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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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Expression of CaZF in chickpea varieties BGD72 and ICCV2 under different drought conditions.Samples harvested at day post-irrigation (DPI) is mentioned. Total RNA (20 µg/lane) from chickpea seedlings were hybridized with probe prepared from CaZF cDNA as described under “Experimental Procedures”. Ribosomal RNAs (rRNA) are shown as loading control.
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pone-0005154-g001: Expression of CaZF in chickpea varieties BGD72 and ICCV2 under different drought conditions.Samples harvested at day post-irrigation (DPI) is mentioned. Total RNA (20 µg/lane) from chickpea seedlings were hybridized with probe prepared from CaZF cDNA as described under “Experimental Procedures”. Ribosomal RNAs (rRNA) are shown as loading control.

Mentions: Subtracted cDNA libraries constructed between two chickpea (Cicer arietinum) cultivars at different points of drought-stress resulted in a number of EST clones expressing higher in the drought tolerant BGD72 than in the sensitive ICCV2 in response to drought. An EST encoding a putative zinc finger protein expressing more in BGD72 than in ICCV2 at different points of stress (Figure 1) was taken for further studies. Full-length cDNA (CaZF) constructed by 5′ RACE was 1185 bp in length (GenBank accession EU513298). Sequence analysis revealed an 843 bp open reading frame (ORF) of 280 amino acid, 139 bp long 5′ and 203 bp long 3′ untranslated region. Deduced amino acid sequence shows (Figure 2A) CaZF is an EPF type C2H2 zinc finger protein having two canonical TFIIIA-type zinc finger motifs (CX2CX3FX5LX2HX3H). Both the zinc finger motifs contain conserved QALGGH sequence. A short spacer sequence of 28 amino acids separates two zinc fingers. Among the studied proteins PIF1 (GB: AAQ54302), a pathogen inducible zinc finger protein from capsicum shows maximum sequence similarity with CaZF of only about 55% homology (expect = 5e-45). Notably, PIF1 is also highly expressed in a pathogen tolerant variety compared to a sensitive one in response to infection [40]. Detailed comparisons of the amino acid sequences among plant zinc finger proteins revealed three conserved regions other than the zinc fingers. CaZF contains a short basic region with a consensus of KXKRSKRXR (B-box), near the N-terminus, which may function as a potential nuclear localization signal (NLS) and/or may participate in DNA binding. Another is a region, consisting of three acidic residues followed by hydrophobic residues rich in leucine, with a consensus of EXEXXAXCLXXL (L-box) located between B-box and the first zinc-finger. The other is a short hydrophobic region containing a highly conserved DLNL sequence as a core (DLN-box) close to the C-terminus. The latter two may play a role in protein-protein interactions or in maintaining the folded structure. CaZF possesses a serine-glutamine rich region at the N-terminus, between L-box and first zinc-finger, which might function as a transactivation domain as suggested for ZPT2-1 and Pszf1 [41], [42] or might be a phosphorylation site for post-translational modification; and an asparagine rich stretch after the second zinc finger at the C-terminus. Similar asparagine-rich domains are also present in some stress-inducible zinc finger proteins such as SCOF-1, EPF2-5, and STZ [43]. Like STO and STZ, the Arabidopsis cDNAs, which increase salt tolerance in yeast in a Calcineurin independent manner, SCOF-1 and EPF2-5, CaZF contains highly basic region followed by acidic amino acids near the C-terminus. But, CaZF has two such combinations of basic and acidic amino acid stretches. Phylogenetic analysis showed that CaZF and one Arachis protein (ZFP248) shares the same clad (Figure 2B).


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)

Expression of CaZF in chickpea varieties BGD72 and ICCV2 under different drought conditions.Samples harvested at day post-irrigation (DPI) is mentioned. Total RNA (20 µg/lane) from chickpea seedlings were hybridized with probe prepared from CaZF cDNA as described under “Experimental Procedures”. Ribosomal RNAs (rRNA) are shown as loading control.
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Related In: Results  -  Collection

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pone-0005154-g001: Expression of CaZF in chickpea varieties BGD72 and ICCV2 under different drought conditions.Samples harvested at day post-irrigation (DPI) is mentioned. Total RNA (20 µg/lane) from chickpea seedlings were hybridized with probe prepared from CaZF cDNA as described under “Experimental Procedures”. Ribosomal RNAs (rRNA) are shown as loading control.
Mentions: Subtracted cDNA libraries constructed between two chickpea (Cicer arietinum) cultivars at different points of drought-stress resulted in a number of EST clones expressing higher in the drought tolerant BGD72 than in the sensitive ICCV2 in response to drought. An EST encoding a putative zinc finger protein expressing more in BGD72 than in ICCV2 at different points of stress (Figure 1) was taken for further studies. Full-length cDNA (CaZF) constructed by 5′ RACE was 1185 bp in length (GenBank accession EU513298). Sequence analysis revealed an 843 bp open reading frame (ORF) of 280 amino acid, 139 bp long 5′ and 203 bp long 3′ untranslated region. Deduced amino acid sequence shows (Figure 2A) CaZF is an EPF type C2H2 zinc finger protein having two canonical TFIIIA-type zinc finger motifs (CX2CX3FX5LX2HX3H). Both the zinc finger motifs contain conserved QALGGH sequence. A short spacer sequence of 28 amino acids separates two zinc fingers. Among the studied proteins PIF1 (GB: AAQ54302), a pathogen inducible zinc finger protein from capsicum shows maximum sequence similarity with CaZF of only about 55% homology (expect = 5e-45). Notably, PIF1 is also highly expressed in a pathogen tolerant variety compared to a sensitive one in response to infection [40]. Detailed comparisons of the amino acid sequences among plant zinc finger proteins revealed three conserved regions other than the zinc fingers. CaZF contains a short basic region with a consensus of KXKRSKRXR (B-box), near the N-terminus, which may function as a potential nuclear localization signal (NLS) and/or may participate in DNA binding. Another is a region, consisting of three acidic residues followed by hydrophobic residues rich in leucine, with a consensus of EXEXXAXCLXXL (L-box) located between B-box and the first zinc-finger. The other is a short hydrophobic region containing a highly conserved DLNL sequence as a core (DLN-box) close to the C-terminus. The latter two may play a role in protein-protein interactions or in maintaining the folded structure. CaZF possesses a serine-glutamine rich region at the N-terminus, between L-box and first zinc-finger, which might function as a transactivation domain as suggested for ZPT2-1 and Pszf1 [41], [42] or might be a phosphorylation site for post-translational modification; and an asparagine rich stretch after the second zinc finger at the C-terminus. Similar asparagine-rich domains are also present in some stress-inducible zinc finger proteins such as SCOF-1, EPF2-5, and STZ [43]. Like STO and STZ, the Arabidopsis cDNAs, which increase salt tolerance in yeast in a Calcineurin independent manner, SCOF-1 and EPF2-5, CaZF contains highly basic region followed by acidic amino acids near the C-terminus. But, CaZF has two such combinations of basic and acidic amino acid stretches. Phylogenetic analysis showed that CaZF and one Arachis protein (ZFP248) shares the same clad (Figure 2B).

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