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. 2014 Jun;165(2):688-704.
doi: 10.1104/pp.113.230268. Epub 2014 May 1.

Overexpression of a Calcium-Dependent Protein Kinase Confers Salt and Drought Tolerance in Rice by Preventing Membrane Lipid Peroxidation

Sonia Campo  1 , Patricia Baldrich  1 , Joaquima Messeguer  1 , Eric Lalanne  1 , María Coca  1 , Blanca San Segundo  2
Affiliations

Overexpression of a Calcium-Dependent Protein Kinase Confers Salt and Drought Tolerance in Rice by Preventing Membrane Lipid Peroxidation

Sonia Campo et al. Plant Physiol. 2014 Jun.

Abstract

The OsCPK4 gene is a member of the complex gene family of calcium-dependent protein kinases in rice (Oryza sativa). Here, we report that OsCPK4 expression is induced by high salinity, drought, and the phytohormone abscisic acid. Moreover, a plasma membrane localization of OsCPK4 was observed by transient expression assays of green fluorescent protein-tagged OsCPK4 in onion (Allium cepa) epidermal cells. Overexpression of OsCPK4 in rice plants significantly enhances tolerance to salt and drought stress. Knockdown rice plants, however, are severely impaired in growth and development. Compared with control plants, OsCPK4 overexpressor plants exhibit stronger water-holding capability and reduced levels of membrane lipid peroxidation and electrolyte leakage under drought or salt stress conditions. Also, salt-treated OsCPK4 seedlings accumulate less Na+ in their roots. We carried out microarray analysis of transgenic rice overexpressing OsCPK4 and found that overexpression of OsCPK4 has a low impact on the rice transcriptome. Moreover, no genes were found to be commonly regulated by OsCPK4 in roots and leaves of rice plants. A significant number of genes involved in lipid metabolism and protection against oxidative stress appear to be up-regulated by OsCPK4 in roots of overexpressor plants. Meanwhile, OsCPK4 overexpression has no effect on the expression of well-characterized abiotic stress-associated transcriptional regulatory networks (i.e. ORYZA SATIVA DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN1 and ORYZA SATIVA No Apical Meristem, Arabidopsis Transcription Activation Factor1-2, Cup-Shaped Cotyledon6 genes) and LATE EMBRYOGENESIS ABUNDANT genes in their roots. Taken together, our data show that OsCPK4 functions as a positive regulator of the salt and drought stress responses in rice via the protection of cellular membranes from stress-induced oxidative damage.

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Figures

Figure 1.
Figure 1.
Expression of OsCPK4 in response to salt and drought stress. Transcript levels were determined by qRT-PCR and normalized to OsCYP and OsUBI mRNAs. A, Structure of OsCPK4 showing the typical domains of plant CPKs: a kinase catalytic domain joined via a junction (J) domain sequence to a calmodulin-like domain (CaM) with four calcium-binding domains (rhombi in the calmodulin domain). The N-terminal amino acid sequence of OsCPK4 is indicated at the top. The bar indicates the DNA fragment used as a probe in northern-blot analyses of OsCPK4 in Supplemental Figure S3. Arrows indicate the positions of primers used for qRT-PCR. UT, Untranslated region. B, Expression of OsCPK4 in leaves and roots of rice plants at the indicated developmental stages. C and D, Expression of OsCPK4 (C) and OsLEA23 (D) in roots of rice plants in response to salt and drought stress. Seven-day-old seedlings were exposed to 100 mm NaCl and air dried or treated with 20% PEG 8000 for the indicated periods of time. The same RNA samples were used in C and D. Values represent means ± se of three replicates (*P ≤ 0.05, **P ≤ 0.001).
Figure 2.
Figure 2.
OsCPK4 expression in response to ABA and accumulation of OsCPK4 in response to ABA treatment and salt and drought stress. A, Diagram of the OsCPK4 promoter showing the distribution of abiotic stress-related cis-elements. UTR, Untranslated region. B, OsCPK4 and OsLEA26 expression in rice roots in response to ABA. The OsCYP and OsUBI mRNAs were used as internal controls for normalization. Significant differences are indicated (*P < 0.05, **P < 0.001). C, Accumulation of OsCPK4 in protein extracts of rice roots that were subjected to salt stress, ABA treatment, or drought stress (air drying) for 1 h. Protein extracts (20 µg each) were probed with an antiserum prepared against the N-terminal domain of OsCPK4. Top, protein extracts from control (C) and salt-stressed (S) roots. Bottom, protein extracts from control (C for ABA treatment, C′ for drought treatment), ABA-treated (A) and drought-stressed (D) rice roots. Ponceau S staining of western blots was used as the loading control.
Figure 3.
Figure 3.
Plasma membrane localization of OsCPK4. Onion epidermal cells were transformed with the OsCPK4-GFP gene via particle bombardment. Confocal images were taken 5 h after bombardment. Projection (A and B) and individual (C and D) sections are shown. A, Localization of the OsCPK4 fusion protein. Fluorescence and transmission images are shown at left and middle, respectively. A merged image of the green fluorescence channel with the corresponding light micrographs is shown at right. B, Onion epithelial cell expressing GFP. C, Onion cell transformed with the OsCPK4-GFP gene after plasmolysis with mannitol (15 min of treatment). Light micrographs show the shrinkage of the protoplast. D, Higher magnification of a plasmolyzed onion cell showing the Hechtian strands (h). Treatment with mannitol renders the Hechtian strands attaching the plasma membrane (pm) to the cell wall (cw). Bars = 20 µm.
Figure 4.
Figure 4.
Salt tolerance of rice plants overexpressing OsCPK4. A, Diagram showing the experimental design for salt tolerance assays. Salt stress was imposed on both transgenic and control plants grown in hydroponic culture until they reached the three-leaf stage (D0). Plants were then transferred to nutrient solution containing 60 mm NaCl or to fresh nutrient solution. In each experiment, wild-type (WT; cv Nipponbare), vector control (pC; three independent lines), and OsCPK4 overexpressor (OsCPK4-OX; five independent lines) plants were assayed. B, Phenotypes of OsCPK4-OX rice plants under salt stress conditions. Whereas control plants (wild type and vector control) grew poorly and exhibited chlorosis, the OsCPK4-OX lines grew in the presence of NaCl. Photographs were taken after 18 d of salt stress (D18). C, Survival rates of OsCPK4-OX and vector control plants at different times of salt treatment. D, OsCPK4 expression in roots of transgenic and control plants used in salt tolerance assays as determined by qRT-PCR analysis. Specific primers for the analysis of OsCPK4 transgene expression were used (Supplemental Table S4). Samples were taken at the D0 time point (onset of salt treatment). Salt tolerance assays were carried out three times and in two successive generations (T2 and T3). In each experiment, at least 30 plants per line were assayed. Asterisks denote significant differences between the OsCPK4-OX and control (wild type and vector control) groups (*P ≤ 0.05, **P ≤ 0.001). [See online article for color version of this figure.]
Figure 5.
Figure 5.
Drought tolerance of rice plants overexpressing OsCPK4. A, Diagram showing the experimental design for drought tolerance assays. Transgenic and control (wild-type and transgenic lines expressing the empty vector) plants were grown for 2 weeks (D0), subjected to 17 d of drought stress (D17), followed by 9 d of rewatering (RW) in the greenhouse. B, Phenotypes of OsCPK4 overexpressor (OsCPK4-OX) and vector control (pC) plants during drought stress. Photographs were taken at day 0 (onset of drought treatment), at 17 d of drought stress (D17), and after 9 d of rewatering (RW). C, Survival rates of OsCPK4-OX and vector control plants exposed to drought stress. D and E, Water loss (D) and water retention rates (E) of detached leaves of OsCPK4-OX and vector control plants at the three-leaf stage. Drought tolerance assays were repeated three times using five independent OsCPK4 transgenic lines and two independent vector control lines (at least 30 plants per line). Values represent means ± se of three replicates (*P ≤ 0.05). [See online article for color version of this figure.]
Figure 6.
Figure 6.
Analysis of cpk4 mutants. A, OsCPK4 gene structure showing the T-DNA insertion sites for the cpk4 mutants identified in the POSTECH collection (2D-00040 and 1D-03351 mutants). Exons and introns are indicated by black boxes and lines, respectively. Gene-specific primers used for the analysis of T-DNA integration are indicated by arrows. B, Analysis of T-DNA integration in the 2D-00040 cpk4 mutant. Ho, he, and Az indicate homozygous, hemizygous, and azygous plants, respectively. C, qRT-PCR analysis of OsCPK4 expression in leaves of 2D-00040 cpk4 mutant plants. Asterisks denote significant differences (**P ≤ 0.001). D, Appearance of the homozygous and hemizygous 2D-00040 cpk4 mutant plants. Similar results were obtained for the 1D-03351 cpk4 mutant. [See online article for color version of this figure.]
Figure 7.
Figure 7.
Differentially expressed genes in OsCPK4 rice plants. A, Functional categories of up-regulated genes in roots of OsCPK4-OX plants as determined by microarray analysis. B, Expression of THIOREDOXIN and LACCASE genes in roots of OsCPK4-OX plants. C, Functional categories of misregulated genes in leaves of OsCPK4-OX plants. D, Expression of WRKY71, WRKY76, and ASPARTIC PROTEINASE genes in leaves of OsCPK4-OX plants. Transcript levels were determined by qRT-PCR using total RNA. Representative results are shown for two OsCPK4 lines (lines 1 and 14) and two vector control lines (lines 3 and 11) from a total of five and three independent OsCPK4 and vector control lines, respectively, analyzed in this work.
Figure 8.
Figure 8.
Effect of salt treatment on lipid peroxidation, electrolyte leakage, and Na+ content. A and B, Detection of lipid peroxidation (A) and electrolyte leakage (B) in leaves of 3-week-old plants, both vector control (pC; two independent lines) and OsCPK4 overexpressor (OsCPK4-OX; three independent lines) plants, treated with 100 mm NaCl for 14 d. Data shown correspond to unstressed (gray bars) and salt-stressed (blue and orange bars) OsCPK4-OX and vector control lines. FW, Fresh weight. C, Imaging of Na+ content in salt-stressed vector control (top row) and OsCPK4-OX (bottom row) roots. Confocal images from roots of 5-d-old seedlings that had been treated with 150 mm NaCl for 16 h and stained with CoroNa-Green (green) and propidium iodide (red) are shown. Bars = 20 μm. D, Quantitative comparison of CoroNa-Green fluorescence intensities. Two independent lines and three individual plants per line were examined for each genotype. Values represent means ± se of average intensities of root sections at 700 μm distance to the tip. Asterisks indicate significant differences (**P ≤ 0.001, ANOVA).
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