Limits...
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.

Show MeSH

Related in: MedlinePlus

CaZF directly binds to STRE and activates CTT1 promoter.A, Gel-shift assay demonstrating that CaZF binds to the STRE sequence containing probe. Either wild-type or mutant versions (M1 and M2) are used. STRE sequences are underlined and modified bases are in bold small case letters (left panel). B, Transactivation assay of CTT1-LacZ construct by CaZF. Full length or truncated CaZF protein and LacZ reporter fused to CTT1 promoter fragment or its mutant were cointroduced in wild type (WT) or hog1 BCY123 yeast strains. The transformed yeast strains were treated with/without 500 mM NaCl for 30 min. Activity of β-galactosidase of each sample (average of three independent transformants) as mentioned in table (left panel) was determined and presented in the form of fold induction in activity (right panel).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2664467&req=5

pone-0005154-g009: CaZF directly binds to STRE and activates CTT1 promoter.A, Gel-shift assay demonstrating that CaZF binds to the STRE sequence containing probe. Either wild-type or mutant versions (M1 and M2) are used. STRE sequences are underlined and modified bases are in bold small case letters (left panel). B, Transactivation assay of CTT1-LacZ construct by CaZF. Full length or truncated CaZF protein and LacZ reporter fused to CTT1 promoter fragment or its mutant were cointroduced in wild type (WT) or hog1 BCY123 yeast strains. The transformed yeast strains were treated with/without 500 mM NaCl for 30 min. Activity of β-galactosidase of each sample (average of three independent transformants) as mentioned in table (left panel) was determined and presented in the form of fold induction in activity (right panel).

Mentions: CTT1, HSP12, GPD1 and GPP2, the well-studied general stress response genes regulated by HOG pathway possess stress tolerance responsive elements in their upstream activating sequence. Msn2p and Msn4p, two C2H2 zinc finger proteins bind to STRE of CTT1 and HSP12 to activate their expressions. To determine the mechanism of action of CaZF in yeast we, therefore, tested the ability of CaZF to bind STRE. CaZF protein fused to glutathione-S-transferase (GST) was purified from bacteria and used for gel mobility shift assay using a radiolabeled probe derived from CTT1 promoter having tetramer of STRE core element (AAGGGG). Figure 9A clearly shows that CaZF bound to STRE in a sequence specific manner as replacement of a ‘G’ residue with ‘A’ residue in the core element of the probe (M1) totally abolished the binding while another replacement outside the core element (M2) maintained the binding efficiency.


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 directly binds to STRE and activates CTT1 promoter.A, Gel-shift assay demonstrating that CaZF binds to the STRE sequence containing probe. Either wild-type or mutant versions (M1 and M2) are used. STRE sequences are underlined and modified bases are in bold small case letters (left panel). B, Transactivation assay of CTT1-LacZ construct by CaZF. Full length or truncated CaZF protein and LacZ reporter fused to CTT1 promoter fragment or its mutant were cointroduced in wild type (WT) or hog1 BCY123 yeast strains. The transformed yeast strains were treated with/without 500 mM NaCl for 30 min. Activity of β-galactosidase of each sample (average of three independent transformants) as mentioned in table (left panel) was determined and presented in the form of fold induction in activity (right panel).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0005154-g009: CaZF directly binds to STRE and activates CTT1 promoter.A, Gel-shift assay demonstrating that CaZF binds to the STRE sequence containing probe. Either wild-type or mutant versions (M1 and M2) are used. STRE sequences are underlined and modified bases are in bold small case letters (left panel). B, Transactivation assay of CTT1-LacZ construct by CaZF. Full length or truncated CaZF protein and LacZ reporter fused to CTT1 promoter fragment or its mutant were cointroduced in wild type (WT) or hog1 BCY123 yeast strains. The transformed yeast strains were treated with/without 500 mM NaCl for 30 min. Activity of β-galactosidase of each sample (average of three independent transformants) as mentioned in table (left panel) was determined and presented in the form of fold induction in activity (right panel).
Mentions: CTT1, HSP12, GPD1 and GPP2, the well-studied general stress response genes regulated by HOG pathway possess stress tolerance responsive elements in their upstream activating sequence. Msn2p and Msn4p, two C2H2 zinc finger proteins bind to STRE of CTT1 and HSP12 to activate their expressions. To determine the mechanism of action of CaZF in yeast we, therefore, tested the ability of CaZF to bind STRE. CaZF protein fused to glutathione-S-transferase (GST) was purified from bacteria and used for gel mobility shift assay using a radiolabeled probe derived from CTT1 promoter having tetramer of STRE core element (AAGGGG). Figure 9A clearly shows that CaZF bound to STRE in a sequence specific manner as replacement of a ‘G’ residue with ‘A’ residue in the core element of the probe (M1) totally abolished the binding while another replacement outside the core element (M2) maintained the binding efficiency.

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