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The sugar beet gene encoding the sodium/proton exchanger 1 (BvNHX1) is regulated by a MYB transcription factor.

Adler G, Blumwald E, Bar-Zvi D - Planta (2010)

Bottom Line: NHXs can be localized in the tonoplast or plasma membranes, where they exchange sodium ions for protons, resulting in sodium ions being removed from the cytosol into the vacuole or extracellular space.The 5 UTR of BvNHX1, including its intron, does not modulate the activity of the promoter.Gel mobility shift assay confirmed that the WT but not the mutated MYB binding site is bound by nuclear protein extracted from salt-stressed Beta vulgaris leaves.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, The Doris and Bertie Black Center for Bioenergetics in Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel.

ABSTRACT
Sodium/proton exchangers (NHX) are key players in the plant response to salinity and have a central role in establishing ion homeostasis. NHXs can be localized in the tonoplast or plasma membranes, where they exchange sodium ions for protons, resulting in sodium ions being removed from the cytosol into the vacuole or extracellular space. The expression of most plant NHX genes is modulated by exposure of the organisms to salt stress or water stress. We explored the regulation of the vacuolar NHX1 gene from the salt-tolerant sugar beet plant (BvNHX1) using Arabidopsis plants transformed with an array of constructs of BvHNX1::GUS, and the expression patterns were characterized using histological and quantitative assays. The 5 UTR of BvNHX1, including its intron, does not modulate the activity of the promoter. Serial deletions show that a 337 bp promoter fragment sufficed for driving activity that indistinguishable from that of the full-length (2,464 bp) promoter. Mutating four putative cis-acting elements within the 337 bp promoter fragment revealed that MYB transcription factor(s) are involved in the activation of the expression of BvNHX1 upon exposure to salt and water stresses. Gel mobility shift assay confirmed that the WT but not the mutated MYB binding site is bound by nuclear protein extracted from salt-stressed Beta vulgaris leaves.

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Activities of mutated 337 bp BvNHX1 promoter fragments. a Putative cis-acting elements in the S5 promoter fragment. Sites in forward or reverse modes are marked above and below the line, respectively. Mutated sequences are marked by asterisks. b GUS activity was assayed in homogenates made from five homozygous lines of Arabidopsis containing S5::GUS constructs with the indicated mutations (see also Table 1). Seeds were sowed on 0.5× MS (white bars) supplemented with 50 mM NaCl (black bars) or 100 mM mannitol (dashed bars), and seedlings harvested and analyzed 2 weeks later
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Fig5: Activities of mutated 337 bp BvNHX1 promoter fragments. a Putative cis-acting elements in the S5 promoter fragment. Sites in forward or reverse modes are marked above and below the line, respectively. Mutated sequences are marked by asterisks. b GUS activity was assayed in homogenates made from five homozygous lines of Arabidopsis containing S5::GUS constructs with the indicated mutations (see also Table 1). Seeds were sowed on 0.5× MS (white bars) supplemented with 50 mM NaCl (black bars) or 100 mM mannitol (dashed bars), and seedlings harvested and analyzed 2 weeks later

Mentions: The DNA sequence of the S5 construct was used to search for potential binding sites of known trans-acting factors through the Plant cis-acting Regulatory DNA Elements (PLACE) database (Higo et al. 1999) http://www.dna.affrc.go.jp/PLACE/ (Fig. 5a). Four of these potential sites were further studied using site-directed mutagenesis (Table 1). The sequences are putative binding sites for MYB 1AT, MYC, MYB ST1, and napA transcription factors. Transcription factors from the MYC and MYB families are known to be also involved in the modulation of salt stress and water stress regulated genes (Zhu 2002; Shinozaki et al. 2003), whereas napA was shown to function in seeds (Stalberg et al. 1996). Site-directed mutagenesis was performed using Stratagene QuikChange II®, where the putative cis-acting sequence was changed into a restriction enzyme recognition sequence (Table 1). Mutated sequences were confirmed by restriction digests and DNA sequence analyses, and were used for the genetic transformation of Arabidopsis plants. Homozygous transgenic plants were selected and assayed for GUS activity. The basal expression levels in non-stressed plants were not affected by the mutation of any of the potential cis-acting elements (Fig. 5b). Mutation in the MYB 1AT box (position −1362) almost abolished the induction of promoter activity by NaCl and mannitol, suggesting that the BvNHX1 promoter is regulated mainly by a MYB 1AT transcription factor. Other mutations did not affect the expression of the GUS reporter gene compared to the non-mutated S5 promoter fragment.Fig. 5


The sugar beet gene encoding the sodium/proton exchanger 1 (BvNHX1) is regulated by a MYB transcription factor.

Adler G, Blumwald E, Bar-Zvi D - Planta (2010)

Activities of mutated 337 bp BvNHX1 promoter fragments. a Putative cis-acting elements in the S5 promoter fragment. Sites in forward or reverse modes are marked above and below the line, respectively. Mutated sequences are marked by asterisks. b GUS activity was assayed in homogenates made from five homozygous lines of Arabidopsis containing S5::GUS constructs with the indicated mutations (see also Table 1). Seeds were sowed on 0.5× MS (white bars) supplemented with 50 mM NaCl (black bars) or 100 mM mannitol (dashed bars), and seedlings harvested and analyzed 2 weeks later
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2872020&req=5

Fig5: Activities of mutated 337 bp BvNHX1 promoter fragments. a Putative cis-acting elements in the S5 promoter fragment. Sites in forward or reverse modes are marked above and below the line, respectively. Mutated sequences are marked by asterisks. b GUS activity was assayed in homogenates made from five homozygous lines of Arabidopsis containing S5::GUS constructs with the indicated mutations (see also Table 1). Seeds were sowed on 0.5× MS (white bars) supplemented with 50 mM NaCl (black bars) or 100 mM mannitol (dashed bars), and seedlings harvested and analyzed 2 weeks later
Mentions: The DNA sequence of the S5 construct was used to search for potential binding sites of known trans-acting factors through the Plant cis-acting Regulatory DNA Elements (PLACE) database (Higo et al. 1999) http://www.dna.affrc.go.jp/PLACE/ (Fig. 5a). Four of these potential sites were further studied using site-directed mutagenesis (Table 1). The sequences are putative binding sites for MYB 1AT, MYC, MYB ST1, and napA transcription factors. Transcription factors from the MYC and MYB families are known to be also involved in the modulation of salt stress and water stress regulated genes (Zhu 2002; Shinozaki et al. 2003), whereas napA was shown to function in seeds (Stalberg et al. 1996). Site-directed mutagenesis was performed using Stratagene QuikChange II®, where the putative cis-acting sequence was changed into a restriction enzyme recognition sequence (Table 1). Mutated sequences were confirmed by restriction digests and DNA sequence analyses, and were used for the genetic transformation of Arabidopsis plants. Homozygous transgenic plants were selected and assayed for GUS activity. The basal expression levels in non-stressed plants were not affected by the mutation of any of the potential cis-acting elements (Fig. 5b). Mutation in the MYB 1AT box (position −1362) almost abolished the induction of promoter activity by NaCl and mannitol, suggesting that the BvNHX1 promoter is regulated mainly by a MYB 1AT transcription factor. Other mutations did not affect the expression of the GUS reporter gene compared to the non-mutated S5 promoter fragment.Fig. 5

Bottom Line: NHXs can be localized in the tonoplast or plasma membranes, where they exchange sodium ions for protons, resulting in sodium ions being removed from the cytosol into the vacuole or extracellular space.The 5 UTR of BvNHX1, including its intron, does not modulate the activity of the promoter.Gel mobility shift assay confirmed that the WT but not the mutated MYB binding site is bound by nuclear protein extracted from salt-stressed Beta vulgaris leaves.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, The Doris and Bertie Black Center for Bioenergetics in Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel.

ABSTRACT
Sodium/proton exchangers (NHX) are key players in the plant response to salinity and have a central role in establishing ion homeostasis. NHXs can be localized in the tonoplast or plasma membranes, where they exchange sodium ions for protons, resulting in sodium ions being removed from the cytosol into the vacuole or extracellular space. The expression of most plant NHX genes is modulated by exposure of the organisms to salt stress or water stress. We explored the regulation of the vacuolar NHX1 gene from the salt-tolerant sugar beet plant (BvNHX1) using Arabidopsis plants transformed with an array of constructs of BvHNX1::GUS, and the expression patterns were characterized using histological and quantitative assays. The 5 UTR of BvNHX1, including its intron, does not modulate the activity of the promoter. Serial deletions show that a 337 bp promoter fragment sufficed for driving activity that indistinguishable from that of the full-length (2,464 bp) promoter. Mutating four putative cis-acting elements within the 337 bp promoter fragment revealed that MYB transcription factor(s) are involved in the activation of the expression of BvNHX1 upon exposure to salt and water stresses. Gel mobility shift assay confirmed that the WT but not the mutated MYB binding site is bound by nuclear protein extracted from salt-stressed Beta vulgaris leaves.

Show MeSH
Related in: MedlinePlus