A Pollen Protein,NaPCCP, That Binds Pistil Arabinogalactan Proteins Also Binds Phosphatidylinositol 3-Phosphate and Associates with the Pollen Tube Endomembrane System

As pollen tubes grow toward the ovary, they are in constant contact with the pistil extracellular matrix (ECM). ECM components are taken up during growth, and some pistil molecules exert their effect inside the pollen tube. For instance, the Nicotiana alata 120-kD glycoprotein (120K) is an abundant arabinogalactan protein that is taken up from the ECM; it has been detected in association with pollen tube vacuoles, but the transport pathway between these compartments is unknown. We recently identified a pollen C2 domain-containing protein (NaPCCP) that binds to the carboxyl-terminal domain of 120K. As C2 domain proteins mediate protein-lipid interactions, NaPCCP could function in intracellular transport of 120K in pollen tubes. Here, we describe binding studies showing that the NaPCCP C2 domain is functional and that binding is specific for phosphatidylinositol 3-phosphate. Subcellular fractionation, immunolocalization, and live imaging results show that NaPCCP is associated with the plasma membrane and internal pollen tube vesicles. Colocalization between an NaPCCP∷green fluorescent protein fusion and internalized FM4-64 suggest an association with the endosomal system. NaPCCP localization is altered in pollen tubes rejected by the self-incompatibility mechanism, but our hypothesis is that it has a general function in the transport of endocytic cargo rather than a specific function in self-incompatibility. NaPCCP represents a bifunctional protein with both phosphatidylinositol 3-phosphate- and arabinogalactan protein-binding domains. Therefore, it could function in the transport of pistil ECM proteins in the pollen tube endomembrane system.Angiosperm sexual reproduction requires pollen transfer to a receptive stigma followed by its hydration, germination, and pollen tube growth. Pollen tubes grow through the stigma and style toward the ovule, where the sperm cells are discharged for fertilization. Pollen tubes do not divide; rather, they extend through tip growth while periodically producing callose plugs, separating highly vacuolated distal regions from the actively growing tip (Taylor and Hepler, 1997). The tip region shows strong zonation. An apical region or clear zone, a subapical, organelle-rich zone, a nuclear zone, and a distal vacuolated zone or plug region that may extend several centimeters are easily recognized (Mascarenhas, 1993). Proper deposition of wall material and rapid tube extension require coordination between GTPase-regulated trafficking pathways, the cytoskeleton, signaling pathways, and oscillatory ion and water fluxes (Li et al., 1999; Fu et al., 2001; Zonia et al., 2002; Camacho and Malhó, 2003; Chen et al., 2003; de Graaf et al., 2005; Gu et al., 2005).Pollen tube endomembrane system dynamics are critical for growth: wall materials are deposited by exocytosis, and the membrane is recovered by endocytosis (Picton and Steer, 1983; Cheung and Wu, 2008). Exocytosis of material synthesized in the Golgi occurs near the tip (Cheung et al., 2002). Additional wall material is produced by membrane-bound callose synthase, but this occurs behind the tip (Brownfield et al., 2007). Distinct endocytosis zones have been identified by pulse-chase membrane labeling, observations of charged nanoparticles, and electron microscopy (Derksen et al., 1995; Moscatelli et al., 2007; Zonia and Munnik, 2008). Clathrin-independent endocytosis occurs at the pollen tube apex; endocytic vesicles clearly contribute to vesicle populations in the clear zone once thought to be composed entirely of exocytic vesicles (Moscatelli et al., 2007; Bove et al., 2008; Zonia and Munnik, 2008). Inhibitor studies suggest that clathrin-dependent endocytosis occurs in the organelle-rich zone a few micrometers back from the tip (Moscatelli et al., 2007). Furthermore, coated vesicles have been observed from 6 to 15 μm from the tip by electron microscopy (Derksen et al., 1995).Pollen-pistil interactions influence pollen tube growth either positively or negatively. Positive effects are evident from the observation that pollen tubes grow as much as 10 times faster and achieve much greater lengths in planta than in culture (Cheung et al., 2000). Self-incompatibility (SI) systems provide the best understood examples of negative effects. In SI, pollen-pistil interactions cause rejection of closely related pollen tubes (de Nettancourt, 2001).Arabinogalactan proteins (AGPs) secreted into the pistil extracellular matrix (ECM) play key roles in both positive and negative interactions, but the underlying molecular interactions with pollen tubes are just beginning to be understood. The transmitting tract-specific (TTS) glycoprotein (Cheung et al., 1995; Wu et al., 1995, 2000) and the 120-kD glycoprotein (120K; Hancock et al., 2005) are pistil AGPs implicated in pollination in Nicotiana. Both are abundant components of the pistil ECM (Cheung et al., 1995; Lind et al., 1996) and share a conserved Cys-rich C-terminal domain (CTD). TTS was first described in Nicotiana tabacum (i.e. NtTTS) as a pollen tube attractant. Pollen tubes grow toward TTS in culture, and its glycosylation levels progressively increase closer to the ovary (Cheung et al., 1995). Pollen tubes deglycosylate TTS, which suggests that TTS may act as a nutritive factor (Wu et al., 1995) and, thus, positively affect pollen tube growth.120K is implicated in SI in Nicotiana alata (Cruz-Garcia et al., 2005; Hancock et al., 2005), a species that displays S-RNase-based gametophytic SI (McClure et al., 1989). In SI, compatibility is controlled by the polymorphic S-locus; pollen is rejected if its S-haplotype matches either of the two S-haplotypes in the diploid pistil (de Nettancourt, 2001). Each S-haplotype is unique and encodes separate pollen- and pistil-specificity genes (Kao and Tsukamoto, 2004). S-RNases determine specificity on the pistil side and directly inhibit the growth of closely related pollen tubes (McClure et al., 1989). S-locus F-box proteins (SLF/SFB) control specificity on the pollen side (Sijacic et al., 2004). SLF/SFB proteins bind S-RNase in vitro and appear to form several distinct complexes with other pollen proteins (Qiao et al., 2004; Hua and Kao, 2006; Huang et al., 2006). SI, therefore, is a clear example of inhibitory pollen-pistil interactions: interaction between a pistil protein, S-RNase, and a pollen protein, SLF/SFB, determines compatibility. However, other pistil factors are also required for SI (McClure et al., 1999; Hancock et al., 2005; McClure and Franklin-Tong, 2006). 120K, for example, is required for SI but does not directly contribute to S-specificity (Hancock et al., 2005).120K was first identified as an abundant component of the transmitting tract ECM that contains both arabinogalactan and extensin-like carbohydrate moieties (Lind et al., 1994). 120K is an S-RNase-binding protein that is taken up by growing pollen tubes (Lind et al., 1996; Cruz-Garcia et al., 2005; Goldraij et al., 2006). Immunolocalization studies show 120K in the pollen tube cytoplasm and associated with pollen tube tonoplast membranes (Lind et al., 1996; Goldraij et al., 2006). Goldraij et al. (2006) also found S-RNase in the lumen of pollen tube vacuoles. In many cases, S-RNase was found in vacuoles with 120K apparently embedded in the surrounding membrane. S-RNase is also found in vacuoles of incompatible pollen tubes, but the breakdown of these vacuoles late in SI and the concomitant release of S-RNase may contribute to the rejection mechanism. Other pistil proteins are also taken up by growing pollen tubes; for example, endocytosis of biotinylated stigma/style Cys-rich adhesin has been reported in lily (Lilium longiflorum) pollen tubes (Kim et al., 2006). Although the uptake of pistil proteins such as 120K and S-RNase has not been well characterized, it is likely that endocytosis and retrograde transport of ECM components occurs on a large scale. Thus, it is important to identify pollen proteins that interact with endocytic cargo from the pistil ECM and that could participate in transport through the pollen tube endomembrane system.We recently described a pollen-specific C2 domain-containing protein, NaPCCP, that interacts with the CTD of the potential cargo proteins, NaTTS and 120K. NaPCCP consists of a short N-terminal domain, an 80-residue C2 domain, and a 79-residue C-terminal region. In vitro pull-down assays showed that the C-terminal region of NaPCCP is sufficient for binding the AGP CTDs (Lee et al., 2008b). Originally implicated in binding mammalian protein kinase C to phosphatidylserine in a calcium-dependent manner (Bazzi and Nelsestuen, 1987, 1990; Brose et al., 1992), C2 domains are now known to contribute to transient membrane association of a variety of proteins with functions that include vesicular transport, lipid modification, GTPase regulation, ubiquitylation, and protein phosphorylation (Coussens et al., 1986; Clark et al., 1991; Brose et al., 1992; Cullen et al., 1995; Dunn et al., 2004). Calcium-independent lipid binding of C2 domain-containing proteins has also been reported (Damer and Creutz, 1994; Fukuda et al., 1994).Here, we report the lipid-binding properties of NaPCCP and its association with the pollen tube endomembrane system. Lipid overlay and liposome-binding experiments show that NaPCCP specifically binds to phosphatidylinositol 3-phosphate (PI3P). Immunolocalization and live imaging studies of compatible pollen tubes show that NaPCCP is associated with the pollen tube plasma membrane (PM) and with punctate structures in the cytoplasm. In SI, incompatible pollen tubes show altered NaPCCP distributions. We speculate that NaPCCP is involved in the uptake and transport of proteins from the ECM.     

SUGAR-INSENSITIVE3, a RING E3 Ligase,Is a New Player in Plant Sugar Response

Sugars, such as sucrose and glucose, have been implicated in the regulation of diverse developmental events in plants and other organisms. We isolated an Arabidopsis (Arabidopsis thaliana) mutant, sugar-insensitive3 (sis3), that is resistant to the inhibitory effects of high concentrations of exogenous glucose and sucrose on early seedling development. In contrast to wild-type plants, sis3 mutants develop green, expanded cotyledons and true leaves when sown on medium containing high concentrations (e.g. 270 mm) of sucrose. Unlike some other sugar response mutants, sis3 exhibits wild-type responses to the inhibitory effects of abscisic acid and paclobutrazol, a gibberellic acid biosynthesis inhibitor, on seed germination. Map-based cloning revealed that SIS3 encodes a RING finger protein. Complementation of the sis3-2 mutant with a genomic SIS3 clone restored sugar sensitivity of sis3-2, confirming the identity of the SIS3 gene. Biochemical analyses demonstrated that SIS3 is functional in an in vitro ubiquitination assay and that the RING motif is sufficient for its activity. Our results indicate that SIS3 encodes a ubiquitin E3 ligase that is a positive regulator of sugar signaling during early seedling development.Almost all living organisms rely on the products of plant photosynthesis for sustenance, either directly or indirectly. Carbohydrates, the major photosynthates, provide both energy and carbon skeletons for fungi, plants, and animals. In addition, sugars, such as Suc and Glc, function as signaling molecules to regulate plant growth, development, gene expression, and metabolic processes. Sugar response pathways are integrated with other signaling pathways, such as those for light, phytohormones, stress, and nitrogen (Dijkwel et al., 1997; Zhou et al., 1998; Roitsch, 1999; Arenas-Huertero et al., 2000; Huijser et al., 2000; Laby et al., 2000; Coruzzi and Zhou, 2001; Rook et al., 2001; Rolland et al., 2006).Several components of plant sugar response pathways have been identified based on the conservation of sugar-sensing mechanisms among eukaryotic cells (Rolland et al., 2001, 2006) or by mutant screens. Yeast HEXOKINASE2 functions in the Glc-mediated catabolite repression pathway (Entian, 1980). In Arabidopsis (Arabidopsis thaliana), mutations in HEXOKINASE1 (HXK1) cause a Glc-insensitive phenotype, and HXK1 demonstrates dual functions in Glc sensing and metabolism (Moore et al., 2003; Cho et al., 2006). Recent studies revealed the involvement of G-protein-coupled receptor systems in sugar response in yeast and Arabidopsis (Chen et al., 2003; Lemaire et al., 2004). Arabidopsis regulator of G-protein signaling1 (rgs1) mutant seedlings are insensitive to 6% Glc (Chen and Jones, 2004), whereas G-protein α-subunit (gpa1) null mutant seedlings are hypersensitive to Glc (Chen et al., 2003). The SNF1/AMPK/SnRK1 protein kinases are postulated to be global regulators of energy control (Polge and Thomas, 2007). Studies conducted on two members of the Arabidopsis SnRK1 (for SNF1-Related Protein Kinases1) family, AKIN10 and AKIN11, have revealed their pivotal roles in stress and sugar signaling (Baena-González et al., 2007). A genetic screen for reduced seedling growth on 175 mm Suc identified the pleiotropic regulatory locus1 (prl1) mutant, which encodes a nuclear WD protein. Further analyses revealed that PRL1 functions in Glc and phytohormone responses (Németh et al., 1998). Interestingly, PRL1 negatively regulates the Arabidopsis SnRK1s AKIN10 and AKIN11 in vitro (Bhalerao et al., 1999).Isolation of additional mutants defective in sugar response has revealed cross talk between sugar and phytohormone response pathways. For example, abscisic acid (ABA) biosynthesis and signaling mutants have been isolated by several genetic screens for seedlings with reduced responses to the inhibitory effects of high levels of Suc or Glc on seedling development. These mutants include abscisic acid-deficient1 (aba1), aba2, aba3, salt-tolerant1/nine-cis-epoxycarotenoid dioxygenase3, abscisic acid-insensitive3 (abi3), and abi4 (Arenas-Huertero et al., 2000; Huijser et al., 2000; Laby et al., 2000; Rook et al., 2001; Cheng et al., 2002; Rolland et al., 2002; Huang et al., 2008), indicating interplay between ABA- and sugar-mediated signaling. Ethylene also exhibits interactions with sugars in controlling seedling development. Both the ethylene overproduction mutant eto1 and the constitutive ethylene response mutant ctr1 exhibit Glc (Zhou et al., 1998) and Suc (Gibson et al., 2001) insensitivity, whereas the ethylene-insensitive mutants etr1, ein2, and ein4 show sugar hypersensitivity (Zhou et al., 1998; Gibson et al., 2001; Cheng et al., 2002).Further characterization of sugar response factors has suggested that ubiquitin-mediated protein degradation may play a role in sugar response. In particular, the PRL1-binding domains of SnRK1s have been shown to recruit SKP1/ASK1, a conserved SCF ubiquitin ligase subunit, as well as the α4/PAD1 proteasomal subunit, indicating a role for SnRK1s in mediating proteasomal binding of SCF ubiquitin ligases (Farrás et al., 2001). In addition, recent studies indicate that PRL1 is part of a CUL4-based E3 ligase and that AKIN10 exhibits decreased rates of degradation in prl1 than in wild-type extracts (Lee et al., 2008). The ubiquitin/26S proteasome pathway plays important roles in many cellular processes and signal transduction pathways in yeast, animals, and plants (Hochstrasser, 1996; Hershko and Ciechanover, 1998; Smalle and Vierstra, 2004). The key task of the pathway is to selectively ubiquitinate substrate proteins and target them for degradation by the 26S proteasome. In short, the multistep ubiquitination process starts with the formation of a thiol-ester linkage between ubiquitin and a ubiquitin-activating enzyme (E1). The activated ubiquitin is then transferred to a ubiquitin-conjugating enzyme (E2), and a ubiquitin protein ligase (E3) then mediates the covalent attachment of ubiquitin to the substrate protein. The specificity of the pathway is largely realized by the E3s, which recognize the substrates that should be ubiquitinated. In Arabidopsis, more than 1,300 genes encode putative E3 subunits and the E3 ligases can be grouped into defined families based upon the presence of HECT (for Homology to E6-AP C Terminus), RING (for Really Interesting New Gene), or U-box domains (Smalle and Vierstra, 2004). The RING-type E3s can be subdivided into single-subunit E3s, which contain the substrate recognition and RING finger domains on the same protein, and multisubunit E3s, which include the SCF (for Skp1-Cullin-F-box), CUL3-BTB (for Broad-complex, Tramtrack, Bric-a-Brac), and APC (for Anaphase-Promoting Complex) complexes (Weissman, 2001; Moon et al., 2004).The Cys-rich RING finger was first described in the early 1990s (Freemont et al., 1991). It is defined as a linear series of conserved Cys and His residues (C3HC/HC3) that bind two zinc atoms in a cross-brace arrangement. RING fingers can be divided into two types, C3HC4 (RING-HC) and C3H2C3 (RING-H2), depending on the presence of either a Cys or a His residue in the fifth position of the motif (Lovering et al., 1993; Freemont, 2000). A recent study of the RING finger ubiquitin ligase family encoded by the Arabidopsis genome resulted in the identification of 469 predicted proteins containing one or more RING domains (Stone et al., 2005). However, the in vivo biological functions of all but a few of the RING proteins remain unknown. Recent studies have implicated several Arabidopsis RING proteins in a variety biological processes, including COP1 and CIP8 (photomorphogenesis; Hardtke et al., 2002; Seo et al., 2004), SINAT5 (auxin signaling; Xie et al., 2002), ATL2 (defense signaling; Serrano and Guzman, 2004), BRH1 (brassinosteroid response; Molnár et al., 2002), RIE1 (seed development; Xu and Li, 2003), NLA (nitrogen limitation adaptation; Peng et al., 2007), HOS1 (cold response; Dong et al., 2006), AIP2 (ABA signaling; Zhang et al., 2005), KEG (ABA signaling; Stone et al., 2006), and SDIR1 (ABA signaling; Zhang et al., 2007).Here, we report the isolation, identification, and characterization of an Arabidopsis mutant, sugar-insensitive3 (sis3), which is resistant to the early seedling developmental arrest caused by high exogenous sugar levels. The responsible locus, SIS3, was identified through a map-based cloning approach and confirmed with additional T-DNA insertional mutants and complementation tests. The SIS3 gene encodes a protein with a RING-H2 domain and three putative transmembrane domains. Glutathione S-transferase (GST)-SIS3 recombinant proteins exhibit in vitro ubiquitin E3 ligase activity. Together, these results indicate that a ubiquitination pathway involving the SIS3 RING protein is required to mediate the sugar response during early seedling development.     

Phosphorylation of the C Terminus of RHD3 Has a Critical Role in Homotypic ER Membrane Fusion in Arabidopsis

The endoplasmic reticulum (ER) consists of dynamically changing tubules and cisternae. In animals and yeast, homotypic ER membrane fusion is mediated by fusogens (atlastin and Sey1p, respectively) that are membrane-associated dynamin-like GTPases. In Arabidopsis (Arabidopsis thaliana), another dynamin-like GTPase, ROOT HAIR DEFECTIVE3 (RHD3), has been proposed as an ER membrane fusogen, but direct evidence is lacking. Here, we show that RHD3 has an ER membrane fusion activity that is enhanced by phosphorylation of its C terminus. The ER network was RHD3-dependently reconstituted from the cytosol and microsome fraction of tobacco (Nicotiana tabacum) cultured cells by exogenously adding GTP, ATP, and F-actin. We next established an in vitro assay system of ER tubule formation with Arabidopsis ER vesicles, in which addition of GTP caused ER sac formation from the ER vesicles. Subsequent application of a shearing force to this system triggered the formation of tubules from the ER sacs in an RHD-dependent manner. Unexpectedly, in the absence of a shearing force, Ser/Thr kinase treatment triggered RHD3-dependent tubule formation. Mass spectrometry showed that RHD3 was phosphorylated at multiple Ser and Thr residues in the C terminus. An antibody against the RHD3 C-terminal peptide abolished kinase-triggered tubule formation. When the Ser cluster was deleted or when the Ser residues were replaced with Ala residues, kinase treatment had no effect on tubule formation. Kinase treatment induced the oligomerization of RHD3. Neither phosphorylation-dependent modulation of membrane fusion nor oligomerization has been reported for atlastin or Sey1p. Taken together, we propose that phosphorylation-stimulated oligomerization of RHD3 enhances ER membrane fusion to form the ER network.In eukaryotic cells, the endoplasmic reticulum (ER) is the organelle with the largest membrane area. The ER consists of an elaborate network of interconnected membrane tubules and cisternae that is continually moving and being remodeled (Friedman and Voeltz, 2011). In plant cells, ER movement and remodeling is primarily driven by the actin-myosin XI cytoskeleton (Sparkes et al., 2009; Ueda et al., 2010; Yokota et al., 2011; Griffing et al., 2014) and secondarily by the microtubule cytoskeleton (Hamada et al., 2014). Several factors involved in creating the ER architecture have been also identified (Anwar et al., 2012; Chen et al., 2012; Goyal and Blackstone, 2013; Sackmann, 2014; Stefano et al., 2014a; Westrate et al., 2015). Among them, ER membrane-bound GTPases, animal atlastins and yeast Sey1p (Synthetic Enhancement of Yop1), function as ER fusogens to form the interconnected tubular network (Hu et al., 2009; Orso et al., 2009; Anwar et al., 2012). Atlastin molecules on the two opposed membranes have been proposed to transiently dimerize to attract the two membranes to each other (Bian et al., 2011; Byrnes and Sondermann, 2011; Morin-Leisk et al., 2011; Moss et al., 2011; Lin et al., 2012; Byrnes et al., 2013). Closely attracted lipid bilayers are supposed to be destabilized by an amphipathic helical domain at the atlastin C terminus to facilitate membrane fusion (Bian et al., 2011; Liu et al., 2012; Faust et al., 2015). Knockdown of atlastins leads to fragmentation of the ER and unbranched ER tubules, while overexpression of atlastins enhances ER membrane fusion, which enlarges the ER profiles (Hu et al., 2009; Orso et al., 2009).An Arabidopsis (Arabidopsis thaliana) protein, ROOT HAIR DEFECTIVE3 (RHD3), has been proposed as a fusogen because (1) when it is disrupted, the ER network is modified into large cable-like strands of poorly branched membranes (Zheng et al., 2004; Chen et al., 2011; Stefano et al., 2012), (2) it shares sequence similarity with the above-mentioned fusogen Sey1p (Hu et al., 2009), and (3) it has structural similarity to atlastin and Sey1p, with a functional GTPase domain at the N-terminal cytosolic domain (Stefano et al., 2012) followed by two transmembrane domains and a cytosolic tail. RHD3 has a longer cytosolic C-terminal tail than do atlastin and Sey1p (Stefano and Brandizzi, 2014). It contains not only an amphipathic region but also a Ser/Thr-rich C terminus.Arabidopsis has two RHD3 isoforms called RHD3-Like 1 and RHD3-Like 2. Fluorescently tagged RHD3 and RHD3-Like 2 localize to the ER (Chen et al., 2011; Stefano et al., 2012; Lee et al., 2013). RHD3 and the two RHD3-Like proteins likely have redundant roles in ER membrane fusion (Zhang et al., 2013). Overexpression of either RHD3 or RHD3-Like 2 with a defective GTPase domain phenocopies the aberrant ER morphology in rhd3-deficient mutants (Chen et al., 2011; Lee et al., 2013).In this study, we show that the Ser/Thr-rich C terminus enhances ER membrane fusion following phosphorylation of its C terminus. We propose a model in which phosphorylation and oligomerization of RHD3 is required for efficient ER membrane fusion. Our findings clarify the mechanisms that regulate RHD3 and consequently the homeostasis of membrane fusion in the ER.     

Calcineurin B-Like Protein-Interacting Protein Kinase CIPK21 Regulates Osmotic and Salt Stress Responses in Arabidopsis

The role of calcium-mediated signaling has been extensively studied in plant responses to abiotic stress signals. Calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs) constitute a complex signaling network acting in diverse plant stress responses. Osmotic stress imposed by soil salinity and drought is a major abiotic stress that impedes plant growth and development and involves calcium-signaling processes. In this study, we report the functional analysis of CIPK21, an Arabidopsis (Arabidopsis thaliana) CBL-interacting protein kinase, ubiquitously expressed in plant tissues and up-regulated under multiple abiotic stress conditions. The growth of a loss-of-function mutant of CIPK21, cipk21, was hypersensitive to high salt and osmotic stress conditions. The calcium sensors CBL2 and CBL3 were found to physically interact with CIPK21 and target this kinase to the tonoplast. Moreover, preferential localization of CIPK21 to the tonoplast was detected under salt stress condition when coexpressed with CBL2 or CBL3. These findings suggest that CIPK21 mediates responses to salt stress condition in Arabidopsis, at least in part, by regulating ion and water homeostasis across the vacuolar membranes.Drought and salinity cause osmotic stress in plants and severely affect crop productivity throughout the world. Plants respond to osmotic stress by changing a number of cellular processes (Xiong et al., 1999; Xiong and Zhu, 2002; Bartels and Sunkar, 2005; Boudsocq and Lauriére, 2005). Some of these changes include activation of stress-responsive genes, regulation of membrane transport at both plasma membrane (PM) and vacuolar membrane (tonoplast) to maintain water and ionic homeostasis, and metabolic changes to produce compatible osmolytes such as Pro (Stewart and Lee, 1974; Krasensky and Jonak, 2012). It has been well established that a specific calcium (Ca²⁺) signature is generated in response to a particular environmental stimulus (Trewavas and Malhó, 1998; Scrase-Field and Knight, 2003; Luan, 2009; Kudla et al., 2010). The Ca²⁺ changes are primarily perceived by several Ca²⁺ sensors such as calmodulin (Reddy, 2001; Luan et al., 2002), Ca²⁺-dependent protein kinases (Harper and Harmon, 2005), calcineurin B-like proteins (CBLs; Luan et al., 2002; Batistič and Kudla, 2004; Pandey, 2008; Luan, 2009; Sanyal et al., 2015), and other Ca²⁺-binding proteins (Reddy, 2001; Shao et al., 2008) to initiate various cellular responses.Plant CBL-type Ca²⁺ sensors interact with and activate CBL-interacting protein kinases (CIPKs) that phosphorylate downstream components to transduce Ca²⁺ signals (Liu et al., 2000; Luan et al., 2002; Batistič and Kudla, 2004; Luan, 2009). In several plant species, multiple members have been identified in the CBL and CIPK family (Luan et al., 2002; Kolukisaoglu et al., 2004; Pandey, 2008; Batistič and Kudla, 2009; Weinl and Kudla, 2009; Pandey et al., 2014). Involvement of specific CBL-CIPK pair to decode a particular type of signal entails the alternative and selective complex formation leading to stimulus-response coupling (D’Angelo et al., 2006; Batistič et al., 2010).Several CBL and CIPK family members have been implicated in plant responses to drought, salinity, and osmotic stress based on genetic analysis of Arabidopsis (Arabidopsis thaliana) mutants (Zhu, 2002; Cheong et al., 2003, 2007; Kim et al., 2003; Pandey et al., 2004, 2008; D’Angelo et al., 2006; Qin et al., 2008; Tripathi et al., 2009; Held et al., 2011; Tang et al., 2012; Drerup et al., 2013; Eckert et al., 2014). A few CIPKs have also been functionally characterized by gain-of-function approach in crop plants such as rice (Oryza sativa), pea (Pisum sativum), and maize (Zea mays) and were found to be involved in osmotic stress responses (Mahajan et al., 2006; Xiang et al., 2007; Yang et al., 2008; Tripathi et al., 2009; Zhao et al., 2009; Cuéllar et al., 2010).In this report, we examined the role of the Arabidopsis CIPK21 gene in osmotic stress response by reverse genetic analysis. The loss-of-function mutant plants became hypersensitive to salt and mannitol stress conditions, suggesting that CIPK21 is involved in the regulation of osmotic stress response in Arabidopsis. These findings are further supported by an enhanced tonoplast targeting of the cytoplasmic CIPK21 through interaction with the vacuolar Ca²⁺ sensors CBL2 and CBL3 under salt stress condition.