂ accumulation in detached leaves using DAB staining. The third or fourth fully expanded leaves were collected from the top of 5-week-old WT and SA plants.