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Reproductive organ and vascular specific promoter of the rice plasma membrane Ca2+ATPase mediates environmental stress responses in plants.

Huda KM, Banu MS, Pathi KM, Tuteja N - PLoS ONE (2013)

Bottom Line: The -1210 bp deletion significantly responded to exogenous methyl viologen and abscisic acid induction.The -1210 and -886 bp flanking region resulted in increased GUS activity in leaves under methyl jasmonate treatments, whereas in shoots the -886 bp and -519 bp deletion gave higher expression.Salicylic acid failed to induce GUS activities in leaves for all the constructs.

View Article: PubMed Central - PubMed

Affiliation: Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India.

ABSTRACT

Background: Plasma membrane Ca(2+)ATPase is a transport protein in the plasma membrane of cells and helps in removal of calcium (Ca(2+)) from the cell, hence regulating Ca(2+) level within cells. Though plant Ca(2+)ATPases have been shown to be involved in plant stress responses but their promoter regions have not been well studied.

Results: The 1478 bp promoter sequence of rice plasma membrane Ca(2+)ATPase contains cis-acting elements responsive to stresses and plant hormones. To identify the functional region, serial deletions of the promoter were fused with the GUS sequence and four constructs were obtained. These were differentially activated under NaCl, PEG cold, methyl viologen, abscisic acid and methyl jasmonate treatments. We demonstrated that the rice plasma membrane Ca(2+)ATPase promoter is responsible for vascular-specific and multiple stress-inducible gene expression. Only full-length promoter showed specific GUS expression under stress conditions in floral parts. High GUS activity was observed in roots with all the promoter constructs. The -1478 to -886 bp flanking region responded well upon treatment with salt and drought. Only the full-length promoter presented cold-induced GUS expression in leaves, while in shoots slight expression was observed for -1210 and -886 bp flanking region. The -1210 bp deletion significantly responded to exogenous methyl viologen and abscisic acid induction. The -1210 and -886 bp flanking region resulted in increased GUS activity in leaves under methyl jasmonate treatments, whereas in shoots the -886 bp and -519 bp deletion gave higher expression. Salicylic acid failed to induce GUS activities in leaves for all the constructs.

Conclusions: The rice plasma membrane Ca(2+)ATPase promoter is a reproductive organ-specific as well as vascular-specific. This promoter contains drought, salt, cold, methyl viologen, abscisic acid and methyl jasmonate related cis-elements, which regulated gene expression. Overall, the tissue-specificity and inducible nature of this promoter could grant wide applicability in plant biotechnology.

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Quantification of GUS relative activity of OsPMCa2+ATPase promoter deletions under drought, salt cold and MV induced stress in leaves shoots and roots of tobacco.The transgenic tobacco plants driven by promoter-GUS fusion constructs full-length (−1478 bp), D1 (−1210 bp) and D3 (−519 bp) were chosen for quantification assays. The deletion construct D2 was also used for the analysis but the data of D2 was similar to the D1, therefore the D2 data is omitted in this figure because of the space constraint. Wild type (WT) tobacco plants with same treatment were used as the control. Data of three independent transgenic lines were measured, and each experiment was replicated three times. Error bars on the graphic represent SE with three replicates.
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pone-0057803-g004: Quantification of GUS relative activity of OsPMCa2+ATPase promoter deletions under drought, salt cold and MV induced stress in leaves shoots and roots of tobacco.The transgenic tobacco plants driven by promoter-GUS fusion constructs full-length (−1478 bp), D1 (−1210 bp) and D3 (−519 bp) were chosen for quantification assays. The deletion construct D2 was also used for the analysis but the data of D2 was similar to the D1, therefore the D2 data is omitted in this figure because of the space constraint. Wild type (WT) tobacco plants with same treatment were used as the control. Data of three independent transgenic lines were measured, and each experiment was replicated three times. Error bars on the graphic represent SE with three replicates.

Mentions: Since it was shown that abiotic stresses can regulate OsPMCa2+ATPase gene expression, its promoter activity was studied in tobacco transgenic plants submitted to drought, salt and cold treatments. The presence of abiotic-stress-responsive elements inside the promoter sequence encouraged the research. In consequence, GUS activity was measured through fluorometric assay. Distinct GUS expression was evidenced for each of the four (full-length, −1478 bp; D1, −1210 bp; D3, −519 bp and wild type) OsPMCa2+ATPase promoter segments (Figure 4). The results of deletion construct D2 were similar to the D1, therefore the data of D2 is omitted in the figures 4 because of the space constraint. In roots, high GUS expression was observed in full-length and in all the deletions (∼2–3.5 fold increase) after treatment with stress. Reckonable increase of GUS activity in leaves (∼4.5 fold) and shoots (∼3 fold) was evidenced for full-length promoter, while −1210 bp showed ∼2.8 fold increase in leaves and ∼2 fold increase in shoots compared with control upon treatment with drought, while no activity was observed in −519 bp deletion. This may be due to presence of some inhibitory elements upstream of −519 bp segment that affect its expression. Similar results were presented in case of salt-induced stress. In leaves and shoots cold shock induced ∼2.8 fold increase GUS expression only for full-length promoter while in −1210 bp and −519 bp no expression was observed in leaves. Under same conditions, a slight induction was present in −1210 bp (∼2.8 fold increase GUS activity) deletions in shoots (Figure 4). Thus, we conclude that the full OsPMCa2+ATPase promoter positively responded to drought, salt and cold. Since deletion −1210 bp and −519 bp did not respond to cold whereas the full-length promoter did, it might result from the absence of cold-related cis-acting elements in these segments. We suggest that cold-related cis-elements may be required in response to cold in leaves outside the region contained in the −1210 bp deletion.


Reproductive organ and vascular specific promoter of the rice plasma membrane Ca2+ATPase mediates environmental stress responses in plants.

Huda KM, Banu MS, Pathi KM, Tuteja N - PLoS ONE (2013)

Quantification of GUS relative activity of OsPMCa2+ATPase promoter deletions under drought, salt cold and MV induced stress in leaves shoots and roots of tobacco.The transgenic tobacco plants driven by promoter-GUS fusion constructs full-length (−1478 bp), D1 (−1210 bp) and D3 (−519 bp) were chosen for quantification assays. The deletion construct D2 was also used for the analysis but the data of D2 was similar to the D1, therefore the D2 data is omitted in this figure because of the space constraint. Wild type (WT) tobacco plants with same treatment were used as the control. Data of three independent transgenic lines were measured, and each experiment was replicated three times. Error bars on the graphic represent SE with three replicates.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585799&req=5

pone-0057803-g004: Quantification of GUS relative activity of OsPMCa2+ATPase promoter deletions under drought, salt cold and MV induced stress in leaves shoots and roots of tobacco.The transgenic tobacco plants driven by promoter-GUS fusion constructs full-length (−1478 bp), D1 (−1210 bp) and D3 (−519 bp) were chosen for quantification assays. The deletion construct D2 was also used for the analysis but the data of D2 was similar to the D1, therefore the D2 data is omitted in this figure because of the space constraint. Wild type (WT) tobacco plants with same treatment were used as the control. Data of three independent transgenic lines were measured, and each experiment was replicated three times. Error bars on the graphic represent SE with three replicates.
Mentions: Since it was shown that abiotic stresses can regulate OsPMCa2+ATPase gene expression, its promoter activity was studied in tobacco transgenic plants submitted to drought, salt and cold treatments. The presence of abiotic-stress-responsive elements inside the promoter sequence encouraged the research. In consequence, GUS activity was measured through fluorometric assay. Distinct GUS expression was evidenced for each of the four (full-length, −1478 bp; D1, −1210 bp; D3, −519 bp and wild type) OsPMCa2+ATPase promoter segments (Figure 4). The results of deletion construct D2 were similar to the D1, therefore the data of D2 is omitted in the figures 4 because of the space constraint. In roots, high GUS expression was observed in full-length and in all the deletions (∼2–3.5 fold increase) after treatment with stress. Reckonable increase of GUS activity in leaves (∼4.5 fold) and shoots (∼3 fold) was evidenced for full-length promoter, while −1210 bp showed ∼2.8 fold increase in leaves and ∼2 fold increase in shoots compared with control upon treatment with drought, while no activity was observed in −519 bp deletion. This may be due to presence of some inhibitory elements upstream of −519 bp segment that affect its expression. Similar results were presented in case of salt-induced stress. In leaves and shoots cold shock induced ∼2.8 fold increase GUS expression only for full-length promoter while in −1210 bp and −519 bp no expression was observed in leaves. Under same conditions, a slight induction was present in −1210 bp (∼2.8 fold increase GUS activity) deletions in shoots (Figure 4). Thus, we conclude that the full OsPMCa2+ATPase promoter positively responded to drought, salt and cold. Since deletion −1210 bp and −519 bp did not respond to cold whereas the full-length promoter did, it might result from the absence of cold-related cis-acting elements in these segments. We suggest that cold-related cis-elements may be required in response to cold in leaves outside the region contained in the −1210 bp deletion.

Bottom Line: The -1210 bp deletion significantly responded to exogenous methyl viologen and abscisic acid induction.The -1210 and -886 bp flanking region resulted in increased GUS activity in leaves under methyl jasmonate treatments, whereas in shoots the -886 bp and -519 bp deletion gave higher expression.Salicylic acid failed to induce GUS activities in leaves for all the constructs.

View Article: PubMed Central - PubMed

Affiliation: Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India.

ABSTRACT

Background: Plasma membrane Ca(2+)ATPase is a transport protein in the plasma membrane of cells and helps in removal of calcium (Ca(2+)) from the cell, hence regulating Ca(2+) level within cells. Though plant Ca(2+)ATPases have been shown to be involved in plant stress responses but their promoter regions have not been well studied.

Results: The 1478 bp promoter sequence of rice plasma membrane Ca(2+)ATPase contains cis-acting elements responsive to stresses and plant hormones. To identify the functional region, serial deletions of the promoter were fused with the GUS sequence and four constructs were obtained. These were differentially activated under NaCl, PEG cold, methyl viologen, abscisic acid and methyl jasmonate treatments. We demonstrated that the rice plasma membrane Ca(2+)ATPase promoter is responsible for vascular-specific and multiple stress-inducible gene expression. Only full-length promoter showed specific GUS expression under stress conditions in floral parts. High GUS activity was observed in roots with all the promoter constructs. The -1478 to -886 bp flanking region responded well upon treatment with salt and drought. Only the full-length promoter presented cold-induced GUS expression in leaves, while in shoots slight expression was observed for -1210 and -886 bp flanking region. The -1210 bp deletion significantly responded to exogenous methyl viologen and abscisic acid induction. The -1210 and -886 bp flanking region resulted in increased GUS activity in leaves under methyl jasmonate treatments, whereas in shoots the -886 bp and -519 bp deletion gave higher expression. Salicylic acid failed to induce GUS activities in leaves for all the constructs.

Conclusions: The rice plasma membrane Ca(2+)ATPase promoter is a reproductive organ-specific as well as vascular-specific. This promoter contains drought, salt, cold, methyl viologen, abscisic acid and methyl jasmonate related cis-elements, which regulated gene expression. Overall, the tissue-specificity and inducible nature of this promoter could grant wide applicability in plant biotechnology.

Show MeSH
Related in: MedlinePlus