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.     

Enhanced Abscisic Acid-Mediated Responses in nia1nia2noa1-2 Triple Mutant Impaired in NIA/NR- and AtNOA1-Dependent Nitric Oxide Biosynthesis in Arabidopsis

Nitric oxide (NO) regulates a wide range of plant processes from development to environmental adaptation. Despite its reported regulatory functions, it remains unclear how NO is synthesized in plants. We have generated a triple nia1nia2noa1-2 mutant that is impaired in nitrate reductase (NIA/NR)- and Nitric Oxide-Associated1 (AtNOA1)-mediated NO biosynthetic pathways. NO content in roots of nia1nia2 and noa1-2 plants was lower than in wild-type plants and below the detection limit in nia1nia2noa1-2 plants. NIA/NR- and AtNOA1-mediated biosynthesis of NO were thus active and responsible for most of the NO production in Arabidopsis (Arabidopsis thaliana). The nia1nia2noa1-2 plants displayed reduced size, fertility, and seed germination potential but increased dormancy and resistance to water deficit. The increasing deficiency in NO of nia1nia2, noa1-2, and nia1nia2noa1-2 plants correlated with increased seed dormancy, hypersensitivity to abscisic acid (ABA) in seed germination and establishment, as well as dehydration resistance. In nia1nia2noa1-2 plants, enhanced drought tolerance was due to a very efficient stomata closure and inhibition of opening by ABA, thus uncoupling NO from ABA-triggered responses in NO-deficient guard cells. The NO-deficient mutants in NIA/NR- and AtNOA1-mediated pathways in combination with the triple mutant will be useful tools to functionally characterize the role of NO and the contribution of both biosynthetic pathways in regulating plant development and defense.Nitric oxide (NO) is a small ubiquitous molecule derived from nitrogen-containing precursors that is one of the earliest and most widespread signaling molecules in living organisms from metazoans to mammals (Torreilles, 2001). The regulatory functions of NO have been extensively studied in mammals, where it is synthesized from Arg through the activity of NO synthases (Knowles and Moncada, 1994). By contrast, the biosynthesis and function of this molecule in plants are largely unknown. During the last 10 years, NO biosynthesis in plants has been one of the most controversial topics in plant biology (Durner and Klessig, 1999; Wendehenne et al., 2001; del Río et al., 2004; Zeier et al., 2004; Lamotte et al., 2005; Meyer et al., 2005; Modolo et al., 2005; Crawford, 2006; Crawford et al., 2006; Zemojtel et al., 2006a). Despite the controversy about its biosynthesis, it is now clear that NO regulates many physiological processes of plants, including seed germination, cell death, defense responses against pathogens, stomata function, senescence, and flowering (Beligni and Lamattina, 2000; Pedroso et al., 2000; Neill et al., 2002; Lamattina et al., 2003; He et al., 2004; Romero-Puertas et al., 2004; Wendehenne et al., 2004; Delledonne, 2005; Guo and Crawford, 2005; Simpson, 2005; Grün et al., 2006; Melotto et al., 2006; Planchet et al., 2006; Ali et al., 2007; Mishina et al., 2007).The molecular mechanisms underlying the control of seed dormancy and germination are still poorly characterized. Genetic data support a central role of abscisic acid (ABA) in regulating seed dormancy, whereas gibberellins promote germination (Finkelstein et al., 2008; Holdsworth et al., 2008). In addition, NO has been lately characterized as a new component in the signaling pathway leading to dormancy breakage. NO-releasing compounds reduce dormancy in a NO-dependent manner in Arabidopsis (Arabidopsis thaliana), some warm-season grasses, and certain barley (Hordeum vulgare) cultivars (Bethke et al., 2004; Sarath et al., 2006). More recently, the aleurone layer cells have been characterized as responsive to NO, gibberellins, and ABA, thus becoming a primary determinant of seed dormancy in Arabidopsis (Bethke et al., 2007).Two main enzyme-based pathways have been proposed to be functional for NO biosynthesis in plants. One is based on the activity of nitrate reductases (Meyer et al., 2005; Modolo et al., 2005), and another one, yet undefined, is based on the direct or indirect function of the Nitric Oxide-Associated1/Resistant to Inhibition by Fosfidomycin1 (AtNOA1/RIF1) protein. It has been also reported that NO synthesis from nitrite occurs in mitochondria associated with mitochondrial electron transport (Planchet et al., 2005) and also that this pathway is mainly functioning in roots under anoxia (Gupta et al., 2005). Moreover, the balance between mitochondrial nitrite reduction and superoxide-dependent NO degradation seems to be derived from factors controlling NO levels in Arabidopsis (Wulff et al., 2009). It has been recently reported that the synthesis of NO in floral organs requires nitrate reductase activity (Seligman et al., 2008) and also that homologues of AtNOA1 participate in NO biosynthesis in diatoms (Vardi et al., 2008), mammals (Zemojtel et al., 2006b; Parihar et al., 2008a, 2008b), and Nicotiana benthamiana (Kato et al., 2008). Recently, the identification of the rif1 mutant, carrying a null mutation in the AtNOA1 locus (At3g47450), allowed uncovering of a function for AtNOA1/RIF1 in the expression of plastome-encoded proteins (Flores-Pérez et al., 2008). Moreover, another recent report claims that AtNOA1 is not a NO synthase but a cGTPase (Moreau et al., 2008), likely playing a role in ribosome assembly and subsequent mRNA translation to proteins in the chloroplasts.To date, it is not clear if both pathways coexist in plants and, if so, the corresponding contributions of each pathway to NO biosynthesis. In this work, we have addressed the functions of both pathways in Arabidopsis by generating a triple mutant in both nitrate reductases and AtNOA1 that is severely impaired in NO production. Further characterization of NO-deficient plants allowed us to identify a functional cross talk between NO and ABA in controlling seed germination and dormancy as well as plant resistance to water deficit.