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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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Activity of the 337 bp upstream promoter fragment. Homozygous plants expressing the S5 construct (Fig. 3, 337 bp promoter fragment fused to GUS) were assayed for the activity of the reporter gene. a–i Histochemical staining of non-stressed plants. a Shoot of soil grown plant. b Mature leaf of soil grown plant. c Trichome. d Cross-section of inflorescence stem. e Roots. f Emerging lateral root. g Root hairs. h Flower. i Silique. j Quantitative analysis using homogenates (five independent lines, 50 seedling each) from seedlings grown for 2 weeks in the 0.5× MS solid medium without (white bar) or containing in addition 50 mM NaCl (black bar), 25 mM Na2SO4 (diagonally dashed), 50 mM KCl (dotted), 100 mM mannitol (horizontally dashed), or 20 μM ABA (vertically dashed)
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Fig4: Activity of the 337 bp upstream promoter fragment. Homozygous plants expressing the S5 construct (Fig. 3, 337 bp promoter fragment fused to GUS) were assayed for the activity of the reporter gene. a–i Histochemical staining of non-stressed plants. a Shoot of soil grown plant. b Mature leaf of soil grown plant. c Trichome. d Cross-section of inflorescence stem. e Roots. f Emerging lateral root. g Root hairs. h Flower. i Silique. j Quantitative analysis using homogenates (five independent lines, 50 seedling each) from seedlings grown for 2 weeks in the 0.5× MS solid medium without (white bar) or containing in addition 50 mM NaCl (black bar), 25 mM Na2SO4 (diagonally dashed), 50 mM KCl (dotted), 100 mM mannitol (horizontally dashed), or 20 μM ABA (vertically dashed)

Mentions: Five constructs comprising upstream sequences fused to reporter gene GUS were made. These constructs, designated S1::GUS to S5::GUS, contained 1,378, 1,108, 844, 576, and 337 bp promoter sequences upstream of the 5′ UTR. T2 and T3 generation plants from homozygous lines containing a single insertion were selected and at least five independent lines of each construct were tested. GUS activity in homogenates prepared from non-stressed and salt-treated seedlings was determined (Fig. 3). Deletions of 270 and 534 bp from the 5′ of the promoter sequence (S2 and S3, respectively) did not affect the promoter activity. Deletion of an additional 268 bp fragment inhibited the activation capacity of the resulting 567 bp promoter fragment (construct S4) both in the absence and presence of salt. On the other hand, the S5::GUS construct containing the shortest, 337 bp long, promoter fragment was sufficient to drive GUS expression to similar levels to that obtained by the S1::GUS construct both in the presence and absence of salt (Fig. 4). These results suggested that the transactivation capacity of the S5 promoter fragment was essentially identical to that of the full-length S1.Fig. 3


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)

Activity of the 337 bp upstream promoter fragment. Homozygous plants expressing the S5 construct (Fig. 3, 337 bp promoter fragment fused to GUS) were assayed for the activity of the reporter gene. a–i Histochemical staining of non-stressed plants. a Shoot of soil grown plant. b Mature leaf of soil grown plant. c Trichome. d Cross-section of inflorescence stem. e Roots. f Emerging lateral root. g Root hairs. h Flower. i Silique. j Quantitative analysis using homogenates (five independent lines, 50 seedling each) from seedlings grown for 2 weeks in the 0.5× MS solid medium without (white bar) or containing in addition 50 mM NaCl (black bar), 25 mM Na2SO4 (diagonally dashed), 50 mM KCl (dotted), 100 mM mannitol (horizontally dashed), or 20 μM ABA (vertically dashed)
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2872020&req=5

Fig4: Activity of the 337 bp upstream promoter fragment. Homozygous plants expressing the S5 construct (Fig. 3, 337 bp promoter fragment fused to GUS) were assayed for the activity of the reporter gene. a–i Histochemical staining of non-stressed plants. a Shoot of soil grown plant. b Mature leaf of soil grown plant. c Trichome. d Cross-section of inflorescence stem. e Roots. f Emerging lateral root. g Root hairs. h Flower. i Silique. j Quantitative analysis using homogenates (five independent lines, 50 seedling each) from seedlings grown for 2 weeks in the 0.5× MS solid medium without (white bar) or containing in addition 50 mM NaCl (black bar), 25 mM Na2SO4 (diagonally dashed), 50 mM KCl (dotted), 100 mM mannitol (horizontally dashed), or 20 μM ABA (vertically dashed)
Mentions: Five constructs comprising upstream sequences fused to reporter gene GUS were made. These constructs, designated S1::GUS to S5::GUS, contained 1,378, 1,108, 844, 576, and 337 bp promoter sequences upstream of the 5′ UTR. T2 and T3 generation plants from homozygous lines containing a single insertion were selected and at least five independent lines of each construct were tested. GUS activity in homogenates prepared from non-stressed and salt-treated seedlings was determined (Fig. 3). Deletions of 270 and 534 bp from the 5′ of the promoter sequence (S2 and S3, respectively) did not affect the promoter activity. Deletion of an additional 268 bp fragment inhibited the activation capacity of the resulting 567 bp promoter fragment (construct S4) both in the absence and presence of salt. On the other hand, the S5::GUS construct containing the shortest, 337 bp long, promoter fragment was sufficient to drive GUS expression to similar levels to that obtained by the S1::GUS construct both in the presence and absence of salt (Fig. 4). These results suggested that the transactivation capacity of the S5 promoter fragment was essentially identical to that of the full-length S1.Fig. 3

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