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.     

The Arabidopsis PHYTOCHROME KINASE SUBSTRATE2 Protein Is a Phototropin Signaling Element That Regulates Leaf Flattening and Leaf Positioning

In Arabidopsis (Arabidopsis thaliana), the blue light photoreceptor phototropins (phot1 and phot2) fine-tune the photosynthetic status of the plant by controlling several important adaptive processes in response to environmental light variations. These processes include stem and petiole phototropism (leaf positioning), leaf flattening, stomatal opening, and chloroplast movements. The PHYTOCHROME KINASE SUBSTRATE (PKS) protein family comprises four members in Arabidopsis (PKS1–PKS4). PKS1 is a novel phot1 signaling element during phototropism, as it interacts with phot1 and the important signaling element NONPHOTOTROPIC HYPOCOTYL3 (NPH3) and is required for normal phot1-mediated phototropism. In this study, we have analyzed more globally the role of three PKS members (PKS1, PKS2, and PKS4). Systematic analysis of mutants reveals that PKS2 (and to a lesser extent PKS1) act in the same subset of phototropin-controlled responses as NPH3, namely leaf flattening and positioning. PKS1, PKS2, and NPH3 coimmunoprecipitate with both phot1-green fluorescent protein and phot2-green fluorescent protein in leaf extracts. Genetic experiments position PKS2 within phot1 and phot2 pathways controlling leaf positioning and leaf flattening, respectively. NPH3 can act in both phot1 and phot2 pathways, and synergistic interactions observed between pks2 and nph3 mutants suggest complementary roles of PKS2 and NPH3 during phototropin signaling. Finally, several observations further suggest that PKS2 may regulate leaf flattening and positioning by controlling auxin homeostasis. Together with previous findings, our results indicate that the PKS proteins represent an important family of phototropin signaling proteins.Plants constantly monitor the properties of light in their natural environment to optimize light capture for photosynthesis and growth (e.g. shade avoidance and phototropism) and to time important developmental transitions (e.g. germination and flowering; Neff et al., 2000; Briggs and Christie, 2002; Franklin and Whitelam, 2005). To do so, plants have a multitude of photoreceptors that allow them to sense changes in light period, direction, wavelength composition, and intensity. The main types of photoreceptors are the red/far-red light-absorbing phytochromes and the UV-A/blue light-sensing phototropins, cryptochromes, and Zeitlupe protein families (Chen et al., 2004; Jiao et al., 2007; Demarsy and Fankhauser, 2009). The signaling pathways triggered by these photoreceptors are integrated to fine-tune responses to ever-changing light environments (Casal, 2000; Franklin and Whitelam, 2004; Iino, 2006).In Arabidopsis (Arabidopsis thaliana), phototropin1 (phot1) and its paralog phot2 were discovered as primary photoreceptors for blue light-induced hypocotyl phototropism and for high light-induced chloroplast avoidance movements, respectively (Liscum and Briggs, 1995; Huala et al., 1997; Jarillo et al., 2001; Kagawa et al., 2001). Subsequent studies have shown that phototropins regulate a wide set of physiological and developmental responses, including chloroplast accumulation under low light, stomatal opening, leaf flattening, and phototropism of the root, inflorescence stem, and petiole (Sakai et al., 2001). Thus, phototropins are proposed to optimize the photosynthetic potential of plants, particularly under unfavorable environments such as extremely high light, weak illumination, and drought (Kasahara et al., 2002; Takemiya et al., 2005; Galen et al., 2007).Phot1 and phot2 regulate these processes selectively and in a fluence-dependent manner. Phot1 mediates the chloroplast accumulation, leaf positioning, and phototropic responses under very low light (Demarsy and Fankhauser, 2009). Under higher light intensities, the phot2 pathway becomes activated and acts redundantly with phot1 in these processes (Sakai et al., 2001). Phot2 also specifically controls the chloroplast avoidance response induced by high light (Jarillo et al., 2001; Kagawa et al., 2001). For stomatal opening, phot1 and phot2 act redundantly over a broad range of light intensity (Kinoshita et al., 2001; Doi et al., 2004).Phototropins are Ser/Thr kinases belonging to the AGC family (for cAMP-dependent protein kinase, cGMP-dependent protein kinase, and phospholipids-dependent protein kinase C; Bogre et al., 2003). Two LOV (for light, oxygen, or voltage) photosensory domains that bind to the blue light-absorbing chromophore FMN regulate the kinase activity (Christie, 2007). Phototropin activation and early signaling events at the level of the photoreceptor itself have been extensively studied (Tokutomi et al., 2008; Demarsy and Fankhauser, 2009). However, downstream signaling is less well understood. Light-induced phot1 autophosphorylation has recently been shown to be an essential signaling event, but apart from the photoreceptor itself, no direct substrate for the kinase activity has been identified in planta (Inoue et al., 2008b; Sullivan et al., 2008). Nonetheless, several proteins are known to interact with phot1. These include Broad-Complex, Tramtrack, Bric-à-Brac (BTB) proteins belonging to the 33-member NONPHOTOTROPIC HYPOCOTYL3 (NPH3)/ROOT PHOTOTROPISM2-LIKE (NRL) subfamily, 14-3-3 proteins, and ADP-ribosylation factors (members of the Ras superfamily of GTP-binding proteins that play important roles in the assembly and disassembly of coat proteins associated with driving vesicle budding and fusion; Motchoulski and Liscum, 1999; Sullivan et al., 2009).Genetic experiments showed that NPH3 is required for phot1- and phot2-mediated phototropism and for phot1-controlled leaf positioning but is not involved in stomatal opening or chloroplast movements (Inada et al., 2004; Inoue et al., 2008a). In addition, RPT2 acts in the phot1-induced phototropic response and stomatal opening but not in chloroplast relocation or phot2-induced movements. RPT2 can associate with phot1 in vitro and in vivo, but there is no evidence for a direct interaction with phot2 (Inada et al., 2004). NPH3 is also known to interact with phot1 in vivo, but an interaction with phot2 has not been reported (Motchoulski and Liscum, 1999; Lariguet et al., 2006). Thus, phototropin signaling is believed to branch quickly, and phot1 and phot2 appear to recruit different signaling components to trigger distinct physiological processes. NPH3 and RPT2 are proposed to mediate protein scaffolding using their protein-protein interaction domains (BTB/Pox virus and Zinc finger as well as coiled coil) and by these means may provide signaling specificity via interaction with specific targets in different tissues and subcellular compartments (Celaya and Liscum, 2005). The phototropins may regulate such interactions by modifying the phosphorylation status of the signaling protein (e.g. NPH3 and 14-3-3 proteins; Pedmale and Liscum, 2007; Sullivan et al., 2009).The nature of phototropin-controlled responses is diverse. On the one hand, chloroplast movements and stomatal opening are rapid, cell-autonomous, and reversible processes. On the other hand, phototropic responses and leaf flattening are slower symmetric growth processes coordinated by cell expansion and division. Such growth coordination is under tight hormonal regulation, and the hormone auxin is a central regulator of phototropism (Holland et al., 2009), leaf flattening (Keller and Van Volkenburgh, 1997; Li et al., 2007; Bainbridge et al., 2008; Braun et al., 2008), and leaf positioning (Tao et al., 2008; Millenaar et al., 2009). An important task is to identify points of convergence between phototropin signaling and auxin signaling. Hypocotyl phototropism is triggered by blue light-induced auxin redistribution and signaling across the organ (Esmon et al., 2006; Holland et al., 2009). Recent reports suggest that the phototropins achieve this by directly regulating the activity of auxin transporters. First, the three main classes of auxin transporters (AUXIN RESISTANT1 [AUX1]/LIKE AUX1, PIN-FORMED [PIN], and P-glycoproteins [PGP]) are involved in the regulation of phototropism (Friml et al., 2002; Noh et al., 2003; Blakeslee et al., 2004; Nagashima et al., 2008; Stone et al., 2008). Second, phot1 is required for the relocalization of PIN1 upon blue light exposure (Blakeslee et al., 2004). Third, the phototropin-related AGC kinase PINOID (PID) is a crucial regulator of PIN1 intracellular cycling, which suggests an important role for AGC kinases in the regulation of auxin transport polarity (Michniewicz et al., 2007; Robert and Offringa, 2008). The link between the phototropins and auxin has not been firmly established in the cases of leaf flattening and leaf positioning.NPH3 is a strong candidate to provide a link between phototropins and auxin transport. First, NPH3 acts specifically in phototropin-controlled processes that involve growth regulation. Second, the rice (Oryza sativa) homolog of NPH3 called COLEOPTILE PHOTOTROPISM1 (CPT1) is an essential mediator of auxin redistribution in coleoptiles during the phototropin response (Haga et al., 2005). Third, an Arabidopsis homolog of NPH3 named MACCHIBOU4/ENHANCER OF PINOID/NAKED PINS IN YUC MUTANTS1 (MAB4/ENP/NPY1) is involved in organogenesis synergistically with PID by controlling PIN1 localization in embryo and inflorescence stems (Cheng et al., 2007; Furutani et al., 2007). However, beyond these correlative observations, the mechanisms of auxin transport regulation by phototropin signaling remain poorly understood (Holland et al., 2009).PHYTOCHROME KINASE SUBSTRATE (PKS) proteins were initially identified as phytochrome signaling components that regulate developmental processes such as deetiolation and growth orientation of roots and hypocotyls (Fankhauser et al., 1999; Lariguet et al., 2003; Khanna et al., 2006; Boccalandro et al., 2008; Molas and Kiss, 2008; Schepens et al., 2008). PKS1, PKS2, and PKS4 interact with phytochrome A and PKS1 is phosphorylated by phytochrome A in vitro (Fankhauser et al., 1999; Lariguet et al., 2003; Schepens et al., 2008). Recently, we have shown that PKS1 also interacts with phot1 and NPH3 in vivo and is required for phot1-mediated root and hypocotyl phototropism (Lariguet et al., 2006; Boccalandro et al., 2008). The importance of PKS proteins for phototropism prompted us to test their involvement in phototropin-mediated responses more globally. Here, we show that PKS2 acts in phot1 and phot2 signaling pathways controlling leaf positioning and leaf flattening but not chloroplast movements and stomatal opening. Interestingly, PKS2 and NPH3 selectively control phototropin-mediated growth responses and interact genetically. Several lines of evidence, including auxin transport assays in mesophyll protoplasts, suggest that PKS2 may regulate these developmental light responses by modulating auxin homeostasis.     

A Dominant Mutation in the Light-Oxygen and Voltage2 Domain Vicinity Impairs Phototropin1 Signaling in Tomato

In higher plants, blue light (BL) phototropism is primarily controlled by the phototropins, which are also involved in stomatal movement and chloroplast relocation. These photoresponses are mediated by two phototropins, phot1 and phot2. Phot1 mediates responses with higher sensitivity than phot2, and phot2 specifically mediates chloroplast avoidance and dark positioning responses. Here, we report the isolation and characterization of a Nonphototropic seedling1 (Nps1) mutant of tomato (Solanum lycopersicum). The mutant is impaired in low-fluence BL responses, including chloroplast accumulation and stomatal opening. Genetic analyses show that the mutant locus is dominant negative in nature. In dark-grown seedlings of the Nps1 mutant, phot1 protein accumulates at a highly reduced level relative to the wild type and lacks BL-induced autophosphorylation. The mutant harbors a single glycine-1484-to-alanine transition in the Hinge1 region of a phot1 homolog, resulting in an arginine-to-histidine substitution (R495H) in a highly conserved A′α helix proximal to the light-oxygen and voltage2 domain of the translated gene product. Significantly, the R495H substitution occurring in the Hinge1 region of PHOT1 abolishes its regulatory activity in Nps1 seedlings, thereby highlighting the functional significance of the A′α helix region in phototropic signaling of tomato.Being sessile in nature, plants have developed diverse sets of sensory mechanisms, integrating external cues such as light, water, and temperature to adapt their growth and development to the ambient environment. Plants have evolved a cohort of photoreceptors such as red/far-red light-sensing phytochromes (Chen and Chory, 2011), UV-A/blue light (BL)-sensing phototropins (Christie, 2007; Holland et al., 2009; Suetsugu and Wada, 2013), cryptochromes (Yu et al., 2010; Liu et al., 2011), Zeitlupe (ZTL)/Flavin-binding, Kelch repeat, F-box protein1/light-oxygen and voltage (LOV)-kelch protein2 members of the ZTL/ADAGIO putative family of photoreceptors (Suetsugu and Wada, 2013), and UV-B light-sensing UV-B resistance8 (Heijde and Ulm, 2012), enabling them to sense nearly the full range of the solar spectrum. One of the most visually obvious photoresponses of flowering plants involves the growth and orientation of organs toward or away from light, particularly during the early stages of growth and the establishment of seedlings (Iino, 1990) and during gap-filling situations in dense canopy conditions (Ballaré, 1999) for optimizing photosynthesis and interspecies/intraspecies competition. Several studies involving the relative effectiveness of different wavelengths of the solar spectrum as well as monitoring of lateral differences in light intensity revealed that the directional growth of plants is specifically mediated by the UV-A/blue region of the visible spectrum. Molecular genetic analysis of Arabidopsis (Arabidopsis thaliana) mutants inhibited in hypocotyl curvature toward BL revealed that, among the UV-A light-/BL-specific photoreceptors, the phototropins perceive ambient light as a cue for directional growth (Liscum and Briggs, 1995; Kagawa et al., 2001).Phototropins have been identified in several plant species, ranging from the green alga Chlamydomonas reinhardtii to higher plants (Briggs et al., 2001). To date, two members of the phototropins have been reported from higher plants, phot1 and phot2, which share sequence homology (Sakai et al., 2001). Physiological analyses with Arabidopsis mutants lacking phot1 and phot2 have revealed that, in addition to regulating the hypocotyl curvature of seedlings toward BL (Huala et al., 1997; Christie et al., 1998), phototropins also regulate a diverse range of responses in flowering plants (Christie and Murphy, 2013; Hohm et al., 2013). These responses include chloroplast movements (Sakai et al., 2001), nuclear positioning (Iwabuchi et al., 2007), stomatal opening (Kinoshita et al., 2001), sun tracking (Inoue et al., 2008b), leaf expansion (Ohgishi et al., 2004), leaf movements (Inoue et al., 2005), leaf photomorphogenesis (Kozuka et al., 2011), leaf flattening (Sakamoto and Briggs, 2002), and the rapid inhibition of the growth of etiolated hypocotyls (Folta and Spalding, 2001).While both phot1 and phot2 overlap in function in regulating phototropism, chloroplast accumulation, leaf expansion, and stomatal opening, they also exhibit differential photosensitivity to BL, where phot1 is more sensitive to low-fluence BL than phot2. Both phot1 and phot2 redundantly regulate the chloroplast accumulation toward low-fluence BL, and phot2 exclusively regulates the chloroplast avoidance from high-fluence BL (Jarillo et al., 2001; Kagawa et al., 2001), while phot1 solely mediates the rapid inhibition of the elongation of etiolated hypocotyls (Folta and Spalding, 2001). Analysis of mutants downstream of blue light perception by phototropins revealed that the phototropin signaling branches out at an early step, and phot1 and phot2 trigger distinct photoresponses recruiting multiple signaling partners (Christie and Murphy, 2013; Hohm et al., 2013).Molecular characterizations have shown that phototropins are plasma membrane-associated Ser/Thr kinases containing a photosensory domain (Briggs and Christie, 2002) in the N-terminal region composed of two LOV domains (LOV1 and LOV2) and the kinase domain at the C-terminal end. The LOV1 and LOV2 domains bind the FMN as chromophore and are responsible for BL sensing by phototropin. Although phototropins characteristically possess two LOV domains, the photoregulation of phototropin activity is predominantly mediated by LOV2 (Christie, 2007). The exposure to BL also causes adduct formation between the FMN and the Cys residue in LOV domains and leads to the phosphorylation of phototropin, which is believed to be the primary step in the transmission of phototropic signals (Christie et al., 1998; Sakai et al., 2000). To decipher the functions of different domains of phototropins, many different substitution mutants of phototropins have been generated, which have enabled the elucidation of the functional significance of the different domains (Matsuoka and Tokutomi, 2005; Jones et al., 2007; Kong et al., 2007; Inoue et al., 2008a). Inoue et al. (2008a) showed that the BL-induced autophosphorylation of Ser-851 in the C-terminal kinase domain of phototropin is the primary step for initiating stomatal opening, phototropism, chloroplast accumulation, and leaf flattening. Mutational studies also revealed that the photosensory N-terminal domain of phototropin acts as a kinase inhibitor, where the LOV2 domain inhibits the activity of kinase domain by binding to it, and BL exposure is required for the dissociation of the LOV2 domain, enabling phosphorylation of the kinase domain (Matsuoka and Tokutomi, 2005; Jones et al., 2007).While our current understanding of phototropism has been greatly facilitated by the isolation of phototropins and their signaling mutants, the phot mutants identified to date are loss-of-function alleles. The lack of dominant-negative alleles or alleles with increased sensitivity to phototropic stimulus has hindered exploration into the roles of different domains of phot proteins in regulating phototropic signaling. In addition, the dearth of mutants defective in phototropin or phototropin-mediated responses has been a major bottleneck in furthering our understanding of the function of phototropins in crop species. Although phototropin homologs have been identified from a variety of crop species, including oat (Avena sativa; Zacherl et al., 1998), rice (Oryza sativa; Kanegae et al., 2000), and tomato (Solanum lycopersicum; Sharma et al., 2007; Sharma and Sharma, 2007), only the coleoptile phototropism1 mutant of rice has been isolated, which is defective in BL phototropism (Haga et al., 2005).Here, we report that in a mutant screen for nonphototropic seedlings under continuous BL, we recovered a strong dominant-negative mutation of phot1. The dominant-negative mutations are useful to elucidate redundant functions, as mutant protein in addition to suppressing its own functions can also suppress the function of its partners. The characterization of this new phot1 mutant revealed that the dominant activity is caused by the substitution of an Arg residue located in the A′α helix in the Hinge1 region between the LOV1 and LOV2 domains. Our study shows the functional importance of the A′α helix (Halavaty and Moffat, 2007) in regulating phot1-mediated signaling in tomato.     

Natural Variation in Sensitivity to a Loss of Chloroplast Translation in Arabidopsis

Mutations that eliminate chloroplast translation in Arabidopsis (Arabidopsis thaliana) result in embryo lethality. The stage of embryo arrest, however, can be influenced by genetic background. To identify genes responsible for improved growth in the absence of chloroplast translation, we examined seedling responses of different Arabidopsis accessions on spectinomycin, an inhibitor of chloroplast translation, and crossed the most tolerant accessions with embryo-defective mutants disrupted in chloroplast ribosomal proteins generated in a sensitive background. The results indicate that tolerance is mediated by ACC2, a duplicated nuclear gene that targets homomeric acetyl-coenzyme A carboxylase to plastids, where the multidomain protein can participate in fatty acid biosynthesis. In the presence of functional ACC2, tolerance is enhanced by a second locus that maps to chromosome 5 and heightened by additional genetic modifiers present in the most tolerant accessions. Notably, some of the most sensitive accessions contain nonsense mutations in ACC2, including the “Nossen” line used to generate several of the mutants studied here. Functional ACC2 protein is therefore not required for survival in natural environments, where heteromeric acetyl-coenzyme A carboxylase encoded in part by the chloroplast genome can function instead. This work highlights an interesting example of a tandem gene duplication in Arabidopsis, helps to explain the range of embryo phenotypes found in Arabidopsis mutants disrupted in essential chloroplast functions, addresses the nature of essential proteins encoded by the chloroplast genome, and underscores the value of using natural variation to study the relationship between chloroplast translation, plant metabolism, protein import, and plant development.Embryo development in Arabidopsis (Arabidopsis thaliana) requires the coordinated expression of a large number of essential genes (Muralla et al., 2011). Recessive mutations that disrupt these nuclear genes result in an embryo-defective (emb) mutant phenotype (Meinke, 2013). Many EMB genes of Arabidopsis encode chloroplast-localized proteins involved in basic metabolism, protein import, and chloroplast gene expression (Hsu et al., 2010; Bryant et al., 2011; Savage et al., 2013). Functional plastids are therefore required for embryo development in Arabidopsis. Mutations that disrupt photosynthesis alone interfere with embryo and seedling pigmentation, not embryo development. Multiple examples of EMB genes that encode chloroplast-localized aminoacyl-tRNA synthetases, RNA-binding proteins, translation factors, and ribosomal proteins have been described in the literature (Berg et al., 2005; Bryant et al., 2011; Muralla et al., 2011; Romani et al., 2012; Tiller and Bock, 2014). Translation of some chloroplast-encoded mRNAs is therefore essential for seed development. This raises a basic question: which chloroplast genes are required? In this report, we used natural variation and genetic analysis to evaluate the model (Bryant et al., 2011) that a single chloroplast gene, acetyl-coenzyme A carboxylase D (accD), needed for the initial stages of fatty acid biosynthesis, underlies the requirement for chloroplast translation during heterotrophic growth and embryo development in Arabidopsis.Targeted gene disruptions in tobacco (Nicotiana tabacum) have identified four chloroplast genes with essential functions that extend beyond photosynthesis: accD, caseinolytic protease P1 (clpP1), hypothetical chloroplast open reading frame1 (ycf1), and ycf2 (Drescher et al., 2000; Kuroda and Maliga, 2003; Kode et al., 2005). Comparative genomics have shown that all four genes are retained in the plastid genomes of most angiosperms, including chlorophyll-deficient, parasitic species (dePamphilis and Palmer, 1990; Funk et al., 2007; Jansen et al., 2007). Several examples of essential chloroplast genes that relocated to the nucleus have also been described (Magee et al., 2010; Rousseau-Gueutin et al., 2013). The absence of ycf1 and ycf2 in grasses (Jansen et al., 2007) and the replacement of accD with a nuclear gene that targets functional protein back to the chloroplast (Konishi and Sasaki, 1994; Chalupska et al., 2008) remain to be explained.The accD gene in Arabidopsis (AtCg00500) encodes one subunit of the chloroplast-localized heteromeric acetyl-coenzyme A carboxylase (ACCase), an essential enzyme in fatty acid biosynthesis that converts acetyl-CoA to malonyl-CoA. Three other subunits are encoded by nuclear genes, one of which is also known to be required for embryo development (Li et al., 2011). Disruptions of three additional genes (At3g25860, At1g34430, and At2g30200) associated with the reactions that precede and follow the step catalyzed by heteromeric ACCase also result in embryo lethality (Lin et al., 2003; Bryant et al., 2011; Muralla et al., 2011). Embryo lethality is also encountered in auxotrophic mutants unable to produce biotin, an essential vitamin required for ACCase function (Schneider et al., 1989; Patton et al., 1998; Muralla et al., 2008). The conversion of acetyl-CoA to malonyl-CoA during fatty acid biosynthesis within the plastid is therefore required for embryo development in Arabidopsis.In addition to the chloroplast-localized, heteromeric ACCase found in most angiosperms, there is also a cytosolic, homomeric ACCase involved in later stages of fatty acid biosynthesis. In both Arabidopsis and Brassica napus, the gene that encodes this homomeric enzyme is duplicated (Yanai et al., 1995; Schulte et al., 1997). One copy (ACC1; At1g36160) encodes an essential protein localized to the cytosol. Disruption of this gene in Arabidopsis (EMB22, GURKE, and PASTICCINO3 [PAS3]) results in an embryo-defective phenotype distinct from that seen following a loss of chloroplast translation (Meinke, 1985; Baud et al., 2004). Weak alleles exhibit cold sensitivity and glossy inflorescence stems resulting from changes in cuticular wax composition (Lü et al., 2011; Amid et al., 2012). The adjacent copy (ACC2; At1g36180) is expressed at low levels and is predicted to encode a chloroplast-localized protein (Yanai et al., 1995; Baud et al., 2003; Babiychuk et al., 2011). Knockouts of this gene exhibit no obvious phenotype under normal growth conditions (Babiychuk et al., 2011).In Brassica spp., plants with albino leaves devoid of chloroplast ribosomes have been produced by germinating seeds on spectinomycin, an inhibitor of chloroplast translation, and then transplanting the young seedlings to basal medium (Zubko and Day, 1998). This experimental approach was initially described as a promising system for generating stable albinism without mutagenesis. However, different results were obtained with tobacco and Arabidopsis seedlings, which were much more sensitive to spectinomycin. In light of this reported variation in seedling responses to spectinomycin and the known duplication of ACC1 in the Brassicaceae, we decided to explore whether natural accessions of Arabidopsis differed in their ability to tolerate a loss of chloroplast translation and whether genetic analysis in Arabidopsis could uncover some of the genes involved. The results described here confirm the value of this approach, provide insights into the phenotypes of mutants defective in essential chloroplast functions, and help to explain the requirement of chloroplast translation for plant growth and development.     

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.     

FIBRILLIN4 Is Required for Plastoglobule Development and Stress Resistance in Apple and Arabidopsis

The fibrillins are a large family of chloroplast proteins that have been linked with stress tolerance and disease resistance. FIBRILLIN4 (FIB4) is found associated with the photosystem II light-harvesting complex, thylakoids, and plastoglobules, which are chloroplast compartments rich in lipophilic antioxidants. For this study, FIB4 expression was knocked down in apple (Malus 3 domestica) using RNA interference. Plastoglobule osmiophilicity was decreased in fib4 knockdown (fib4 KD) tree chloroplasts compared with the wild type, while total plastoglobule number was unchanged. Compared with the wild type, net photosynthetic CO₂ fixation in fib4 KD trees was decreased at high light intensity but was increased at low light intensity. Furthermore, fib4 KD trees produced more anthocyanins than the wild type when transferred from low to high light intensity, indicating greater sensitivity to high light stress. Relative to the wild type, fib4 KD apples were more sensitive to methyl viologen and had higher superoxide levels during methyl viologen treatment. Arabidopsis (Arabidopsis thaliana) fib4 mutants and fib4 KD apples were more susceptible than their wild-type counterparts to the bacterial pathogens Pseudomonas syringae pathovar tomato and Erwinia amylovora, respectively, and were more sensitive to ozone-induced tissue damage. Following ozone stress, plastoglobule osmiophilicity decreased in wild-type apple and remained low in fib4 KD trees; total plastoglobule number increased in fib4 KD apples but not in the wild type. These results indicate that FIB4 is required for plastoglobule development and resistance to multiple stresses. This study suggests that FIB4 is involved in regulating plastoglobule content and that defective regulation of plastoglobule content leads to broad stress sensitivity and altered photosynthetic activity.Increased production of reactive oxygen species (ROS) is among the first biochemical responses of plants when challenged by pathogens and harsh environmental conditions (Mehdy, 1994; Lamb and Dixon, 1997; Joo et al., 2005). ROS are implicated in tissue damage during environmental stress and in the promotion of disease development by necrotrophic and hemibiotrophic pathogens (Venisse et al., 2001; Apel and Hirt, 2004; Shetty et al., 2008). For example, ROS production is critical for host colonization and pathogenesis by the bacterium Erwinia amylovora, which causes fire blight disease in rosaceous plants such as apple (Malus 3 domestica) and pear (Pyrus communis; Venisse et al., 2001).The chloroplast is a site of ROS production during biotic and abiotic stress (Joo et al., 2005; Liu et al., 2007). The chloroplast has a battery of enzymes such as superoxide dismutase and ascorbate peroxidase, and antioxidants such as ascorbate, glutathione, and tocopherols, for protection against ROS (Noctor and Foyer, 1998; Asada, 2006). Plastoglobules are lipoprotein bodies attached to the thylakoids (Austin et al., 2006) that store lipids, including antioxidants such as tocopherols, carotenes, and plastoquinones (Steinmüller and Tevini, 1985; Tevini and Steinmüller, 1985). In addition to antioxidants, plastoglobules contain tocopherol cyclase, which is involved in γ-tocopherol synthesis (Austin et al., 2006; Vidi et al., 2006). The antioxidant content of plastoglobules and their apparent involvement in tocopherol biosynthesis imply that they could play a role in plant responses to oxidative stress.Plastoglobules contain fibrillins, which were initially described as protein components of chromoplast fibrils with a molecular mass of approximately 30 kD (Winkenbach et al., 1976; Knoth et al., 1986; Emter et al., 1990; Deruère et al., 1994). Fibrillins are ubiquitous proteins present from cyanobacteria to plants (Laizet et al., 2004). Fibrillins maintain plastoglobule structural integrity (Deruère et al., 1994; Pozueta-Romero et al., 1997; Langenkämper et al., 2001; Vidi et al., 2006; Bréhélin et al., 2007) and stabilize the photosynthetic apparatus during photooxidative stress (Gillet et al., 1998; Yang et al., 2006; Youssef et al., 2010), osmotic stress (Gillet et al., 1998), drought (Pruvot et al., 1996; Rey et al., 2000), and low temperature (Rorat et al., 2001). Fibrillins are involved in abscisic acid-mediated protection from photoinhibition (Yang et al., 2006), and a subfamily of Arabidopsis (Arabidopsis thaliana) fibrillins (FIB1a, -1b, and -2) conditions jasmonate production during low-temperature, photooxidative stress (Youssef et al., 2010). Arabidopsis plants lacking one fibrillin (At4g22240) and tomato (Solanum lycopersicum) plants with suppressed expression of a fibrillin (LeCHRC) are susceptible to Pseudomonas syringae and Botrytis cinerea, respectively (Cooper et al., 2003; Leitner-Dagan et al., 2006), indicating that fibrillins play a role in disease resistance.The Arabidopsis fibrillin encoded by At3g23400 has received various appellations, including FIBRILLIN4 (FIB4; Laizet et al., 2004), Harpin-Binding Protein1 (Song et al., 2002), AtPGL 30.4 (Vidi et al., 2006), and Fibrillin6 (Galetskiy et al., 2008); here, it will be referred to by its earliest published name, FIB4. FIB4 is found associated with the PSII light-harvesting complex (Galetskiy et al., 2008). FIB4 has also been detected in plastoglobules (Vidi et al., 2006; Ytterberg et al., 2006) and thylakoids (Friso et al., 2004; Peltier et al., 2004). However, the specific function of FIB4 is unknown. Several lines of evidence suggest that FIB4 may be involved in plant disease resistance responses: pathogen-associated molecular patterns trigger its phosphorylation (Jones et al., 2006); pathogen-associated molecular patterns stimulate the expression of its ortholog in tobacco (Nicotiana tabacum; Jones et al., 2006; Sanabria and Dubery, 2006); and it can physically interact with the HrpN (harpin) virulence protein of the fire blight pathogen E. amylovora in a yeast two-hybrid assay, suggesting that it could be a receptor or target of HrpN (Song et al., 2002). In addition, it is thought that FIB4 may be involved in the transport of small, hydrophobic molecules because it contains a conserved lipocalin signature (Jones et al., 2006). Here, we report a genetic analysis of FIB4 function in apple and Arabidopsis in terms of its role in plastoglobule development and plant resistance to biotic and abiotic stresses.     

Autophagy Plays a Role in Chloroplast Degradation during Senescence in Individually Darkened Leaves

Chloroplasts contain approximately 80% of total leaf nitrogen and represent a major source of recycled nitrogen during leaf senescence. While bulk degradation of the cytosol and organelles in plants is mediated by autophagy, its role in chloroplast catabolism is largely unknown. We investigated the effects of autophagy disruption on the number and size of chloroplasts during senescence. When leaves were individually darkened, senescence was promoted similarly in both wild-type Arabidopsis (Arabidopsis thaliana) and in an autophagy-defective mutant, atg4a4b-1. The number and size of chloroplasts decreased in darkened leaves of wild type, while the number remained constant and the size decrease was suppressed in atg4a4b-1. When leaves of transgenic plants expressing stroma-targeted DsRed were individually darkened, a large accumulation of fluorescence in the vacuolar lumen was observed. Chloroplasts exhibiting chlorophyll fluorescence, as well as Rubisco-containing bodies, were also observed in the vacuole. No accumulation of stroma-targeted DsRed, chloroplasts, or Rubisco-containing bodies was observed in the vacuoles of the autophagy-defective mutant. We have succeeded in demonstrating chloroplast autophagy in living cells and provide direct evidence of chloroplast transportation into the vacuole.Chloroplasts contain 75% to 80% of total leaf nitrogen mainly as proteins (Makino and Osmond, 1991). During leaf senescence, chloroplast proteins are gradually degraded as a major source of nitrogen for new growth (Wittenbach, 1978; Friedrich and Huffaker, 1980; Mae et al., 1984), correlating with a decline in photosynthetic activity, while chloroplasts gradually shrink and transform into gerontoplasts, characterized by the disintegration of the thylakoid membranes and accumulation of plastoglobuli (for a recent review, see Krupinska, 2006). Concomitantly, a decline in the cellular population of chloroplasts is also evident in many cases, for example, during natural (Kura-Hotta et al., 1990; Inada et al., 1998), dark-induced (Wittenbach et al., 1982), and nutrient-limited senescence (Mae et al., 1984; Ono et al., 1995), suggesting the existence of a whole chloroplast degradation system. Some electron microscopic studies have shown whole chloroplasts in the central vacuole, which is rich in lytic hydrolases (Wittenbach et al., 1982; Minamikawa et al., 2001). However, there is no direct evidence of chloroplasts moving into the vacuole in living cells and the mechanism of transport is not yet understood (Hörtensteiner and Feller, 2002; Krupinska, 2006).The most abundant chloroplast protein is Rubisco (EC 4.1.1.39), comprising approximately 50% of the soluble protein (Wittenbach, 1978). The amount of Rubisco decreases rapidly in the early phase of leaf senescence, although more slowly in the later phase (Friedrich and Huffaker, 1980; Mae et al., 1984). In contrast, the chloroplast number remains relatively constant, making it impossible to explain Rubisco loss solely by whole chloroplast degradation. However, the mechanism of intrachloroplastic Rubisco degradation is still unknown (for review, see Feller et al., 2008). Using immunoelectron microscopy, we previously demonstrated in naturally senescing wheat (Triticum aestivum) leaves that Rubisco is released from chloroplasts into the cytoplasm and transported to the vacuole for subsequent degradation in small spherical bodies, named Rubisco-containing bodies (RCBs; Chiba et al., 2003). Similar chloroplast-derived structures were also subsequently confirmed in senescent leaves of soybean (Glycine max) and/or Arabidopsis (Arabidopsis thaliana) by electron microscopy (Otegui et al., 2005), and recently in tobacco (Nicotiana tabacum) leaves by immunoelectron microscopy, although the authors gave them a different name, Rubisco vesicular bodies (Prins et al., 2008). RCBs have double membranes, which seem to be derived from the chloroplast envelope; thus, the RCB-mediated degradation of stromal proteins represents a potential mechanism for chloroplast shrinkage during senescence. We recently demonstrated that Rubisco and stroma-targeted fluorescent proteins can be mobilized to the vacuole by ATG-dependent autophagy via RCBs, using leaves treated with concanamycin A, a vacuolar H⁺-ATPase inhibitor (Ishida et al., 2008). To investigate further, we wished to observe chloroplast autophagy and degradation directly in living cells to determine whether autophagy is responsible for chloroplast shrinkage and whether it is involved in the vacuolar degradation of whole chloroplasts during leaf senescence.Autophagy is known to be a major system for the bulk degradation of intracellular proteins and organelles in the vacuole in yeast and plants, or the lysosome in animals (for detailed mechanisms, see reviews by Ohsumi, 2001; Levine and Klionsky, 2004; Thompson and Vierstra, 2005; Bassham et al., 2006). In those systems, a portion of the cytoplasm, including entire organelles, is engulfed in membrane-bound vesicles and delivered to the vacuole/lysosome. A recent genome-wide search confirmed that Arabidopsis has many genes homologous to the yeast autophagy genes (ATGs; Doelling et al., 2002; Hanaoka et al., 2002; for detailed functions of ATGs, see the reviews noted above). Using knockout mutants of ATGs and a monitoring system with an autophagy marker, GFP-ATG8, numerous studies have demonstrated the presence of the autophagy system in plants and its importance in several biological processes (Yoshimoto et al., 2004; Liu et al., 2005; Suzuki et al., 2005; Thompson et al., 2005; Xiong et al., 2005, 2007; Fujiki et al., 2007; Phillips et al., 2008). These articles suggest that autophagy plays an important role in nutrient recycling during senescence, especially in nutrient-starved plants. The atg mutants exhibited an accelerated loss of some chloroplast proteins, but not all, under nutrient-starved conditions and during senescence, suggesting that autophagy is not the sole mechanism for the degradation of chloroplast proteins; other, as yet unidentified systems must be responsible for the degradation of chloroplast contents when the ATG system is compromised (Levine and Klionsky, 2004; Bassham et al., 2006). However, it still remains likely that autophagy is responsible for the vacuolar degradation of chloroplasts in wild-type plants.Prolonged observation is generally required to follow leaf senescence events in naturally aging leaves and senescence-associated processes tend to become chaotic over time. To observe chloroplast degradation over a short period, and to draw clear conclusions, a suitable experimental model of leaf senescence is required. Weaver and Amasino (2001) reported that senescence is rapidly induced in individually darkened leaves (IDLs) of Arabidopsis, but retarded in plants subjected to full darkness. In addition, Keech et al. (2007) observed a significant decrease of both the number and size of chloroplasts in IDLs within 6 d.In this study, using IDLs as a senescence model, we aimed to investigate the involvement of autophagy in chloroplast degradation. We show direct evidence for the transport of whole chloroplasts and RCBs to the vacuole by autophagy.     

Patterns of Metabolite Changes Identified from Large-Scale Gene Perturbations in Arabidopsis Using a Genome-Scale Metabolic Network

Metabolomics enables quantitative evaluation of metabolic changes caused by genetic or environmental perturbations. However, little is known about how perturbing a single gene changes the metabolic system as a whole and which network and functional properties are involved in this response. To answer this question, we investigated the metabolite profiles from 136 mutants with single gene perturbations of functionally diverse Arabidopsis (Arabidopsis thaliana) genes. Fewer than 10 metabolites were changed significantly relative to the wild type in most of the mutants, indicating that the metabolic network was robust to perturbations of single metabolic genes. These changed metabolites were closer to each other in a genome-scale metabolic network than expected by chance, supporting the notion that the genetic perturbations changed the network more locally than globally. Surprisingly, the changed metabolites were close to the perturbed reactions in only 30% of the mutants of the well-characterized genes. To determine the factors that contributed to the distance between the observed metabolic changes and the perturbation site in the network, we examined nine network and functional properties of the perturbed genes. Only the isozyme number affected the distance between the perturbed reactions and changed metabolites. This study revealed patterns of metabolic changes from large-scale gene perturbations and relationships between characteristics of the perturbed genes and metabolic changes.Rational and quantitative assessment of metabolic changes in response to genetic modification (GM) is an open question and in need of innovative solutions. Nontargeted metabolite profiling can detect thousands of compounds, but it is not easy to understand the significance of the changed metabolites in the biochemical and biological context of the organism. To better assess the changes in metabolites from nontargeted metabolomics studies, it is important to examine the changed metabolites in the context of the genome-scale metabolic network of the organism.Metabolomics is a technique that aims to quantify all the metabolites in a biological system (Nikolau and Wurtele, 2007; Nicholson and Lindon, 2008; Roessner and Bowne, 2009). It has been used widely in studies ranging from disease diagnosis (Holmes et al., 2008; DeBerardinis and Thompson, 2012) and drug discovery (Cascante et al., 2002; Kell, 2006) to metabolic reconstruction (Feist et al., 2009; Kim et al., 2012) and metabolic engineering (Keasling, 2010; Lee et al., 2011). Metabolomic studies have demonstrated the possibility of identifying gene functions from changes in the relative concentrations of metabolites (metabotypes or metabolic signatures; Ebbels et al., 2004) in various species including yeast (Saccharomyces cerevisiae; Raamsdonk et al., 2001; Allen et al., 2003), Arabidopsis (Arabidopsis thaliana; Brotman et al., 2011), tomato (Solanum lycopersicum; Schauer et al., 2006), and maize (Zea mays; Riedelsheimer et al., 2012). Metabolomics has also been used to better understand how plants interact with their environments (Field and Lake, 2011), including their responses to biotic and abiotic stresses (Dixon et al., 2006; Arbona et al., 2013), and to predict important agronomic traits (Riedelsheimer et al., 2012). Metabolite profiling has been performed on many plant species, including angiosperms such as Arabidopsis, poplar (Populus trichocarpa), and Catharanthus roseus (Sumner et al., 2003; Rischer et al., 2006), basal land plants such as Selaginella moellendorffii and Physcomitrella patens (Erxleben et al., 2012; Yobi et al., 2012), and Chlamydomonas reinhardtii (Fernie et al., 2012; Davis et al., 2013). With the availability of whole genome sequences of various species, metabolomics has the potential to become a useful tool for elucidating the functions of genes using large-scale systematic analyses (Fiehn et al., 2000; Saito and Matsuda, 2010; Hur et al., 2013).Although metabolomics data have the potential for identifying the roles of genes that are associated with metabolic phenotypes, the biochemical mechanisms that link functions of genes with metabolic phenotypes are still poorly characterized. For example, we do not yet know the principles behind how perturbing the expression of a single gene changes the metabolic system as a whole. Large-scale metabolomics data have provided useful resources for linking phenotypes to genotypes (Fiehn et al., 2000; Roessner et al., 2001; Tikunov et al., 2005; Schauer et al., 2006; Lu et al., 2011; Fukushima et al., 2014). For example, Lu et al. (2011) compared morphological and metabolic phenotypes from more than 5,000 Arabidopsis chloroplast mutants using gas chromatography (GC)- and liquid chromatography (LC)-mass spectrometry (MS). Fukushima et al. (2014) generated metabolite profiles from various characterized and uncharacterized mutant plants and clustered the mutants with similar metabolic phenotypes by conducting multidimensional scaling with quantified metabolic phenotypes. Nonetheless, representation and analysis of such a large amount of data remains a challenge for scientific discovery (Lu et al., 2011). In addition, these studies do not examine the topological and functional characteristics of metabolic changes in the context of a genome-scale metabolic network. To understand the relationship between genotype and metabolic phenotype, we need to investigate the metabolic changes caused by perturbing the expression of a gene in a genome-scale metabolic network perspective, because metabolic pathways are not independent biochemical factories but are components of a complex network (Berg et al., 2002; Merico et al., 2009).Much progress has been made in the last 2 decades to represent metabolism at a genome scale (Terzer et al., 2009). The advances in genome sequencing and emerging fields such as biocuration and bioinformatics enabled the representation of genome-scale metabolic network reconstructions for model organisms (Bassel et al., 2012). Genome-scale metabolic models have been built and applied broadly from microbes to plants. The first step toward modeling a genome-scale metabolism in a plant species started with developing a genome-scale metabolic pathway database for Arabidopsis (AraCyc; Mueller et al., 2003) from reference pathway databases (Kanehisa and Goto, 2000; Karp et al., 2002; Zhang et al., 2010). Genome-scale metabolic pathway databases have been built for several plant species (Mueller et al., 2005; Zhang et al., 2005, 2010; Urbanczyk-Wochniak and Sumner, 2007; May et al., 2009; Dharmawardhana et al., 2013; Monaco et al., 2013, 2014; Van Moerkercke et al., 2013; Chae et al., 2014; Jung et al., 2014). Efforts have been made to develop predictive genome-scale metabolic models using enzyme kinetics and stoichiometric flux-balance approaches (Sweetlove et al., 2008). de Oliveira Dal’Molin et al. (2010) developed a genome-scale metabolic model for Arabidopsis and successfully validated the model by predicting the classical photorespiratory cycle as well as known key differences between redox metabolism in photosynthetic and nonphotosynthetic plant cells. Other genome-scale models have been developed for Arabidopsis (Poolman et al., 2009; Radrich et al., 2010; Mintz-Oron et al., 2012), C. reinhardtii (Chang et al., 2011; Dal’Molin et al., 2011), maize (Dal’Molin et al., 2010; Saha et al., 2011), sorghum (Sorghum bicolor; Dal’Molin et al., 2010), and sugarcane (Saccharum officinarum; Dal’Molin et al., 2010). These predictive models have the potential to be applied broadly in fields such as metabolic engineering, drug target discovery, identification of gene function, study of evolutionary processes, risk assessment of genetically modified crops, and interpretations of mutant phenotypes (Feist and Palsson, 2008; Ricroch et al., 2011).Here, we interrogate the metabotypes caused by 136 single gene perturbations of Arabidopsis by analyzing the relative concentration changes of 1,348 chemically identified metabolites using a reconstructed genome-scale metabolic network. We examine the characteristics of the changed metabolites (the metabolites whose relative concentrations were significantly different in mutants relative to the wild type) in the metabolic network to uncover biological and topological consequences of the perturbed genes.