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Overexpression of the soybean GmERF3 gene, an AP2/ERF type transcription factor for increased tolerances to salt, drought, and diseases in transgenic tobacco.

Zhang G, Chen M, Li L, Xu Z, Chen X, Guo J, Ma Y - J. Exp. Bot. (2009)

Bottom Line: The GmERF3 protein fused to the GAL4 DNA-binding domain to activate transcription of reporter genes in yeast.Furthermore, overexpression of GmERF3 in transgenic tobacco led to higher levels of free proline and soluble carbohydrates compared to wild-type plants under drought conditions.The overall results suggested that GmERF3 as an AP2/ERF transcription factor may play dual roles in response to biotic and abiotic stresses in plants.

View Article: PubMed Central - PubMed

Affiliation: The National Key Facility for Crop Genetic Resources and Genetic Improvement, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China.

ABSTRACT
A new member of the AP2/ERF transcription factor family, GmERF3, was isolated from soybean. Sequence analysis showed that GmERF3 contained an AP2/ERF domain of 58 amino acids and two putative nuclear localization signal (NLS) domains. It belonged to a group IV protein in the ERF (ethylene response factor) subfamily as typified by a conserved N-terminal motif [MCGGAI(I/L)]. Expression of GmERF3 was induced by treatments with high salinity, drought, abscisic acid (ABA), salicylic acid (SA), jasmonic acid (JA), ethylene (ET), and soybean mosaic virus (SMV), whereas there was no significant GmERF3 mRNA accumulation under cold stress treatment. GmERF3 could bind to the GCC box and DRE/CRT element, and was targeted to the nucleus when transiently expressed in onion epidermal cells. The GmERF3 protein fused to the GAL4 DNA-binding domain to activate transcription of reporter genes in yeast. Ectopic expression of the GmERF3 gene in transgenic tobacco plants induced the expression of some PR genes and enhanced resistance against infection by Ralstonia solanacearum, Alternaria alternata, and tobacco mosaic virus (TMV), and gave tolerance to high salinity and dehydration stresses. Furthermore, overexpression of GmERF3 in transgenic tobacco led to higher levels of free proline and soluble carbohydrates compared to wild-type plants under drought conditions. The overall results suggested that GmERF3 as an AP2/ERF transcription factor may play dual roles in response to biotic and abiotic stresses in plants.

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Sequence-specific binding activity of GmERF3 to the GCC box and DRE/CRT element. (A) Nucleotide sequences of GCC, mutated GCC (mGCC), DRE/CRT, and mutated DRE/CRT (mDRE/CRT) probes. The core sequences of GCC and DRE/CRT elements are underlined and mutated nucleotides in mGCC and mDRE/CRT probes are expressed in bold italic. (B) Gel retardation assay showing sequence-specific binding to the GCC box of the recombinant GmERF3 protein. 0.2 μg GST-GmERF3 protein was added to each reaction mixture. Lane 1 contained only the free GCC probes, and lane 2 contained free GCC probe and GST protein. Lane 3, radiolabelled GCC probe; lanes 4 and 5, titration with a cold GCC sequence as a competitor; lane 6, radiolabelled mutated GCC (mGCC) probe; lanes 7 and 8, titration with cold mGCC sequence as a competitor. (C) Gel retardation assay showing sequence-specific binding to the DRE/CRT element of the recombinant GmERF3 protein. 0.2 μg GST-GmERF3 protein was added to each reaction mixture. Lane 1 contained only the free DRE/CRT probe, and lane 2 contained free DRE/CRT probe; GST protein; lane 3, radiolabelled DRE/CRT probe; lanes 4 and 5, titration with a cold DRE/CRT sequence as a competitor; lane 6, radiolabelled mutated DRE/CRT (mDRE/CRT) probe; lanes 7 and 8, titration with a cold mDRE/CRT sequence as a competitor.
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fig3: Sequence-specific binding activity of GmERF3 to the GCC box and DRE/CRT element. (A) Nucleotide sequences of GCC, mutated GCC (mGCC), DRE/CRT, and mutated DRE/CRT (mDRE/CRT) probes. The core sequences of GCC and DRE/CRT elements are underlined and mutated nucleotides in mGCC and mDRE/CRT probes are expressed in bold italic. (B) Gel retardation assay showing sequence-specific binding to the GCC box of the recombinant GmERF3 protein. 0.2 μg GST-GmERF3 protein was added to each reaction mixture. Lane 1 contained only the free GCC probes, and lane 2 contained free GCC probe and GST protein. Lane 3, radiolabelled GCC probe; lanes 4 and 5, titration with a cold GCC sequence as a competitor; lane 6, radiolabelled mutated GCC (mGCC) probe; lanes 7 and 8, titration with cold mGCC sequence as a competitor. (C) Gel retardation assay showing sequence-specific binding to the DRE/CRT element of the recombinant GmERF3 protein. 0.2 μg GST-GmERF3 protein was added to each reaction mixture. Lane 1 contained only the free DRE/CRT probe, and lane 2 contained free DRE/CRT probe; GST protein; lane 3, radiolabelled DRE/CRT probe; lanes 4 and 5, titration with a cold DRE/CRT sequence as a competitor; lane 6, radiolabelled mutated DRE/CRT (mDRE/CRT) probe; lanes 7 and 8, titration with a cold mDRE/CRT sequence as a competitor.

Mentions: Some proteins in the ERF subfamily have dual binding activities with the GCC box and DRE/CRT sequence (Park et al., 2001; Hao et al., 2002; Zhang et al., 2004; Lee et al., 2005; Tang et al., 2007). To test whether GmERF3 binds to the cis-acting elements of the GCC box and DRE/CRT element in vitro, gel retardation assays were performed. Purified proteins from E. coli cultures harbouring the appropriate vector without the GmERF3 gene were isolated as a control (GST control). The sequences of GCC, mutated GCC (mGCC), DRE/CRT, and mutated DRE/CRT (mDRE/CRT) are shown in Fig. 3A. When the GCC box was used as a probe, the soybean GmERF3 protein caused a mobility shift in the radioactively labelled GCC probe (Fig. 3B, lane 3), which migrated more slowly than the free probe (Fig. 3B, lane 1). This shift was specific to the soybean protein and did not occur in the GST control (Fig. 3B, lane 2). Furthermore, when a mutated version of the GCC box (mGCC) was used in the assay, the mobility shift was not observed (Fig. 3B, lane 6). Competition experiments were conducted to examine the specificity of the mobility shift. The purified soybean GmERF3 protein was mixed with the radiolabelled GCC probe, and an unlabelled competitor (either the GCC probe or the mGCC probe) was then added to the mixture in varying amounts. As the levels of unlabelled GCC increased, the amount of bound labelled probe decreased. A 50-fold excess of a cold GCC sequence was sufficient to displace the labelled probe (Fig. 3B, lane 4). When the ratio of unlabelled to labelled GCC probe reached approximately 100:1, virtually no labelled probe was bound (Fig. 3B, lane 5). When the unlabelled mGCC probe was used as the competitor, no binding competition was observed, even at a ratio of 100:1 unlabelled mGCC to labelled GCC (Fig. 3B, lanes 7 and 8). Similar experiments were carried out with DRE/CRT and its mutated sequence. Recombinant GmERF3 was also able to bind the DRE/CRT sequence in a specific manner (Fig. 3C), although this binding capacity seemed to be lower than to the GCC box.


Overexpression of the soybean GmERF3 gene, an AP2/ERF type transcription factor for increased tolerances to salt, drought, and diseases in transgenic tobacco.

Zhang G, Chen M, Li L, Xu Z, Chen X, Guo J, Ma Y - J. Exp. Bot. (2009)

Sequence-specific binding activity of GmERF3 to the GCC box and DRE/CRT element. (A) Nucleotide sequences of GCC, mutated GCC (mGCC), DRE/CRT, and mutated DRE/CRT (mDRE/CRT) probes. The core sequences of GCC and DRE/CRT elements are underlined and mutated nucleotides in mGCC and mDRE/CRT probes are expressed in bold italic. (B) Gel retardation assay showing sequence-specific binding to the GCC box of the recombinant GmERF3 protein. 0.2 μg GST-GmERF3 protein was added to each reaction mixture. Lane 1 contained only the free GCC probes, and lane 2 contained free GCC probe and GST protein. Lane 3, radiolabelled GCC probe; lanes 4 and 5, titration with a cold GCC sequence as a competitor; lane 6, radiolabelled mutated GCC (mGCC) probe; lanes 7 and 8, titration with cold mGCC sequence as a competitor. (C) Gel retardation assay showing sequence-specific binding to the DRE/CRT element of the recombinant GmERF3 protein. 0.2 μg GST-GmERF3 protein was added to each reaction mixture. Lane 1 contained only the free DRE/CRT probe, and lane 2 contained free DRE/CRT probe; GST protein; lane 3, radiolabelled DRE/CRT probe; lanes 4 and 5, titration with a cold DRE/CRT sequence as a competitor; lane 6, radiolabelled mutated DRE/CRT (mDRE/CRT) probe; lanes 7 and 8, titration with a cold mDRE/CRT sequence as a competitor.
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fig3: Sequence-specific binding activity of GmERF3 to the GCC box and DRE/CRT element. (A) Nucleotide sequences of GCC, mutated GCC (mGCC), DRE/CRT, and mutated DRE/CRT (mDRE/CRT) probes. The core sequences of GCC and DRE/CRT elements are underlined and mutated nucleotides in mGCC and mDRE/CRT probes are expressed in bold italic. (B) Gel retardation assay showing sequence-specific binding to the GCC box of the recombinant GmERF3 protein. 0.2 μg GST-GmERF3 protein was added to each reaction mixture. Lane 1 contained only the free GCC probes, and lane 2 contained free GCC probe and GST protein. Lane 3, radiolabelled GCC probe; lanes 4 and 5, titration with a cold GCC sequence as a competitor; lane 6, radiolabelled mutated GCC (mGCC) probe; lanes 7 and 8, titration with cold mGCC sequence as a competitor. (C) Gel retardation assay showing sequence-specific binding to the DRE/CRT element of the recombinant GmERF3 protein. 0.2 μg GST-GmERF3 protein was added to each reaction mixture. Lane 1 contained only the free DRE/CRT probe, and lane 2 contained free DRE/CRT probe; GST protein; lane 3, radiolabelled DRE/CRT probe; lanes 4 and 5, titration with a cold DRE/CRT sequence as a competitor; lane 6, radiolabelled mutated DRE/CRT (mDRE/CRT) probe; lanes 7 and 8, titration with a cold mDRE/CRT sequence as a competitor.
Mentions: Some proteins in the ERF subfamily have dual binding activities with the GCC box and DRE/CRT sequence (Park et al., 2001; Hao et al., 2002; Zhang et al., 2004; Lee et al., 2005; Tang et al., 2007). To test whether GmERF3 binds to the cis-acting elements of the GCC box and DRE/CRT element in vitro, gel retardation assays were performed. Purified proteins from E. coli cultures harbouring the appropriate vector without the GmERF3 gene were isolated as a control (GST control). The sequences of GCC, mutated GCC (mGCC), DRE/CRT, and mutated DRE/CRT (mDRE/CRT) are shown in Fig. 3A. When the GCC box was used as a probe, the soybean GmERF3 protein caused a mobility shift in the radioactively labelled GCC probe (Fig. 3B, lane 3), which migrated more slowly than the free probe (Fig. 3B, lane 1). This shift was specific to the soybean protein and did not occur in the GST control (Fig. 3B, lane 2). Furthermore, when a mutated version of the GCC box (mGCC) was used in the assay, the mobility shift was not observed (Fig. 3B, lane 6). Competition experiments were conducted to examine the specificity of the mobility shift. The purified soybean GmERF3 protein was mixed with the radiolabelled GCC probe, and an unlabelled competitor (either the GCC probe or the mGCC probe) was then added to the mixture in varying amounts. As the levels of unlabelled GCC increased, the amount of bound labelled probe decreased. A 50-fold excess of a cold GCC sequence was sufficient to displace the labelled probe (Fig. 3B, lane 4). When the ratio of unlabelled to labelled GCC probe reached approximately 100:1, virtually no labelled probe was bound (Fig. 3B, lane 5). When the unlabelled mGCC probe was used as the competitor, no binding competition was observed, even at a ratio of 100:1 unlabelled mGCC to labelled GCC (Fig. 3B, lanes 7 and 8). Similar experiments were carried out with DRE/CRT and its mutated sequence. Recombinant GmERF3 was also able to bind the DRE/CRT sequence in a specific manner (Fig. 3C), although this binding capacity seemed to be lower than to the GCC box.

Bottom Line: The GmERF3 protein fused to the GAL4 DNA-binding domain to activate transcription of reporter genes in yeast.Furthermore, overexpression of GmERF3 in transgenic tobacco led to higher levels of free proline and soluble carbohydrates compared to wild-type plants under drought conditions.The overall results suggested that GmERF3 as an AP2/ERF transcription factor may play dual roles in response to biotic and abiotic stresses in plants.

View Article: PubMed Central - PubMed

Affiliation: The National Key Facility for Crop Genetic Resources and Genetic Improvement, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China.

ABSTRACT
A new member of the AP2/ERF transcription factor family, GmERF3, was isolated from soybean. Sequence analysis showed that GmERF3 contained an AP2/ERF domain of 58 amino acids and two putative nuclear localization signal (NLS) domains. It belonged to a group IV protein in the ERF (ethylene response factor) subfamily as typified by a conserved N-terminal motif [MCGGAI(I/L)]. Expression of GmERF3 was induced by treatments with high salinity, drought, abscisic acid (ABA), salicylic acid (SA), jasmonic acid (JA), ethylene (ET), and soybean mosaic virus (SMV), whereas there was no significant GmERF3 mRNA accumulation under cold stress treatment. GmERF3 could bind to the GCC box and DRE/CRT element, and was targeted to the nucleus when transiently expressed in onion epidermal cells. The GmERF3 protein fused to the GAL4 DNA-binding domain to activate transcription of reporter genes in yeast. Ectopic expression of the GmERF3 gene in transgenic tobacco plants induced the expression of some PR genes and enhanced resistance against infection by Ralstonia solanacearum, Alternaria alternata, and tobacco mosaic virus (TMV), and gave tolerance to high salinity and dehydration stresses. Furthermore, overexpression of GmERF3 in transgenic tobacco led to higher levels of free proline and soluble carbohydrates compared to wild-type plants under drought conditions. The overall results suggested that GmERF3 as an AP2/ERF transcription factor may play dual roles in response to biotic and abiotic stresses in plants.

Show MeSH
Related in: MedlinePlus