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A novel Glycine soja cysteine proteinase inhibitor GsCPI14, interacting with the calcium/calmodulin-binding receptor-like kinase GsCBRLK, regulated plant tolerance to alkali stress

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Abstract

It has been well demonstrated that cystatins regulated plant stress tolerance through inhibiting the cysteine proteinase activity under environmental stress. However, there was limited information about the role of cystatins in plant alkali stress response, especially in wild soybean. Here, in this study, we focused on the biological characterization of a novel Glycine soja cystatin protein GsCPI14, which interacted with the calcium/calmodulin-binding receptor-like kinase GsCBRLK and positively regulated plant alkali stress tolerance. The protein–protein interaction between GsCBRLK and GsCPI14 was confirmed by using split-ubiquitin based membrane yeast two-hybrid analysis and bimolecular fluorescence complementation assay. Expression of GsCPI14 was greatly induced by salt, ABA and alkali stress in G. soja, and GsCBRLK overexpression (OX) in Glycine max promoted the stress induction of GmCPI14 expression under stress conditions. Furthermore, we found that GsCPI14-eGFP fusion protein localized in the entire Arabidopsis protoplast and onion epidermal cell, and GsCPI14 showed ubiquitous expression in different tissues of G. soja. In addition, we gave evidence that the GST-GsCPI14 fusion protein inhibited the proteolytic activity of papain in vitro. At last, we demonstrated that OX of GsCPI14 in Arabidopsis promoted the seed germination under alkali stress, as evidenced by higher germination rates. GsCPI14 transgenic Arabidopsis seedlings also displayed better growth performance and physiological index under alkali stress. Taken together, results presented in this study demonstrated that the G. soja cysteine proteinase inhibitor GsCPI14 interacted with the calcium/calmodulin-binding receptor-like kinase GsCBRLK and regulated plant tolerance to alkali stress.

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Acknowledgments

This work was supported by Heilongjiang Provincial Higher School Science and Technology Innovation Team Building Program (2011TD005), National Natural Science Foundation of China (31171578), National Major Project for Cultivation of Transgenic Crops (2011ZX08004-002) and Heilongjiang Provincial Graduate Student Innovation Research Projects (YJSCX2012-047HLJ).

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  1. Key Laboratory of Agricultural Biological Functional Genes, Northeast Agricultural University, Harbin, 150030, People’s Republic of China

    Xiaoli Sun, Shanshan Yang, Mingzhe Sun, Sunting Wang, Xiaodong Ding, Dan Zhu, Wei Ji, Hua Cai, Chaoyue Zhao, Xuedong Wang & Yanming Zhu

  2. Department of Neurology, The University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA

    Xiaodong Ding

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  1. Xiaoli Sun
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  2. Shanshan Yang
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Correspondence to Yanming Zhu.

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Xiaoli Sun and Shanshan Yang are co-first authors.

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11103_2013_167_MOESM1_ESM.tif

Genomic organization and cystatin domain distribution of the 21 soybean cystatin genes. Exons were indicated by blue boxes and introns by lines. The cystatin domains were marked as yellow boxes (TIFF 832 kb)

11103_2013_167_MOESM2_ESM.tif

Schematic alignment of the 21 soybean cystatins based on amino acid sequences. The predicted signal peptide (SP) and the C-terminal tails (C-term) were shaded. The approximate locations of the five β-sheets and the single α-helix were indicated (TIFF 1132 kb)

11103_2013_167_MOESM3_ESM.tif

Purified GST-GsCPI14 fusion protein. The full-length GsCPI14 gene was fused to the C-terminus of GST tag. The GST-fused GsCPI14 protein was induced by 1 mM IPTG for 6 h at 30 °C in E. coli strain Rosetta cells and purified by the glutathione agarose (TIFF 960 kb)

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Sun, X., Yang, S., Sun, M. et al. A novel Glycine soja cysteine proteinase inhibitor GsCPI14, interacting with the calcium/calmodulin-binding receptor-like kinase GsCBRLK, regulated plant tolerance to alkali stress. Plant Mol Biol 85, 33–48 (2014). https://doi.org/10.1007/s11103-013-0167-4

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