Abstract
A novel CBL-interacting protein kinase (CIPK) gene, ZmCIPK16, was isolated from maize (Zea mays), which has been certified to have two copies in the genome. The ZmCIPK16 is strongly induced in maize seedlings by PEG, NaCl, ABA, dehydration, heat and drought, but not by cold. A yeast two-hybrid assay demonstrated that ZmCIPK16 interacted with ZmCBL3, ZmCBL4, ZmCBL5, and ZmCBL8. Bimolecular fluorescence complementation (BiFC) assays prove that ZmCIPK16 can interact with ZmCBL3, ZmCBL4, ZmCBL5, and ZmCBL8 in vivo. Subcellular localization showed that ZmCIPK16 is distributed in the nucleus, plasma membrane and cytoplasm; this is different from the specific localization of ZmCBL3, ZmCBL4, and ZmCBL5, which are found in the plasma membrane. The results also showed that overexpression of ZmCIPK16 in the Arabidopsis sos2 mutant induced the expression of the SOS1 gene and enhanced salt tolerance. These findings indicate that ZmCIPK16 may be involved in the CBL-CIPK signaling network in maize responses to salt stress.
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Abbreviations
- ABA:
-
Abscisic acid
- BiFC:
-
Bimolecular fluorescence complementation
- CBL:
-
Calcineurin B-like protein
- CIPK:
-
CBL-interacting protein kinase
- ORF:
-
Open reading frame
- PEG:
-
Polyethylene glycol
- RACE:
-
Rapid amplification of cDNA ends
- SOS:
-
Salt overly sensitive
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Acknowledgments
The authors thank Dr. Zhizhong Chen (China Agricultural University) for providing Arabidopsis sos2 mutants, Dr. Klaus Harter (Botanisches Institut, Universitat zu Koln, Germany) for the vectors used in the BiFC assay, Liping Liang and Xiping Shi for help with the confocal microscope. This work was supported by the National High-tech Program of China (2006AA10Z103), the Natural Science Foundation of China (30500286) and the research grant from the Institute of Crop Sciences, CAAS (082060302–18).
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Jinfeng Zhao and Zhenfei Sun are contributed equally to this work.
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11103_2008_9445_MOESM3_ESM.doc
Subcellular localization of GFP, ZmCIPK16-GFP, ZmCBL3-GFP,ZmCBL4-GFP and ZmCBL5-GFP in onion cells. Left, fluorescence images; right, Bright-field; they were taken from the same cell for each protein localization assay a/b, pBI221-GFP; c/d, ZmCIPK16-GFP; e/f, ZmCBL3-GFP; g/h, ZmCBL4-GFP; i/j, ZmCBL5-GFP. (DOC 27 kb)
11103_2008_9445_MOESM4_ESM.tif
Southern analysis of ZmCIPK16 in transgenic events of Arabidopsis T1 generation. Genomic DNA (10 μg) of different T1 transgenic line (9–2、17–3、19–1、20–4) was digested with EcoRI respectively, separated by electrophoresed on 0.8% (w/v) agarose gel and transferred onto Hybond-N+ nylon membrane. A 523 bp cDNA fragment located in the 5’ region of the ZmCIPK16 open reading frame was labeled with α-32p-dCTP as probe. (TIFF 11561 kb)
11103_2008_9445_MOESM5_ESM.tif
The expression of ZmCIPK16 in Arabidopsis wild type and transgenic plants. 10 μg total RNA from two-week-old seedlings were loading. A 523 bp cDNA fragment located in the 5’ region of the ZmCIPK16 open reading frame was labeled with α-32p-dCTP as probe. rRNA was visualized by staining with methylene blue as loading control. (TIFF 12788 kb)
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Zhao, J., Sun, Z., Zheng, J. et al. Cloning and characterization of a novel CBL-interacting protein kinase from maize. Plant Mol Biol 69, 661–674 (2009). https://doi.org/10.1007/s11103-008-9445-y
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DOI: https://doi.org/10.1007/s11103-008-9445-y