Control of vacuolar dynamics and regulation of stomatal aperture by tonoplast potassium uptake.

Stomatal movements rely on alterations in guard cell turgor. This requires massive K(+) bidirectional fluxes across the plasma and tonoplast membranes. Surprisingly, given their physiological importance, the transporters mediating the energetically uphill transport of K(+) into the vacuole remain to be identified. Here, we report that, in Arabidopsis guard cells, the tonoplast-localized K(+)/H(+) exchangers NHX1 and NHX2 are pivotal in the vacuolar accumulation of K(+) and that nhx1 nhx2 mutant lines are dysfunctional in stomatal regulation. Hypomorphic and complete-loss-of-function double mutants exhibited significantly impaired stomatal opening and closure responses. Disruption of K(+) accumulation in guard cells correlated with more acidic vacuoles and the disappearance of the highly dynamic remodelling of vacuolar structure associated with stomatal movements. Our results show that guard cell vacuolar accumulation of K(+) is a requirement for stomatal opening and a critical component in the overall K(+) homeostasis essential for stomatal closure, and suggest that vacuolar K(+) fluxes are also of decisive importance in the regulation of vacuolar dynamics and luminal pH that underlie stomatal movements.

Wounding-Induced Stomatal Closure Requires Jasmonate-Mediated Activation of GORK K+ Channels by a Ca2+ Sensor-Kinase CBL1-CIPK5 Complex.

Guard cells integrate various hormone signals and environmental cues to balance plant gas exchange and transpiration. The wounding-associated hormone jasmonic acid (JA) and the drought hormone abscisic acid (ABA) both trigger stomatal closure. In contrast to ABA however, the molecular mechanisms of JA-induced stomatal closure have remained largely elusive. Here, we identify a fast signaling pathway for JA targeting the K+ efflux channel GORK. Wounding triggers both local and systemic stomatal closure by activation of the JA signaling cascade followed by GORK phosphorylation and activation through CBL1-CIPK5 Ca2+ sensor-kinase complexes. GORK activation strictly depends on plasma membrane targeting and Ca2+ binding of CBL1-CIPK5 complexes. Accordingly, in gork, cbl1, and cipk5 mutants, JA-induced stomatal closure is specifically abolished. The ABA-coreceptor ABI2 counteracts CBL1-CIPK5-dependent GORK activation. Hence, JA-induced Ca2+ signaling in response to biotic stress converges with the ABA-mediated drought stress pathway to facilitate GORK-mediated stomatal closure upon wounding.

A plastid protein crucial for Ca2+-regulated stomatal responses.

* Guard cell movements are regulated by environmental cues including, for example, elevations in extracellular Ca(2+) concentration. Here, the subcellular localization and physiological function of the Ca(2+)-sensing receptor (CAS) protein was investigated. * CAS protein localization was ascertained by microscopic analyses of green fluorescent protein (GFP) fusion proteins and biochemical fractionation assays. Comparative guard cell movement investigations were performed in wild-type and cas loss-of-function mutant lines of Arabidopsis thaliana. Cytoplasmic Ca(2+) dynamics were addressed in plants expressing the yellow cameleon reporter protein YC3.6. * This study identified CAS as a chloroplast-localized protein that is crucial for proper stomatal regulation in response to elevations of external Ca(2+). CAS fulfils this role through modulation of the cytoplasmic Ca(2+) concentration. * This work reveals a novel role of the chloroplast in cellular Ca(2+) signal transduction.

CBL1-CIPK26-mediated phosphorylation enhances activity of the NADPH oxidase RBOHC, but is dispensable for root hair growth.

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 Ca2+ -dependent phosphorylation has been implemented in these processes. However, the kinases regulating RBOHC remained enigmatic. Here we identify CBL1-CIPK26 Ca2+ sensor-kinase complexes as modulators of RBOHC activity. Combined genetic, cell biological and biochemical analyses reveal synergistic function of CIPK26-mediated phosphorylation and Ca2+ 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.

Protein fragment bimolecular fluorescence complementation analyses for the in vivo study of protein-protein interactions and cellular protein complex localizations.

The analyses of protein-protein interactions are crucial for understanding cellular processes including signal transduction, protein trafficking, and movement. Protein fragment complementation assays are based on the reconstitution of protein function when non-active protein fragments are brought together by interacting proteins that were genetically fused to these protein fragments. Bimolecular fluorescence complementation (BiFC) relies on the reconstitution of fluorescent proteins and enables both the analysis of protein-protein interactions and the visualization of protein complex formations in vivo. Transient expression of proteins is a convenient approach to study protein functions in planta or in other organisms and minimizes the need for time-consuming generation of stably expressing transgenic organisms. Here we describe protocols for BiFC analyses in Nicotiana benthamiana and Arabidopsis thaliana leaves transiently transformed by Agrobacterium infiltration. Further, we discuss different BiFC applications and provide examples for proper BiFC analyses in planta.

The Calcineurin B-like calcium sensors CBL1 and CBL9 together with their interacting protein kinase CIPK26 regulate the Arabidopsis NADPH oxidase RBOHF.

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.

A new ß-estradiol-inducible vector set that facilitates easy construction and efficient expression of transgenes reveals CBL3-dependent cytoplasm to tonoplast translocation of CIPK5.

Transient and stable expression of transgenes is central to many investigations in plant biology research. Chemical regulation of expression can circumvent problems of plant lethality caused by constitutive overexpression or allow inducible knock (out/down) approaches. Several chemically inducible or repressible systems have been described and successfully applied. However, cloning and application-specific modification of most available inducible expression systems have been limited and remained complicated due to restricted cloning options. Here we describe a new set of 57 vectors that enable transgene expression in transiently or stably transformed cells. All vectors harbor a synthetically optimized XVE expression cassette, allowing ß-estradiol mediated protein expression. Plasmids are equipped with the reporter genes GUS, GFP, mCherry, or with HA and StrepII epitope tags and harbor an optimized multiple cloning site for flexible and simple cloning strategies. Moreover, the vector design allows simple substitution of the driving promoter to achieve tissue-specificity or to modulate expression ranges of inducible transgene expression. We report details of the kinetics and dose-dependence of expression induction in Arabidopsis leaf mesophyll protoplasts, transiently transformed Nicotiana benthamiana leaves, and stably transformed Arabidopsis plants. Using these vectors, we investigated the influence of CBL (Calcineurin B-like) protein expression on the subcellular localization of CIPKs (Calcineurin B-like interacting protein kinases). These analyses uncovered that induced co-expression of CBL3 is fully sufficient for dynamic translocation of CIPK5 from the cytoplasm to the tonoplast. Thus, the vector system presented here facilitates a broad range of research applications.

S-acylation-dependent association of the calcium sensor CBL2 with the vacuolar membrane is essential for proper abscisic acid responses.

Calcineurin B-like (CBL) proteins contribute to decoding calcium signals by interacting with CBL-interacting protein kinases (CIPKs). Currently, there is still very little information about the function and specific targeting mechanisms of CBL proteins that are localized at the vacuolar membrane. In this study, we focus on CBL2, an abundant vacuolar membrane-localized calcium sensor of unknown function from Arabidopsis thaliana. We show that vacuolar targeting of CBL2 is specifically brought about by S-acylation of three cysteine residues in its N-terminus and that CBL2 S-acylation and targeting occur by a Brefeldin A-insensitive pathway. Loss of CBL2 function renders plants hypersensitive to the phytohormone abscisic acid (ABA) during seed germination and only fully S-acylated and properly vacuolar-targeted CBL2 proteins can complement this mutant phenotype. These findings define an S-acylation-dependent vacuolar membrane targeting pathway for proteins and uncover a crucial role of vacuolar calcium sensors in ABA responses.