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The Translation Initiation Factor 1A ( TheIF1A ) from Tamarix hispida Is Regulated by a Dof Transcription Factor and Increased Abiotic Stress Tolerance

View Article: PubMed Central - PubMed

ABSTRACT

Eukaryotic translation initiation factor 1A (eIF1A) functions as an mRNA scanner and AUG initiation codon locator. However, few studies have clarified the role of eIF1A in abiotic stress. In this study, we cloned eIF1A (TheIF1A) from Tamarix hispida and found its expression to be induced by NaCl and polyethylene glycol (PEG) in roots, stems, and leaves. Compared to control, TheIF1A root expression was increased 187.63-fold when exposed to NaCl for 6 h, suggesting a potential abiotic stress response for this gene. Furthermore, transgenic tobacco plants overexpressing TheIF1A exhibited enhanced seed germination and a higher total chlorophyll content under salt and mannitol stresses. Increased superoxide dismutase, peroxidase, glutathione transferase and glutathione peroxidase activities, as well as decreased electrolyte leakage rates and malondialdehyde contents, were observed in TheIF1A-transgenic tobacco and T. hispida seedlings under salt and mannitol stresses. Histochemical staining suggested that TheIF1A improves reactive oxygen species (ROS) scavenging in plants. Moreover, TheIF1A may regulate expression of stress-related genes, including TOBLTP, GST, MnSOD, NtMPK9, poxN1, and CDPK15. Moreover, a 1352-bp promoter fragment of TheIF1A was isolated, and cis-elements were identified. Yeast one-hybrid assays showed that ThDof can specifically bind to the Dof motif present in the promoter. In addition, ThDof showed expression patterns similar to those of TheIF1A under NaCl and PEG stresses. These findings suggest the potential mechanism and physiological roles of TheIF1A. ThDof may be an upstream regulator of TheIF1A, and TheIF1A may function as a stress response regulator to improve plant salt and osmotic stress tolerance via regulation of associated enzymes and ROS scavenging, thereby reducing cell damage under stress conditions.

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Yeast one-hybrid analyses of upstream regulators of TheIF1A.(A) P, positive control, the pGADT7-Rec2 vector encoding murine p53 fused with GAL4 AD. N, negative control, the pHIS2 reporter vector containing the cis-acting DNA consensus sequence recognized by p53. Dof, Dof motif. M, indicates a mutated Dof motif. S, The TheIF1A promoter fragment containing the Dof motif. M1, The TheIF1A promoter fragment not containing the Dof motif. M2, The TheIF1A promoter fragment containing the mutated Dof motif. Transformants spotted onto SD/-Leu/-Trp (DDO) were used as positive controls for transformant growth. Positive transformants were further confirmed by spotting serial dilutions (1/1, 1/10, 1/100, 1/1000, 1/10000) onto SD/-His/-Leu/-Trp plates with 50 mM 3-AT (TDO/+50 mM 3-AT); the triangle indicates the dilutions from 1 to 10000. (B) Results of transient reporter experiments for effector overexpression in Arabidopsis. p1301 and pCAMBIA1301 were used as positive controls. Dof, M, M1, and M2 were consistent with (A). (C) GUS activity according to (B). ∗indicates significant differences between the ‘M’ and other lines (p < 0.05).
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Figure 3: Yeast one-hybrid analyses of upstream regulators of TheIF1A.(A) P, positive control, the pGADT7-Rec2 vector encoding murine p53 fused with GAL4 AD. N, negative control, the pHIS2 reporter vector containing the cis-acting DNA consensus sequence recognized by p53. Dof, Dof motif. M, indicates a mutated Dof motif. S, The TheIF1A promoter fragment containing the Dof motif. M1, The TheIF1A promoter fragment not containing the Dof motif. M2, The TheIF1A promoter fragment containing the mutated Dof motif. Transformants spotted onto SD/-Leu/-Trp (DDO) were used as positive controls for transformant growth. Positive transformants were further confirmed by spotting serial dilutions (1/1, 1/10, 1/100, 1/1000, 1/10000) onto SD/-His/-Leu/-Trp plates with 50 mM 3-AT (TDO/+50 mM 3-AT); the triangle indicates the dilutions from 1 to 10000. (B) Results of transient reporter experiments for effector overexpression in Arabidopsis. p1301 and pCAMBIA1301 were used as positive controls. Dof, M, M1, and M2 were consistent with (A). (C) GUS activity according to (B). ∗indicates significant differences between the ‘M’ and other lines (p < 0.05).

Mentions: Twelve Dof motifs were identified in the TheIF1A promoter, suggesting that TheIF1A is likely regulated by TFs that interact with this motif. A yeast one-hybrid assay was conducted to identify upstream regulators using the reporter vector pHIS2-cis (containing triple tandem Dof motif repeats) as bait to screen a Tamarix TF cDNA library. One protein (ThDof, KF896302) was found to bind to the Dof motif but failed to bind to mutant Dof motifs (Figure 3A). In addition, ThDof could bind to a truncated TheIF1A promoter retaining the Dof motif but could not bind to promoter fragments lacking the motif or with a mutated Dof motif (Figure 3A). These findings suggest that ThDof may specifically bind to the Dof motif and may regulate TheIF1A by binding to this motif in the TheIF1A promoter.


The Translation Initiation Factor 1A ( TheIF1A ) from Tamarix hispida Is Regulated by a Dof Transcription Factor and Increased Abiotic Stress Tolerance
Yeast one-hybrid analyses of upstream regulators of TheIF1A.(A) P, positive control, the pGADT7-Rec2 vector encoding murine p53 fused with GAL4 AD. N, negative control, the pHIS2 reporter vector containing the cis-acting DNA consensus sequence recognized by p53. Dof, Dof motif. M, indicates a mutated Dof motif. S, The TheIF1A promoter fragment containing the Dof motif. M1, The TheIF1A promoter fragment not containing the Dof motif. M2, The TheIF1A promoter fragment containing the mutated Dof motif. Transformants spotted onto SD/-Leu/-Trp (DDO) were used as positive controls for transformant growth. Positive transformants were further confirmed by spotting serial dilutions (1/1, 1/10, 1/100, 1/1000, 1/10000) onto SD/-His/-Leu/-Trp plates with 50 mM 3-AT (TDO/+50 mM 3-AT); the triangle indicates the dilutions from 1 to 10000. (B) Results of transient reporter experiments for effector overexpression in Arabidopsis. p1301 and pCAMBIA1301 were used as positive controls. Dof, M, M1, and M2 were consistent with (A). (C) GUS activity according to (B). ∗indicates significant differences between the ‘M’ and other lines (p < 0.05).
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Figure 3: Yeast one-hybrid analyses of upstream regulators of TheIF1A.(A) P, positive control, the pGADT7-Rec2 vector encoding murine p53 fused with GAL4 AD. N, negative control, the pHIS2 reporter vector containing the cis-acting DNA consensus sequence recognized by p53. Dof, Dof motif. M, indicates a mutated Dof motif. S, The TheIF1A promoter fragment containing the Dof motif. M1, The TheIF1A promoter fragment not containing the Dof motif. M2, The TheIF1A promoter fragment containing the mutated Dof motif. Transformants spotted onto SD/-Leu/-Trp (DDO) were used as positive controls for transformant growth. Positive transformants were further confirmed by spotting serial dilutions (1/1, 1/10, 1/100, 1/1000, 1/10000) onto SD/-His/-Leu/-Trp plates with 50 mM 3-AT (TDO/+50 mM 3-AT); the triangle indicates the dilutions from 1 to 10000. (B) Results of transient reporter experiments for effector overexpression in Arabidopsis. p1301 and pCAMBIA1301 were used as positive controls. Dof, M, M1, and M2 were consistent with (A). (C) GUS activity according to (B). ∗indicates significant differences between the ‘M’ and other lines (p < 0.05).
Mentions: Twelve Dof motifs were identified in the TheIF1A promoter, suggesting that TheIF1A is likely regulated by TFs that interact with this motif. A yeast one-hybrid assay was conducted to identify upstream regulators using the reporter vector pHIS2-cis (containing triple tandem Dof motif repeats) as bait to screen a Tamarix TF cDNA library. One protein (ThDof, KF896302) was found to bind to the Dof motif but failed to bind to mutant Dof motifs (Figure 3A). In addition, ThDof could bind to a truncated TheIF1A promoter retaining the Dof motif but could not bind to promoter fragments lacking the motif or with a mutated Dof motif (Figure 3A). These findings suggest that ThDof may specifically bind to the Dof motif and may regulate TheIF1A by binding to this motif in the TheIF1A promoter.

View Article: PubMed Central - PubMed

ABSTRACT

Eukaryotic translation initiation factor 1A (eIF1A) functions as an mRNA scanner and AUG initiation codon locator. However, few studies have clarified the role of eIF1A in abiotic stress. In this study, we cloned eIF1A (TheIF1A) from Tamarix hispida and found its expression to be induced by NaCl and polyethylene glycol (PEG) in roots, stems, and leaves. Compared to control, TheIF1A root expression was increased 187.63-fold when exposed to NaCl for 6 h, suggesting a potential abiotic stress response for this gene. Furthermore, transgenic tobacco plants overexpressing TheIF1A exhibited enhanced seed germination and a higher total chlorophyll content under salt and mannitol stresses. Increased superoxide dismutase, peroxidase, glutathione transferase and glutathione peroxidase activities, as well as decreased electrolyte leakage rates and malondialdehyde contents, were observed in TheIF1A-transgenic tobacco and T. hispida seedlings under salt and mannitol stresses. Histochemical staining suggested that TheIF1A improves reactive oxygen species (ROS) scavenging in plants. Moreover, TheIF1A may regulate expression of stress-related genes, including TOBLTP, GST, MnSOD, NtMPK9, poxN1, and CDPK15. Moreover, a 1352-bp promoter fragment of TheIF1A was isolated, and cis-elements were identified. Yeast one-hybrid assays showed that ThDof can specifically bind to the Dof motif present in the promoter. In addition, ThDof showed expression patterns similar to those of TheIF1A under NaCl and PEG stresses. These findings suggest the potential mechanism and physiological roles of TheIF1A. ThDof may be an upstream regulator of TheIF1A, and TheIF1A may function as a stress response regulator to improve plant salt and osmotic stress tolerance via regulation of associated enzymes and ROS scavenging, thereby reducing cell damage under stress conditions.

No MeSH data available.


Related in: MedlinePlus