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, 169 (1), 780-92

Calcineurin B-Like Protein-Interacting Protein Kinase CIPK21 Regulates Osmotic and Salt Stress Responses in Arabidopsis

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Calcineurin B-Like Protein-Interacting Protein Kinase CIPK21 Regulates Osmotic and Salt Stress Responses in Arabidopsis

Girdhar K Pandey et al. Plant Physiol.

Abstract

The role of calcium-mediated signaling has been extensively studied in plant responses to abiotic stress signals. Calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs) constitute a complex signaling network acting in diverse plant stress responses. Osmotic stress imposed by soil salinity and drought is a major abiotic stress that impedes plant growth and development and involves calcium-signaling processes. In this study, we report the functional analysis of CIPK21, an Arabidopsis (Arabidopsis thaliana) CBL-interacting protein kinase, ubiquitously expressed in plant tissues and up-regulated under multiple abiotic stress conditions. The growth of a loss-of-function mutant of CIPK21, cipk21, was hypersensitive to high salt and osmotic stress conditions. The calcium sensors CBL2 and CBL3 were found to physically interact with CIPK21 and target this kinase to the tonoplast. Moreover, preferential localization of CIPK21 to the tonoplast was detected under salt stress condition when coexpressed with CBL2 or CBL3. These findings suggest that CIPK21 mediates responses to salt stress condition in Arabidopsis, at least in part, by regulating ion and water homeostasis across the vacuolar membranes.

Figures

Figure 1.
Figure 1.
Expression analysis of CIPK21. A, RT-PCR analysis of CIPK21 expression in different organs and during seed germination of Arabidopsis plants. Total RNA was isolated from various tissues (root, stem, leaf, flower, and silique) of the wild-type (Col-0) plants growing under long-day conditions or from germinating seeds and young seedlings (3 and 21 d after sowing). RT-PCR was performed with CIPK21-specific primers and ACTIN2-specific primers. B, Quantitative RT-PCR of CIPK21 in wild-type seedlings under salt and mannitol treatments. Salt (300 mm) and mannitol (400 mm) treatments were given to 3-week-old MS-grown wild-type seedlings in the Petri plate. Tissue was collected at different time intervals, and RNA was isolated to make first-strand cDNA. Quantitative PCR was done using gene-specific forward and reverse primers. ACTIN2 was used to normalize the variance among the samples. Change in transcript level was calculated as fold change with respect to water control, indicated at y axis. ses in the replicates are indicated by error bars. C, Expression of the CIPK21-promoter-β-glucuronidase (uidA) fusion seedlings 1, 2, 4, and 10 d after germination. D, Histochemical GUS activity of transgenic plants expressing uidA reporter gene driven by CIPK21 promoter. Expression pattern in 3-week-old seedlings (1) and individual organ of adult plant (2–4), mature leaves (2), and root (3 and 4, enlarged view).
Figure 2.
Figure 2.
Isolation and complementation of the cipk21 T-DNA insertional mutant. A, Scheme representing the Arabidopsis CIPK21 gene. Exons (black boxes) and introns (lines) are indicated. The position of the T-DNA insertion is indicated by a triangle (not represented to scale). B, Genomic DNA fragment used for complementation. A 4.5-kb fragment including the CIPK21 coding region and 1.6 kb of the 5′ flanking DNA upstream of the start codon and 3′ untranslated region was amplified by PCR and cloned into the pCAMBIA 1300 for plant transformation. C, RT-PCR analysis of CIPK21 gene expression from wild-type (WT), cipk21, and cipk21/CIPK21 plants. Expression of ACTIN2 was analyzed as a loading control.
Figure 3.
Figure 3.
Phenotypic analysis of cipk21 mutant. A, Inhibition of germination and growth of young seedlings in wild-type (WT), cipk21, and cipk21/CIPK21 plants grown on normal MS agar and MS agar containing different concentration of NaCl and mannitol. Seeds were incubated at 4°C for 6 d before transfer to 23°C for germination. The photographs were taken on day 12 after transfer to 23°C. B, Seed germination rate of the wild type, cipk21, and cipk21/CIPK21 in different concentrations of NaCl and mannitol. Germination was scored at 2 d (for NaCl) and 3 d (for mannitol) after incubation at 23°C. C, Kinetics of seed germination for wild-type, cipk21, and cipk21/CIPK21 plants on medium containing 175 mm NaCl or 400 mm mannitol. Results in B and C are presented as average values along with ses from three experiments. The wild type has been indicated by rhombuses, cipk21 by squares, and cipk21/CIPK21 by triangles. *, Significant differences (one-way ANOVA) between the wild type and cipk21 (P < 0.05) in B and C.
Figure 4.
Figure 4.
Adult cipk21 mutant plants are hypersensitive to NaCl and mannitol. A, Rosette leaf stage of mature plants of wild-type (WT), cipk21, and cipk21/CIPK21 plants treated with water (top and third rows), 400 mm mannitol (bottom row), or 300 mm NaCl (second row). B, Quantification of death of stem after 300 mm NaCl and 400 mm mannitol treatment. C, Chlorophyll content of plants treated with NaCl and mannitol. Results depicted in B and C are average values ± se of three independent experiments. Three-week-old plants were treated with 300 mm NaCl and 400 mm mannitol three times after every 3 d. Photographs were taken on the 10th day after treatment. *, Significant differences (one-way ANOVA) between the wild type and cipk21 (P < 0.05) in B and C.
Figure 5.
Figure 5.
CIPK21 interacts with CBL2 and CBL3 in yeast two-hybrid assay. A, Dilution series of yeast AH109 strains transformed with AD-CIPK21 and all CBLs proteins in BD vectors. Yeast two-hybrid analysis identified CBL2 and CBL3 as predominant CIPK21 interactors. In addition, a weak interaction was also observed with CBL1 and CBL9. Interaction between the full-length AD-CIPK8/BD-CBL1 and AD-CIPK21/BD used as positive and negative controls, respectively. B, Yeast two-hybrid analysis showing vector swapping of CBL2 and CBL3 interaction with CIPK21. Dilution series of yeast AH109 strains transformed with BD-CIPK21 and CBL2 and CBL3 proteins in AD vectors. The combination of plasmids in A and B is indicated on the left, and decreasing cell densities in the dilution series are illustrated by narrowing triangles. Yeast was grown on SD-L-W medium (first column), SD-L-W-H medium (second column), or SD-L-W-H medium containing 1 mm 3-AT (third column).
Figure 6.
Figure 6.
Colocalization of CIPK21 with CBL2 and CBL3 proteins in the epidermal peel cells of N. benthamiana. CIPK21:GFP coexpressed with either CBL2:mCherry (top row, red) or CBL3:OFP (bottom row, red) display the formation of CBL2/CIPK21 and CBL3/CIPK21 complexes preferentially at the vacuolar membrane, as indicated by the yellow color in the overlay image (right column). Bar = 40 µm.
Figure 7.
Figure 7.
Effects of salt stress on the CBL2/3-CIPK21 BiFC complexes. A, Investigation of interaction of CBL2 and CBL3 with CIPK21 by BiFC in epidermal cells of N. benthamiana. CBL2/CBL3-CIPK21 complexes formed mostly at the vacuolar membrane as reported for individual CBL2 and CBL3 (left columns). Upon stress treatment with 125 mm NaCl, large tonoplastic vesicles were observed compared with control for both CBL2-CIPK21 and CBL3-CIPK21 (right columns) as shown in protoplasts (top row; bars = 10 µm) and intact epidermal cells (bottom row; bars = 40 µm). Plasmid combinations are indicated on the left. B, Quantification of the relative fluorescence intensity to monitor the effects of salt stress treatment on BiFC complexes formed by N-terminal of YFP (YN)-CIPK21/CBL2-C-terminal of YFP (YC; above) and YN-CIPK21/CBL3-YC (below), which were incubated with 125 mm NaCl or control (10 mm MES, pH 5.6, and 10 mm MgCl2) medium. Results are presented as average values along with ses from three experiments.
Figure 8.
Figure 8.
Hypothetical model for CIPK21 function in osmotic and salt stress signaling. Under normal (top left) and salt stress conditions (bottom left), CIPK21 is localized in the cytoplasm and the nucleus. Upon coexpression of CBL2 or CBL3, CIPK21-CBL2/CBL3 complex formation initiates in the vacuolar membrane (top right), which is enhanced under salt stress (bottom right). Possibly under salt stress, CBL2/CBL3 detects the rise in cytoplasmic Ca2+, activates CIPKs (CIPK21), and targets them to the tonoplast. Possible targets of CIPK21 may be vacuolar membrane Na+-channels/transporters facilitating Na+ sequestration from cytosol into the vacuole under salt stress, which also might be responsible for the osmoregulation in plant cell.

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