ABA Inducible Rice Protein Phosphatase 2C Confers ABA Insensitivity and Abiotic Stress Tolerance in <i>Arabidopsis</i>

<div><p><i>Arabidopsis</i> PP2C belonging to group A have been extensively worked out and known to negatively regulate ABA signaling. However, rice (<i>Oryza sativa</i>) orthologs of <i>Arabidopsis</i> 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 <i>Arabidopsis</i> 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 <i>Arabidopsis</i> 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.</p></div

Stomata movement of OsPP108 overexpression transgenic lines under ABA treatment.

<p>(A) Stomata aperture of 4 week old WT and OsPP108^OX lines, L1, L2 and L4, in mock treatment (Stomata Opening Solution; SOS) (upper panel) and 100μM ABA (lower panel). 30 stomata were analyzed for each genotype and representation of those are shown in pictures (n = 3). Pictures were taken at 60x magnification in Olympus BX53 fluorescence microscope in bright field. Scale bar = 5μM. (B) Graph depicting percentage of different type of stomata (closed, partially open and open) in WT and OsPP108^OX transgenic in mock and ABA treatments. Stomata were grouped based on apertures sizes, i.e., open- 2.5–5.0μM, partially open- 1.0–2.5μM and closed- < 1 μM. Percentage of stomata is calculated based on the average of three independent replicate experiments.</p

Expression profile of <i>OsPP108</i> under ABA and abiotic stresses.

<p>qPCR analysis was performed to generate the expression profile of <i>OsPP108</i> under ABA, salt, drought and cold treatment in rice seedlings. Different time period of treatment in hours is indicated on X-axis and fold change in expression level w.r.t. untreated control sample is indicated on Y-axis. Each bar represents mean value of two replicates. Standard error among the samples is indicated by error bars. * represents p-value < 0.05 and ** p-value < 0.01 for treated samples w.r.t. untreated control (0h).</p

Salt and mannitol stress tolerance of adult OsPP108OX plants.

<p>(A) Phenotype of 3 week old WT and OsPP108^OX lines L1, L2 and L4 without stress treatment (upper row), after salt treatment (middle row) and after mannitol treatment (lower row). (B) Survival percentage of WT and different OsPP108^OX lines without stress and after salt and mannitol stress for 10 days. Values are mean ± SD (n = 24 plants). *p-value <0.05 and **p-value < 0.01 for transgenic lines w.r.t. respective WT in different stresses (C) Fv/Fm ratios after stress treatment. **p-value < 0.01 and ***p-value < 0.005 for transgenic lines w.r.t. respective WT, in different stresses, and (D) Total chlorophyll content after stress treatment. Data presented is mean ± SD (n = 3). *p-value < 0.05 and **p-value < 0.01 for transgenic lines w.r.t. respective WT, in different stresses.</p

Germination and seedling growth analysis of OsPP108 overexpression <i>Arabidopsis</i> lines on salt and mannitol containing media.

<p>(A) Germination and vertical growth of WT (Col-0) and three OsPP108^OX transgenic lines L1, L2 and L4 on 1/2MS (control) and different concentrations of NaCl (150 and 175mM) and mannitol (375 and 400mM) after 7 days. (B) Horizontal germination and growth pattern and seedling establishment of approximately 100 seeds from WT and transgenic lines on different MS media supplemented with different concentrations of NaCl (150 and 175mM) and mannitol (350 and 375mM) after 7 days growth. (C) Seed germination percentage of WT and three transgenic lines on 150mM, 175mM NaCl and 350mM, 375mM mannitol supplemented MS media. 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 stress media, at different time points.</p

Sub-cellular localization of OsPP108 in <i>Nicotiana benthamiana</i>.

<p>Agrobacterium-infiltrated tobacco leaves expressing the GFP-OsPP108 fusion protein driven by the 2XCaMV35S promoter. Confocal images of fluorescence (green) for cell expressing OsPP108 (35S:OsPP108-GFP) are showing its distribution predominantly in the nucleus and partly in cytoplasm (upper panel). Fluorescence is distributed throughout the cells, transformed with vector only (35S: GFP) (middle panel). Co-localization of green (GFP) signal with blue DAPI organelle marker confirms the localization of OsPP108 in nucleus (lower panel). Detection of fluorescence was performed under a confocal laser-scanning microscope (wavelength: 488nm for GFP and 405nm for DAPI). Scale bar = 20 μm.</p

Expression profile of stress marker genes.

<p>qPCR analysis was done to generate expression profile of different stress marker genes such as <i>RD29A</i>, <i>RD29B</i>, <i>RAB18</i> and <i>KIN1</i> in WT (Col-0) and two OsPP108 overexpression lines L1 and L2 after ABA (50μM), salt (300mM) and mannitol (400mM) stress treatment. Various time points of stress treatment are indicated at X-axis and relative expression value (fold change) is indicated on Y-axis. Data from mean of two replicates is presented as columns and standard deviation among the samples is denoted by error bar. *p-value < 0.05 and **p-value- < 0.01 indicate statistically significant expression change of transgenic lines w.r.t. respective WT in different treatment and different time points.</p

Germination and seedling growth analysis of OsPP108 overexpression transgenic <i>Arabidopsis</i> lines on ABA containing media.

<p>(A) RT-PCR analysis showing expression level of <i>OsPP108</i> in WT (Col-0) and transgenic lines (L1, L2 and L4) using gene-specific primers after 24 PCR cycles. <i>ACTIN2</i> is expressed as endogenous control in all cDNA samples. (B) Germination and vertical growth of WT (Col-0) and three OsPP108^OX 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.</p

Quantitative analysis of abiotic stress tolerance phenotype.

<p>(A) Cotyledon emergence percentage of WT and OsPP108^OX lines L1, L2, L4 on MS (control) media and MS media supplemented with different concentrations of NaCl (150mM and 175mM) and (B) mannitol (350mM and 375mM). Approximately 100 seeds were counted for each genotype. * p-value < 0.05, ** p-value <0.01 and *** p-value < 0.005 shows statistically significant cotyledon emergence for transgenic lines w.r.t WT on different stress media. (C) Fresh weight of 7 days old seedling grown on MS and different NaCl and (D) mannitol 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 shows statistically significant fresh weight of transgenic lines w.r.t WT on different stress media.</p

The <i>unc-53</i> gene negatively regulates <i>rac</i> GTPases to inhibit <i>unc-5</i> activity during Distal tip cell migrations in <i>C. elegans</i>

<p>The <i>unc-53</i>/NAV2 gene encodes for an adaptor protein required for cell migrations along the anteroposterior (AP) axes of <i>C. elegans</i>. This study identifies <i>unc-53</i> as a novel component of signaling pathways regulating Distal tip cell (DTC) migrations along the AP and dorsoventral (DV) axes. <i>unc-53</i> negatively regulates and functions downstream of <i>ced-10</i>/Rac pathway genes; <i>ced-10</i>/Rac and <i>mig-</i>2/RhoG, which are required for proper DTC migration. Moreover, <i>unc-53</i> exhibits genetic interaction with <i>abl-1</i> and <i>unc-5</i>, the 2 known negative regulators of <i>ced-10</i>/Rac signaling. Our genetic analysis supports the model, where <i>abl-1</i> negatively regulates <i>unc-53</i> during DTC migrations and requirement of <i>unc-53</i> function during both AP and DV DTC migrations could be due to <i>unc-53</i> mediated regulation of <i>unc-5</i> activity.</p