A CBL-Interacting Protein Kinase TaCIPK2 Confers Drought Tolerance in Transgenic Tobacco Plants through Regulating the Stomatal Movement

<div><p>In plants, the CBL-CIPK signaling pathways play key roles in the response to abiotic stresses. However, functional studies of CIPKs in the important staple crop wheat are very rare. In this study, we identified a <i>CIPK</i> gene from wheat, designated <i>TaCIPK2</i>. Expression analysis results showed that <i>TaCIPK2</i> could be up-regulated in wheat leaves by polyethylene glycol, abscisic acid and H₂O₂ treatments. Subcellular localization analyses revealed that TaCIPK2 was present in whole wheat epidermal cells. A yeast two-hybrid assay indicated that TaCIPK2 interacted with TaCBL1, 2, 3 and 4 in vitro. Transgenic tobacco plants over-expressing <i>TaCIPK2</i> exhibited increased drought tolerance, indicated by a larger proportion of green cotyledons and higher survival rates under the osmotic and drought stress conditions compared with control plants. Additionally, physiological index analyses revealed that the transgenic tobacco plants had lower water loss rates and ion leakage, accumulated less malondialdehyde and H₂O₂, and had higher catalase and superoxide dismutase activities than the control plants. The transgenic plants also exhibited faster stomatal closure following exposure to osmotic stress conditions. The seed germination rates and stomatal aperture of <i>TaCIPK2</i>-overexpressing tobacco plants decreased after exogenous abscisic acid treatment was applied, implying that the transgenic tobacco plants were more sensitive to exogenous abscisic acid than the control plants. Our results indicate that TaCIPK2 plays a positive regulatory role in drought stress responses in transgenic tobacco plants.</p></div

The analysis of green cotyledon rates in the controls (VC and WT) and <i>TaCIPK2</i>-overexpressing lines at early developmental stages.

<p>(a) The controls and the T₃ generation of <i>TaCIPK2</i>-overexpressing tobacco seeds were germinated on MS media with or without 0.5 μM ABA, 200 mM mannitol and 300 mM mannitol and photographed after two weeks. (b) The green cotyledon rates were counted after germination. Vertical bars indicate ± SD calculated from three independent biological replicates with similar results.</p

Annexin-V staining suggests that the 4 microplasma-treated HeLa cells show apoptotic response.

<p>Annexin-V staining suggests that the 4 microplasma-treated HeLa cells show apoptotic response.</p

Physiological indices of control and <i>TaCIPK2</i>-overexpressing transgenic plants under drought stress conditions.

<p>Analysis of H₂O₂ content (a), CAT (b), SOD (c), and POD (d) activities, MDA content (e) and ion leakage (f) in control and <i>TaCIPK2</i>-overexpressing (OE) lines under normal and drought stress conditions. Values shown are means ± SE of three replicates. Asterisks indicate statistically significant differences from control (**<i>P < 0</i>.<i>01</i>).</p

Subcellular localization of GFP and TaCIPK2::GFP fusion protein in wheat epidermal cells.

<p>The fusion protein CIPK2-GFP and GFP (control) were transiently expressed in wheat epidermal cells. Scale bar = 200 μm. The green fluorescence signals were determined by fluorescence microscopy (LX71, Olympus, Japan).</p

Expression patterns of <i>TaCIPK2</i> under normal conditions and stress treatments with NaCl, PEG, ABA, and H₂O₂ in wheat leaves by qRT-PCR analysis.

<p>The 2-week-old wheat leaves were treated with NaCl (a)., PEG 6000 (b), ABA (c) and H₂O₂ (d). The 2^−ΔΔCT method was used to analyze the relative expression of TaCIPK2. Asterisks indicate statistically significant differences (*<i>P < 0</i>.<i>05</i>; **<i>P < 0</i>.<i>01</i>) compared to untreated wheat at 0 h. Three independent experiments were performed and error bars show the SD.</p

Current-voltage waveforms of the plasma discharge.

<p>Current-voltage waveforms of the plasma discharge.</p

Yeast two-hybrid analysis of TaCIPK2-TaCBLs interaction.

<p>The pGBKT7-TaCBLs and pGADT7-TaCIPK2 plasmids were co-transformed into the AH109 yeast strain and the transformants were selected on SD/-Trp/-Leu, SD/-Trp/-Leu/-Ade and SD/-Ade/-His/-Trp/-Leu with or without X-α-gal.</p

Schematic of the microplasma jet setup and a sketch of the biomedical treatment; the micro-manipulated tip has a diameter of ∼1 µm.

<p>Schematic of the microplasma jet setup and a sketch of the biomedical treatment; the micro-manipulated tip has a diameter of ∼1 µm.</p

Real-time monitoring of the apoptotic membrane changes of the single HepG2 cell treated with the microplasma for 15 s. Annexin-V fluorescent staining was performed to visualize these changes.

<p>The cell labeling is the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101299#pone-0101299-g004" target="_blank">Figure 4</a>.</p