Large-Scale Phosphoprotein Analysis in Medicago truncatula Roots Provides Insight into in Vivo Kinase Activity in Legumes

Nitrogen fixation in legumes requires the development of root organs called nodules and their infection by symbiotic rhizobia. Over the last decade, Medicago truncatula has emerged as a major model plant for the analysis of plant-microbe symbioses and for addressing questions pertaining to legume biology. While the initiation of symbiosis and the development of nitrogen-fixing root nodules depend on the activation of a protein phosphorylation-mediated signal transduction cascade in response to symbiotic signals produced by the rhizobia, few sites of in vivo phosphorylation have previously been identified in M. truncatula. We have characterized sites of phosphorylation on proteins from M. truncatula roots, from both whole cell lysates and membrane-enriched fractions, using immobilized metal affinity chromatography and tandem mass spectrometry. Here, we report 3,457 unique phosphopeptides spanning 3,404 nonredundant sites of in vivo phosphorylation on 829 proteins in M. truncatula Jemalong A17 roots, identified using the complementary tandem mass spectrometry fragmentation methods electron transfer dissociation and collision-activated dissociation. With this being, to our knowledge, the first large-scale plant phosphoproteomic study to utilize electron transfer dissociation, analysis of the identified phosphorylation sites revealed phosphorylation motifs not previously observed in plants. Furthermore, several of the phosphorylation motifs, including LxKxxs and RxxSxxxs, have yet to be reported as kinase specificities for in vivo substrates in any species, to our knowledge. Multiple sites of phosphorylation were identified on several key proteins involved in initiating rhizobial symbiosis, including SICKLE, NUCLEOPORIN133, and INTERACTING PROTEIN OF DMI3. Finally, we used these data to create an open-access online database for M. truncatula phosphoproteomic data.Medicago truncatula has become a model for studying the biology of leguminous plants such as soybean (Glycine max), alfalfa (Medicago sativa), and clover (Trifolium spp.; Singh et al., 2007). Most members of this vast family have the ability to fix atmospheric nitrogen by virtue of an endosymbiotic association with rhizobial bacteria, through which legumes undergo nodulation, the process of forming root nodules (Jones et al., 2007). Legumes are central to modern agriculture and civilization because of their ability to grow in nitrogen-depleted soils and replenish nitrogen through crop rotation. Consequently, there is great interest in understanding the molecular events that allow legumes to recognize their symbionts, develop root nodules, and fix nitrogen. Nod factors are lipochitooligosaccharidic signals secreted by the rhizobia and are required, in most legumes, for intracellular infection and nodule development. In recent decades, an elegant combination of genetics, biochemistry, and cell biology has shown that Nod factors activate intricate signaling events within cells of legume roots, including protein phosphorylation cascades and intracellular ion fluxes (Oldroyd and Downie, 2008).Protein phosphorylation is a central mechanism of signal transfer in cells (Laugesen et al., 2006; Peck, 2006; Huber, 2007). Several characterized protein kinases are required for symbiosis signal transduction in M. truncatula roots (Lévy et al., 2004; Yoshida and Parniske, 2005; Smit et al., 2007). A recent antibody-based study of cultured M. truncatula cells observed protein phosphorylation changes at the proteomic level in response to fungal infection (Trapphoff et al., 2009); however, the target residues of the phosphorylation events were not determined. A variety of studies have determined in vitro phosphorylation sites on legume proteins and demonstrated the biological importance of the target residues by mutagenesis (Yoshida and Parniske, 2005; Arrighi et al., 2006; Lima et al., 2006; Miyahara et al., 2008; Yano et al., 2008). To our knowledge, only six sites of in vivo protein phosphorylation have been detected for M. truncatula (Laugesen et al., 2006; Lima et al., 2006; Wienkoop et al., 2008), demonstrating the need for the identification of endogenous protein phosphorylation sites in legume model organisms on a proteome-wide scale.While considerable advancements have been made in the global analysis of protein phosphorylation (Nita-Lazar et al., 2008; Macek et al., 2009; Piggee, 2009; Thingholm et al., 2009), phosphoproteomics in plants has lagged years behind that of the mammalian systems (Kersten et al., 2006, 2009; Peck, 2006), which have more fully sequenced genomes and better annotated protein predictions. Arabidopsis (Arabidopsis thaliana), the first plant genome sequenced (Arabidopsis Genome Initiative, 2000), is now predicted to have over 1,000 protein kinases (Finn et al., 2008), approximately twice as many as in human (Manning et al., 2002). Because many of the kinases in the commonly studied mammalian systems are not conserved in the plant kingdom, there is significant need for large-scale phosphoproteomic technologies to discern the intricacies of phosphorylation-mediated cell signaling in plants. With the high mass accuracy afforded by the linear ion trap-orbitrap hybrid mass spectrometer (Makarov et al., 2006; Yates et al., 2006), recent studies in Arabidopsis have reported 2,597 phosphopeptides from suspension cell culture (Sugiyama et al., 2008) and 3,029 phosphopeptides from seedlings (Reiland et al., 2009).All previous large-scale plant phosphoproteomic studies have relied solely on collision-activated dissociation (CAD) during tandem mass spectrometry (MS/MS) and have not taken advantage of the more recently developed methods (Kersten et al., 2009) electron capture dissociation (Kelleher et al., 1999) or electron transfer dissociation (ETD; Coon et al., 2004; Syka et al., 2004). Mapping sites of posttranslational modifications, such as phosphorylation, is often more straightforward using electron-based fragmentation methods, as they frequently produce a full spectrum of sequence-informative ions without causing neutral loss of the modifying functional groups (Meng et al., 2005; Chi et al., 2007; Khidekel et al., 2007; Molina et al., 2007; Wiesner et al., 2008; Chalkley et al., 2009; Swaney et al., 2009). With an ETD-enabled hybrid orbitrap mass spectrometer (McAlister et al., 2007, 2008), we previously compared the performance of CAD and ETD tandem MS for large-scale identification of phosphopeptides (Swaney et al., 2009). ETD identified a greater percentage of unique phosphopeptides and more frequently localized phosphorylation sites. Still, the low overlap of identified phosphopeptides indicates that the two methods are highly complementary. With this in mind, we recently developed a decision tree-driven tandem MS algorithm to select the optimal fragmentation method for each precursor (Swaney et al., 2008).Here, we utilize this technology to map sites of in vivo protein phosphorylation in roots of M. truncatula Jemalong A17 plants. Phosphoproteins, from both whole-cell lysate and membrane-enriched fractions, were analyzed after digestion with a variety of different enzymes individually. Utilizing the complementary fragmentation methods of ETD and CAD, we report 3,404 nonredundant phosphorylation sites at an estimated false discovery rate (FDR) of 1%. Analysis of these data revealed several phosphorylation motifs not previously observed in plants. The phosphorylation sites identified provide insight into the potential regulation of key proteins involved in rhizobial symbiosis, potential consensus sequences by which kinases recognize their substrates, and critical phosphorylation events that are conserved between plant species.     

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.     

STATE TRANSITION7-Dependent Phosphorylation Is Modulated by Changing Environmental Conditions,and Its Absence Triggers Remodeling of Photosynthetic Protein Complexes

In plants and algae, the serine/threonine kinase STN7/STT7, orthologous protein kinases in Chlamydomonas reinhardtii and Arabidopsis (Arabidopsis thaliana), respectively, is an important regulator in acclimation to changing light environments. In this work, we assessed STT7-dependent protein phosphorylation under high light in C. reinhardtii, known to fully induce the expression of LIGHT-HARVESTING COMPLEX STRESS-RELATED PROTEIN3 (LHCSR3) and a nonphotochemical quenching mechanism, in relationship to anoxia where the activity of cyclic electron flow is stimulated. Our quantitative proteomics data revealed numerous unique STT7 protein substrates and STT7-dependent protein phosphorylation variations that were reliant on the environmental condition. These results indicate that STT7-dependent phosphorylation is modulated by the environment and point to an intricate chloroplast phosphorylation network responding in a highly sensitive and dynamic manner to environmental cues and alterations in kinase function. Functionally, the absence of the STT7 kinase triggered changes in protein expression and photoinhibition of photosystem I (PSI) and resulted in the remodeling of photosynthetic complexes. This remodeling initiated a pronounced association of LHCSR3 with PSI-LIGHT HARVESTING COMPLEX I (LHCI)-ferredoxin-NADPH oxidoreductase supercomplexes. Lack of STT7 kinase strongly diminished PSII-LHCII supercomplexes, while PSII core complex phosphorylation and accumulation were significantly enhanced. In conclusion, our study provides strong evidence that the regulation of protein phosphorylation is critical for driving successful acclimation to high light and anoxic growth environments and gives new insights into acclimation strategies to these environmental conditions.Oxygenic photosynthesis converts solar energy into chemical energy. This energy is utilized for carbon dioxide assimilation, allowing the formation of complex organic material. Plant photosynthesis is performed by a series of reactions in and at the thylakoid membrane, resulting in light-dependent water oxidation, NADP reduction, and ATP formation (Whatley et al., 1963). These light reactions are catalyzed by two photosystems (PSI and PSII). A third multiprotein complex, also embedded in the thylakoid membrane, is the cytochrome b₆f (cyt b₆f) complex that links photosynthetic electron transfer processes between the two photosystems and functions in proton translocation. The ATP synthase takes advantage of the proton-motive force that is generated by the light reactions (Mitchell, 1961) to produce ATP. ATP and NADPH, generated through linear electron flow from PSII to PSI, drive the Calvin-Benson-Bassham cycle (Bassham et al., 1950) to fix CO₂. Alternatively, cyclic electron flow (CEF) between PSI and the cyt b₆f complex solely produces ATP (Arnon, 1959).Under normal growth conditions, CEF provides additionally required ATP for CO₂ fixation (Lucker and Kramer, 2013), counteracts overreduction of the PSI acceptor side under stressful environmental cues, and readjusts the ATP poise, leading to increased lumen acidification important for photoprotection (Alric, 2010; Peltier et al., 2010; Leister and Shikanai, 2013; Shikanai, 2014). In microalgae and vascular plants, CEF relies on the NAD(P)H dehydrogenase-dependent and/or PROTON GRADIENT REGULATION5 (PGR5)-related pathways (Munekage et al., 2002, 2004; Petroutsos et al., 2009; Tolleter et al., 2011; Johnson et al., 2014). For both pathways, supercomplexes consisting of PSI-LIGHT HARVESTING COMPLEX I (LHCI) and components of the respective electron transfer routes have been identified. In Arabidopsis (Arabidopsis thaliana), a unique NAD(P)H dehydrogenase-PSI supercomplex with a molecular mass of more than 1,000 kD was discovered (Peng et al., 2008). From Chlamydomonas reinhardtii, Iwai et al. (2010) isolated a protein supercomplex composed of PSI-LHCI, LHCII, the cyt b₆/f complex, ferredoxin-NADPH oxidoreductase (FNR), and PROTON GRADIENT REGULATION-LIKE1 (PGRL1).PGRL1 and PGR5 interact physically in Arabidopsis and associate with PSI to allow the operation of CEF (DalCorso et al., 2008). Functional data suggest that PGRL1 might operate as a ferredoxin-plastoquinone reductase (Hertle et al., 2013). The PGRL1-containing CEF supercomplex isolated from C. reinhardtii is capable of CEF under in vitro conditions in the presence of exogenously added soluble plastocyanin and ferredoxin (Iwai et al., 2010). Terashima et al. (2012) isolated a CEF supercomplex of similar composition from anaerobic growth conditions that was active in vitro and contained proteins such as the chloroplast-localized Ca²⁺ sensor CAS and ANAEROBIC RESPONSE1 (ANR1), which were also shown to be functionally important for efficient CEF in the alga. Notably, it was suggested that the onset of CEF in C. reinhardtii is redox controlled (Takahashi et al., 2013).It has been demonstrated that efficient CEF is crucial for successful acclimation to excess light (Munekage et al., 2004; Dang et al., 2014; Johnson et al., 2014; Kukuczka et al., 2014). The most rapid response to excess light, however, relies on a mechanism called nonphotochemical quenching (NPQ). The fastest constituent of NPQ is energy-dependent (qE) quenching, which operates at a time scale of seconds to minutes and regulates the thermal dissipation of excess absorbed light energy, thereby providing effective photoprotection. In vascular plants, the PSII protein PSII SUBUNIT S is essential for qE (Li et al., 2000), whereas qE induction in the green alga C. reinhardtii is mediated by LIGHT-HARVESTING COMPLEX STRESS-RELATED PROTEIN3 (LHCSR3), an ancient light-harvesting protein that is missing in vascular plants (Peers et al., 2009). CEF and qE are complementary for acclimation to excess light, as double mutants deficient in both mechanisms possess additive phenotypes and are highly sensitive to light (Kukuczka et al., 2014). Another constituent of NPQ is the quenching by state transitions. State transitions are important to balance the excitation energy between PSI and PSII (Bonaventura and Myers, 1969; Murata, 1969). Under light conditions where PSII is preferentially excited, both PSII core and LHCII proteins become phosphorylated (Lemeille and Rochaix, 2010). As a consequence, phosphorylated LHCII proteins detach from PSII and partly connect to PSI (state 2). Under conditions where PSI excitation is predominant, this process is reversed. LHCII proteins are dephosphorylated and associate with PSII (state 1). The extent of state transition between vascular plants such as Arabidopsis and C. reinhardtii differs significantly. The proportion of mobile LHCII antenna is about 80% in the alga, whereas in Arabidopsis, only 15% to 20% of LHCII is transferred to PSI under state 2 conditions (Lemeille and Rochaix, 2010). However, the large increase in PSI antenna size in C. reinhardtii has recently been challenged (Nagy et al., 2014; Ünlü et al., 2014): while 70% to 80% of mobile LHCII detached from PSII in response to transition to state 2 conditions, only a fraction of about 20% functionally attached to PSI.Phosphorylation of LHC proteins requires the function of the STT7 kinase or its ortholog STN7 in C. reinhardtii or Arabidopsis, respectively. In the absence of the STT7/STN7 kinase, the initiation of state transitions is blocked (Depège et al., 2003; Bellafiore et al., 2005). The mobile LHCII fraction of C. reinhardtii includes the two monomeric minor LHCII antenna proteins, CP26 and CP29 (encoded by lhcb5 and lhcb4 genes), and the major chlorophyll a/b binding protein of LHCII, LHCBM5 (Takahashi et al., 2006), but also the LHCSR3 protein was suggested to migrate during state transitions (Allorent et al., 2013). Takahashi et al. (2014) suggested that only CP29 and LHCBM5 directly associate with PSI to form the PSI-LHCI-LHCII supercomplex, while the binding of CP26 could occur indirectly or via the other two proteins. However, it is not yet known whether STT7 directly phosphorylates the LHCII proteins or if this takes place as part of a kinase cascade (Rochaix, 2007). Nevertheless, the direct interaction between STT7 and the LHCII proteins is quite likely, since none of the other chloroplast kinases was found to be specifically required for LHCII phosphorylation (Rochaix, 2014). The activity of the STT7 kinase is mainly determined by the redox status of the plastoquinone pool (Vener et al., 1997; Zito et al., 1999). The identification of a PROTEIN PHOSPHATASE 2C (PP2C)-type phosphatase responsible for the dephosphorylation of the LHCII proteins in Arabidopsis has been described by two studies in parallel pointing to the fact that this enzyme, called PROTEIN PHOSPHATASE1/THYLAKOID-ASSOCIATED PHOSPHATASE38, acts directly on phosphorylated LHCII proteins, in particular when they are associated with the PSI-LHCI supercomplex (Pribil et al., 2010; Shapiguzov et al., 2010). Moreover, it is not known whether these phosphatases are constitutively active or if they are regulated by other means, for example through the redox state of the plastoquinone pool. Nonetheless, both enzymes are conserved in land plants and exhibit orthologous proteins in C. reinhardtii (Rochaix et al., 2012).Another kinase related to STN7/STT7 is encoded in the Arabidopsis and C. reinhardtii genomes and named STN8 and STATE TRANSITION-LIKE1 (STL1), respectively. STN8 is involved in PSII core subunit phosphorylation and influences the repair of PSII after photodamage (Bonardi et al., 2005; Vainonen et al., 2005). Remarkably, the disassembly of the PSII holocomplex is inhibited in STN7/STN8 double mutants (Tikkanen et al., 2008; Fristedt et al., 2009; Dietzel et al., 2011; Nath et al., 2013), suggesting that the phosphorylation of core subunits is required for PSII disassembly. It was further suggested that STN8 controls the transition between linear electron flow and CEF by the phosphorylation of PGRL1 in Arabidopsis (Reiland et al., 2011). As described for STN7, the activity of STN8 is probably regulated via the redox state of the plastoquinone pool (Bennett, 1991; Fristedt et al., 2009). Notably, the action of STN8 is counteracted by a chloroplast PP2C phosphatase (Samol et al., 2012), allowing for the fast reversibility of STN8-mediated acclimation responses. Thus, it appears that an intricate regulatory network of chloroplast protein kinases and phosphatases evolved in vascular plants and algae that drives the acclimation response to various environmental cues, including excess and changing light settings (Rochaix et al., 2012). As STN7/STT7 and STN8/STL1 kinase activities appear to be controlled by the redox poise of the plastoquinone pool, the plastoquinone pool would be a central player in these acclimation responses. On the other hand, the kinases themselves are subjected to phosphorylation (Reiland et al., 2009, 2011; Lemeille et al., 2010; Wang et al., 2013). However, the functional consequences of this phosphorylation are unknown.Recent comparative analyses revealed the presence of at least 15 distinct chloroplast protein kinases, suggesting an intricate kinase phosphorylation network in the chloroplast (Bayer et al., 2012). Generally, the phosphorylation of proteins is one of the most abundant posttranslational modifications. In complex eukaryotic systems, protein phosphorylation occurs most frequently on Ser followed by Thr residues, whereas protein phosphorylation of Tyr residues (1,800:200:1) is comparatively rare (Hunter, 1998; Mann et al., 2002). Protein phosphorylation is a general phenomenon in vivo; it is assumed that about one-third of all proteins are phosphorylated at a given time (Cohen, 2000; Ahn and Resing, 2001; Venter et al., 2001; Manning et al., 2002; Knight et al., 2003). A recent large-scale quantitative evaluation of human proteomic data strengthened the importance of protein phosphorylation for cellular function and human biology (Wilhelm et al., 2014). The C. reinhardtii and Arabidopsis genomes encode large kinase families (Arabidopsis Genome Initiative, 2000; Kerk et al., 2002; Merchant et al., 2007), supporting the view that protein phosphorylation also plays an important role in a plant’s life cycle. It is thus evident that the understanding of protein phosphorylation, including the specificity of residues phosphorylated or dephosphorylated in response to cellular as well as environmental factors, is one key to understanding the complex functional biological networks at the whole-system level. Likewise, it is crucial to design experimental setups allowing the linkage between phosphorylation events and particular physiological consequences to be elucidated.In this regard, we designed experiments to investigate STT7 kinase-dependent phosphorylation dynamics in C. reinhardtii in response to high light and anoxia, employing quantitative proteomics in conjunction with in-depth physiological characterization. These conditions are particularly interesting, as high light conditions are known to fully induce LHCSR3 protein expression and qE, while anoxia promotes CEF activity. Recently, it was demonstrated that qE and CEF are complementary and crucial in acclimation to these environmental cues (Kukuczka et al., 2014). Notably, LHCSR3 phosphorylation was suggested to depend on STT7 function (Bonente et al., 2011), while CEF supercomplex formation was found to be independent of STT7 kinase function (Takahashi et al., 2013), indicating that STT7 function might impact the acclimation to high light and anoxia in different ways. However, our quantitative proteomics and physiological data reveal that STT7-dependent variations in protein phosphorylation profiles have similar dramatic phenotypic consequences in both conditions, strongly suggesting that the regulation of protein phosphorylation is critical for driving successful acclimation to high light and anoxic growth environments.     

Role of the Rice Hexokinases OsHXK5 and OsHXK6 as Glucose Sensors

The Arabidopsis (Arabidopsis thaliana) hexokinase 1 (AtHXK1) is recognized as an important glucose (Glc) sensor. However, the function of hexokinases as Glc sensors has not been clearly demonstrated in other plant species, including rice (Oryza sativa). To investigate the functions of rice hexokinase isoforms, we characterized OsHXK5 and OsHXK6, which are evolutionarily related to AtHXK1. Transient expression analyses using GFP fusion constructs revealed that OsHXK5 and OsHXK6 are associated with mitochondria. Interestingly, the OsHXK5ΔmTP-GFP and OsHXK6ΔmTP-GFP fusion proteins, which lack N-terminal mitochondrial targeting peptides, were present mainly in the nucleus with a small amount of the proteins seen in the cytosol. In addition, the OsHXK5NLS-GFP and OsHXK6NLS-GFP fusion proteins harboring nuclear localization signals were targeted predominantly in the nucleus, suggesting that these OsHXKs retain a dual-targeting ability to mitochondria and nuclei. In transient expression assays using promoter∷luciferase fusion constructs, these two OsHXKs and their catalytically inactive alleles dramatically enhanced the Glc-dependent repression of the maize (Zea mays) Rubisco small subunit (RbcS) and rice α-amylase genes in mesophyll protoplasts of maize and rice. Notably, the expression of OsHXK5, OsHXK6, or their mutant alleles complemented the Arabidopsis glucose insensitive2-1 mutant, thereby resulting in wild-type characteristics in seedling development, Glc-dependent gene expression, and plant growth. Furthermore, transgenic rice plants overexpressing OsHXK5 or OsHXK6 exhibited hypersensitive plant growth retardation and enhanced repression of the photosynthetic gene RbcS in response to Glc treatment. These results provide evidence that rice OsHXK5 and OsHXK6 can function as Glc sensors.In higher plants, sugars are known to function as signaling molecules in addition to being a fundamental source of fuel for carbon and energy metabolism. Indeed, sugars have been shown to regulate physiological processes during the entire plant life cycle, from germination to flowering and senescence, and to function during defense responses to biotic and abiotic stresses (Jang and Sheen, 1994; Jang et al., 1997; Perata et al., 1997; Smeekens and Rook, 1997; Smeekens, 1998; Wingler et al., 1998; Rolland et al., 2001, 2006; Leon and Sheen, 2003; Gibson, 2005; Biemelt and Sonnewald, 2006; Seo et al., 2007). Therefore, to sustain normal plant growth and development, rigorous sugar sensing and signaling systems are important for coordinating and modulating many essential metabolic pathways.Glc, one of the main products of photosynthesis, is the most widely recognized sugar molecule that regulates plant signaling pathways (Koch, 1996; Yu et al., 1996; Ho et al., 2001; Chen, 2007). Yeast (Saccharomyces cerevisiae) has several Glc sensors, including the hexokinase ScHXK2, Glc transporter-like proteins Sucrose nonfermenting 3 (Snf3) and Restores glucose transport 2 (Rgt2), and G protein-coupled receptor Gpr1. These sensors have been reported to sense the internal and external Glc status as part of mechanisms controlling cell growth and gene expression (Rolland et al., 2001; Lemaire et al., 2004; Santangelo, 2006). Similarly, recent studies in plants have unveiled sugar sensing and signaling systems mediated by hexokinase as a Glc sensor or G protein-coupled receptors in a hexokinase-independent way (Rolland et al., 2001, 2002, 2006; Chen et al., 2003; Moore et al., 2003; Holsbeeks et al., 2004; Cho et al., 2006b; Huang et al., 2006). In addition, plant Snf1-related protein kinase 1 (SnRK1), which is an ortholog of the yeast Snf1, plays important roles linking sugar signal, as well as stress and developmental signals, for the global regulation of plant metabolism, energy balance, growth, and survival (Baena-González et al., 2007; Lu et al., 2007; Baena-González and Sheen, 2008).In addition to the catalytic role of hexokinase in plants, which is to facilitate hexose phosphorylation to form hexose-6-P, the role of hexokinase as an evolutionarily conserved Glc sensor was first recognized from biochemical, genetic, and molecular studies of Arabidopsis (Arabidopsis thaliana) hexokinase 1 (AtHXK1) transgenic plants and glucose insensitive2 (gin2) mutants (Jang et al., 1997; Rolland et al., 2002; Harrington and Bush, 2003; Moore et al., 2003; Cho et al., 2006b). Transgenic plants expressing catalytically inactive AtHXK1 mutant alleles in the gin2 mutant background have provided compelling evidence that the catalytic and sensory functions of AtHXK1 are uncoupled in the Arabidopsis plant (Moore et al., 2003). Furthermore, proteomics and yeast two-hybrid interaction experiments have revealed that in the nucleus, AtHXK1 interacts with two partners, the vacuolar H⁺-ATPase B1 and the 19S regulatory particle of proteasome subunit, to directly control the expression of specific photosynthetic genes (Cho et al., 2006b; Chen, 2007). In these studies, the interactions between AtHXK1 and vacuolar H⁺-ATPase B1 or 19S regulatory particle of proteasome subunit appeared not to require the enzymatic activity of AtHXK1. In the tomato (Solanum lycopersicum) plant, AtHXK1 expression causes a reduction in photosynthesis, growth inhibition, and the induction of rapid senescence (Dai et al., 1999), which are all characteristics of sugar sensing and signaling in photosynthetic tissues. With the exception of Arabidopsis HXK1, the role of hexokinases as Glc sensors has yet to be demonstrated in other plant species (Halford et al., 1999; Veramendi et al., 2002; Rolland et al., 2006).Hexokinases have been shown to associate with various subcellular compartments, including mitochondria, chloroplasts, Golgi complexes, endoplasmic reticula, plasma membranes, and cytosols, suggesting numerous distinct intracellular functions (Schleucher et al., 1998; Wiese et al., 1999; Frommer et al., 2003; Olsson et al., 2003; Giese et al., 2005; Cho et al., 2006a; Kandel-Kfir et al., 2006; Rezende et al., 2006; Damari-Weissler et al., 2007). In yeast, the Glc sensor ScHXK2 has a nuclear localization signal (NLS) within its N-terminal domain and resides partly in the nucleus in addition to the cytosol (Herrero et al., 1998; Randez-Gil et al., 1998). Furthermore, the nuclear localization of ScHXK2 is required for Glc repression of several genes, such as SUC2, HXK1, and GLK1 (Herrero et al., 1998; Rodríguez et al., 2001). A portion of cellular AtHXK1, which is predominantly associated with mitochondria, was also found to reside in the nucleus (Yanagisawa et al., 2003; Cho et al., 2006b). Under conditions of Glc excess, it has thus been hypothesized that nuclear AtHXK1 binds its substrate Glc, resulting in the suppression of target gene expression (Cho et al., 2006b; Chen, 2007).We have previously isolated 10 rice (Oryza sativa) hexokinases, OsHXK1 through OsHXK10, and demonstrated that all of these subtypes possess hexokinase activity (Cho et al., 2006a). The results of this previous study showed that OsHXK4 and OsHXK7 reside in the chloroplast stroma and cytosol, respectively. Based on sequence similarity and subcellular localization, we have identified two rice hexokinases homologous to AtHXK1, OsHXK5 and OsHXK6. The subcellular localization of OsHXK5 and OsHXK6, observed with GFP fusion constructs, suggested that OsHXK5 and OsHXK6 retain a dual-targeting ability to mitochondria and nuclei. This finding prompted us to examine whether these homologues play a role in Glc sensing and signaling in rice. To address this question, we observed the function of OsHXK5 and OsHXK6 in mesophyll protoplasts of maize (Zea mays) and rice and in transgenic rice plants. In addition, we transformed the Arabidopsis gin2-1 mutant with either wild-type or catalytically inactive alleles of OsHXK5 and OsHXK6 and analyzed their sugar sensing and signaling characteristics. Finally, the conserved role of hexokinase as a Glc sensor in Arabidopsis and rice plants is discussed.