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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

Quantitative analysis of ABA insensitive phenotype.(A) Cotyledon emergence percentage of WT and OsPP108OX lines L1, L2, L4 on MS (control) media and MS media supplemented with different concentrations of ABA (2μM, 5μM, 10μM). Approximately 100 seeds were counted for each genotype and average of three observations is plotted on the graph ± SD. * p-value < 0.05, ** p-value < 0.01 shows statistically significant cotyledon emergence for transgenic lines w.r.t WT on different ABA concentrations (B) Fresh weight of 7 days old seedling grown on MS and different ABA concentrations. 15 seedlings of each genotype were recorded and average of three observations is plotted on the graph ± SD. * p-value < 0.05, ** p-value <0.01 statistically significant fresh weight of transgenic lines w.r.t WT.
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pone.0125168.g005: Quantitative analysis of ABA insensitive phenotype.(A) Cotyledon emergence percentage of WT and OsPP108OX lines L1, L2, L4 on MS (control) media and MS media supplemented with different concentrations of ABA (2μM, 5μM, 10μM). Approximately 100 seeds were counted for each genotype and average of three observations is plotted on the graph ± SD. * p-value < 0.05, ** p-value < 0.01 shows statistically significant cotyledon emergence for transgenic lines w.r.t WT on different ABA concentrations (B) Fresh weight of 7 days old seedling grown on MS and different ABA concentrations. 15 seedlings of each genotype were recorded and average of three observations is plotted on the graph ± SD. * p-value < 0.05, ** p-value <0.01 statistically significant fresh weight of transgenic lines w.r.t WT.

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)

Quantitative analysis of ABA insensitive phenotype.(A) Cotyledon emergence percentage of WT and OsPP108OX lines L1, L2, L4 on MS (control) media and MS media supplemented with different concentrations of ABA (2μM, 5μM, 10μM). Approximately 100 seeds were counted for each genotype and average of three observations is plotted on the graph ± SD. * p-value < 0.05, ** p-value < 0.01 shows statistically significant cotyledon emergence for transgenic lines w.r.t WT on different ABA concentrations (B) Fresh weight of 7 days old seedling grown on MS and different ABA concentrations. 15 seedlings of each genotype were recorded and average of three observations is plotted on the graph ± SD. * p-value < 0.05, ** p-value <0.01 statistically significant fresh weight of transgenic lines w.r.t WT.
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Related In: Results  -  Collection

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pone.0125168.g005: Quantitative analysis of ABA insensitive phenotype.(A) Cotyledon emergence percentage of WT and OsPP108OX lines L1, L2, L4 on MS (control) media and MS media supplemented with different concentrations of ABA (2μM, 5μM, 10μM). Approximately 100 seeds were counted for each genotype and average of three observations is plotted on the graph ± SD. * p-value < 0.05, ** p-value < 0.01 shows statistically significant cotyledon emergence for transgenic lines w.r.t WT on different ABA concentrations (B) Fresh weight of 7 days old seedling grown on MS and different ABA concentrations. 15 seedlings of each genotype were recorded and average of three observations is plotted on the graph ± SD. * p-value < 0.05, ** p-value <0.01 statistically significant fresh weight of transgenic lines w.r.t WT.
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