?A specific calcium-dependent protein kinase is associated to cytokinin signalling pathways in Medicago truncatula

RITA M. ULLOA; PABLO GARGANTINI; SILVINA GONZALEZ-RIZZO; DELPHINE CHINCHILLA; MARCELA RAICES; VERÓNICA GIAMMARIA; FLORIAN FRUGIER; MARTIN CRESPI

Amsterdam, Holanda

Congreso; PlantGems 4; 2005

Centre for BioSystems Genomics.

The induction of the legume symbiotic nodule, a process that has recently been shown to implicate calcium signalling, is initiated by de-differentiation of root cortical cells into a primordium that accumulates amyloplasts. This morphogenetic response can be mimicked by cytokinin treatments of legume roots. CDPKs (Calcium Dependent Protein Kinases) are natural transducers of calcium action. We have studied the expression of CDPK genes in roots and various nodule types induced by several rhizobium strains in alfalfa using conserved oligonucleotides for all members of the CDPK gene family. When specific oligonucleotides directed against the N-terminal domain of MsCDPK3 were used, an induction of this gene was observed in roots carrying nodule primordia but not in roots or mature nodules. In spot-inoculated roots, expression of MsCDPK3 was induced at 4 days post infection, once nodule primordia are formed, both in M. sativa and the model legume M. truncatula. This correlated with an increase of CDPK activity in the particulate fraction of S. meliloti-infected roots. MsCDPK3 amino-terminal myristoylation and palmitoylation consensus may regulate subcellular localization of this gene. Upon transient expression in epidermal onion cells and M. truncatula roots, a fusion of MsCDPK3-GFP was targeted to the plasma membrane. Both, myristoylation and palmitoylation of MsCDPK3 were critical for this targeting suggesting that a myristoyl/palmitoyl switch mechanism could be involved in regulating CDPK activity. Real-time PCR assays specific for MtCDPK3 performed on M truncatula roots showed that this gene is induced by abiotic stresses but also after a 6 h treatment with different citokinin concentrations (10-8 M to 10-6 M BAP). Using an antibody directed against CDPK3, we could show that this cytokinin induction was also detected at protein level. The role of MtCDPK3 in root signalling pathways was explored using RNAi constructs. Western analysis indicates extensive reduction of gene expression in composite plants. Though no major phenotype could be detected in these roots in response to cytokinins, the total number of nodules was higher in the RNAi-CDPK hairy roots compared to control GUS-silenced composite plants, suggesting that MtCDPK3 was acting as a negative regulator in this process.