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Rice bifunctional phytocystatin is a dual modulator of legumain and papain-like proteases

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Abstract

Phytocystatins are well-known inhibitors of C1A cysteine proteinases. However, previous research has revealed legumain (C13) protease inhibition via a carboxy-extended phytocystatin. Among the 12 phytocystatins genes in rice, OcXII is the only gene possessing this carboxy-terminal extension. The specific legumain inhibition activity was confirmed, in our work, using a recombinant OcXII harboring only the carboxy-terminal domain and this part did not exhibit any effect on papain-like activities. Meanwhile, rice plants silenced at the whole OcXII gene presented higher legumain and papain-like proteolytic activities, resulting in a faster initial seedling growth. However, when germinated under stressful alkaline conditions, OcXII-silenced plants exhibited impaired root formation and delayed shoot growth. Interestingly, the activity of OcXII promoter gene was detected in the rice seed scutellum region, and decreases with seedling growth. Seeds from these plants also exhibited slower growth at germination under ABA or alkaline conditions, while maintaining very high levels of OcXII transcriptional activation. This likely reinforces the proteolytic control necessary for seed germination and growth. In addition, increased legumain activity was detected in OcXII RNAi plants subjected to a fungal elicitor. Overall, the results of this study highlight the association of OcXII with not only plant development processes, but also with stress response pathways. The results of this study reinforce the bifunctional ability of carboxy-extended phytocystatins in regulating legumain proteases via its carboxy-extended domain and papain-like proteases by its amino-terminal domain.

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Acknowledgments

This work was supported by a CNPq Grant 478417/2012-8. R. Margis received a research fellowship 307868/2011-7 from CNPq and A.P. Christoff a CAPES PhD fellowship. The authors also would like to thank A. Caverzan, Oliveira L.F.V, D.J. Messeder, F.M. Bianchi, and N.E. Junqueira for experimental support.

Author contributions

A.P.C. and R.M designed the research, analyzed the data and wrote the paper; A.P.C performed the experiments; A.P.C and G.P performed plant transformation experiments; A.P.C and C.S. performed enzymatic assays experiments, M.M-P and M.A-F helped with the experimental structure and reviewed the manuscript.

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Authors and Affiliations

  1. PPGBM, Departamento de Genetica, Universidade Federal do Rio Grande do Sul–UFRGS, Porto Alegre, Rs, Brazil

    Ana Paula Christoff, Gisele Passaia, Marcia Margis-Pinheiro & Rogerio Margis

  2. PPGBCM, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul–UFRGS, Sala 213, Prédio 43431, PO Box 15005, Porto Alegre, Rs, CEP 91501-970, Brazil

    Caroline Salvati, Marcia Margis-Pinheiro & Rogerio Margis

  3. Departamento de Genética, Universidade Federal do Rio de Janeiro–UFRJ, Rio de Janeiro, Rj, Brazil

    Márcio Alves-Ferreira

  4. Departamento de Biofísica, Universidade Federal do Rio Grande do Sul–UFRGS, Porto Alegre, Rs, Brazil

    Rogerio Margis

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  1. Ana Paula Christoff
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  2. Gisele Passaia
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Correspondence to Rogerio Margis.

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Christoff, A.P., Passaia, G., Salvati, C. et al. Rice bifunctional phytocystatin is a dual modulator of legumain and papain-like proteases. Plant Mol Biol 92, 193–207 (2016). https://doi.org/10.1007/s11103-016-0504-5

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