Annexin 1 regulates the H2O2-induced calcium signature in Arabidopsis thaliana roots

Hydrogen peroxide is the most stable of the reactive oxygen species (ROS) and is a regulator of development, immunity and adaptation to stress. It frequently acts by elevating cytosolic free Ca²⁺ ([Ca²⁺]_cyt) as a second messenger, with activation of plasma membrane Ca²⁺-permeable influx channels as a fundamental part of this process. At the genetic level, to date only the Ca²⁺-permeable Stelar K⁺ Outward Rectifier (SKOR) channel has been identified as being responsive to hydrogen peroxide. We show here that the ROS-regulated Ca²⁺ transport protein Annexin 1 in <i>Arabidopsis thaliana</i> (AtANN1) is involved in regulating the root epidermal [Ca²⁺]_cyt response to stress levels of extracellular hydrogen peroxide. Peroxide-stimulated [Ca²⁺]_cyt elevation (determined using aequorin luminometry) was aberrant in roots and root epidermal protoplasts of the <i>Atann1</i> knockout mutant. Similarly, peroxide-stimulated net Ca²⁺ influx and K⁺ efflux were aberrant in <i>Atann1</i> root mature epidermis, determined using extracellular vibrating ion-selective microelectrodes. Peroxide induction of <i>GSTU1</i> (<i>Glutathione-S-Transferase1 Tau 1</i>), which is known to be [Ca²⁺]_cyt-dependent was impaired in mutant roots, consistent with a lesion in signalling. Expression of <i>AtANN1</i> in roots was suppressed by peroxide, consistent with the need to restrict further Ca²⁺ influx. Differential regulation of annexin expression was evident, with <i>AtANN2</i> down-regulation but up-regulation of <i>AtANN3</i> and <i>AtANN4</i>. Overall the results point to involvement of AtANN1 in shaping the root peroxide-induced [Ca²⁺]_cyt signature and downstream signalling.