Limits...
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.

Show MeSH

Related in: MedlinePlus

Gus expression analysis and estimation of relative GUS activity of various OsPMCa2+ATPase promoter deletions under ABA and methyl jasmonate (MeJA)-induced stress. A)Gus activity in leaves, B) Gus activity in shoots and C) Gus activity in roots. Gus was detected in X-Gluc solution using mature leaf disc, shoots cutting and roots from transgenic tobacco plant, directed by promoter constructs full-length (−1478 bp), D1 (−1210 bp), D2 (−886 bp) and D3 (−519 bp). For positive and negative controls 35S and WT were used. D) The transgenic tobacco plants driven by promoter-GUS fusion constructs full-length, D1 and D3 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 control. Leaf, stem and root samples from transgenic lines were used. Data were measured in three independent transgenic lines, and each experiment was replicated three times. Error bars on the graphic represent SE within the three replicates.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3585799&req=5

pone-0057803-g005: Gus expression analysis and estimation of relative GUS activity of various OsPMCa2+ATPase promoter deletions under ABA and methyl jasmonate (MeJA)-induced stress. A)Gus activity in leaves, B) Gus activity in shoots and C) Gus activity in roots. Gus was detected in X-Gluc solution using mature leaf disc, shoots cutting and roots from transgenic tobacco plant, directed by promoter constructs full-length (−1478 bp), D1 (−1210 bp), D2 (−886 bp) and D3 (−519 bp). For positive and negative controls 35S and WT were used. D) The transgenic tobacco plants driven by promoter-GUS fusion constructs full-length, D1 and D3 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 control. Leaf, stem and root samples from transgenic lines were used. Data were measured in three independent transgenic lines, and each experiment was replicated three times. Error bars on the graphic represent SE within the three replicates.

Mentions: Leaves, shoot and roots were separated from transgenic plants and hormones were applied to examine GUS expression for each of the four OsPMCa2+ATPase promoter segments. GUS expression responded differently to ABA, SA and MeJA-induced treatments are shown in Figure 5. In leaves, among all the promoter segments, only −519 bp deletion significantly responded to exogenous ABA induction whereas full-length (−1478 bp) promoter and −886 bp deletion showed less GUS expression when compared to −1210 bp deletion (Figure 5A). Similar pattern was evidenced in stems (Figure 5B); while in roots all the segments respond well as compared to the 35S-GUS construct (Figure 5C). GUS activity was also measured through fluorometric assay and the results are shown in Figure 5D, but due to similar expression pattern of D1 and D2, the data of D2 is omitted in the Figure 5D because of the space constraint. Approx. 5–6.5 fold increase GUS expression was observed in roots for full-length, −1210 bp and −519 bp construct compared to control when induced by ABA. While, in leaves and shoots ∼3.5 fold GUS induction was observed only for −1210 bp. It is interesting that the full-length, −1210 bp and −886 bp promoter segments positively responded to ABA induction, while no response was detected in leaves and shoots for −519 bp promoter segment under ABA treatment (Figure 5A, B & D). This implied that an ABA-related cis-acting element might exist outside the region of −519 bp construct. We also expose transgenic leaves to SA which failed to induce GUS activities for all constructs (data not showed).


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)

Gus expression analysis and estimation of relative GUS activity of various OsPMCa2+ATPase promoter deletions under ABA and methyl jasmonate (MeJA)-induced stress. A)Gus activity in leaves, B) Gus activity in shoots and C) Gus activity in roots. Gus was detected in X-Gluc solution using mature leaf disc, shoots cutting and roots from transgenic tobacco plant, directed by promoter constructs full-length (−1478 bp), D1 (−1210 bp), D2 (−886 bp) and D3 (−519 bp). For positive and negative controls 35S and WT were used. D) The transgenic tobacco plants driven by promoter-GUS fusion constructs full-length, D1 and D3 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 control. Leaf, stem and root samples from transgenic lines were used. Data were measured in three independent transgenic lines, and each experiment was replicated three times. Error bars on the graphic represent SE within the three replicates.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0057803-g005: Gus expression analysis and estimation of relative GUS activity of various OsPMCa2+ATPase promoter deletions under ABA and methyl jasmonate (MeJA)-induced stress. A)Gus activity in leaves, B) Gus activity in shoots and C) Gus activity in roots. Gus was detected in X-Gluc solution using mature leaf disc, shoots cutting and roots from transgenic tobacco plant, directed by promoter constructs full-length (−1478 bp), D1 (−1210 bp), D2 (−886 bp) and D3 (−519 bp). For positive and negative controls 35S and WT were used. D) The transgenic tobacco plants driven by promoter-GUS fusion constructs full-length, D1 and D3 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 control. Leaf, stem and root samples from transgenic lines were used. Data were measured in three independent transgenic lines, and each experiment was replicated three times. Error bars on the graphic represent SE within the three replicates.
Mentions: Leaves, shoot and roots were separated from transgenic plants and hormones were applied to examine GUS expression for each of the four OsPMCa2+ATPase promoter segments. GUS expression responded differently to ABA, SA and MeJA-induced treatments are shown in Figure 5. In leaves, among all the promoter segments, only −519 bp deletion significantly responded to exogenous ABA induction whereas full-length (−1478 bp) promoter and −886 bp deletion showed less GUS expression when compared to −1210 bp deletion (Figure 5A). Similar pattern was evidenced in stems (Figure 5B); while in roots all the segments respond well as compared to the 35S-GUS construct (Figure 5C). GUS activity was also measured through fluorometric assay and the results are shown in Figure 5D, but due to similar expression pattern of D1 and D2, the data of D2 is omitted in the Figure 5D because of the space constraint. Approx. 5–6.5 fold increase GUS expression was observed in roots for full-length, −1210 bp and −519 bp construct compared to control when induced by ABA. While, in leaves and shoots ∼3.5 fold GUS induction was observed only for −1210 bp. It is interesting that the full-length, −1210 bp and −886 bp promoter segments positively responded to ABA induction, while no response was detected in leaves and shoots for −519 bp promoter segment under ABA treatment (Figure 5A, B & D). This implied that an ABA-related cis-acting element might exist outside the region of −519 bp construct. We also expose transgenic leaves to SA which failed to induce GUS activities for all constructs (data not showed).

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