Study On The Mechanism Of Rice Ca²⁺-OsCBL-OsMTN Signal System

Calcineurin B-like (CBL) proteins represent a unique family of calcium sensors in plant cells. As the sensor relays, the CBL family members are currently known to interact exclusively with a group of Ser/Thr protein kinases called CIPKs (for CBL-interacting protein kinases), thereby mediating the calcium signals elicited by various stimuli, including salinity, drought, cold, low K+concentration, high pH, abscisic acid, gibberellic acid and osmotic stress. However, the Arabidopsis CBL3protein member of this family has a novel interaction partner,5’-methylthioadenosine nucleosidase AtMTNl, which is one key enzyme in Methionine-recycling pathways. CBL3-AtMTN1mediated calcium signaling regulates the biosynthesis of ethylene and polyamines, which are involved in plant growth and development as well as various stress responses.In our study, one full-length cDNAs containing complete ORF, which encode unique rice5’-methylthioadenosine nucleosidase, were isolated from rice by RT-PCR and named as OsMTN. The results of real-time PCR analysis indicated that OsMTN was up-regulated by salt, PEG6000, HgCl2, ABA and bacterial blight. These results suggested that OsMTN might function as an early stress-responsive gene.To unravel the interaction of rice OsMTN proteins with the complete set of rice CBLs, we studied OsMTN interactions with OsCBL1to OsCBL10in yeast two hybrid interaction analyses. OsMTN exhibited a significant interaction with OsCBL2, OsCBL3and some degree of interaction with OsCBL6, whereas other seven OsCBLs did not show any affinity toward OsMTN. To investigate the interaction localization of rice OsMTN with three OsCBLs, we performed the bimolecular fluorescence complementation assay and subcellular localization assay. We observed a ring-like distribution of OsMTN::GFP at the plasma membrane and OsCBL2::GFP, OsCBL3::GFP and CBL6::GFP at the plasma membrane and tonoplast. Our findings suggest that OsMTN-OsCBLs decode calcium signals not only at the plasma membrane, but also in the tonoplast. To explore the function of OsMTN under stresses, its open reading frames (ORFs) was cloned into the vector pCABMIA1300S and transformed into the calli of rice variety Nipponbare by Agrobacterium tumefaciens-mediated method. The transgenic regenerated seedlings screened by Hygromycin have been confirmed through PCR assay, and their functional identification will be carried out.