Fine‐tuning of RBOHF activity is achieved by differential phosphorylation and Ca²⁺ binding

Abstract

Summary RBOHF from Arabidopsis thaliana represents a multifunctional NADPH oxidase regulating biotic and abiotic stress tolerance, developmental processes and guard cell aperture. The molecular components and mechanisms determining RBOHF activity remain to be elucidated. Here we combined protein interaction studies, biochemical and genetic approaches, and pathway reconstitution analyses to identify and characterize proteins that confer RBOHF regulation and elucidated mechanisms that adjust RBOHF activity. While the Ca2+ sensor‐activated kinases CIPK11 and CIPK26 constitute alternative paths for RBOHF activation, the combined activity of CIPKs and the kinase open stomata 1 (OST1) triggers complementary activation of this NADPH oxidase, which is efficiently counteracted through dephosphorylation by the phosphatase ABI1. Within RBOHF, several distinct phosphorylation sites (p‐sites) in the N‐terminus of RBOHF appear to contribute individually to activity regulation. These findings identify RBOHF as a convergence point targeted by a complex regulatory network of kinases and phosphatases. We propose that this allows for fine‐tuning of plant reactive oxygen species (ROS) production by RBOHF in response to different stimuli and in diverse physiological processes.

An Arabidopsis Mutant Over-Expressing Subtilase SBT4.13 Uncovers the Role of Oxidative Stress in the Inhibition of Growth by Intracellular Acidification

“…Known regulation of NOX enzymes in plants occurs by phosphorylation and through increase of cytosolic free calcium, the latter acting both directly on EF motifs of the oxidases and through Ca 2+ sensor-activated kinases [41,42]. The in vivo activation of NOX by intracellular acidification identified in the present work could occur by either direct effect of acidic pH on the enzymes or by an increase of free cytosolic Ca 2+ triggered by intracellular acidification, as described by Felle [45].…”

“…Cytosolic pH [25,26,39] and NOX enzymes [40][41][42][43][44] have both complex regulations and multiple physiological roles and we found a connection between these two important actors. Future studies should identify the mechanism of activation of NOXs by low pHc and the cellular targets regulating growth.…”

Two SnRK2-Interacting Calcium Sensor Isoforms Negatively Regulate SnRK2 Activity by Different Mechanisms

“…They are known as calcium-independent enzymes; however, several data indicate their interplay with calcium signaling pathways. Calcium-dependent kinases, CIPKs, together with SnRK2.6, phosphorylate and regulate activity of NADPH oxidase (respiratory burst oxidase homolog F; Sirichandra et al, 2009 andHan et al, 2019), whereas CDPKs along with SnRK2s activate the guard cell S-type anion channel in response to ABA (Geiger et al, 2010;Brandt et al, 2012). Importantly, in the snrk2.2/snrk2.3/snrk2.6 knockout mutant, the Ca 21dependent S-type anion channel regulation was impaired (Brandt et al, 2015), indicating interconnection between SnRK2s and calcium signaling pathways.…”

Dual-sensing genetically encoded fluorescent indicators resolve the spatiotemporal coordination of cytosolic abscisic acid and second messenger dynamics in Arabidopsis

“…HEK293T cells contain neglectable ABA levels, and are therefore well suited for ABA indicator screenings with a potential to facilitate the heterologous characterization of ABA transporters using ABA indicators. Successful characterizations of plasma membrane proteins in HEK293T cells has been demonstrated for RBOHs and CYCLIC NUCLEOTIDE-GATED ION CHANNEL (CNGC)-type Ca 2+ channels (Gao et al, 2016; Han et al, 2019). The differences of ABAleon characteristics between HEK293T cell and in vitro analyses might be due to a lower stability of ABAleon2.15 in HEK293T cells.…”

“…RBOH-derived extracellular ROS propagate to adjacent cells to activate plasma membrane localized Ca 2+ channels (Evans et al, 2016; Choi et al, 2016). Ca 2+ -dependent activation of RBOHs is well established (Kadota et al, 2015; Han et al, 2019). However, whether RBOH-dependent ROS contribute to Ca 2+ channel activation, has only been inferred from pharmacological- and genetic analyses (Kwak et al, 2003; Evans et al, 2016).…”