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CaZF, a plant transcription factor functions through and parallel to HOG and calcineurin pathways in Saccharomyces cerevisiae to provide osmotolerance.

Jain D, Roy N, Chattopadhyay D - PLoS ONE (2009)

Bottom Line: In Saccharomyces cerevisiae most of the cellular responses to hyper-osmotic stress is regulated by two interconnected pathways involving high osmolarity glycerol mitogen-activated protein kinase (Hog1p) and Calcineurin (CAN), a Ca(2+)/calmodulin-regulated protein phosphatase 2B.In this study, we report that heterologous expression of CaZF provides osmotolerance in S. cerevisiae through Hog1p and Calcineurin dependent as well as independent pathways.CaZF directly binds to stress response element (STRE) to activate STRE-containing promoter in yeast.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi, India.

ABSTRACT
Salt-sensitive yeast mutants were deployed to characterize a gene encoding a C2H2 zinc finger protein (CaZF) that is differentially expressed in a drought-tolerant variety of chickpea (Cicer arietinum) and provides salinity-tolerance in transgenic tobacco. In Saccharomyces cerevisiae most of the cellular responses to hyper-osmotic stress is regulated by two interconnected pathways involving high osmolarity glycerol mitogen-activated protein kinase (Hog1p) and Calcineurin (CAN), a Ca(2+)/calmodulin-regulated protein phosphatase 2B. In this study, we report that heterologous expression of CaZF provides osmotolerance in S. cerevisiae through Hog1p and Calcineurin dependent as well as independent pathways. CaZF partially suppresses salt-hypersensitive phenotypes of hog1, can and hog1can mutants and in conjunction, stimulates HOG and CAN pathway genes with subsequent accumulation of glycerol in absence of Hog1p and CAN. CaZF directly binds to stress response element (STRE) to activate STRE-containing promoter in yeast. Transactivation and salt tolerance assays of CaZF deletion mutants showed that other than the transactivation domain a C-terminal domain composed of acidic and basic amino acids is also required for its function. Altogether, results from this study suggests that CaZF is a potential plant salt-tolerance determinant and also provide evidence that in budding yeast expression of HOG and CAN pathway genes can be stimulated in absence of their regulatory enzymes to provide osmotolerance.

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CaZF suppresses salt sensitive phenotype of Hog and Calcineurin mutants.A, Wild type and mutant BCY123 cells harbouring either only vector (pYES2.1) or CaZF were spotted onto YPGalRaf plates, containing 0.4 M NaCl. Right panel demonstrated the CaZF expression level in WT and mutant BCY123 yeast strain. rRNA was taken as loading control. B, Wild type BCY123 and hog1 or cnb mutants expressing CaZF were spotted onto YPGalRaf medium containing 0.5 M sorbitol. All spotting experiments were performed, as described in Figure. 4. Representative figures from three independent experiments are shown.
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pone-0005154-g006: CaZF suppresses salt sensitive phenotype of Hog and Calcineurin mutants.A, Wild type and mutant BCY123 cells harbouring either only vector (pYES2.1) or CaZF were spotted onto YPGalRaf plates, containing 0.4 M NaCl. Right panel demonstrated the CaZF expression level in WT and mutant BCY123 yeast strain. rRNA was taken as loading control. B, Wild type BCY123 and hog1 or cnb mutants expressing CaZF were spotted onto YPGalRaf medium containing 0.5 M sorbitol. All spotting experiments were performed, as described in Figure. 4. Representative figures from three independent experiments are shown.

Mentions: Exposure to high salinity causes hypercationic and hyperosmotic stresses to eukaryotic cells [31]. Inter-connected pathways regulated by Hog1p MAP kinase and CAN protein phosphatase determine most of the responses to hyperosmotic stress. A number of salt tolerance determinants in plants have been isolated by their ability to suppress salt sensitivity of the yeast mutants [49]. Therefore, CaZF was tested for its ability to provide osmotic tolerance to some of the well-studied yeast mutants. CaZF suppressed the salt sensitivity when expressed in cna1cna2 double mutant, lacking both the redundant catalytic subunits and cnb mutant lacking the regulatory subunit of Calcineurin on 400 mM NaCl (Figure 6A). However, CaZF could not protect the cnb mutant against the ionic osmolytes KCl (data not shown) and NaCl to the extent as it did for the wild type cells. This was also reflected when growth rates were measured in liquid medium. cnb mutant cells expressing CaZF grew faster (doubling time 7.2±0.16 h) than that carrying only vector (doubling time 11.8±0.3 h) but grew at a much slower rate than the wild type cells expressing CaZF (doubling time 4.8±0.2 h). These data indicate that CaZF functions through a pathway, which is additive to but independent of Calcineurin pathway. Alternatively, CaZF can partially complement salt sensitivity of the can mutants but requires the CAN pathway for its full function. Interestingly, growth rates of the cnb mutant and the wild type cells harboring CaZF in non-ionic osmolyte sorbitol containing medium were comparable (Figure 6B). The probable explanation is Calcineurin pathway protects the cells against toxicity of only ionic osmolytes while HOG pathway protects against hypertonic stress due to both ionic and nonionic osmolytes [50].


CaZF, a plant transcription factor functions through and parallel to HOG and calcineurin pathways in Saccharomyces cerevisiae to provide osmotolerance.

Jain D, Roy N, Chattopadhyay D - PLoS ONE (2009)

CaZF suppresses salt sensitive phenotype of Hog and Calcineurin mutants.A, Wild type and mutant BCY123 cells harbouring either only vector (pYES2.1) or CaZF were spotted onto YPGalRaf plates, containing 0.4 M NaCl. Right panel demonstrated the CaZF expression level in WT and mutant BCY123 yeast strain. rRNA was taken as loading control. B, Wild type BCY123 and hog1 or cnb mutants expressing CaZF were spotted onto YPGalRaf medium containing 0.5 M sorbitol. All spotting experiments were performed, as described in Figure. 4. Representative figures from three independent experiments are shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2664467&req=5

pone-0005154-g006: CaZF suppresses salt sensitive phenotype of Hog and Calcineurin mutants.A, Wild type and mutant BCY123 cells harbouring either only vector (pYES2.1) or CaZF were spotted onto YPGalRaf plates, containing 0.4 M NaCl. Right panel demonstrated the CaZF expression level in WT and mutant BCY123 yeast strain. rRNA was taken as loading control. B, Wild type BCY123 and hog1 or cnb mutants expressing CaZF were spotted onto YPGalRaf medium containing 0.5 M sorbitol. All spotting experiments were performed, as described in Figure. 4. Representative figures from three independent experiments are shown.
Mentions: Exposure to high salinity causes hypercationic and hyperosmotic stresses to eukaryotic cells [31]. Inter-connected pathways regulated by Hog1p MAP kinase and CAN protein phosphatase determine most of the responses to hyperosmotic stress. A number of salt tolerance determinants in plants have been isolated by their ability to suppress salt sensitivity of the yeast mutants [49]. Therefore, CaZF was tested for its ability to provide osmotic tolerance to some of the well-studied yeast mutants. CaZF suppressed the salt sensitivity when expressed in cna1cna2 double mutant, lacking both the redundant catalytic subunits and cnb mutant lacking the regulatory subunit of Calcineurin on 400 mM NaCl (Figure 6A). However, CaZF could not protect the cnb mutant against the ionic osmolytes KCl (data not shown) and NaCl to the extent as it did for the wild type cells. This was also reflected when growth rates were measured in liquid medium. cnb mutant cells expressing CaZF grew faster (doubling time 7.2±0.16 h) than that carrying only vector (doubling time 11.8±0.3 h) but grew at a much slower rate than the wild type cells expressing CaZF (doubling time 4.8±0.2 h). These data indicate that CaZF functions through a pathway, which is additive to but independent of Calcineurin pathway. Alternatively, CaZF can partially complement salt sensitivity of the can mutants but requires the CAN pathway for its full function. Interestingly, growth rates of the cnb mutant and the wild type cells harboring CaZF in non-ionic osmolyte sorbitol containing medium were comparable (Figure 6B). The probable explanation is Calcineurin pathway protects the cells against toxicity of only ionic osmolytes while HOG pathway protects against hypertonic stress due to both ionic and nonionic osmolytes [50].

Bottom Line: In Saccharomyces cerevisiae most of the cellular responses to hyper-osmotic stress is regulated by two interconnected pathways involving high osmolarity glycerol mitogen-activated protein kinase (Hog1p) and Calcineurin (CAN), a Ca(2+)/calmodulin-regulated protein phosphatase 2B.In this study, we report that heterologous expression of CaZF provides osmotolerance in S. cerevisiae through Hog1p and Calcineurin dependent as well as independent pathways.CaZF directly binds to stress response element (STRE) to activate STRE-containing promoter in yeast.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi, India.

ABSTRACT
Salt-sensitive yeast mutants were deployed to characterize a gene encoding a C2H2 zinc finger protein (CaZF) that is differentially expressed in a drought-tolerant variety of chickpea (Cicer arietinum) and provides salinity-tolerance in transgenic tobacco. In Saccharomyces cerevisiae most of the cellular responses to hyper-osmotic stress is regulated by two interconnected pathways involving high osmolarity glycerol mitogen-activated protein kinase (Hog1p) and Calcineurin (CAN), a Ca(2+)/calmodulin-regulated protein phosphatase 2B. In this study, we report that heterologous expression of CaZF provides osmotolerance in S. cerevisiae through Hog1p and Calcineurin dependent as well as independent pathways. CaZF partially suppresses salt-hypersensitive phenotypes of hog1, can and hog1can mutants and in conjunction, stimulates HOG and CAN pathway genes with subsequent accumulation of glycerol in absence of Hog1p and CAN. CaZF directly binds to stress response element (STRE) to activate STRE-containing promoter in yeast. Transactivation and salt tolerance assays of CaZF deletion mutants showed that other than the transactivation domain a C-terminal domain composed of acidic and basic amino acids is also required for its function. Altogether, results from this study suggests that CaZF is a potential plant salt-tolerance determinant and also provide evidence that in budding yeast expression of HOG and CAN pathway genes can be stimulated in absence of their regulatory enzymes to provide osmotolerance.

Show MeSH
Related in: MedlinePlus