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Enhancement of stress tolerance in transgenic tobacco plants constitutively expressing AtIpk2beta, an inositol polyphosphate 6-/3-kinase from Arabidopsis thaliana.

Yang L, Tang R, Zhu J, Liu H, Mueller-Roeber B, Xia H, Zhang H - Plant Mol. Biol. (2007)

Bottom Line: Here we expressed Arabidopsis inositol polyphosphate 6-/3-kinase (AtIpk2beta) in two heterologous systems, i.e. the yeast Saccharomyces cerevisiae and in tobacco (Nicotiana tabacum), and tested the effect on abiotic stress tolerance.Transgenic tobacco plants constitutively expressing AtIpk2beta under the control of the Cauliflower Mosaic Virus 35S promoter were generated and found to exhibit improved tolerance to diverse abiotic stresses when compared to wild type plants.Expression patterns of various stress responsive genes were enhanced, and the activities of anti-oxidative enzymes were elevated in transgenic plants, suggesting a possible involvement of AtIpk2beta in plant stress responses.

View Article: PubMed Central - PubMed

Affiliation: National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China.

ABSTRACT
Inositol phosphates (IPs) and their turnover products have been implicated to play important roles in stress signaling in eukaryotic cells. In higher plants genes encoding inositol polyphosphate kinases have been identified previously, but their physiological functions have not been fully resolved. Here we expressed Arabidopsis inositol polyphosphate 6-/3-kinase (AtIpk2beta) in two heterologous systems, i.e. the yeast Saccharomyces cerevisiae and in tobacco (Nicotiana tabacum), and tested the effect on abiotic stress tolerance. Expression of AtIpk2beta rescued the salt-, osmotic- and temperature-sensitive growth defects of a yeast mutant strain (arg82Delta) that lacks inositol polyphosphate multikinase activity encoded by the ARG82/IPK2 gene. Transgenic tobacco plants constitutively expressing AtIpk2beta under the control of the Cauliflower Mosaic Virus 35S promoter were generated and found to exhibit improved tolerance to diverse abiotic stresses when compared to wild type plants. Expression patterns of various stress responsive genes were enhanced, and the activities of anti-oxidative enzymes were elevated in transgenic plants, suggesting a possible involvement of AtIpk2beta in plant stress responses.

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Construct used for tobacco transformation and Western blot analysis of AtIpk2β in wild type and transgenic tobacco plants. (a) Schematic map of the binary construct used for tobacco transformation. Expression of AtIpk2β is driven by the Cauliflower Mosaic Virus 35S promoter. Ocs, ocs terminator; RB and LB, right and left border of T-DNA, respectively. (b) Western blot analysis. Lane 1, wild type Arabidopsis; lane 2, wild type tobacco; lanes 3–7, transgenic AtIpk2β expressing tobacco lines S11, S13, S14, S15, and S18. Coomassie Bright Blue stained gel is shown below the Western blot to demonstrate equal loading of protein
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Fig2: Construct used for tobacco transformation and Western blot analysis of AtIpk2β in wild type and transgenic tobacco plants. (a) Schematic map of the binary construct used for tobacco transformation. Expression of AtIpk2β is driven by the Cauliflower Mosaic Virus 35S promoter. Ocs, ocs terminator; RB and LB, right and left border of T-DNA, respectively. (b) Western blot analysis. Lane 1, wild type Arabidopsis; lane 2, wild type tobacco; lanes 3–7, transgenic AtIpk2β expressing tobacco lines S11, S13, S14, S15, and S18. Coomassie Bright Blue stained gel is shown below the Western blot to demonstrate equal loading of protein

Mentions: To test whether constitutive expression of AtIpk2β affects abiotic stress tolerance in plants, we introduced its open reading frame (Fig. 2a) into the genome of tobacco (Nicotiana tabacum cv. SR-1) by Agrobacterium tumefaciens-mediated transformation. Expression of AtIpk2β in the transgenic plant was controlled by the Cauliflower Mosaic Virus (CaMV) 35S promoter. Forty-six independent transgenic plants (T0 generation) were obtained; six lines were grown to produce seeds. Transgenic plants homozygous for the 35S:AtIpk2β transgene were obtained and three lines (S13, S14 and S15) were chosen for further experiments. Constitutive expression of AtIpk2β did not affect overall plant morphology although the transgenic plants grew slightly faster than wild type plants under normal growth condition. Western blot analyses were performed to test for the presence of AtIpk2β protein. A 33-kDa band corresponding to AtIpk2β was identified in Arabidopsis plants; similarly, 33-kDa AtIpk2β protein was detected in the transgenic but not the wild type tobacco lines (Fig. 2b), indicating that it was successfully expressed in the genetically modified plants.Fig. 2


Enhancement of stress tolerance in transgenic tobacco plants constitutively expressing AtIpk2beta, an inositol polyphosphate 6-/3-kinase from Arabidopsis thaliana.

Yang L, Tang R, Zhu J, Liu H, Mueller-Roeber B, Xia H, Zhang H - Plant Mol. Biol. (2007)

Construct used for tobacco transformation and Western blot analysis of AtIpk2β in wild type and transgenic tobacco plants. (a) Schematic map of the binary construct used for tobacco transformation. Expression of AtIpk2β is driven by the Cauliflower Mosaic Virus 35S promoter. Ocs, ocs terminator; RB and LB, right and left border of T-DNA, respectively. (b) Western blot analysis. Lane 1, wild type Arabidopsis; lane 2, wild type tobacco; lanes 3–7, transgenic AtIpk2β expressing tobacco lines S11, S13, S14, S15, and S18. Coomassie Bright Blue stained gel is shown below the Western blot to demonstrate equal loading of protein
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2238787&req=5

Fig2: Construct used for tobacco transformation and Western blot analysis of AtIpk2β in wild type and transgenic tobacco plants. (a) Schematic map of the binary construct used for tobacco transformation. Expression of AtIpk2β is driven by the Cauliflower Mosaic Virus 35S promoter. Ocs, ocs terminator; RB and LB, right and left border of T-DNA, respectively. (b) Western blot analysis. Lane 1, wild type Arabidopsis; lane 2, wild type tobacco; lanes 3–7, transgenic AtIpk2β expressing tobacco lines S11, S13, S14, S15, and S18. Coomassie Bright Blue stained gel is shown below the Western blot to demonstrate equal loading of protein
Mentions: To test whether constitutive expression of AtIpk2β affects abiotic stress tolerance in plants, we introduced its open reading frame (Fig. 2a) into the genome of tobacco (Nicotiana tabacum cv. SR-1) by Agrobacterium tumefaciens-mediated transformation. Expression of AtIpk2β in the transgenic plant was controlled by the Cauliflower Mosaic Virus (CaMV) 35S promoter. Forty-six independent transgenic plants (T0 generation) were obtained; six lines were grown to produce seeds. Transgenic plants homozygous for the 35S:AtIpk2β transgene were obtained and three lines (S13, S14 and S15) were chosen for further experiments. Constitutive expression of AtIpk2β did not affect overall plant morphology although the transgenic plants grew slightly faster than wild type plants under normal growth condition. Western blot analyses were performed to test for the presence of AtIpk2β protein. A 33-kDa band corresponding to AtIpk2β was identified in Arabidopsis plants; similarly, 33-kDa AtIpk2β protein was detected in the transgenic but not the wild type tobacco lines (Fig. 2b), indicating that it was successfully expressed in the genetically modified plants.Fig. 2

Bottom Line: Here we expressed Arabidopsis inositol polyphosphate 6-/3-kinase (AtIpk2beta) in two heterologous systems, i.e. the yeast Saccharomyces cerevisiae and in tobacco (Nicotiana tabacum), and tested the effect on abiotic stress tolerance.Transgenic tobacco plants constitutively expressing AtIpk2beta under the control of the Cauliflower Mosaic Virus 35S promoter were generated and found to exhibit improved tolerance to diverse abiotic stresses when compared to wild type plants.Expression patterns of various stress responsive genes were enhanced, and the activities of anti-oxidative enzymes were elevated in transgenic plants, suggesting a possible involvement of AtIpk2beta in plant stress responses.

View Article: PubMed Central - PubMed

Affiliation: National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China.

ABSTRACT
Inositol phosphates (IPs) and their turnover products have been implicated to play important roles in stress signaling in eukaryotic cells. In higher plants genes encoding inositol polyphosphate kinases have been identified previously, but their physiological functions have not been fully resolved. Here we expressed Arabidopsis inositol polyphosphate 6-/3-kinase (AtIpk2beta) in two heterologous systems, i.e. the yeast Saccharomyces cerevisiae and in tobacco (Nicotiana tabacum), and tested the effect on abiotic stress tolerance. Expression of AtIpk2beta rescued the salt-, osmotic- and temperature-sensitive growth defects of a yeast mutant strain (arg82Delta) that lacks inositol polyphosphate multikinase activity encoded by the ARG82/IPK2 gene. Transgenic tobacco plants constitutively expressing AtIpk2beta under the control of the Cauliflower Mosaic Virus 35S promoter were generated and found to exhibit improved tolerance to diverse abiotic stresses when compared to wild type plants. Expression patterns of various stress responsive genes were enhanced, and the activities of anti-oxidative enzymes were elevated in transgenic plants, suggesting a possible involvement of AtIpk2beta in plant stress responses.

Show MeSH
Related in: MedlinePlus