Abstract
Plant responses to developmental and environmental cues are often mediated by calcium (Ca2+) signals that are transmitted by diverse calcium sensors. The calcineurin B-like (CBL) protein family represents calcium sensors that decode calcium signals through specific interactions with a group of CBL-interacting protein kinases. We report functional analysis of Arabidopsis CBL2 and CBL3, two closely related CBL members that are localized to the vacuolar membrane through the N-terminal tonoplast-targeting sequence. While cbl2 or cbl3 single mutant did not show any phenotypic difference from the wild type, the cbl2 cbl3 double mutant was stunted with leaf tip necrosis, underdeveloped roots, shorter siliques and fewer seeds. These defects were reminiscent of those in the vha-a2 vha-a3 double mutant deficient in vacuolar H+-ATPase (V-ATPase). Indeed, the V-ATPase activity was reduced in the cbl2 cbl3 double mutant, connecting tonoplast CBL-type calcium sensors to the regulation of V-ATPase. Furthermore, cbl2 cbl3 double mutant was compromised in ionic tolerance and micronutrient accumulation, consistent with the defect in V-ATPase activity that has been shown to function in ion compartmentalization. Our results suggest that calcium sensors CBL2 and CBL3 serve as molecular links between calcium signaling and V-ATPase, a central regulator of intracellular ion homeostasis.
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Acknowledgements
We thank Prof Karin Schumacher and Dr Melanie Krebs (University of Heidelberg, Germany) for providing the vha-a2 vha-a3 mutant seeds. Special thanks would go to Chen Zhong (Shanghai Institutes for Biological Sciences, CAS) for the assistance in ionomic analysis. This work is supported by the grants from the National Natural Science Foundation of China (31171169, 30872044), the National mega project of GMO crops (2011ZX08001-003, 2011ZX08004-002 and 2011ZX08010-004) to HXZ and National Science Foundation to SL.
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Supplementary information
Supplementary information, Figure S1
Expression of CBL2 and CBL3 in response to various abiotic stresses. (PDF 92 kb)
Supplementary information, Figure S2
The N-terminal 16-aa of CBL2 is required but not sufficient for its tonoplast targeting. (PDF 562 kb)
Supplementary information, Figure S3
The first 19-aa in the N-terminus of CBL2 determines the tonoplast localization. (PDF 719 kb)
Supplementary information, Figure S4
The efficacy of CBL2 Tonoplast Targeting Sequence (TTS) is independent of its N-terminal location. (PDF 353 kb)
Supplementary information, Figure S5
TTS targets several soluble proteins but not transmembrane proteins to the tonoplast. (PDF 459 kb)
Supplementary information, Figure S6
Purification of recombinant CBL2 and CBL3 proteins from E-Coli cells. (PDF 191 kb)
Supplementary information, Figure S7
Complementation of the cbl2 cbl3 double mutant by CBL2 or CBL3. (PDF 60 kb)
Supplementary information, Figure S8
Phenotype of cbl2 cbl3 and vha-a2 vha-a3 plants grown in the soil. (PDF 365 kb)
Supplementary information, Figure S9
In situ calibration of BCECF-AM in protoplast vacuoles. (PDF 228 kb)
Supplementary information, Figure S10
Growth phenotype of cbl2, cbl3 single mutants and cbl2 cbl3 double mutant under various ionic stress conditions. (PDF 580 kb)
Supplementary information, Figure S11
Yeast two-hybrid assays on CBL2/3 in combination with VHA subunits. (PDF 58 kb)
Supplementary information, Figure S12
CBL2 and CBL3 interact with an array of CIPK proteins. (PDF 116 kb)
Supplementary information, Table S1
Primers used in this study. (PDF 9 kb)
Supplementary information, Data S1
The flanking sequence of the T-DNA insertion lines in this study. (PDF 11 kb)
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Tang, RJ., Liu, H., Yang, Y. et al. Tonoplast calcium sensors CBL2 and CBL3 control plant growth and ion homeostasis through regulating V-ATPase activity in Arabidopsis. Cell Res 22, 1650–1665 (2012). https://doi.org/10.1038/cr.2012.161
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DOI: https://doi.org/10.1038/cr.2012.161
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