The Role of Arabidopsis thaliana Annexins 1, 2 and 4 in Extracellular ATP Signalling

Abstract

Extracellular ATP (eATP) is an important signalling molecule in animals but its importance is less understood in plants. Accumulation of eATP in plants occurs in response to biotic and abiotic stresses. eATP causes downstream responses such as increase in cytosolic free calcium ([Ca$^{2+}$]${cyt}$), reactive oxygen species (ROS), nitric oxide (NO) and phosphatidic acid (PA). The identification of $\it{Arabidopsis}$ $\it{thaliana}$ AtDORN1 (Does Not Respond to Nucleotides) as the first higher plant purinoreceptor has confirmed eATP in plant signalling systems. AtDORN1 is a plasma membrane receptor kinase that appears to command the eATP-induced transient increase in [Ca$^{2+}$]${cyt}$ and directs the translational response to wounding. The identity of the plasma membrane Ca$^{2+}$-permeable channels involved in eATP-induced [Ca$^{2+}$]${cyt}$ increases remain unknown. Patch clamp electrophysiology has shown that a plasma membrane Ca$^{2+}$ influx conductance lies downstream of the AtRBOHC NADPH oxidase in the response to eATP. As $\it{Arabidopsis}$ $\it{thaliana}$ ANNEXIN 1 (AtANN1) underpins ROS-activated plasma membrane Ca$^{2+}$ influx conductance, it has been considered here as operating downstream of AtDORN1 in the eATP-induced increase in [Ca$^{2+}$]${cyt}$. In this thesis, the role of AtANN1, AtANN2 and AtANN4 in eATP signalling was tested in $\it{A.}$ $\it{thaliana}$. Using (apo)aequorin, AtANN1 and ANNEXIN 2 (AtANN2) have been found to be involved in the root′s [Ca$^{2+}$]${cyt}$ response to both eATP and eADP whereas ANNEXIN 4 (AtANN4) might be acting as a negative regulator of AtANN1 (Chapters 3 and 4). Application of Gd$^{3+}$ as a plasma membrane Ca$^{2+}$ channel blocker indicated the possibility of the release of Ca$^{2+}$ from intracellular stores (Chapters 3 and 4). AtANN1 is confirmed to be downstream of the AtDORN1 receptor and also possibly downstream of AtRBOH NADPH Oxidase based on [Ca$^{2+}$]${cyt}$ measurement and ROS assays (Chapter 3). Loss${-}$of${-}$function mutants of AtANN1, AtANN2 and AtANN4 altered the eATP${-}$induced gene expression of $\it{AtACS6}$ but not $\it{AtWRKY40}$ demonstrating a possible link between eATP signalling and hormone responses in plants (Chapters 3 and 4). Unlike AtANN1 and AtANN4, fewer studies regarding the involvement of AtANN2 in salinity stress and biotic stress were reported. In Chapter 5, results suggest that AtANN2 might not be part of the components mediating salinity stress and biotic stress in plants.