Gene Regulatory Variation Mediates Flowering Responses to Vernalization along an Altitudinal Gradient in Arabidopsis

Steep environmental gradients provide ideal settings for studies of potentially adaptive phenotypic and genetic variation in plants. The accurate timing of flowering is crucial for reproductive success and is regulated by several pathways, including the vernalization pathway. Among the numerous genes known to enable flowering in response to vernalization, the most prominent is FLOWERING LOCUS C (FLC). FLC and other genes of the vernalization pathway vary extensively among natural populations and are thus candidates for the adaptation of flowering time to environmental gradients such as altitude. We used 15 natural Arabidopsis (Arabidopsis thaliana) genotypes originating from an altitudinal gradient (800–2,700 m above sea level) in the Swiss Alps to test whether flowering time correlated with altitude under different vernalization scenarios. Additionally, we measured the expression of 12 genes of the vernalization pathway and its downstream targets. Flowering time correlated with altitude in a nonlinear manner for vernalized plants. Flowering time could be explained by the expression and regulation of the vernalization pathway, most notably by AGAMOUS LIKE19 (AGL19), FLOWERING LOCUS T (FT), and FLC. The expression of AGL19, FT, and VERNALIZATION INSENSITIVE3 was associated with altitude, and the regulation of MADS AFFECTING FLOWERING2 (MAF2) and MAF3 differed between low- and high-altitude genotypes. In conclusion, we found clinal variation across an altitudinal gradient both in flowering time and the expression and regulation of genes in the flowering time control network, often independent of FLC, suggesting that the timing of flowering may contribute to altitudinal adaptation.Environmental gradients, such as temperature or water availability, provide an ideal setting to study how species adapt to contrasting environmental scenarios (Reich et al., 2003; Keller et al., 2013). Many studies have shown that phenotypic plant traits such as leaf number, allocation to reproductive biomass, and height change along environmental gradients (Etterson, 2004; Leger and Rice, 2007; Fischer et al., 2011), and some studies could correlate environmental clines to changes in allelic frequencies at specific candidate genes (Manel et al., 2010; Poncet et al., 2010; Fischer et al., 2013).Although allelic variation at genes with major effects may explain variation in some phenotypes, fine-tuning of other quantitative traits along an environmental gradient may require an adjustment of larger regulatory networks (Whitehead and Crawford, 2006; Hodgins-Davis and Townsend, 2009; Hodgins et al., 2013). In Arabidopsis (Arabidopsis thaliana), numerous genetic pathways have been studied extensively, mainly using laboratory accessions (Shinozaki and Yamaguchi-Shinozaki, 2007; Wellmer and Riechmann, 2010; Ó’Maoiléidigh et al., 2014). However, how consistently such pathways are expressed in natural populations, and how they respond to different environmental conditions, often remains unclear. Studying the expression of genetic pathways in natural genotypes originating from an environmental cline under a variety of climatic scenarios provides an ideal approach to understanding how plants can adapt to contrasting environments along a climatic gradient.Across altitudes, environmental gradients are particularly steep: climatic conditions, including temperature, solar radiation, and precipitation, may change dramatically on a small geographic scale (Körner, 2007), while daylength and other factors remain constant. Many phenotypic traits, such as height, total seed weight, leaf size, and allocation to vegetative reproduction, have been found to change along altitudinal gradients in plants (Byars et al., 2007; Gonzalo-Turpin and Hazard, 2009; Fischer et al., 2011). Among these, the timing of flowering (i.e. the transition from vegetative growth to the reproductive phase) is a key developmental phase transition in seasonal alpine environments, as its accuracy is crucial for reproductive success: too-early flowering increases the risk of encountering detrimental frost (Kollas et al., 2013), whereas time for seed maturation may run out if flowering starts too late (Inouye and Wielgolaski, 2003; Chuine, 2010). These contrasting selective pressures may change along an altitudinal gradient, where the vegetation period becomes shorter with increasing altitude.In Arabidopsis, an annual weed native to Eurasia and northern Africa, two different life cycles have been described (Koornneef et al., 2004; Alonso-Blanco et al., 2009): summer annuals germinate and flower within one growing season and do not require winter to initiate flowering; winter annuals germinate usually in autumn, overwinter as vegetative rosettes, and flower in the following spring. Accessions expressing a winter-annual life cycle need vernalization (a prolonged cold period) in order to initiate flowering; otherwise, they remain in a vegetative rosette stage for an extended period of time.On the molecular level, the transition to flowering is among the best-studied processes in plants (Wellmer and Riechmann, 2010; Andrés and Coupland, 2012), and in Arabidopsis, several genetic pathways controlling flowering are known. Signals from the vernalization pathway, photoperiod pathway, autonomous pathway, GA pathway, and plant age all contribute to ensuring the correct timing of flowering (Ehrenreich et al., 2009; Wellmer and Riechmann, 2010; Srikanth and Schmid, 2011). Within the vernalization pathway, a number of key players have been identified (Andrés and Coupland, 2012; Schmitz and Amasino, 2012; Song et al., 2012; Zografos and Sung, 2012). In winter annuals, a functional FRIGIDA (FRI) allele is required to activate FLOWERING LOCUS C (FLC). FLC strongly suppresses the flowering promoters FLOWERING LOCUS T (FT) and AGAMOUS LIKE20 (AGL20; also referred to as SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1) and thus inhibits flowering. During vernalization, VERNALIZATION INSENSITIVE3 (VIN3) represses FLC, and the repressed state is maintained in subsequent warm periods by epigenetic silencing (Crevillén and Dean, 2011; Zografos and Sung, 2012), allowing FT, AGL20, and, through positive feedback with AGL20, AGL24 (Liu et al., 2008) to initiate flowering. Many natural populations and most laboratory accessions, among them Columbia-0 (Col-0), carry nonfunctional FRI or FLC alleles and thus respond only weakly to vernalization, resulting in fast flowering, summer-annual life cycles (Johanson et al., 2000; Gazzani et al., 2003; Shindo et al., 2005).In addition to the well-studied FLC branch of the vernalization pathway, FLC-independent components of the vernalization response have been identified. For example, AGL19 has been found to promote flowering following vernalization without interacting with FLC (Schönrock et al., 2006), and relatives of FLC, the MADS AFFECTING FLOWERING genes (MAF1–MAF5; MAF1 is also referred to as FLOWERING LOCUS M [FLM]; De Bodt et al., 2003), have been shown to inhibit flowering in a similar way to FLC (Ratcliffe et al., 2003; Scortecci et al., 2003; Werner et al., 2005; Sung et al., 2006; Gu et al., 2013). Genes MAF2 to MAF5 (Ratcliffe et al., 2003) are arranged in a tandem gene array and vary extensively among natural populations (Caicedo et al., 2009; Rosloski et al., 2010), and several recent studies have associated this polymorphic region with natural variation in flowering time (Salomé et al., 2011; Silady et al., 2011; Lasky et al., 2012; Fournier-Level et al., 2013; Grillo et al., 2013), making these genes interesting candidates for studying associations between flowering time and ecological parameters.Associating genetic variation at a single gene with latitude or altitude has often proven to be difficult (Shindo et al., 2005; Stinchcombe et al., 2005; Méndez-Vigo et al., 2011), although Caicedo et al. (2004) found evidence for epistatic interactions between FRI and FLC alleles associated with latitude. Interestingly, some recent studies suggest that regulatory processes within the vernalization pathway may contribute to natural phenotypic variability (Shindo et al., 2006; Strange et al., 2011). Overall, the response to vernalization appears to be a complex process in natural populations, potentially involving epigenetic regulation of a number of genes. Therefore, to gain a better understanding of the involvement of this complex genetic network in the response to ecological parameters, it is essential to study multiple interacting genes of the vernalization pathway simultaneously.Here, we used 15 natural Arabidopsis genotypes originating from an altitudinal cline (800–2,700 m) in the Swiss Alps to study the associations between vernalization, flowering initiation, gene expression and regulation, and altitude. Importantly, all genotypes originated from a restricted geographic range; thus, confounding effects such as differences in daylength, as found along latitudinal clines, can be excluded. We measured flowering time and the expression of 12 genes of the vernalization pathway under different vernalization scenarios to assess whether the response to vernalization is associated with altitude. In particular, we tested the hypotheses that (1) flowering time correlates with altitude; (2) genotypes from high altitudes need longer vernalization periods to initiate flowering reliably; (3) gene expression and regulation of the vernalization pathway can explain flowering time; (4) gene expression and regulation of the vernalization pathway is associated with altitude; and (5) FLC-independent branches of the vernalization pathway are important for initiating flowering and, thus, for altitudinal adaptation in natural populations.     

Evidence That High Activity of Vacuolar Invertase Is Required for Cotton Fiber and Arabidopsis Root Elongation through Osmotic Dependent and Independent Pathways,Respectively

Vacuolar invertase (VIN) has long been considered as a major player in cell expansion. However, direct evidence for this view is lacking due, in part, to the complexity of multicellular plant tissues. Here, we used cotton (Gossypium spp.) fibers, fast-growing single-celled seed trichomes, to address this issue. VIN activity in elongating fibers was approximately 4-6-fold higher than that in leaves, stems, and roots. It was undetectable in fiberless cotton seed epidermis but became evident in initiating fibers and remained high during their fast elongation and dropped when elongation slowed. Furthermore, a genotype with faster fiber elongation had significantly higher fiber VIN activity and hexose levels than a slow-elongating genotype. By contrast, cell wall or cytoplasmic invertase activities did not show correlation with fiber elongation. To unravel the molecular basis of VIN-mediated fiber elongation, we cloned GhVIN1, which displayed VIN sequence features and localized to the vacuole. Once introduced to Arabidopsis (Arabidopsis thaliana), GhVIN1 complemented the short-root phenotype of a VIN T-DNA mutant and enhanced the elongation of root cells in the wild type. This demonstrates that GhVIN1 functions as VIN in vivo. In cotton fiber, GhVIN1 expression level matched closely with VIN activity and fiber elongation rate. Indeed, transformation of cotton fiber with GhVIN1 RNA interference or overexpression constructs reduced or enhanced fiber elongation, respectively. Together, these analyses provide evidence on the role of VIN in cotton fiber elongation mediated by GhVIN1. Based on the relative contributions of sugars to sap osmolality in cotton fiber and Arabidopsis root, we conclude that VIN regulates their elongation in an osmotic dependent and independent manner, respectively.Suc is the principal end product of photosynthesis in higher plants and the major carbohydrate translocated from source to sink tissues through phloem. Suc cleavage, serving as a starting point for various carbohydrate metabolic pathways, is catalyzed by Suc synthase (EC 2.4.1.13) and invertase (β-fructofuranosidase; EC 3.2.1.26). In contrast to the reversible reaction of Suc synthase, invertase irreversibly hydrolyzes Suc to Fru and Glc. This hydrolysis step is required for the development of many sink tissues and their responses to various stresses (Sturm, 1999; Weschke et al., 2003; Roitsch and González, 2004; Huang et al., 2007; Essmann et al., 2008; Jin et al., 2009; for a recent review, see Ruan et al., 2010).Based on their pH optimums and subcellular localizations, invertases are classified into three isoforms: a nonglycosylated cytosolic invertase (CIN), with an optimal pH of 7.0 to 7.8, and highly glycosylated acid invertases with an optimum pH of 3.5 to 5.5 either tightly bound to cell wall (CWIN) or appearing as a soluble form inside the vacuole (VIN; Roitsch and González, 2004). Mutational and transgenic studies have established the critical roles of CWIN in the development of seed (Cheng et al., 1996; Ruan et al., 2003), pollen (Roitsch et al., 2003), root (Tang et al., 1999), and leaf and fruit (Jin et al., 2009). By contrast, much less is known about the function of VIN or CIN (Ruan et al., 2010).High VIN expression or activity has been observed in a range of expanding tissues, including maize (Zea mays) ovaries (Andersen et al., 2002; McLaughlin and Boyer, 2004), grape (Vitis vinifera) berry (Davies and Robinson, 1996), carrot (Daucus carota) taproot (Tang et al., 1999), and sugar beet (Beta vulgaris) petioles (González et al., 2005). It is hypothesized that VIN may play a major role in plant cell expansion, a key step in plant cell development (González et al., 2005). However, progress in determining the roles of VIN in cell expansion suffers from several experimental limitations. Most notably, the multicellular nature of plant tissues makes it difficult to quantitatively evaluate the contribution of VIN in specific cell types. For example, decrease of VIN expression is associated with maize ovary abortion or reduction in its expansion (Andersen et al., 2002; McLaughlin and Boyer, 2004). The VIN gene Ivr2, however, is expressed in nucellus and vascular bundles of the pedicel deeply embedded within the pericarp (Andersen et al., 2002). This inherent anatomical feature makes it challenging to experimentally assess the role of invertase in these cells.In this context, developing cotton (Gossypium hirsutum) fiber offers a tractable experimental system to study the role of invertase in cell expansion for the following reasons. First, after initiation from the ovule epidermis at anthesis, the single-celled cotton fibers undergo rapid and synchronized unidirectional expansion to several centimeters long by approximately 18 d after anthesis (DAA; Ruan et al., 2001). Hence, a large quantity of homogenous single cells can be readily harvested for studying the control of cell expansion (Ruan, 2007). Second, compelling evidence has indicated a major role of osmotically active solutes in fiber elongation through the generation of cell turgor (Ruan et al., 2004). To this end, Suc moves into fibers symplasmically early in elongation (Ruan et al., 2001), and hexoses accumulated in the vacuole are major osmotically active solutes in the fiber sap (Dhindsa et al., 1975; Ruan et al., 1997), where VIN activity has been reported (Wäfler and Meier, 1994). These observations raise the possibility that VIN may be a central player in osmotically driven fiber expansion (Andersen et al., 2002; Ruan, 2005). Finally, elucidating the role of VIN in cotton fiber could help us not only better understand the control of rapid cell expansion but also identify novel ways to increase fiber length, a key quality and yield determinant of cotton, the most important textile crop worldwide (Ruan, 2005).This study aims to examine the role of VIN in cell expansion by using cotton fiber as a model, coupled with integrative analyses on elongating root of Arabidopsis (Arabidopsis thaliana). A combination of cellular, biochemical, and molecular genetic analyses show that (1) rapid fiber expansion requires high activity of VIN, which is probably exerted by the expression of GhVIN1, and (2) the impact on cotton fiber and Arabidopsis root elongation by VIN is most likely achieved through an osmotic dependent and independent manner, respectively.     

Characterization of the Possible Roles for B Class MADS Box Genes in Regulation of Perianth Formation in Orchid

To investigate sepal/petal/lip formation in Oncidium Gower Ramsey, three paleoAPETALA3 genes, O. Gower Ramsey MADS box gene5 (OMADS5; clade 1), OMADS3 (clade 2), and OMADS9 (clade 3), and one PISTILLATA gene, OMADS8, were characterized. The OMADS8 and OMADS3 mRNAs were expressed in all four floral organs as well as in vegetative leaves. The OMADS9 mRNA was only strongly detected in petals and lips. The mRNA for OMADS5 was only strongly detected in sepals and petals and was significantly down-regulated in lip-like petals and lip-like sepals of peloric mutant flowers. This result revealed a possible negative role for OMADS5 in regulating lip formation. Yeast two-hybrid analysis indicated that OMADS5 formed homodimers and heterodimers with OMADS3 and OMADS9. OMADS8 only formed heterodimers with OMADS3, whereas OMADS3 and OMADS9 formed homodimers and heterodimers with each other. We proposed that sepal/petal/lip formation needs the presence of OMADS3/8 and/or OMADS9. The determination of the final organ identity for the sepal/petal/lip likely depended on the presence or absence of OMADS5. The presence of OMADS5 caused short sepal/petal formation. When OMADS5 was absent, cells could proliferate, resulting in the possible formation of large lips and the conversion of the sepal/petal into lips in peloric mutants. Further analysis indicated that only ectopic expression of OMADS8 but not OMADS5/9 caused the conversion of the sepal into an expanded petal-like structure in transgenic Arabidopsis (Arabidopsis thaliana) plants.The ABCDE model predicts the formation of any flower organ by the interaction of five classes of homeotic genes in plants (Yanofsky et al., 1990; Jack et al., 1992; Mandel et al., 1992; Goto and Meyerowitz, 1994; Jofuku et al., 1994; Pelaz et al., 2000, 2001; Theißen and Saedler, 2001; Pinyopich et al., 2003; Ditta et al., 2004; Jack, 2004). The A class genes control sepal formation. The A, B, and E class genes work together to regulate petal formation. The B, C, and E class genes control stamen formation. The C and E class genes work to regulate carpel formation, whereas the D class gene is involved in ovule development. MADS box genes seem to have a central role in flower development, because most ABCDE genes encode MADS box proteins (Coen and Meyerowitz, 1991; Weigel and Meyerowitz, 1994; Purugganan et al., 1995; Rounsley et al., 1995; Theißen and Saedler, 1995; Theißen et al., 2000; Theißen, 2001).The function of B group genes, such as APETALA3 (AP3) and PISTILLATA (PI), has been thought to have a major role in specifying petal and stamen development (Jack et al., 1992; Goto and Meyerowitz, 1994; Krizek and Meyerowitz, 1996; Kramer et al., 1998; Hernandez-Hernandez et al., 2007; Kanno et al., 2007; Whipple et al., 2007; Irish, 2009). In Arabidopsis (Arabidopsis thaliana), mutation in AP3 or PI caused identical phenotypes of second whorl petal conversion into a sepal structure and third flower whorl stamen into a carpel structure (Bowman et al., 1989; Jack et al., 1992; Goto and Meyerowitz, 1994). Similar homeotic conversions for petal and stamen were observed in the mutants of the AP3 and PI orthologs from a number of core eudicots such as Antirrhinum majus, Petunia hybrida, Gerbera hybrida, Solanum lycopersicum, and Nicotiana benthamiana (Sommer et al., 1990; Tröbner et al., 1992; Angenent et al., 1993; van der Krol et al., 1993; Yu et al., 1999; Liu et al., 2004; Vandenbussche et al., 2004; de Martino et al., 2006), from basal eudicot species such as Papaver somniferum and Aquilegia vulgaris (Drea et al., 2007; Kramer et al., 2007), as well as from monocot species such as Zea mays and Oryza sativa (Ambrose et al., 2000; Nagasawa et al., 2003; Prasad and Vijayraghavan, 2003; Yadav et al., 2007; Yao et al., 2008). This indicated that the function of the B class genes AP3 and PI is highly conserved during evolution.It has been thought that B group genes may have arisen from an ancestral gene through multiple gene duplication events (Doyle, 1994; Theißen et al., 1996, 2000; Purugganan, 1997; Kramer et al., 1998; Kramer and Irish, 1999; Lamb and Irish, 2003; Kim et al., 2004; Stellari et al., 2004; Zahn et al., 2005; Hernandez-Hernandez et al., 2007). In the gymnosperms, there was a single putative B class lineage that duplicated to generate the paleoAP3 and PI lineages in angiosperms (Kramer et al., 1998; Theißen et al., 2000; Irish, 2009). The paleoAP3 lineage is composed of AP3 orthologs identified in lower eudicots, magnolid dicots, and monocots (Kramer et al., 1998). Genes in this lineage contain the conserved paleoAP3- and PI-derived motifs in the C-terminal end of the proteins, which have been thought to be characteristics of the B class ancestral gene (Kramer et al., 1998; Tzeng and Yang, 2001; Hsu and Yang, 2002). The PI lineage is composed of PI orthologs that contain a highly conserved PI motif identified in most plant species (Kramer et al., 1998). Subsequently, there was a second duplication at the base of the core eudicots that produced the euAP3 and TM6 lineages, which have been subject to substantial sequence changes in eudicots during evolution (Kramer et al., 1998; Kramer and Irish, 1999). The paleoAP3 motif in the C-terminal end of the proteins was retained in the TM6 lineage and replaced by a conserved euAP3 motif in the euAP3 lineage of most eudicot species (Kramer et al., 1998). In addition, many lineage-specific duplications for paleoAP3 lineage have occurred in plants such as orchids (Hsu and Yang, 2002; Tsai et al., 2004; Kim et al., 2007; Mondragón-Palomino and Theißen, 2008, 2009; Mondragón-Palomino et al., 2009), Ranunculaceae, and Ranunculales (Kramer et al., 2003; Di Stilio et al., 2005; Shan et al., 2006; Kramer, 2009).Unlike the A or C class MADS box proteins, which form homodimers that regulate flower development, the ability of B class proteins to form homodimers has only been reported in gymnosperms and in the paleoAP3 and PI lineages of some monocots. For example, LMADS1 of the lily Lilium longiflorum (Tzeng and Yang, 2001), OMADS3 of the orchid Oncidium Gower Ramsey (Hsu and Yang, 2002), and PeMADS4 of the orchid Phalaenopsis equestris (Tsai et al., 2004) in the paleoAP3 lineage, LRGLOA and LRGLOB of the lily Lilium regale (Winter et al., 2002), TGGLO of the tulip Tulipa gesneriana (Kanno et al., 2003), and PeMADS6 of the orchid P. equestris (Tsai et al., 2005) in the PI lineage, and GGM2 of the gymnosperm Gnetum gnemon (Winter et al., 1999) were able to form homodimers that regulate flower development. Proteins in the euAP3 lineage and in most paleoAP3 lineages were not able to form homodimers and had to interact with PI to form heterodimers in order to regulate petal and stamen development in various plant species (Schwarz-Sommer et al., 1992; Tröbner et al., 1992; Riechmann et al., 1996; Moon et al., 1999; Winter et al., 2002; Kanno et al., 2003; Vandenbussche et al., 2004; Yao et al., 2008). In addition to forming dimers, AP3 and PI were able to interact with other MADS box proteins, such as SEPALLATA1 (SEP1), SEP2, and SEP3, to regulate petal and stamen development (Pelaz et al., 2000; Honma and Goto, 2001; Theißen and Saedler, 2001; Castillejo et al., 2005).Orchids are among the most important plants in the flower market around the world, and research on MADS box genes has been reported for several species of orchids during the past few years (Lu et al., 1993, 2007; Yu and Goh, 2000; Hsu and Yang, 2002; Yu et al., 2002; Hsu et al., 2003; Tsai et al., 2004, 2008; Xu et al., 2006; Guo et al., 2007; Kim et al., 2007; Chang et al., 2009). Unlike the flowers in eudicots, the nearly identical shape of the sepals and petals as well as the production of a unique lip in orchid flowers make them a very special plant species for the study of flower development. Four clades (1–4) of genes in the paleoAP3 lineage have been identified in several orchids (Hsu and Yang, 2002; Tsai et al., 2004; Kim et al., 2007; Mondragón-Palomino and Theißen, 2008, 2009; Mondragón-Palomino et al., 2009). Several works have described the possible interactions among these four clades of paleoAP3 genes and one PI gene that are involved in regulating the differentiation and formation of the sepal/petal/lip of orchids (Tsai et al., 2004; Kim et al., 2007; Mondragón-Palomino and Theißen, 2008, 2009). However, the exact mechanism that involves the orchid B class genes remains unclear and needs to be clarified by more experimental investigations.O. Gower Ramsey is a popular orchid with important economic value in cut flower markets. Only a few studies have been reported on the role of MADS box genes in regulating flower formation in this plant species (Hsu and Yang, 2002; Hsu et al., 2003; Chang et al., 2009). An AP3-like MADS gene that regulates both floral formation and initiation in transgenic Arabidopsis has been reported (Hsu and Yang, 2002). In addition, four AP1/AGAMOUS-LIKE9 (AGL9)-like MADS box genes have been characterized that show novel expression patterns and cause different effects on floral transition and formation in Arabidopsis (Hsu et al., 2003; Chang et al., 2009). Compared with other orchids, the production of a large and well-expanded lip and five small identical sepals/petals makes O. Gower Ramsey a special case for the study of the diverse functions of B class MADS box genes during evolution. Therefore, the isolation of more B class MADS box genes and further study of their roles in the regulation of perianth (sepal/petal/lip) formation during O. Gower Ramsey flower development are necessary. In addition to the clade 2 paleoAP3 gene OMADS3, which was previously characterized in our laboratory (Hsu and Yang, 2002), three more B class MADS box genes, OMADS5, OMADS8, and OMADS9, were characterized from O. Gower Ramsey in this study. Based on the different expression patterns and the protein interactions among these four orchid B class genes, we propose that the presence of OMADS3/8 and/or OMADS9 is required for sepal/petal/lip formation. Further sepal and petal formation at least requires the additional presence of OMADS5, whereas large lip formation was seen when OMADS5 expression was absent. Our results provide a new finding and information pertaining to the roles for orchid B class MADS box genes in the regulation of sepal/petal/lip formation.     

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.