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The positive regulatory roles of the TIFY10 proteins in plant responses to alkaline stress.

Zhu D, Li R, Liu X, Sun M, Wu J, Zhang N, Zhu Y - PLoS ONE (2014)

Bottom Line: We also revealed that ectopic expression of GsTIFY10a in Medicago sativa promoted plant growth, and increased the NADP-ME activity, citric acid content and free proline content but decreased the MDA content of transgenic plants under alkaline stress.In addition, we showed that neither GsTIFY10a nor GsTIFY10e exhibited transcriptional activity in yeast cells.Taken together, our results provided direct evidence supporting the positive regulatory roles of the TIFY10 proteins in plant responses to alkaline stress.

View Article: PubMed Central - PubMed

Affiliation: College of Life Science, Qingdao Agricultural University, Qingdao, P.R. China; Plant Bioengineering Laboratory, Northeast Agricultural University, Harbin, P.R. China.

ABSTRACT
The TIFY family is a novel plant-specific protein family, and is characterized by a conserved TIFY motif (TIFF/YXG). Our previous studies indicated the potential roles of TIFY10/11 proteins in plant responses to alkaline stress. In the current study, we focused on the regulatory roles and possible physiological and molecular basis of the TIFY10 proteins in plant responses to alkaline stress. We demonstrated the positive function of TIFY10s in alkaline responses by using the AtTIFY10a and AtTIFY10b knockout Arabidopsis, as evidenced by the relatively lower germination rates of attify10a and attify10b mutant seeds under alkaline stress. We also revealed that ectopic expression of GsTIFY10a in Medicago sativa promoted plant growth, and increased the NADP-ME activity, citric acid content and free proline content but decreased the MDA content of transgenic plants under alkaline stress. Furthermore, expression levels of the stress responsive genes including NADP-ME, CS, H+-ppase and P5CS were also up-regulated in GsTIFY10a transgenic plants under alkaline stress. Interestingly, GsTIFY10a overexpression increased the jasmonate content of the transgenic alfalfa. In addition, we showed that neither GsTIFY10a nor GsTIFY10e exhibited transcriptional activity in yeast cells. However, through Y2H and BiFc assays, we demonstrated that GsTIFY10a, not GsTIFY10e, could form homodimers in yeast cells and in living plant cells. As expected, we also demonstrated that GsTIFY10a and GsTIFY10e could heterodimerize with each other in both yeast and plant cells. Taken together, our results provided direct evidence supporting the positive regulatory roles of the TIFY10 proteins in plant responses to alkaline stress.

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Transcriptional activity analysis of the GsTIFY10a and GsTIFY10e proteins.a. Transcriptional activity analysis of GsTIFY10a. b. Transcriptional activity analysis of GsTIFY10e. The pGBKT7-GsTIFY10a and pGBKT7-GsTIFY10e vectors were transformed into the yeast reporter strain AH109, and LacZ activity was assessed by using the β-galactosidase filter lift assays. The AtDREB1A transcription factor was used as a positive control.
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pone-0111984-g008: Transcriptional activity analysis of the GsTIFY10a and GsTIFY10e proteins.a. Transcriptional activity analysis of GsTIFY10a. b. Transcriptional activity analysis of GsTIFY10e. The pGBKT7-GsTIFY10a and pGBKT7-GsTIFY10e vectors were transformed into the yeast reporter strain AH109, and LacZ activity was assessed by using the β-galactosidase filter lift assays. The AtDREB1A transcription factor was used as a positive control.

Mentions: Our previous studies demonstrated that two of the Glycine soja TIFY10 proteins GsTIFY10a and GsTIFY10e positively regulated plant tolerance to alkaline stress. Both of them were found to localize at the nuclei of plant cells, indicating that they might act as transcriptional regulators. To further investigate the molecular basis of the TIFY10 proteins in alkaline responses, we identified the transcriptional activity of the GsTIFY10a and GsTIFY10e proteins. The full-length GsTIFY10a and GsTIFY10e genes were fused to the GAL4 DNA-binding domain in the pGBKT7 vector, and then introduced to the yeast reporter strain AH109. The AtbZIP1 transcription factor, which showed transcriptional activity in yeast cells, was used as a positive control [45], [46]. LacZ activity was assessed by using the β-galactosidase filter lift assays. As shown in Fig.8, only the recombinant yeast cells carrying the AtbZIP1-BD vector displayed LacZ activity. These results implied that neither GsTIFY10a nor GsTIFY10e showed transcriptional activity in yeast cells.


The positive regulatory roles of the TIFY10 proteins in plant responses to alkaline stress.

Zhu D, Li R, Liu X, Sun M, Wu J, Zhang N, Zhu Y - PLoS ONE (2014)

Transcriptional activity analysis of the GsTIFY10a and GsTIFY10e proteins.a. Transcriptional activity analysis of GsTIFY10a. b. Transcriptional activity analysis of GsTIFY10e. The pGBKT7-GsTIFY10a and pGBKT7-GsTIFY10e vectors were transformed into the yeast reporter strain AH109, and LacZ activity was assessed by using the β-galactosidase filter lift assays. The AtDREB1A transcription factor was used as a positive control.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4222965&req=5

pone-0111984-g008: Transcriptional activity analysis of the GsTIFY10a and GsTIFY10e proteins.a. Transcriptional activity analysis of GsTIFY10a. b. Transcriptional activity analysis of GsTIFY10e. The pGBKT7-GsTIFY10a and pGBKT7-GsTIFY10e vectors were transformed into the yeast reporter strain AH109, and LacZ activity was assessed by using the β-galactosidase filter lift assays. The AtDREB1A transcription factor was used as a positive control.
Mentions: Our previous studies demonstrated that two of the Glycine soja TIFY10 proteins GsTIFY10a and GsTIFY10e positively regulated plant tolerance to alkaline stress. Both of them were found to localize at the nuclei of plant cells, indicating that they might act as transcriptional regulators. To further investigate the molecular basis of the TIFY10 proteins in alkaline responses, we identified the transcriptional activity of the GsTIFY10a and GsTIFY10e proteins. The full-length GsTIFY10a and GsTIFY10e genes were fused to the GAL4 DNA-binding domain in the pGBKT7 vector, and then introduced to the yeast reporter strain AH109. The AtbZIP1 transcription factor, which showed transcriptional activity in yeast cells, was used as a positive control [45], [46]. LacZ activity was assessed by using the β-galactosidase filter lift assays. As shown in Fig.8, only the recombinant yeast cells carrying the AtbZIP1-BD vector displayed LacZ activity. These results implied that neither GsTIFY10a nor GsTIFY10e showed transcriptional activity in yeast cells.

Bottom Line: We also revealed that ectopic expression of GsTIFY10a in Medicago sativa promoted plant growth, and increased the NADP-ME activity, citric acid content and free proline content but decreased the MDA content of transgenic plants under alkaline stress.In addition, we showed that neither GsTIFY10a nor GsTIFY10e exhibited transcriptional activity in yeast cells.Taken together, our results provided direct evidence supporting the positive regulatory roles of the TIFY10 proteins in plant responses to alkaline stress.

View Article: PubMed Central - PubMed

Affiliation: College of Life Science, Qingdao Agricultural University, Qingdao, P.R. China; Plant Bioengineering Laboratory, Northeast Agricultural University, Harbin, P.R. China.

ABSTRACT
The TIFY family is a novel plant-specific protein family, and is characterized by a conserved TIFY motif (TIFF/YXG). Our previous studies indicated the potential roles of TIFY10/11 proteins in plant responses to alkaline stress. In the current study, we focused on the regulatory roles and possible physiological and molecular basis of the TIFY10 proteins in plant responses to alkaline stress. We demonstrated the positive function of TIFY10s in alkaline responses by using the AtTIFY10a and AtTIFY10b knockout Arabidopsis, as evidenced by the relatively lower germination rates of attify10a and attify10b mutant seeds under alkaline stress. We also revealed that ectopic expression of GsTIFY10a in Medicago sativa promoted plant growth, and increased the NADP-ME activity, citric acid content and free proline content but decreased the MDA content of transgenic plants under alkaline stress. Furthermore, expression levels of the stress responsive genes including NADP-ME, CS, H+-ppase and P5CS were also up-regulated in GsTIFY10a transgenic plants under alkaline stress. Interestingly, GsTIFY10a overexpression increased the jasmonate content of the transgenic alfalfa. In addition, we showed that neither GsTIFY10a nor GsTIFY10e exhibited transcriptional activity in yeast cells. However, through Y2H and BiFc assays, we demonstrated that GsTIFY10a, not GsTIFY10e, could form homodimers in yeast cells and in living plant cells. As expected, we also demonstrated that GsTIFY10a and GsTIFY10e could heterodimerize with each other in both yeast and plant cells. Taken together, our results provided direct evidence supporting the positive regulatory roles of the TIFY10 proteins in plant responses to alkaline stress.

Show MeSH
Related in: MedlinePlus