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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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Overexpression of GsTIFY10a in alfalfa promoted plant growth under alkaline stress.a. Schematic representation of expression constructs to ectopically express GsTIFY10a in Medicago sativa. b. Semi-quantitative RT-PCR analysis showing the transcript levels of GsTIFY10a in transgenic alfalfa lines. c. Growth performance of WT and transgenic lines under control conditions or NaHCO3 treatments. d. The shoot length of WT and transgenic plants. e. The ground fresh weight of WT and transgenic plants. f. The ground dry weight of WT and transgenic plants. For phenotypic analysis under alkaline stress, the propagated WT and GsTIFY10a transgenic plants with similar sizes (approximately 25 cm high) were treated with 1/8 Hoagland nutrient solution containing either 0, or 100, or 150 mM NaHCO3 every 3 days for a total of 12 days. Photographs were taken 12 days after initial treatment. Thirty plants of each line were used for each experiment. Data are means (±SE) of three replicates. Significant differences were determined by one-way ANOVA (P<0.0001) statistical analysis. Different letters show significant differences between groups as indicated by Dunnett's posttests (P<0.05).
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pone-0111984-g004: Overexpression of GsTIFY10a in alfalfa promoted plant growth under alkaline stress.a. Schematic representation of expression constructs to ectopically express GsTIFY10a in Medicago sativa. b. Semi-quantitative RT-PCR analysis showing the transcript levels of GsTIFY10a in transgenic alfalfa lines. c. Growth performance of WT and transgenic lines under control conditions or NaHCO3 treatments. d. The shoot length of WT and transgenic plants. e. The ground fresh weight of WT and transgenic plants. f. The ground dry weight of WT and transgenic plants. For phenotypic analysis under alkaline stress, the propagated WT and GsTIFY10a transgenic plants with similar sizes (approximately 25 cm high) were treated with 1/8 Hoagland nutrient solution containing either 0, or 100, or 150 mM NaHCO3 every 3 days for a total of 12 days. Photographs were taken 12 days after initial treatment. Thirty plants of each line were used for each experiment. Data are means (±SE) of three replicates. Significant differences were determined by one-way ANOVA (P<0.0001) statistical analysis. Different letters show significant differences between groups as indicated by Dunnett's posttests (P<0.05).

Mentions: In previous studies, we have demonstrated the involvement of three GsTIFY genes in salt-alkaline stress, among which GsTIFY10a could dramatically improve the alkaline tolerance. In an attempt to generate transgenic alfalfa with superior alkaline tolerance, we ectopically expressed GsTIFY10a in the wild type Medicago sativa through the Agrobacterium tumefaciens-mediated transformation strategy. The GsTIFY10a gene was under the control of the cauliflower mosaic virus (CaMV) 35S promoter, with the binding enhancers E12 and omega (Fig. 4A). After glufosinate selection, the regenerated alfalfa seedlings were analyzed by PCR and semi-quantitative RT-PCR assays. We identified a total of six transgenic lines (Fig. 4B), and three of them, with different expression levels (#12, #13 and #28), were used to examine the responses to alkaline stress.


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)

Overexpression of GsTIFY10a in alfalfa promoted plant growth under alkaline stress.a. Schematic representation of expression constructs to ectopically express GsTIFY10a in Medicago sativa. b. Semi-quantitative RT-PCR analysis showing the transcript levels of GsTIFY10a in transgenic alfalfa lines. c. Growth performance of WT and transgenic lines under control conditions or NaHCO3 treatments. d. The shoot length of WT and transgenic plants. e. The ground fresh weight of WT and transgenic plants. f. The ground dry weight of WT and transgenic plants. For phenotypic analysis under alkaline stress, the propagated WT and GsTIFY10a transgenic plants with similar sizes (approximately 25 cm high) were treated with 1/8 Hoagland nutrient solution containing either 0, or 100, or 150 mM NaHCO3 every 3 days for a total of 12 days. Photographs were taken 12 days after initial treatment. Thirty plants of each line were used for each experiment. Data are means (±SE) of three replicates. Significant differences were determined by one-way ANOVA (P<0.0001) statistical analysis. Different letters show significant differences between groups as indicated by Dunnett's posttests (P<0.05).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111984-g004: Overexpression of GsTIFY10a in alfalfa promoted plant growth under alkaline stress.a. Schematic representation of expression constructs to ectopically express GsTIFY10a in Medicago sativa. b. Semi-quantitative RT-PCR analysis showing the transcript levels of GsTIFY10a in transgenic alfalfa lines. c. Growth performance of WT and transgenic lines under control conditions or NaHCO3 treatments. d. The shoot length of WT and transgenic plants. e. The ground fresh weight of WT and transgenic plants. f. The ground dry weight of WT and transgenic plants. For phenotypic analysis under alkaline stress, the propagated WT and GsTIFY10a transgenic plants with similar sizes (approximately 25 cm high) were treated with 1/8 Hoagland nutrient solution containing either 0, or 100, or 150 mM NaHCO3 every 3 days for a total of 12 days. Photographs were taken 12 days after initial treatment. Thirty plants of each line were used for each experiment. Data are means (±SE) of three replicates. Significant differences were determined by one-way ANOVA (P<0.0001) statistical analysis. Different letters show significant differences between groups as indicated by Dunnett's posttests (P<0.05).
Mentions: In previous studies, we have demonstrated the involvement of three GsTIFY genes in salt-alkaline stress, among which GsTIFY10a could dramatically improve the alkaline tolerance. In an attempt to generate transgenic alfalfa with superior alkaline tolerance, we ectopically expressed GsTIFY10a in the wild type Medicago sativa through the Agrobacterium tumefaciens-mediated transformation strategy. The GsTIFY10a gene was under the control of the cauliflower mosaic virus (CaMV) 35S promoter, with the binding enhancers E12 and omega (Fig. 4A). After glufosinate selection, the regenerated alfalfa seedlings were analyzed by PCR and semi-quantitative RT-PCR assays. We identified a total of six transgenic lines (Fig. 4B), and three of them, with different expression levels (#12, #13 and #28), were used to examine the responses to alkaline stress.

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