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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 Ca 2+ 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., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

Root hairs (RH) are tip growing polarized cells aiding the uptake of nutrients and water into plants. RH differentiation involves the interplay of various hormones and second messengers. Tightly regulated production of reactive oxygen species by the NADPH oxidase RBOHC crucially functions in RH differentiation and Ca 2+ -dependent phosphorylation has been implemented in these processes. However, the kinases regulating RBOHC remained enigmatic. Here we identify CBL1-CIPK26 Ca 2+ sensor-kinase complexes as modulators of RBOHC activity. Combined genetic, cell biological and biochemical analyses reveal synergistic function of CIPK26-mediated phosphorylation and Ca 2+ binding for RBOHC activation. Complementation of rbohC mutant RH phenotypes by a S318/322 phosphorylation deficient RBOHC version suggests flexible and alternating phosphorylation patterns as mechanism fine-tuning ROS production in RH development., (© 2018 Federation of European Biochemical Societies.)

Reactive oxygen species (ROS) accumulate at the tip of growing pollen tubes. In Arabidopsis, NADPH oxidases RbohH and RbohJ are localized at the plasma membrane of pollen tube tip and produce ROS in a Ca(2+)-dependent manner. The ROS produced by Rbohs and Ca(2+) presumably play a critical role in the positive feedback regulation that maintains the tip growth. Ultrastructural cytochemical analysis revealed ROS accumulation in the apoplast/cell wall of the pollen grains on the stigmatic papillae in the wild type, but not in the rbohH rbohJ double mutant, suggesting that apoplastic ROS derived from RbohH and RbohJ are involved in pollen tube elongation into the stigmatic papillae by affecting the cell wall metabolism.

In flowering plants, pollen germinates on the stigma and pollen tubes grow through the style to fertilize the ovules. Enzymatic production of reactive oxygen species (ROS) has been suggested to be involved in pollen tube tip growth. Here, we characterized the function and regulation of the NADPH oxidases RbohH and RbohJ (Respiratory burst oxidase homolog H and J) in pollen tubes in Arabidopsis thaliana. In the rbohH and rbohJ single mutants, pollen tube tip growth was comparable to that of the wild type; however, tip growth was severely impaired in the double mutant. In vivo imaging showed that ROS accumulation in the pollen tube was impaired in the double mutant. Both RbohH and RbohJ, which contain Ca(2+) binding EF-hand motifs, possessed Ca(2+)-induced ROS-producing activity and localized at the plasma membrane of the pollen tube tip. Point mutations in the EF-hand motifs impaired Ca(2+)-induced ROS production and complementation of the double mutant phenotype. We also showed that a protein phosphatase inhibitor enhanced the Ca(2+)-induced ROS-producing activity of RbohH and RbohJ, suggesting their synergistic activation by protein phosphorylation and Ca(2+). Our results suggest that ROS production by RbohH and RbohJ is essential for proper pollen tube tip growth, and furthermore, that Ca(2+)-induced ROS positive feedback regulation is conserved in the polarized cell growth to shape the long tubular cell.

Reactive oxygen species (ROS) produced by NADPH oxidases play critical roles in plant environmental responses. Arabidopsis thaliana NADPH oxidase AtRbohF-mediated ROS-production is involved in abiotic stress responses. Because overproduction of ROS is highly toxic to cells, the activity of AtRbohF needs to be tightly regulated in response to diverse stimuli. The ROS-producing activity of AtRbohF is activated by Ca(2+) and protein phosphorylation, but other regulatory factors for AtRbohF are mostly unknown. In this study, we screened for proteins that interact with the N-terminal cytosolic region of AtRbohF by a yeast two-hybrid screen, and isolated AtSRC2, an A. thaliana homolog of SRC2 (soybean gene regulated by cold-2). A co-immunoprecipitation assay revealed that AtSRC2 interacts with the N-terminal region of AtRbohF in plant cells. Intracellular localization of GFP-tagged AtSRC2 was partially overlapped with that of GFP-tagged AtRbohF at the cell periphery. Co-expression of AtSRC2 enhanced the Ca(2+)-dependent ROS-producing activity of AtRbohF in HEK293T cells, but did not affect its phosphorylation-dependent activation. Low-temperature treatment induced expression of the AtSRC2 gene in Arabidopsis roots in proportion to levels of ROS production that was partially dependent on AtRbohF. Our findings suggest that AtSRC2 is a novel activator of Ca(2+)-dependent AtRbohF-mediated ROS production and may play a role in cold responses., (© 2013 Elsevier B.V. All rights reserved.)

The slow vacuolar (SV) channel has been characterized in different dicots by patch-clamp recordings. This channel represents the major cation conductance of the largest organelle in most plant cells. Studies with the tpc1-2 mutant of the model dicot plant Arabidopsis thaliana identified the SV channel as the product of the TPC1 gene. By contrast, research on rice and wheat TPC1 suggested that the monocot gene encodes a plasma membrane calcium-permeable channel. To explore the site of action of grass TPC1 channels, we expressed OsTPC1 from rice (Oryza sativa) and TaTPC1 from wheat (Triticum aestivum) in the background of the Arabidopsis tpc1-2 mutant. Cross-species tpc1 complementation and patch-clamping of vacuoles using Arabidopsis and rice tpc1 null mutants documented that both monocot TPC1 genes were capable of rescuing the SV channel deficit. Vacuoles from wild-type rice but not the tpc1 loss-of-function mutant harbor SV channels exhibiting the hallmark properties of dicot TPC1/SV channels. When expressed in human embryonic kidney (HEK293) cells OsTPC1 was targeted to Lysotracker-Red-positive organelles. The finding that the rice TPC1, just like those from the model plant Arabidopsis and even animal cells, is localized and active in lyso-vacuolar membranes associates this cation channel species with endomembrane function., (© 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.)

Pharmacological evidence suggests that anion channel-mediated plasma membrane anion effluxes are crucial in early defense signaling to induce immune responses and hypersensitive cell death in plants. However, their molecular bases and regulation remain largely unknown. We overexpressed Arabidopsis SLAC1, an S-type anion channel involved in stomatal closure, in cultured tobacco BY-2 cells and analyzed the effect on cryptogein-induced defense responses including fluxes of Cl(-) and other ions, production of reactive oxygen species (ROS), gene expression and hypersensitive responses. The SLAC1-GFP fusion protein was localized at the plasma membrane in BY-2 cells. Overexpression of SLAC1 enhanced cryptogein-induced Cl(-) efflux and extracellular alkalinization as well as rapid/transient and slow/prolonged phases of NADPH oxidase-mediated ROS production, which was suppressed by an anion channel inhibitor, DIDS. The overexpressor also showed enhanced sensitivity to cryptogein to induce downstream immune responses, including the induction of defense marker genes and the hypersensitive cell death. These results suggest that SLAC1 expressed in BY-2 cells mediates cryptogein-induced plasma membrane Cl(-) efflux to positively modulate the elicitor-triggered activation of other ion fluxes, ROS as well as a wide range of defense signaling pathways. These findings shed light on the possible involvement of the SLAC/SLAH family anion channels in cryptogein signaling to trigger the plasma membrane ion channel cascade in the plant defense signal transduction network.

Stimulus-specific accumulation of second messengers like reactive oxygen species (ROS) and Ca(2+) are central to many signaling and regulation processes in plants. However, mechanisms that govern the reciprocal interrelation of Ca(2+) and ROS signaling are only beginning to emerge. NADPH oxidases of the respiratory burst oxidase homolog (RBOH) family are critical components contributing to the generation of ROS while Calcineurin B-like (CBL) Ca(2+) sensor proteins together with their interacting kinases (CIPKs) have been shown to function in many Ca(2+)- signaling processes. In this study, we identify direct functional interactions between both signaling systems. We report that the CBL-interacting protein kinase CIPK26 specifically interacts with the N-terminal domain of RBOHF in yeast two-hybrid analyses and with the full-length RBOHF protein in plant cells. In addition, CIPK26 phosphorylates RBOHF in vitro and co-expression of either CBL1 or CBL9 with CIPK26 strongly enhances ROS production by RBOHF in HEK293T cells. Together, these findings identify a direct interconnection between CBL-CIPK-mediated Ca(2+) signaling and ROS signaling in plants and provide evidence for a synergistic activation of the NADPH oxidase RBOHF by direct Ca(2+)-binding to its EF-hands and Ca(2+)-induced phosphorylation by CBL1/9-CIPK26 complexes.

The plant NADPH oxidases, known as respiratory burst oxidase homologues (Rbohs), play an indispensable role in a wide array of cellular and developmental processes. Arabidopsis thaliana RbohF (AtRbohF)-mediated production of reactive oxygen species (ROS) is involved in biotic and abiotic stress responses. Because of the toxicity of excess amount of ROS, the ROS-producing activity of Rbohs is speculated to be negatively regulated. However, its mechanism is mostly unknown to date. Here, we report the identification of calcineurin B-like protein-interacting protein kinase 26 (CIPK26) as a novel regulatory factor of AtRbohF. We isolated CIPK26 as an AtRbohF-interacting partner by a yeast two-hybrid screen. Our co-immunoprecipitation assay revealed that the CIPK26 protein interacts with the N-terminal region of AtRbohF in Nicotiana benthamiana cell extracts. The fluorescence of both GFP-tagged CIPK26 and AtRbohF was predominantly observed at the cell periphery. We also showed that co-expression of CIPK26 decreases the ROS-producing activity of AtRbohF in HEK293T cells. Together, these results suggest that the direct binding of CIPK26 to AtRbohF negatively modulates ROS production and play a role in the regulation of ROS signalling in plants.

Reactive oxygen species (ROS) produced by NADPH oxidases play critical roles in signalling and development. Given the high toxicity of ROS, their production is tightly regulated. In Arabidopsis, respiratory burst oxidase homologue F (AtrbohF) encodes NADPH oxidase. Here we characterised the activation of AtRbohF using a heterologous expression system. AtRbohF exhibited ROS-producing activity that was synergistically activated by protein phosphorylation and Ca2+. The two EF-hand motifs of AtRbohF in the N-terminal cytosolic region were crucial for its Ca2+-dependent activation. AtrbohD and AtrbohF are involved in stress responses. Although the activation mechanisms for AtRbohD and AtRbohF were similar, AtRbohD had significantly greater ROS-producing activity than AtRbohF, which may reflect their functional diversity, at least in part. We further characterised the interrelationship between Ca2+ and phosphorylation regarding activation and found that protein phosphorylation-induced activation was independent of Ca2+. In contrast, K-252a, a protein kinase inhibitor, inhibited the Ca2+-dependent ROS-producing activity of AtRbohD and AtRbohF in a dose-dependent manner, suggesting that protein phosphorylation is a prerequisite for the Ca2+-dependent activation of Rboh. Positive feedback regulation of Ca2+ and ROS through AtRbohC has been proposed to play a critical role in root hair tip growth. Our findings suggest that Rboh phosphorylation is the initial trigger for the plant Ca2+-ROS signalling network., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Cytosolic calcium homeostasis is pivotal for intracellular signaling and requires sensing of calcium concentrations in the cytosol and accessible stores. Numerous Ca²⁺ binding sites have been characterized in cytosolic proteins. However, little is known about Ca²⁺ binding inside organelles, like the vacuole. The slow vacuolar (SV) channel, encoded by Arabidopsis thaliana TPC1, is regulated by luminal Ca²⁺. However, the D454/fou2 mutation in TPC1 eliminates vacuolar calcium sensitivity and increases store calcium content. In a search for the luminal calcium binding site, structure modeling indicated a possible coordination site formed by residues Glu-450, Asp-454, Glu-456, and Glu-457 on the luminal side of TPC1. Each Glu residue was replaced by Gln, the modified genes were transiently expressed in loss-of-TPC1-function protoplasts, and SV channel responses to luminal calcium were recorded by patch clamp. SV channels lacking any of the four negatively charged residues appeared altered in calcium sensitivity of channel gating. Our results indicate that Glu-450 and Asp-454 are directly involved in Ca²⁺ binding, whereas Glu-456 and Glu-457 are probably involved in connecting the luminal Ca²⁺ binding site to the channel gate. This novel vacuolar calcium binding site represents a potential tool to address calcium storage in plants.

Plant respiratory burst oxidase homolog (rboh) proteins, which are homologous to the mammalian 91-kDa glycoprotein subunit of the phagocyte oxidase (gp91(phox)) or NADPH oxidase 2 (NOX2), have been implicated in the production of reactive oxygen species (ROS) both in stress responses and during development. Unlike mammalian gp91(phox)/NOX2 protein, plant rboh proteins have hydrophilic N-terminal regions containing two EF-hand motifs, suggesting that their activation is dependent on Ca(2+). However, the significance of Ca(2+) binding to the EF-hand motifs on ROS production has been unclear. By employing a heterologous expression system, we showed that ROS production by Arabidopsis thaliana rbohD (AtrbohD) was induced by ionomycin, which is a Ca(2+) ionophore that induces Ca(2+) influx into the cell. This activation required a conformational change in the EF-hand region, as a result of Ca(2+) binding to the EF-hand motifs. We also showed that AtrbohD was directly phosphorylated in vivo, and that this was enhanced by the protein phosphatase inhibitor calyculin A (CA). Moreover, CA itself induced ROS production and dramatically enhanced the ionomycin-induced ROS production of AtrbohD. Our results suggest that Ca(2+) binding and phosphorylation synergistically activate the ROS-producing enzyme activity of AtrbohD.

The specification and maintenance of growth sites are tightly regulated during cell morphogenesis in all organisms. ROOT HAIR DEFECTIVE 2 reduced nicotinamide adenine dinucleotide phosphate (RHD2 NADPH) oxidase-derived reactive oxygen species (ROS) stimulate a Ca2+ influx into the cytoplasm that is required for root hair growth in Arabidopsis thaliana. We found that Ca2+, in turn, activated the RHD2 NADPH oxidase to produce ROS at the growing point in the root hair. Together, these components could establish a means of positive feedback regulation that maintains an active growth site in expanding root hair cells. Because the location and stability of growth sites predict the ultimate form of a plant cell, our findings demonstrate how a positive feedback mechanism involving RHD2, ROS, and Ca2+ can determine cell shape.

Elevations in cytoplasmic calcium ([Ca(2)+](cyt)) are an important component of early abscisic acid (ABA) signal transduction. To determine whether defined mutations in ABA signal transduction affect [Ca(2)+](cyt) signaling, the Ca(2)+-sensitive fluorescent dye fura 2 was loaded into the cytoplasm of Arabidopsis guard cells. Oscillations in [Ca(2)+](cyt) could be induced when the external calcium concentration was increased, showing viable Ca(2)+ homeostasis in these dye-loaded cells. ABA-induced [Ca(2)+](cyt) elevations in wild-type stomata were either transient or sustained, with a mean increase of approximately 300 nM. Interestingly, ABA-induced [Ca(2)+](cyt) increases were significantly reduced but not abolished in guard cells of the ABA-insensitive protein phosphatase mutants abi1 and abi2. Plasma membrane slow anion currents were activated in wild-type, abi1, and abi2 guard cell protoplasts by increasing [Ca(2)+](cyt), demonstrating that the impairment in ABA activation of anion currents in the abi1 and abi2 mutants was bypassed by increasing [Ca(2)+](cyt). Furthermore, increases in external calcium alone (which elevate [Ca(2)+](cyt)) resulted in stomatal closing to the same extent in the abi1 and abi2 mutants as in the wild type. Conversely, stomatal opening assays indicated different interactions of abi1 and abi2, with Ca(2)+-dependent signal transduction pathways controlling stomatal closing versus stomatal opening. Together, [Ca(2)+](cyt) recordings, anion current activation, and stomatal closing assays demonstrate that the abi1 and abi2 mutations impair early ABA signaling events in guard cells upstream or close to ABA-induced [Ca(2)+](cyt) elevations. These results further demonstrate that the mutations can be bypassed during anion channel activation and stomatal closing by experimental elevation of [Ca(2)+](cyt).