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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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MDA level, anti-oxidative enzyme activity, and expression level of stress regulated genes in transgenic plants. (a) MDA levels in wild type and transgenic lines S13, S15 after treatment with 200-mM NaCl. (b–d) One-month-old seedlings were incubated for 12 h in 1% H2O2 or water (control) under continuous white light. SOD, APX and CAT activities were determined. Results are presented as means and standard errors from three independent experiments. * and ** indicate significant differences in comparison to the wild type at P < 0.05 and P < 0.01, respectively (Student’s t-test). (e) Two-week-old seedlings of wild type and transgenic lines (S13, S15) were used for RNA extraction. For the salt stress experiments, seedlings were treated with 300-mM NaCl for 5 h before RNA isolation. The transcriptional levels of five stress genes were determined by RT-PCR analyses. The stress genes used for the tests are as follows: lipid transfer protein (AY562132); fructose-bisphosphate aldolase (AY554169); raffinose synthase family protein/seed imbibition protein (AY554170); group 2 LEA proteins NtERD 10B (AB049336) and NtERD 10C (AB049337)
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Fig7: MDA level, anti-oxidative enzyme activity, and expression level of stress regulated genes in transgenic plants. (a) MDA levels in wild type and transgenic lines S13, S15 after treatment with 200-mM NaCl. (b–d) One-month-old seedlings were incubated for 12 h in 1% H2O2 or water (control) under continuous white light. SOD, APX and CAT activities were determined. Results are presented as means and standard errors from three independent experiments. * and ** indicate significant differences in comparison to the wild type at P < 0.05 and P < 0.01, respectively (Student’s t-test). (e) Two-week-old seedlings of wild type and transgenic lines (S13, S15) were used for RNA extraction. For the salt stress experiments, seedlings were treated with 300-mM NaCl for 5 h before RNA isolation. The transcriptional levels of five stress genes were determined by RT-PCR analyses. The stress genes used for the tests are as follows: lipid transfer protein (AY562132); fructose-bisphosphate aldolase (AY554169); raffinose synthase family protein/seed imbibition protein (AY554170); group 2 LEA proteins NtERD 10B (AB049336) and NtERD 10C (AB049337)

Mentions: Lipid hydroperoxidation is an effective indicator of cellular oxidative damage (Yoshimura et al. 2004). Changes in the rates of lipid hydroperoxide production induced by oxidative stress were measured by determining MDA content in leaf disks. At high salinity (200-mM NaCl), MDA concentration markedly increased in wild type plants, whereas only a marginal increase was observed in the two transgenic lines (Fig. 7a). Although superoxide dismutase (SOD) activity was largely unaffected in wild type and transgenic plants treated with 1% H2O2 (Fig. 7b), a slight elevation of APX activity was observed in transgenic plants (Fig. 7c). Also, catalase (CAT) activity increased in transgenic plants (Fig. 7d). These results indicate that constitutive expression of AtIpk2β enhanced tolerance to oxidative stress-induced membrane hydroperoxidation in transgenic tobacco plants.Fig. 7


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)

MDA level, anti-oxidative enzyme activity, and expression level of stress regulated genes in transgenic plants. (a) MDA levels in wild type and transgenic lines S13, S15 after treatment with 200-mM NaCl. (b–d) One-month-old seedlings were incubated for 12 h in 1% H2O2 or water (control) under continuous white light. SOD, APX and CAT activities were determined. Results are presented as means and standard errors from three independent experiments. * and ** indicate significant differences in comparison to the wild type at P < 0.05 and P < 0.01, respectively (Student’s t-test). (e) Two-week-old seedlings of wild type and transgenic lines (S13, S15) were used for RNA extraction. For the salt stress experiments, seedlings were treated with 300-mM NaCl for 5 h before RNA isolation. The transcriptional levels of five stress genes were determined by RT-PCR analyses. The stress genes used for the tests are as follows: lipid transfer protein (AY562132); fructose-bisphosphate aldolase (AY554169); raffinose synthase family protein/seed imbibition protein (AY554170); group 2 LEA proteins NtERD 10B (AB049336) and NtERD 10C (AB049337)
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Related In: Results  -  Collection

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Fig7: MDA level, anti-oxidative enzyme activity, and expression level of stress regulated genes in transgenic plants. (a) MDA levels in wild type and transgenic lines S13, S15 after treatment with 200-mM NaCl. (b–d) One-month-old seedlings were incubated for 12 h in 1% H2O2 or water (control) under continuous white light. SOD, APX and CAT activities were determined. Results are presented as means and standard errors from three independent experiments. * and ** indicate significant differences in comparison to the wild type at P < 0.05 and P < 0.01, respectively (Student’s t-test). (e) Two-week-old seedlings of wild type and transgenic lines (S13, S15) were used for RNA extraction. For the salt stress experiments, seedlings were treated with 300-mM NaCl for 5 h before RNA isolation. The transcriptional levels of five stress genes were determined by RT-PCR analyses. The stress genes used for the tests are as follows: lipid transfer protein (AY562132); fructose-bisphosphate aldolase (AY554169); raffinose synthase family protein/seed imbibition protein (AY554170); group 2 LEA proteins NtERD 10B (AB049336) and NtERD 10C (AB049337)
Mentions: Lipid hydroperoxidation is an effective indicator of cellular oxidative damage (Yoshimura et al. 2004). Changes in the rates of lipid hydroperoxide production induced by oxidative stress were measured by determining MDA content in leaf disks. At high salinity (200-mM NaCl), MDA concentration markedly increased in wild type plants, whereas only a marginal increase was observed in the two transgenic lines (Fig. 7a). Although superoxide dismutase (SOD) activity was largely unaffected in wild type and transgenic plants treated with 1% H2O2 (Fig. 7b), a slight elevation of APX activity was observed in transgenic plants (Fig. 7c). Also, catalase (CAT) activity increased in transgenic plants (Fig. 7d). These results indicate that constitutive expression of AtIpk2β enhanced tolerance to oxidative stress-induced membrane hydroperoxidation in transgenic tobacco plants.Fig. 7

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