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ABA inducible rice protein phosphatase 2C confers ABA insensitivity and abiotic stress tolerance in Arabidopsis.

Singh A, Jha SK, Bagri J, Pandey GK - PLoS ONE (2015)

Bottom Line: At adult stage, OsPP108 overexpression leads to high tolerance to salt, mannitol and drought stresses with far better physiological parameters such as water loss, fresh weight, chlorophyll content and photosynthetic potential (Fv/Fm) in transgenic Arabidopsis plants.Expression profile of various stress marker genes in OsPP108 overexpressing plants revealed interplay of ABA dependent and independent pathway for abiotic stress tolerance.Overall, this study has identified a potential rice group A PP2C, which regulates ABA signaling negatively and abiotic stress signaling positively.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Molecular Biology, University of Delhi South Campus, Benito Juarez Road, Dhaula Kuan, New Delhi, India.

ABSTRACT
Arabidopsis PP2C belonging to group A have been extensively worked out and known to negatively regulate ABA signaling. However, rice (Oryza sativa) orthologs of Arabidopsis group A PP2C are scarcely characterized functionally. We have identified a group A PP2C from rice (OsPP108), which is highly inducible under ABA, salt and drought stresses and localized predominantly in the nucleus. Genetic analysis revealed that Arabidopsis plants overexpressing OsPP108 are highly insensitive to ABA and tolerant to high salt and mannitol stresses during seed germination, root growth and overall seedling growth. At adult stage, OsPP108 overexpression leads to high tolerance to salt, mannitol and drought stresses with far better physiological parameters such as water loss, fresh weight, chlorophyll content and photosynthetic potential (Fv/Fm) in transgenic Arabidopsis plants. Expression profile of various stress marker genes in OsPP108 overexpressing plants revealed interplay of ABA dependent and independent pathway for abiotic stress tolerance. Overall, this study has identified a potential rice group A PP2C, which regulates ABA signaling negatively and abiotic stress signaling positively. Transgenic rice plants overexpressing this gene might provide an answer to the problem of low crop yield and productivity during adverse environmental conditions.

No MeSH data available.


Related in: MedlinePlus

Germination and seedling growth analysis of OsPP108 overexpression transgenic Arabidopsis lines on ABA containing media.(A) RT-PCR analysis showing expression level of OsPP108 in WT (Col-0) and transgenic lines (L1, L2 and L4) using gene-specific primers after 24 PCR cycles. ACTIN2 is expressed as endogenous control in all cDNA samples. (B) Germination and vertical growth of WT (Col-0) and three OsPP108OX lines L1, L2 and L4 on 1/2MS (control) and different ABA concentrations (5μM, 10μM, 25 μM and 50 μM) after 7 days. (C) Horizontal germination based growth pattern and seedling establishment of approximately 100 seeds from WT and transgenic lines on different ABA concentrations (2μM, 5μM, 10μM) after 7 days growth. (D) Seed germination percentage of WT and three transgenic lines on 2μM ABA, 5μM ABA, 10μM ABA. Germination percentage was counted for approximately 100 seeds for each genotype till 5 days of growth. Experiments were repeated three times and mean value ± SD is plotted on the graph. p-value <0.05 was observed for all the transgenic lines (L1, L2 and L4) w.r.t. WT on different ABA concentrations at different time points.
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pone.0125168.g004: Germination and seedling growth analysis of OsPP108 overexpression transgenic Arabidopsis lines on ABA containing media.(A) RT-PCR analysis showing expression level of OsPP108 in WT (Col-0) and transgenic lines (L1, L2 and L4) using gene-specific primers after 24 PCR cycles. ACTIN2 is expressed as endogenous control in all cDNA samples. (B) Germination and vertical growth of WT (Col-0) and three OsPP108OX lines L1, L2 and L4 on 1/2MS (control) and different ABA concentrations (5μM, 10μM, 25 μM and 50 μM) after 7 days. (C) Horizontal germination based growth pattern and seedling establishment of approximately 100 seeds from WT and transgenic lines on different ABA concentrations (2μM, 5μM, 10μM) after 7 days growth. (D) Seed germination percentage of WT and three transgenic lines on 2μM ABA, 5μM ABA, 10μM ABA. Germination percentage was counted for approximately 100 seeds for each genotype till 5 days of growth. Experiments were repeated three times and mean value ± SD is plotted on the graph. p-value <0.05 was observed for all the transgenic lines (L1, L2 and L4) w.r.t. WT on different ABA concentrations at different time points.

Mentions: The expression pattern under ABA and abiotic stresses suggested significant role of OsPP108 in ABA and abiotic stress triggered signaling in plants and prompted us for its in-planta functional characterization. We performed seed germination and root growth assays for three CaMV35S::OsPP108 (overexpression) lines (Fig 4A) on 1/2 MS media supplemented with different concentration of ABA (Fig 4B). Growth was monitored until 7 days and OsPP108 overexpressing plants showed insensitivity to ABA in seed germination and overall growth in comparison to WT. Insensitivity was observed until a very high ABA level (50μM). The insensitive phenotype was evaluated in term of seeds germination over a period of 5 days and all three transgenic lines could tolerate and grow much better on higher ABA level (upto 10 μM). Only 25–30% of WT seeds could germinate on this ABA concentration and almost none develop into a healthy seedling, whereas > 80% seeds of all the three overexpressing lines were germinated and developed into healthy seedlings (Fig 4C and 4D). Tolerance of OsPP108 overexpressing seeds to ABA was also estimated as full emergence of cotyledon at fourth day of growth, and all three overexpression lines had approximately 85–90% fully emerged cotyledons at 2μM ABA and 75–80% at 10 μM ABA level whereas, ~ 8% WT seeds could develop cotyledons even at 2μM ABA (Fig 5A). Fresh weight estimation after 7 days of growth at 2μM ABA showed that each seedling of three transgenic lines was ~2.4 mg, whereas WT seedling weighed only ~0.3 mg (Fig 5B), and this observation also indicated the strong insensitivity of OsPP108 overexpressing lines on higher ABA concentration. To confirm that this response is due to overexpression of OsPP108 in Arabidopsis, and to rule out the possibility of down-regulation of its Arabidopsis homologs (i.e. HAI2, HAI3, AHG1 and PP2CA) due to heterologous OsPP108 overexpression, expression analysis was performed for all these genes in the OsPP108OX lines and WT, but no significant change in expression was observed for any homolog in OsPP108OX lines (S2 Fig). qPCR primers are listed in S1 Table.


ABA inducible rice protein phosphatase 2C confers ABA insensitivity and abiotic stress tolerance in Arabidopsis.

Singh A, Jha SK, Bagri J, Pandey GK - PLoS ONE (2015)

Germination and seedling growth analysis of OsPP108 overexpression transgenic Arabidopsis lines on ABA containing media.(A) RT-PCR analysis showing expression level of OsPP108 in WT (Col-0) and transgenic lines (L1, L2 and L4) using gene-specific primers after 24 PCR cycles. ACTIN2 is expressed as endogenous control in all cDNA samples. (B) Germination and vertical growth of WT (Col-0) and three OsPP108OX lines L1, L2 and L4 on 1/2MS (control) and different ABA concentrations (5μM, 10μM, 25 μM and 50 μM) after 7 days. (C) Horizontal germination based growth pattern and seedling establishment of approximately 100 seeds from WT and transgenic lines on different ABA concentrations (2μM, 5μM, 10μM) after 7 days growth. (D) Seed germination percentage of WT and three transgenic lines on 2μM ABA, 5μM ABA, 10μM ABA. Germination percentage was counted for approximately 100 seeds for each genotype till 5 days of growth. Experiments were repeated three times and mean value ± SD is plotted on the graph. p-value <0.05 was observed for all the transgenic lines (L1, L2 and L4) w.r.t. WT on different ABA concentrations at different time points.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4401787&req=5

pone.0125168.g004: Germination and seedling growth analysis of OsPP108 overexpression transgenic Arabidopsis lines on ABA containing media.(A) RT-PCR analysis showing expression level of OsPP108 in WT (Col-0) and transgenic lines (L1, L2 and L4) using gene-specific primers after 24 PCR cycles. ACTIN2 is expressed as endogenous control in all cDNA samples. (B) Germination and vertical growth of WT (Col-0) and three OsPP108OX lines L1, L2 and L4 on 1/2MS (control) and different ABA concentrations (5μM, 10μM, 25 μM and 50 μM) after 7 days. (C) Horizontal germination based growth pattern and seedling establishment of approximately 100 seeds from WT and transgenic lines on different ABA concentrations (2μM, 5μM, 10μM) after 7 days growth. (D) Seed germination percentage of WT and three transgenic lines on 2μM ABA, 5μM ABA, 10μM ABA. Germination percentage was counted for approximately 100 seeds for each genotype till 5 days of growth. Experiments were repeated three times and mean value ± SD is plotted on the graph. p-value <0.05 was observed for all the transgenic lines (L1, L2 and L4) w.r.t. WT on different ABA concentrations at different time points.
Mentions: The expression pattern under ABA and abiotic stresses suggested significant role of OsPP108 in ABA and abiotic stress triggered signaling in plants and prompted us for its in-planta functional characterization. We performed seed germination and root growth assays for three CaMV35S::OsPP108 (overexpression) lines (Fig 4A) on 1/2 MS media supplemented with different concentration of ABA (Fig 4B). Growth was monitored until 7 days and OsPP108 overexpressing plants showed insensitivity to ABA in seed germination and overall growth in comparison to WT. Insensitivity was observed until a very high ABA level (50μM). The insensitive phenotype was evaluated in term of seeds germination over a period of 5 days and all three transgenic lines could tolerate and grow much better on higher ABA level (upto 10 μM). Only 25–30% of WT seeds could germinate on this ABA concentration and almost none develop into a healthy seedling, whereas > 80% seeds of all the three overexpressing lines were germinated and developed into healthy seedlings (Fig 4C and 4D). Tolerance of OsPP108 overexpressing seeds to ABA was also estimated as full emergence of cotyledon at fourth day of growth, and all three overexpression lines had approximately 85–90% fully emerged cotyledons at 2μM ABA and 75–80% at 10 μM ABA level whereas, ~ 8% WT seeds could develop cotyledons even at 2μM ABA (Fig 5A). Fresh weight estimation after 7 days of growth at 2μM ABA showed that each seedling of three transgenic lines was ~2.4 mg, whereas WT seedling weighed only ~0.3 mg (Fig 5B), and this observation also indicated the strong insensitivity of OsPP108 overexpressing lines on higher ABA concentration. To confirm that this response is due to overexpression of OsPP108 in Arabidopsis, and to rule out the possibility of down-regulation of its Arabidopsis homologs (i.e. HAI2, HAI3, AHG1 and PP2CA) due to heterologous OsPP108 overexpression, expression analysis was performed for all these genes in the OsPP108OX lines and WT, but no significant change in expression was observed for any homolog in OsPP108OX lines (S2 Fig). qPCR primers are listed in S1 Table.

Bottom Line: At adult stage, OsPP108 overexpression leads to high tolerance to salt, mannitol and drought stresses with far better physiological parameters such as water loss, fresh weight, chlorophyll content and photosynthetic potential (Fv/Fm) in transgenic Arabidopsis plants.Expression profile of various stress marker genes in OsPP108 overexpressing plants revealed interplay of ABA dependent and independent pathway for abiotic stress tolerance.Overall, this study has identified a potential rice group A PP2C, which regulates ABA signaling negatively and abiotic stress signaling positively.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Molecular Biology, University of Delhi South Campus, Benito Juarez Road, Dhaula Kuan, New Delhi, India.

ABSTRACT
Arabidopsis PP2C belonging to group A have been extensively worked out and known to negatively regulate ABA signaling. However, rice (Oryza sativa) orthologs of Arabidopsis group A PP2C are scarcely characterized functionally. We have identified a group A PP2C from rice (OsPP108), which is highly inducible under ABA, salt and drought stresses and localized predominantly in the nucleus. Genetic analysis revealed that Arabidopsis plants overexpressing OsPP108 are highly insensitive to ABA and tolerant to high salt and mannitol stresses during seed germination, root growth and overall seedling growth. At adult stage, OsPP108 overexpression leads to high tolerance to salt, mannitol and drought stresses with far better physiological parameters such as water loss, fresh weight, chlorophyll content and photosynthetic potential (Fv/Fm) in transgenic Arabidopsis plants. Expression profile of various stress marker genes in OsPP108 overexpressing plants revealed interplay of ABA dependent and independent pathway for abiotic stress tolerance. Overall, this study has identified a potential rice group A PP2C, which regulates ABA signaling negatively and abiotic stress signaling positively. Transgenic rice plants overexpressing this gene might provide an answer to the problem of low crop yield and productivity during adverse environmental conditions.

No MeSH data available.


Related in: MedlinePlus