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Simultaneous over-expression of PaSOD and RaAPX in transgenic Arabidopsis thaliana confers cold stress tolerance through increase in vascular lignifications.

Shafi A, Dogra V, Gill T, Ahuja PS, Sreenivasulu Y - PLoS ONE (2014)

Bottom Line: Antioxidant enzymes play a significant role in eliminating toxic levels of reactive oxygen species (ROS), generated during stress from living cells.It was found that the transgenic plants over-expressing both the genes were more tolerant to cold stress than either of the single gene expressing transgenic plants during growth and development.In the present study, Confocal and SEM analysis in conjunction with qPCR data on the expression pattern of lignin biosynthetic pathway genes revealed that the cold stress tolerance of the transgenic plants might be because of the peroxide induced up-regulation of lignin by antioxidant genes mediated triggering.

View Article: PubMed Central - PubMed

Affiliation: Division of Biotechnology, CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India; Academy of Scientific and Innovative Research (AcSIR), CSIR- Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India.

ABSTRACT
Antioxidant enzymes play a significant role in eliminating toxic levels of reactive oxygen species (ROS), generated during stress from living cells. In the present study, two different antioxidant enzymes namely copper-zinc superoxide dismutase derived from Potentilla astrisanguinea (PaSOD) and ascorbate peroxidase (RaAPX) from Rheum austral both of which are high altitude cold niche area plants of Himalaya were cloned and simultaneously over-expressed in Arabidopsis thaliana to alleviate cold stress. It was found that the transgenic plants over-expressing both the genes were more tolerant to cold stress than either of the single gene expressing transgenic plants during growth and development. In both single (PaSOD, RaAPX) and double (PaSOD + RaAPX) transgenic plants higher levels of total antioxidant enzyme activities, chlorophyll content, total soluble sugars, proline content and lower levels of ROS, ion leakage were recorded when compared to the WT during cold stress (4°C), besides increase in yield. In the present study, Confocal and SEM analysis in conjunction with qPCR data on the expression pattern of lignin biosynthetic pathway genes revealed that the cold stress tolerance of the transgenic plants might be because of the peroxide induced up-regulation of lignin by antioxidant genes mediated triggering.

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Related in: MedlinePlus

Confirmation of PaSOD and RaAPX gene expression in transgenic Arabidopsis plants at 20°C.The same plants were used for crossing to obtain the double transgenic plants. Expression of genes was (S26 and S15; A20 and A18) confirmed by estimating their transcripts from the leaf tissue by semi-quantitative RT-PCR. Constitutively expressed GAP-C was used as loading control.
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pone-0110302-g001: Confirmation of PaSOD and RaAPX gene expression in transgenic Arabidopsis plants at 20°C.The same plants were used for crossing to obtain the double transgenic plants. Expression of genes was (S26 and S15; A20 and A18) confirmed by estimating their transcripts from the leaf tissue by semi-quantitative RT-PCR. Constitutively expressed GAP-C was used as loading control.

Mentions: Arabidopsis transgenic plants over expressing PaSOD under the control of CaMV35 S promoter were generated as reported earlier [12]. Similarly transgenic Arabidopsis plants over expressing ascorbate peroxidase gene from Rheum (RaAPX) under the control of CaMV35 S promoter were also developed. Presence of these transgenes was confirmed by PCR by using genomic DNA from these plants as template and their expression by the presence of its transcripts from leaf tissues by semi-quantitative RT-PCR (Fig. 1). On the basis of 3∶1 segregation and with high enzyme activities, homozygous transgenic lines S26 (for PaSOD) and A20 (for RaAPX) were selected for further analysis as well as for crossings for raising the transgenics with both these genes. Transgenic Arabidopsis plants having both PaSOD and RaAPX genes were obtained by crossings of these individual gene transgenic lines. Presence of transcripts for both genes in transgenic plants was confirmed by semi-quantitative RT-PCR (Fig. 2) and considered as transgenic line with both the genes. Two individual transgenic lines of each PaSOD (S15 and S26) and RaAPX (A18 and A20) were analysed and data was presented. Out of a number of transgenic lines having both the genes, the line 18O and 19C was selected for further analysis and data was presented.


Simultaneous over-expression of PaSOD and RaAPX in transgenic Arabidopsis thaliana confers cold stress tolerance through increase in vascular lignifications.

Shafi A, Dogra V, Gill T, Ahuja PS, Sreenivasulu Y - PLoS ONE (2014)

Confirmation of PaSOD and RaAPX gene expression in transgenic Arabidopsis plants at 20°C.The same plants were used for crossing to obtain the double transgenic plants. Expression of genes was (S26 and S15; A20 and A18) confirmed by estimating their transcripts from the leaf tissue by semi-quantitative RT-PCR. Constitutively expressed GAP-C was used as loading control.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4201527&req=5

pone-0110302-g001: Confirmation of PaSOD and RaAPX gene expression in transgenic Arabidopsis plants at 20°C.The same plants were used for crossing to obtain the double transgenic plants. Expression of genes was (S26 and S15; A20 and A18) confirmed by estimating their transcripts from the leaf tissue by semi-quantitative RT-PCR. Constitutively expressed GAP-C was used as loading control.
Mentions: Arabidopsis transgenic plants over expressing PaSOD under the control of CaMV35 S promoter were generated as reported earlier [12]. Similarly transgenic Arabidopsis plants over expressing ascorbate peroxidase gene from Rheum (RaAPX) under the control of CaMV35 S promoter were also developed. Presence of these transgenes was confirmed by PCR by using genomic DNA from these plants as template and their expression by the presence of its transcripts from leaf tissues by semi-quantitative RT-PCR (Fig. 1). On the basis of 3∶1 segregation and with high enzyme activities, homozygous transgenic lines S26 (for PaSOD) and A20 (for RaAPX) were selected for further analysis as well as for crossings for raising the transgenics with both these genes. Transgenic Arabidopsis plants having both PaSOD and RaAPX genes were obtained by crossings of these individual gene transgenic lines. Presence of transcripts for both genes in transgenic plants was confirmed by semi-quantitative RT-PCR (Fig. 2) and considered as transgenic line with both the genes. Two individual transgenic lines of each PaSOD (S15 and S26) and RaAPX (A18 and A20) were analysed and data was presented. Out of a number of transgenic lines having both the genes, the line 18O and 19C was selected for further analysis and data was presented.

Bottom Line: Antioxidant enzymes play a significant role in eliminating toxic levels of reactive oxygen species (ROS), generated during stress from living cells.It was found that the transgenic plants over-expressing both the genes were more tolerant to cold stress than either of the single gene expressing transgenic plants during growth and development.In the present study, Confocal and SEM analysis in conjunction with qPCR data on the expression pattern of lignin biosynthetic pathway genes revealed that the cold stress tolerance of the transgenic plants might be because of the peroxide induced up-regulation of lignin by antioxidant genes mediated triggering.

View Article: PubMed Central - PubMed

Affiliation: Division of Biotechnology, CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India; Academy of Scientific and Innovative Research (AcSIR), CSIR- Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India.

ABSTRACT
Antioxidant enzymes play a significant role in eliminating toxic levels of reactive oxygen species (ROS), generated during stress from living cells. In the present study, two different antioxidant enzymes namely copper-zinc superoxide dismutase derived from Potentilla astrisanguinea (PaSOD) and ascorbate peroxidase (RaAPX) from Rheum austral both of which are high altitude cold niche area plants of Himalaya were cloned and simultaneously over-expressed in Arabidopsis thaliana to alleviate cold stress. It was found that the transgenic plants over-expressing both the genes were more tolerant to cold stress than either of the single gene expressing transgenic plants during growth and development. In both single (PaSOD, RaAPX) and double (PaSOD + RaAPX) transgenic plants higher levels of total antioxidant enzyme activities, chlorophyll content, total soluble sugars, proline content and lower levels of ROS, ion leakage were recorded when compared to the WT during cold stress (4°C), besides increase in yield. In the present study, Confocal and SEM analysis in conjunction with qPCR data on the expression pattern of lignin biosynthetic pathway genes revealed that the cold stress tolerance of the transgenic plants might be because of the peroxide induced up-regulation of lignin by antioxidant genes mediated triggering.

Show MeSH
Related in: MedlinePlus