1 Department of Biology, Concordia University, 7141 Sherbrooke W., Montreal, QC, H4B 1R6, Canada.
2 Institut de Recherche en Biologie Végétale, Université de Montréal, 4101 Sherbrooke Est, Montréal, QC, H1X 2B2, Canada.
3 Department of Genetics, Faculty of Agriculture, Ain-Shams University, Shoubra El-khema, Cairo, Egypt.
4 Department of Biotechnology & Microbiology, Mahatma Jyoti Rao Phoole University, Jaipur, Rajasthan, India.
5 Department of Biology, Concordia University, 7141 Sherbrooke W., Montreal, QC, H4B 1R6, Canada. patrick.gulick@concordia.ca.

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. 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 (GPA1^QL) 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 GPA1^QL 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 GPA1^QL. 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.