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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 and salt tolerance of transgenic tobacco.A, Northern blot showing expression of CaZF in T1 transgenic tobacco lines transformed with pBI121 without (vector) or with CaZF (L21, L1, L17 and L46). Full length CaZF cDNA was used as probe. B, Vector control and T1 transgenic progenies were grown on 0.5 MS (Murashige and Skoog) medium for 10 d. C, Effect of salt stress on tobacco seedlings from vector control and T1 transgenic progenies (CaZFL21, CaZFL1, CaZFL17 and CaZFL46) was demonstrated by germinating seeds on 0.5 MS medium supplemented with 200 mM NaCl for 20 d. Representative figure of three independent experiments are shown. D, Chlorophyll content determined by leaf disc senescence assay for salinity tolerance of 30d-old vector control and transgenic tobacco lines overexpressing CaZF, after incubation in water, 150 mM and 300 mM NaCl solutions for 72 hr under continuous white light at 25±2°C. Results of three independent experiments are shown.
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pone-0005154-g004: Expression of CaZF and salt tolerance of transgenic tobacco.A, Northern blot showing expression of CaZF in T1 transgenic tobacco lines transformed with pBI121 without (vector) or with CaZF (L21, L1, L17 and L46). Full length CaZF cDNA was used as probe. B, Vector control and T1 transgenic progenies were grown on 0.5 MS (Murashige and Skoog) medium for 10 d. C, Effect of salt stress on tobacco seedlings from vector control and T1 transgenic progenies (CaZFL21, CaZFL1, CaZFL17 and CaZFL46) was demonstrated by germinating seeds on 0.5 MS medium supplemented with 200 mM NaCl for 20 d. Representative figure of three independent experiments are shown. D, Chlorophyll content determined by leaf disc senescence assay for salinity tolerance of 30d-old vector control and transgenic tobacco lines overexpressing CaZF, after incubation in water, 150 mM and 300 mM NaCl solutions for 72 hr under continuous white light at 25±2°C. Results of three independent experiments are shown.

Mentions: To establish the functional significance of CaZF in planta the complete ORF of CaZF gene was introduced into tobacco plants using Agrobacterium-mediated transformation. Out of twelve transgenic lines harboring single copy of the transgene, two relatively high expressing and two relatively low expressing lines (Figure 4A) were chosen for salt-tolerance analysis. The vector transformed and the CaZF-expressing T1 transgenic lines were germinated simultaneously and grew normally in 0.5 MS (Murashige and Skoog) medium (Figure 4B). To assess the effect of high salt on the seed germination/growth of the vector control and T1 plants overexpressing CaZF, surface-sterilized seeds were plated on 0.5 MS supplemented with 200 mM NaCl. In the presence of high salt, vector transformed seeds showed almost no germination (only one out of thirty-two seeds in one repeat) until 20 d, while on average 85–95% CaZF overexpressing T1 seeds showed germination within 15 d (Figure 4C). To test for salinity tolerance, leaf disks from all four lines of T1 transgenic plants and vector transformed plants were floated separately on water, 150 mM or 300 mM NaCl for 72 h and subsequently total chlorophyll content was quantitated. Chlorophyll content of the vector-transformed and CaZF-expressing plants was comparable in presence of water. However, salinity-induced loss of chlorophyll was much lower in CaZF overexpressing lines (average 13.2% and 23.1% for L1/L21, and 27.8% and 51.2% for L17/L46 at 150 mM and 300 mM NaCl respectively) compared with that in the vector control (average 62.3% and 76.4% at 150 mM and 300 mM NaCl respectively) (Figure 4D). From the damage caused by salt stress it was evident that CaZF overexpressing transgenic tobacco plants have a better ability to tolerate salinity stress as compared to vector control plants. The degree of bleaching (yellow color) observed in leaf disks after 72 h can reflect the extent of damage caused by stress. CaZF-expressing transgenic tobacco seedlings also exhibited improved drought tolerance (data not shown).


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 and salt tolerance of transgenic tobacco.A, Northern blot showing expression of CaZF in T1 transgenic tobacco lines transformed with pBI121 without (vector) or with CaZF (L21, L1, L17 and L46). Full length CaZF cDNA was used as probe. B, Vector control and T1 transgenic progenies were grown on 0.5 MS (Murashige and Skoog) medium for 10 d. C, Effect of salt stress on tobacco seedlings from vector control and T1 transgenic progenies (CaZFL21, CaZFL1, CaZFL17 and CaZFL46) was demonstrated by germinating seeds on 0.5 MS medium supplemented with 200 mM NaCl for 20 d. Representative figure of three independent experiments are shown. D, Chlorophyll content determined by leaf disc senescence assay for salinity tolerance of 30d-old vector control and transgenic tobacco lines overexpressing CaZF, after incubation in water, 150 mM and 300 mM NaCl solutions for 72 hr under continuous white light at 25±2°C. Results of three independent experiments are shown.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0005154-g004: Expression of CaZF and salt tolerance of transgenic tobacco.A, Northern blot showing expression of CaZF in T1 transgenic tobacco lines transformed with pBI121 without (vector) or with CaZF (L21, L1, L17 and L46). Full length CaZF cDNA was used as probe. B, Vector control and T1 transgenic progenies were grown on 0.5 MS (Murashige and Skoog) medium for 10 d. C, Effect of salt stress on tobacco seedlings from vector control and T1 transgenic progenies (CaZFL21, CaZFL1, CaZFL17 and CaZFL46) was demonstrated by germinating seeds on 0.5 MS medium supplemented with 200 mM NaCl for 20 d. Representative figure of three independent experiments are shown. D, Chlorophyll content determined by leaf disc senescence assay for salinity tolerance of 30d-old vector control and transgenic tobacco lines overexpressing CaZF, after incubation in water, 150 mM and 300 mM NaCl solutions for 72 hr under continuous white light at 25±2°C. Results of three independent experiments are shown.
Mentions: To establish the functional significance of CaZF in planta the complete ORF of CaZF gene was introduced into tobacco plants using Agrobacterium-mediated transformation. Out of twelve transgenic lines harboring single copy of the transgene, two relatively high expressing and two relatively low expressing lines (Figure 4A) were chosen for salt-tolerance analysis. The vector transformed and the CaZF-expressing T1 transgenic lines were germinated simultaneously and grew normally in 0.5 MS (Murashige and Skoog) medium (Figure 4B). To assess the effect of high salt on the seed germination/growth of the vector control and T1 plants overexpressing CaZF, surface-sterilized seeds were plated on 0.5 MS supplemented with 200 mM NaCl. In the presence of high salt, vector transformed seeds showed almost no germination (only one out of thirty-two seeds in one repeat) until 20 d, while on average 85–95% CaZF overexpressing T1 seeds showed germination within 15 d (Figure 4C). To test for salinity tolerance, leaf disks from all four lines of T1 transgenic plants and vector transformed plants were floated separately on water, 150 mM or 300 mM NaCl for 72 h and subsequently total chlorophyll content was quantitated. Chlorophyll content of the vector-transformed and CaZF-expressing plants was comparable in presence of water. However, salinity-induced loss of chlorophyll was much lower in CaZF overexpressing lines (average 13.2% and 23.1% for L1/L21, and 27.8% and 51.2% for L17/L46 at 150 mM and 300 mM NaCl respectively) compared with that in the vector control (average 62.3% and 76.4% at 150 mM and 300 mM NaCl respectively) (Figure 4D). From the damage caused by salt stress it was evident that CaZF overexpressing transgenic tobacco plants have a better ability to tolerate salinity stress as compared to vector control plants. The degree of bleaching (yellow color) observed in leaf disks after 72 h can reflect the extent of damage caused by stress. CaZF-expressing transgenic tobacco seedlings also exhibited improved drought tolerance (data not shown).

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