An Extended AE-Rich N-Terminal Trunk in Secreted Pineapple Cystatin Enhances Inhibition of Fruit Bromelain and Is Posttranslationally Removed during Ripening

Phytocystatins are potent inhibitors of cysteine proteases and have been shown to participate in senescence, seed and organ biogenesis, and plant defense. However, phytocystatins are generally poor inhibitors of the cysteine protease, bromelain, of pineapple (Ananas comosus). Here, we demonstrated that pineapple cystatin, AcCYS1, inhibited (>95%) stem and fruit bromelain. AcCYS1 is a unique cystatin in that it contains an extended N-terminal trunk (NTT) of 63 residues rich in alanine and glutamate. A signal peptide preceding the NTT is processed in vitro by microsomal membranes giving rise to a 27-kD species. AcCYS1 mRNA was present in roots and leaves but was most abundant in fruit. Using immunofluorescence and immunoelectron microscopy with an AcCYS1-specific antiserum, AcCYS1 was found in the apoplasm. Immunoblot analysis identified a 27-kD protein in fruit, roots, and leaves and a 15-kD species in mature ripe fruit. Ripe fruit extracts proteolytically removed the NTT of 27-kD AcCYS1 in vitro to produce the 15-kD species. Mass spectrometry analysis was used to map the primary cleavage site immediately after a conserved critical glycine-94. The AE-rich NTT was required to inhibit fruit and stem bromelain (>95%), whereas its removal decreased inhibition to 20% (fruit) and 80% (stem) and increased the dissociation equilibrium constant by 1.8-fold as determined by surface plasmon resonance assays. We propose that proteolytic removal of the NTT results in the decrease of the inhibitory potency of AcCYS1 against fruit bromelain during fruit ripening to increase tissue proteolysis, softening, and degradation.Phytocystatins are Cys protease inhibitors from plants that reside in the cystatin superfamily and contain a distinctive α-helix-forming sequence, [LVI]-[AGT]-[RKE]-[FY]-[AS]-[VI]-x-[EDQV]-[HYFQ]-N, in the main body (Margis et al., 1998). The most investigated phytocystatin is rice (Oryza sativa) oryzacystatin I (OC-I; Abe et al., 1987). Its three-dimensional structure (Nagata et al., 2000) resembles the structure of chicken egg white cystatin (Bode et al., 1988). These structural features of OC-I include a five-stranded antiparallel β-pleated sheet, which is wrapped around the α-helix. Two regions are predicted to reversibly bind to the active site of papain-like Cys proteases. They are the highly conserved QxVxG motif that is situated on a loop between the second and third β-strand and a conserved W on a loop between the fourth and fifth β-strand (Arai et al., 1991; Urwin et al., 1995). A conserved G immediately precedes the main body at the N terminus. The region preceding the conserved G is referred to as the N-terminal trunk (NTT) and has been shown to interact with Cys protease (Machleidt et al., 1989; Björk et al., 1995; Girard et al., 2007), but the role of the NTT in phytocystatins is less clear.Although the NTT of OC-I did not affect the inhibition of papain (Abe et al., 1988; Chen et al., 1992), the NTTs of other phytocystatins were subsequently shown to modulate the binding affinities to various enzymes (Urwin et al., 1995; Kiggundu et al., 2006). Some phytocystatins were predicted to possess an N-terminal signal peptide for transport into the lumen of the endoplasmic reticulum and/or a C-terminal extension, which may be involved in binding legumain-type Cys proteases (Lim et al., 1996; Womack et al., 2000; Martínez et al., 2005a, 2007; Abraham et al., 2006; Gianotti et al., 2006). Other phytocystatins, designated multicystatins, contain multiple copies of the main body (Kouzuma et al., 2000; Diop et al., 2004; Christova et al., 2006; Girard et al., 2007).Phytocystatins function in diverse biological processes, such as protein turnover during seed development and germination (Kuroda et al., 2001; Martínez et al., 2005c; Abraham et al., 2006; Kiyosaki et al., 2007; Valdés-Rodríguez et al., 2007), organogenesis (Corre-Menguy et al., 2002; Massonneau et al., 2005; Rivard et al., 2007), programmed cell death (Beers et al., 2000; Belenghi et al., 2003), fruit development (Ryan et al., 1998), and defense against a variety of pests and pathogens (Koiwa et al., 2000; Gholizadeh et al., 2005; Christova et al., 2006; Girard et al., 2007). Thus, phytocystatins inhibit both endogenous and exogenous Cys proteases. It is expected that cystatins have a high affinity for their endogenous cognate targets because they have coevolved functionally in the same cellular environment and the cystatin could control potentially damaging proteolytic activity (Otlewski et al., 2005). Similarly, exogenous targets require effective inhibitor-enzyme binding to confer resistance upon pathogen/herbivore attack (Kiggundu et al., 2006). The identification of natural targets of phytocystatins and the elucidation of their regulatory mechanisms are critical to improve our understanding of their roles in plants and for the development of practical applications (Urwin et al., 1997; Arai et al., 1998; Lilley et al., 2004).In pineapple (Ananas comosus), four major Cys proteases have been identified. They are the stem (Ritonja et al., 1989) and fruit bromelains (Yamada et al., 1976; Rowan et al., 1990) and unique ananain (Lee et al., 1997) and comosain (Rowan et al., 1990). Stem and fruit bromelains are encoded by distinct genes (Harrach et al., 1998; Jung et al., 2008) and share 68% sequence identity. They both contain signal peptides for entering the secretory pathway and propeptides for intramolecular inhibition and assisting protein folding. However, the primary species of bromelains that accumulate in plant cells have the propeptide removed (Yamada et al., 1976; Ritonja et al., 1989). Due to their broad substrate specificity and strong proteolytic activity, pineapple Cys proteases have become of considerable economical importance in the food and pharmaceutical industry (Rowan et al., 1990; Maurer, 2001). Fruit and stem bromelains are highly abundant and have been extensively studied (Vanhoof and Cooreman, 1997). Only kiwifruit (Actinidia deliciosa) cystatin has some inhibitory effect on stem bromelain (Rasaam and Laing, 2004). Here, we analyzed a ubiquitously expressed pineapple cystatin, AcCYS1, that we found to be secreted to the apoplast. AcCYS1 is unusual in that it contains an extended NTT of 63 residues that is rich in Ala and Glu. We showed that the NTT is important for complete inhibition of fruit and stem bromelain in the picomolar range and it is cleaved upon fruit ripening. Based on in vitro inhibition analysis against fruit and stem bromelain of three different species of AcCYS1, differing in the length of their NTT, we hypothesize that the cleavage of the NTT enhances the proteolytic activity of fruit bromelain during fruit ripening and senescence.     

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.     

PLASTID MOVEMENT IMPAIRED1 and PLASTID MOVEMENT IMPAIRED1-RELATED1 Mediate Photorelocation Movements of Both Chloroplasts and Nuclei

Organelle movement and positioning play important roles in fundamental cellular activities and adaptive responses to environmental stress in plants. To optimize photosynthetic light utilization, chloroplasts move toward weak blue light (the accumulation response) and escape from strong blue light (the avoidance response). Nuclei also move in response to strong blue light by utilizing the light-induced movement of attached plastids in leaf cells. Blue light receptor phototropins and several factors for chloroplast photorelocation movement have been identified through molecular genetic analysis of Arabidopsis (Arabidopsis thaliana). PLASTID MOVEMENT IMPAIRED1 (PMI1) is a plant-specific C2-domain protein that is required for efficient chloroplast photorelocation movement. There are two PLASTID MOVEMENT IMPAIRED1-RELATED (PMIR) genes, PMIR1 and PMIR2, in the Arabidopsis genome. However, the mechanism in which PMI1 regulates chloroplast and nuclear photorelocation movements and the involvement of PMIR1 and PMIR2 in these organelle movements remained unknown. Here, we analyzed chloroplast and nuclear photorelocation movements in mutant lines of PMI1, PMIR1, and PMIR2. In mesophyll cells, the pmi1 single mutant showed severe defects in both chloroplast and nuclear photorelocation movements resulting from the impaired regulation of chloroplast-actin filaments. In pavement cells, pmi1 mutant plants were partially defective in both plastid and nuclear photorelocation movements, but pmi1pmir1 and pmi1pmir1pmir2 mutant lines lacked the blue light-induced movement responses of plastids and nuclei completely. These results indicated that PMI1 is essential for chloroplast and nuclear photorelocation movements in mesophyll cells and that both PMI1 and PMIR1 are indispensable for photorelocation movements of plastids and thus, nuclei in pavement cells.In plants, organelles move within the cell and become appropriately positioned to accomplish their functions and adapt to the environment (for review, see Wada and Suetsugu, 2004). Light-induced chloroplast movement (chloroplast photorelocation movement) is one of the best characterized organelle movements in plants (Suetsugu and Wada, 2012). Under weak light conditions, chloroplasts move toward light to capture light efficiently (the accumulation response; Zurzycki, 1955). Under strong light conditions, chloroplasts escape from light to avoid photodamage (the avoidance response; Kasahara et al., 2002; Sztatelman et al., 2010; Davis and Hangarter, 2012; Cazzaniga et al., 2013). In most green plant species, these responses are induced primarily by the blue light receptor phototropin (phot) in response to a range of wavelengths from UVA to blue light (approximately 320–500 nm; for review, see Suetsugu and Wada, 2012; Wada and Suetsugu, 2013; Kong and Wada, 2014). Phot-mediated chloroplast movement has been shown in land plants, such as Arabidopsis (Arabidopsis thaliana; Jarillo et al., 2001; Kagawa et al., 2001; Sakai et al., 2001), the fern Adiantum capillus-veneris (Kagawa et al., 2004), the moss Physcomitrella patens (Kasahara et al., 2004), and the liverwort Marchantia polymorpha (Komatsu et al., 2014). Two phots in Arabidopsis, phot1 and phot2, redundantly mediate the accumulation response (Sakai et al., 2001), whereas phot2 primarily regulates the avoidance response (Jarillo et al., 2001; Kagawa et al., 2001; Luesse et al., 2010). M. polymorpha has only one phot that mediates both the accumulation and avoidance responses (Komatsu et al., 2014), although two or more phots mediate chloroplast photorelocation movement in A. capillus-veneris (Kagawa et al., 2004) and P. patens (Kasahara et al., 2004). Thus, duplication and functional diversification of PHOT genes have occurred during land plant evolution, and plants have gained a sophisticated light sensing system for chloroplast photorelocation movement.In general, movements of plant organelles, including chloroplasts, are dependent on actin filaments (for review, see Wada and Suetsugu, 2004). Most organelles common in eukaryotes, such as mitochondria, peroxisomes, and Golgi bodies, use the myosin motor for their movements, but there is no clear evidence that chloroplast movement is myosin dependent (for review, see Suetsugu et al., 2010a). Land plants have innovated a novel actin-based motility system that is specialized for chloroplast movement as well as a photoreceptor system (for review, see Suetsugu et al., 2010a; Wada and Suetsugu, 2013; Kong and Wada, 2014). Chloroplast-actin (cp-actin) filaments, which were first found in Arabidopsis, are short actin filaments specifically localized around the chloroplast periphery at the interface between the chloroplast and the plasma membrane (Kadota et al., 2009). Strong blue light induces the rapid disappearance of cp-actin filaments and then, their subsequent reappearance preferentially at the front region of the moving chloroplasts. This asymmetric distribution of cp-actin filaments is essential for directional chloroplast movement (Kadota et al., 2009; Kong et al., 2013a). The greater the difference in the amount of cp-actin filaments between the front and rear regions of chloroplasts becomes, the faster the chloroplasts move, in which the magnitude of the difference is determined by fluence rate (Kagawa and Wada, 2004; Kadota et al., 2009; Kong et al., 2013a). Strong blue light-induced disappearance of cp-actin filaments is regulated in a phot2-dependent manner before the intensive polymerization of cp-actin filaments at the front region occurs (Kadota et al., 2009; Ichikawa et al., 2011; Kong et al., 2013a). This phot2-dependent response contributes to the greater difference in the amount of cp-actin filaments between the front and rear regions of chloroplasts. Similar behavior of cp-actin filaments has also been observed in A. capillus-veneris (Tsuboi and Wada, 2012) and P. patens (Yamashita et al., 2011).Like chloroplasts, nuclei also show light-mediated movement and positioning (nuclear photorelocation movement) in land plants (for review, see Higa et al., 2014b). In gametophytic cells of A. capillus-veneris, weak light induced the accumulation responses of both chloroplasts and nuclei, whereas strong light induced avoidance responses (Kagawa and Wada, 1993, 1995; Tsuboi et al., 2007). However, in mesophyll cells of Arabidopsis, strong blue light induced both chloroplast and nuclear avoidance responses, but weak blue light induced only the chloroplast accumulation response (Iwabuchi et al., 2007, 2010; Higa et al., 2014a). In Arabidopsis pavement cells, small numbers of tiny plastids were found and showed autofluorescence under the confocal laser-scanning microscopy (Iwabuchi et al., 2010; Higa et al., 2014a). Hereafter, the plastid in the pavement cells is called the pavement cell plastid. Strong blue light-induced avoidance responses of pavement cell plastids and nuclei were induced in a phot2-dependent manner, but the accumulation response was not detected for either organelle (Iwabuchi et al., 2007, 2010; Higa et al., 2014a). In both Arabidopsis and A. capillus-veneris, phots mediate nuclear photorelocation movement, and phot2 mediates the nuclear avoidance response (Iwabuchi et al., 2007, 2010; Tsuboi et al., 2007). The nuclear avoidance response is dependent on actin filaments in both mesophyll and pavement cells of Arabidopsis (Iwabuchi et al., 2010). Recently, it was shown that the nuclear avoidance response relies on cp-actin-dependent movement of pavement cell plastids, where nuclei are associated with pavement cell plastids of Arabidopsis (Higa et al., 2014a). In mesophyll cells, nuclear avoidance response is likely dependent on cp-actin filament-mediated chloroplast movement, because the mutants deficient in chloroplast movement were also defective in nuclear avoidance response (Higa et al., 2014a). Thus, phots mediate both chloroplast (and pavement cell plastid) and nuclear photorelocation movement by regulating cp-actin filaments.Molecular genetic analyses of Arabidopsis mutants deficient in chloroplast photorelocation movement have identified many molecular factors involved in signal transduction and/or motility systems as well as those involved in the photoreceptor system for chloroplast photorelocation movement (and thus, nuclear photorelocation movement; for review, see Suetsugu and Wada, 2012; Wada and Suetsugu, 2013; Kong and Wada, 2014). CHLOROPLAST UNUSUAL POSITIONING1 (CHUP1; Oikawa et al., 2003) and KINESIN-LIKE PROTEIN FOR ACTIN-BASED CHLOROPLAST MOVEMENT (KAC; Suetsugu et al., 2010b) are key factors for generating and/or maintaining cp-actin filaments. Both proteins are highly conserved in land plants and essential for the movement and attachment of chloroplasts to the plasma membrane in Arabidopsis (Oikawa et al., 2003, 2008; Suetsugu et al., 2010b), A. capillus-veneris (Suetsugu et al., 2012), and P. patens (Suetsugu et al., 2012; Usami et al., 2012). CHUP1 is localized on the chloroplast outer membrane and binds to globular and filamentous actins and profilin in vitro (Oikawa et al., 2003, 2008; Schmidt von Braun and Schleiff, 2008). Although KAC is a kinesin-like protein, it lacks microtubule-dependent motor activity but has filamentous actin binding activity (Suetsugu et al., 2010b). An actin-bundling protein THRUMIN1 (THRUM1) is required for efficient chloroplast photorelocation movement (Whippo et al., 2011) and interacts with cp-actin filaments (Kong et al., 2013a). chup1 and kac mutant plants were shown to lack detectable cp-actin filaments (Kadota et al., 2009; Suetsugu et al., 2010b; Ichikawa et al., 2011; Kong et al., 2013a). Similarly, cp-actin filaments were rarely detected in thrum1 mutant plants (Kong et al., 2013a), indicating that THRUM1 also plays an important role in maintaining cp-actin filaments.Other proteins J-DOMAIN PROTEIN REQUIRED FOR CHLOROPLAST ACCUMULATION RESPONSE1 (JAC1; Suetsugu et al., 2005), WEAK CHLOROPLAST MOVEMENT UNDER BLUE LIGHT1 (WEB1; Kodama et al., 2010), and PLASTID MOVEMENT IMPAIRED2 (PMI2; Luesse et al., 2006; Kodama et al., 2010) are involved in the light regulation of cp-actin filaments and chloroplast photorelocation movement. JAC1 is an auxilin-like J-domain protein that mediates the chloroplast accumulation response through its J-domain function (Suetsugu et al., 2005; Takano et al., 2010). WEB1 and PMI2 are coiled-coil proteins that interact with each other (Kodama et al., 2010). Although web1 and pmi2 were partially defective in the avoidance response, the jac1 mutation completely suppressed the phenotype of web1 and pmi2, suggesting that the WEB1/PMI2 complex suppresses JAC1 function (i.e. the accumulation response) under strong light conditions (Kodama et al., 2010). Both web1 and pmi2 showed impaired disappearance of cp-actin filaments in response to strong blue light (Kodama et al., 2010). However, the exact molecular functions of these proteins are unknown.In this study, we characterized mutant plants deficient in the PMI1 gene and two homologous genes PLASTID MOVEMENT IMPAIRED1-RELATED1 (PMIR1) and PMIR2. PMI1 was identified through molecular genetic analyses of pmi1 mutants that showed severe defects in chloroplast accumulation and avoidance responses (DeBlasio et al., 2005). PMI1 is a plant-specific C2-domain protein (DeBlasio et al., 2005; Zhang and Aravind, 2010), but its roles and those of PMIRs in cp-actin-mediated chloroplast and nuclear photorelocation movements remained unclear. Thus, we analyzed chloroplast and nuclear photorelocation movements in the single, double, and triple mutants of pmi1, pmir1, and pmir2.