Doctorate thesis of National Institute of Further Education in Agricultural Science

Wednesday, November 27, 2013

Post-translational regulation of potassium channels by CPKs (Calcium-dependent Protein Kinases) in Arabidopsis thaliana: a role in adaptive response to environmental stresses?

Elsa Ronzier
BPMP, “Electrical and calcium signalling” team

Jury :
Alain VAVASSEUR, DR / CEA Cadarache, Rapporteur
Sébastien THOMINE, DR/ISV, Rapporteur
Sylvie COURSOL, CR/Institut Jean-Pierre Bourgin, Examinateur
Frédéric GAYMARD, DR/B&PMP, Examinateur
Jean-Baptiste THIBAUD, DR/B&PMP, CoDirecteur de thèse
Tou Cheu XIONG, CR/B&PMP, CoDirecteur de thèse

Abstract :
Potassium Shaker channels are major pathways for K+ across plant cell plasma membrane. These channels are implicated in K+ absorption from soil and in its redistribution throughout the plant. They are more particularly implicated in stomatal movement and therefore are finely regulated. They can especially undergo post-translational modifications such as phosphorylation by protein kinases. The aim of this work is to determine the implication of CPKs (Ca2+-dependent Protein Kinases) in Shaker channel post-translational regulation. CPK13 and CPK6 molecular mechanisms of action on Shaker sub-unit KAT2 activity are specifically studied here. First part of this work consisted in cloning, producing and characterizing recombinant proteins and broad screening of CPK effects on Shaker channel activity, using heterologous expression. Second part focuses on the role of CPK13 in stomatal regulation through its effect on KAT2 activity. Over-expression of CPK13 in plant is shown to induce a defect of stomatal aperture and plant growth. KAT2 and CPK13 interaction at the plasma membrane is evidenced by using FRET-FLIM technique. KAT2 phosphorylation by CPK13 is checked on peptide arrays. Finally, a 60% decrease of KAT2 activity by CPK13 is shown using voltage-clamp on xenopus oocyte. Third and last part of this work suggests a role of CPK6 in salt stress resistance through KAT2 channel regulation. Inward potassium channels are indeed known to be activated upon salt stress to contribute keeping a low Na+/K+ ratio. Now, voltage-clamp technique demonstrates that KAT2 activity is increased by CPK6 and salt stress is shown to both increase CPK6 expression and elicit a calcium wave. Using GUS lines evidences KAT2 and CPK6 co-expression in Arabidopsis thaliana (in phloem and guard cells). Physical interaction between these two partners is shown by FRET-FLIM, and KAT2 phosphorylation by CPK13 gets strong support from peptide array assays.