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The stress induced caleosin, RD20/CLO3, acts as a negative regulator of GPA1 in Arabidopsis

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A stress induced calcium-binding protein, RD20/CLO3 interacts with the alpha subunit of the heterotrimeric G-protein complex in Arabidopsis and affects etiolation and leaf morphology.

Abstract

Heterotrimeric G proteins and calcium signaling have both been shown to play a role in the response to environmental abiotic stress in plants; however, the interaction between calcium-binding proteins and G-protein signaling molecules remains elusive. We investigated the interaction between the alpha subunit of the heterotrimeric G-protein complex, GPA1, of Arabidopsis thaliana with the calcium-binding protein, the caleosin RD20/CLO3, a gene strongly induced by drought, salt and abscisic acid. The proteins were found to interact in vivo by bimolecular fluorescent complementation (BiFC); the interaction was localized to the endoplasmic reticulum and to oil bodies within the cell. The constitutively GTP-bound GPA1 (GPA1QL) also interacts with RD20/CLO3 as well as its EF-hand mutant variations and these interactions are localized to the plasma membrane. The N-terminal portion of RD20/CLO3 was found to be responsible for the interaction with GPA1 and GPA1QL using both BiFC and yeast two-hybrid assays. RD20/CLO3 contains a single calcium-binding EF-hand in the N-terminal portion of the protein; disruption of the calcium-binding capacity of the protein obliterates interaction with GPA1 in in vivo assays and decreases the interaction between the caleosin and the constitutively active GPA1QL. Analysis of rd20/clo3 mutants shows that RD20/CLO3 plays a key role in the signaling pathway controlling hypocotyl length in dark grown seedlings and in leaf morphology. Our findings indicate a novel role for RD20/CLO3 as a negative regulator of GPA1.

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Acknowledgements

We would like to thank Dr. Alan Jones, North Carolina State University for the gift of the gpa1 mutant line, Dr. Sarah Assmann (Pennsylvania State U) for the gift of the GPA1 promoter::GUS transgenic Arabidopsis reporter line and for the AGB1/AGG3(γ)-BD fusion for Y2H, and Dr. Elizabeth Blée (Université de Strasbourg) for the gift of the P35S:RD20/rd20 line. We would like to thank Dr. Deborah Maret for editorial assistance. The authors acknowledge the Centre for Microscopy and Cellular Imaging (CMCI) funded by Concordia University, Montreal, Canada and the Canada Foundation for Innovation.

Funding

This work is supported by a grant from the Natural Science and Engineering Research Council of Canada.

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  1. Hala B. Khalil

    Present address: Department of Genetics, Faculty of Agriculture, Ain-Shams University, Shoubra El-khema, Cairo, Egypt

  2. Gajra Garg

    Present address: Department of Biotechnology & Microbiology, Mahatma Jyoti Rao Phoole University, Jaipur, Rajasthan, India

Authors and Affiliations

  1. Department of Biology, Concordia University, 7141 Sherbrooke W., Montreal, QC, H4B 1R6, Canada

    Sabrina C. Brunetti, Michelle K. M. Arseneault, Justin A. Wright, Zhejun Wang, Mohammad-Reza Ehdaeivand, Michael J. Lowden, Hala B. Khalil, Gajra Garg & Patrick J. Gulick

  2. Institut de Recherche en Biologie Végétale, Université de Montréal, 4101 Sherbrooke Est, Montréal, QC, H1X 2B2, Canada

    Jean Rivoal

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Contributions

PJG designed the project. SCB, ZW, JAW, and HK participated in the experimental design. SCB, MKMA, ZW, JAW, HK, GG, ME, JR and MJL performed the experiments and analyzed data. SCB, MKMA, JAW, and PJG contributed to writing and revision of the manuscript.

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Correspondence to Patrick J. Gulick.

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Brunetti, S.C., Arseneault, M.K.M., Wright, J.A. et al. The stress induced caleosin, RD20/CLO3, acts as a negative regulator of GPA1 in Arabidopsis. Plant Mol Biol 107, 159–175 (2021). https://doi.org/10.1007/s11103-021-01189-x

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