The Trehalose 6-Phosphate/SnRK1 Signaling Pathway Primes Growth Recovery following Relief of Sink Limitation

Trehalose 6-P (T6P) is a sugar signal in plants that inhibits SNF1-related protein kinase, SnRK1, thereby altering gene expression and promoting growth processes. This provides a model for the regulation of growth by sugar. However, it is not known how this model operates under sink-limited conditions when tissue sugar content is uncoupled from growth. To test the physiological importance of this model, T6P, SnRK1 activities, sugars, gene expression, and growth were measured in Arabidopsis (Arabidopsis thaliana) seedlings after transfer to cold or zero nitrogen compared with sugar feeding under optimal conditions. Maximum in vitro activities of SnRK1 changed little, but T6P accumulated up to 55-fold, correlating with tissue Suc content in all treatments. SnRK1-induced and -repressed marker gene expression strongly related to T6P above and below a threshold of 0.3 to 0.5 nmol T6P g⁻¹ fresh weight close to the dissociation constant (4 µm) of the T6P/ SnRK1 complex. This occurred irrespective of the growth response to Suc. This implies that T6P is not a growth signal per se, but through SnRK1, T6P primes gene expression for growth in response to Suc accumulation under sink-limited conditions. To test this hypothesis, plants with genetically decreased T6P content and SnRK1 overexpression were transferred from cold to warm to analyze the role of T6P/SnRK1 in relief of growth restriction. Compared with the wild type, these plants were impaired in immediate growth recovery. It is concluded that the T6P/SnRK1 signaling pathway responds to Suc induced by sink restriction that enables growth recovery following relief of limitations such as low temperature.The nonreducing Glc disaccharide, trehalose [α-d-glucopyranosyl-(1→1)-α-d-glucopyranoside] is widespread in nature. In resurrection plants, fungi, bacteria, and nonvertebrate animals, it performs a role as a carbon source and stress protection compound (Elbein et al., 2003; Paul et al., 2008). In the majority of plants, however, amounts of trehalose are too low to perform this function. Instead, the pathway has developed into a specialized system that regulates and integrates metabolism with growth and development (Schluepmann et al., 2003; Lunn et al., 2006; Ramon and Rolland, 2007; Gómez et al., 2010). This system is indispensible throughout seed and vegetative development (Eastmond et al., 2002; van Dijken et al., 2004; Gómez et al., 2010), and evidence suggests that the critical function is performed by the precursor of trehalose, trehalose 6-P (T6P). There is one known trehalose biosynthesis pathway in plants from the intermediates Glc 6-P and UDP-Glc catalyzed by trehalose phosphate synthase (TPS), which synthesizes T6P. T6P is then converted to trehalose by trehalose phosphate phosphatase (TPP). The regulation of T6P content in plants by TPSs and TPPs is not well understood. TPS1 is thought to account for most TPS catalytic activity in plants (Vandesteene et al., 2010). All 10 TPPs are now known to be catalytically active (Vandesteene et al., 2012); however, their specific contribution to T6P homeostasis is not known. Evidence suggests that T6P is a sugar signal in plants. T6P responds strongly to Suc supply when Suc is fed to seedlings grown in culture and in response to an increase in Suc in illuminated leaves (Lunn et al., 2006). Biosynthetic pathways for cell wall (Gómez et al., 2006) and starch synthesis (Kolbe et al., 2005) are regulated by T6P, supporting the observation that T6P promotes carbon utilization and growth of seedlings at high sugar levels when its content is increased through expression of otsA, a TPS-encoding gene from Escherichia coli (Schluepmann et al., 2003; Paul et al., 2010). In contrast, expression of otsB, a corresponding TPP-encoding gene from E. coli, decreases T6P content and inhibits growth in the presence of high sugar (Schluepmann et al., 2003; Paul et al., 2010). Given the importance of T6P in the regulation of growth and end-product synthesis, targets for its interaction have been eagerly sought.Recently, it was found that T6P inhibits the protein kinase SnRK1 in growing tissues of plants (Zhang et al., 2009; Debast et al., 2011; Delatte et al., 2011; Martínez-Barajas et al., 2011) through an intermediary factor. SnRK1 (AKIN10/AKIN11) is a member of the SNF1-related AMPK group of protein kinases that perform central functions in the regulation of responses of cells to endogenous energy and carbon status (Hardie, 2007). Baena-González et al. (2007) established that over 1000 genes are regulated by SnRK1 involved in biosynthetic, growth, and stress responses. It was observed that, in addition to cell wall and starch synthesis, T6P could regulate amino acid metabolism, protein, and nucleotide synthesis (Zhang et al., 2009) and is most likely connected to hormone signaling (Zhang et al., 2009; Paul et al., 2010). A model is proposed where SnRK1 inhibits growth processes when sugar and energy supplies are scarce, thus enabling survival under starvation stress conditions. When sugar supply is plentiful, T6P accumulates and inhibits SnRK1 blocking expression of genes involved in the stress survival response and inducing genes involved in the feast response, including growth processes. Interestingly, plants with altered SnRK1 activity display similar phenotypes to plants with altered T6P in both growth and developmental processes such that plants with genetically decreased T6P content resemble those with overexpressed SnRK1 and vice versa (Schluepmann et al., 2003; Baena-González et al., 2007; Wingler et al., 2012).Sugars fluctuate widely in plants in response to changes in photosynthesis and in response to environmental variables. Sugar starvation conditions, such as those induced by deep shade, limit growth through lack of sugar availability; SnRK1 would be active under such conditions. High sugar availability, however, does not necessarily indicate good conditions for growth and high growth rates. For example, under low-temperature and limiting nutrient supply, growth is limited in spite of abundant sugar availability (Paul and Stitt, 1993; Usadel et al., 2008). This is termed sink-limited growth, when growth is limited by capacity of sinks, i.e. growing regions to use assimilate. It departs from the famine model of growth regulation by SnRK1. The interrelationship between T6P, SnRK1, and growth is not known under such conditions. Here, we vary growth conditions by temperature and nutrient supply to induce sink-limited growth and feed Suc and Glc at physiological levels (15 mm). We show a strong specific interrelationship between T6P and Suc and SnRK1-regulated gene expression under all conditions irrespective of growth rate. This implies that T6P is not a growth signal per se, but through SnRK1, T6P primes gene expression for growth. By priming, we mean being in a prepared state with an advanced capacity to activate growth following relief of a growth limitation, such as low temperature. To test that T6P/SnRK1 enable growth recovery following relief from sink limitation, plants with genetically decreased T6P content and SnRK1 overexpression were transferred from cold to warm. Compared with the wild type, these plants were impaired in immediate growth recovery. It is concluded that T6P responds to Suc induced by growth restriction. This enables growth recovery following relief of limitations downstream of T6P/SnRK1, such as low temperature. Our findings are included in a model for the regulation of growth by the T6P/SnRK1 signaling pathway.     

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.     

The Cauliflower Mosaic Virus Protein P6 Forms Motile Inclusions That Traffic along Actin Microfilaments and Stabilize Microtubules

The gene VI product (P6) of Cauliflower mosaic virus (CaMV) is a multifunctional protein known to be a major component of cytoplasmic inclusion bodies formed during CaMV infection. Although these inclusions are known to contain virions and are thought to be sites of translation from the CaMV 35S polycistronic RNA intermediate, the precise role of these bodies in the CaMV infection cycle remains unclear. Here, we examine the functionality and intracellular location of a fusion between P6 and GFP (P6-GFP). We initially show that the ability of P6-GFP to transactivate translation is comparable to unmodified P6. Consequently, our work has direct application for the large body of literature in which P6 has been expressed ectopically and its functions characterized. We subsequently found that P6-GFP forms highly motile cytoplasmic inclusion bodies and revealed through fluorescence colocalization studies that these P6-GFP bodies associate with the actin/endoplasmic reticulum network as well as microtubules. We demonstrate that while P6-GFP inclusions traffic along microfilaments, those associated with microtubules appear stationary. Additionally, inhibitor studies reveal that the intracellular movement of P6-GFP inclusions is sensitive to the actin inhibitor, latrunculin B, which also inhibits the formation of local lesions by CaMV in Nicotiana edwardsonii leaves. The motility of P6 along microfilaments represents an entirely new property for this protein, and these results imply a role for P6 in intracellular and cell-to-cell movement of CaMV.Cauliflower mosaic virus (CaMV), the type member of the genus Caulimovirus, has a circular double-stranded DNA genome known to encode six open reading frames (ORFs). The gene product of ORF VI (P6) is a multifunctional protein whose ascribed functions have increased in number since its initial characterization over 20 years ago. P6 was originally described as the most abundant CaMV protein in infected plants (Odell and Howell, 1980) and was later shown to be the major constituent of amorphous, electron-dense inclusion bodies that are thought to be the sites of virion assembly (Fujisawa et al., 1967; Rubio-Huertos et al., 1968; Himmelbach et al., 1996; Cecchini et al., 1997). Indeed, despite the detection of other viral proteins in CaMV inclusions, the P6 protein on its own is capable of forming inclusion bodies (Cecchini et al., 1997; Li and Leisner, 2002; Haas et al., 2005).P6 is the major pathogenicity determinant for CaMV (Daubert et al., 1984; Baughman et al., 1988; Stratford and Covey, 1989; Zijlstra and Hohn, 1992) and was recently shown to be a suppressor of RNA silencing (Love et al., 2007). In addition, P6 also functions as an avirulence determinant, as it has been shown to be responsible for eliciting a hypersensitive response in Nicotiana edwardsonii and Datura stramonium, as well as nonnecrotic resistance in Nicotiana bigelovii and Arabidopsis (Arabidopsis thaliana) ectotype Tsu-O (Daubert et al., 1984; Schoelz et al., 1986; Wintermantel et al., 1993; Agama et al., 2002). The portion of the P6 protein recognized by plants is localized to the N-terminal third of the protein (Wintermantel et al., 1993; Palanichelvam et al., 2000; Agama et al., 2002). P6 also has a significant effect on plant metabolism, as it is responsible for down-regulating or inducing expression of several plant genes (Geri et al., 1999), including genes involved in ethylene signaling (Geri et al., 2004).Replication of CaMV involves the production of a polycistronic RNA intermediate, the 35S RNA, and P6 acts as a translational transactivator (TAV) by modifying the host translational machinery to allow for reinitiation of translation on this RNA (Ryabova et al., 2002). To carry out this function, the P6 protein physically interacts with the initiation factor eIF3 (Park et al., 2001), as well as ribosomal proteins L13 (Bureau et al., 2004), L18 (Leh et al., 2000), and L24 (Park et al., 2001). Finally, P6 is also a nucleocytoplasmic shuttle protein whose nuclear export is dependent upon a Leu-rich sequence near its N terminus, a region that is also involved in inclusion body formation (Li and Leisner, 2002; Haas et al., 2005). Although the precise role of the P6 protein''s nucleocytoplasmic shuttle function during infection remains to be elucidated, P6 does have the capacity to bind RNA (De Tapia et al., 1993; Cerritelli et al., 1998) and as such may act to control export of the 35S RNA from the nucleus to the cytoplasm, drawing the 35S RNA into the nascent P6 inclusion bodies where viral proteins are translated.Despite the recognized intracellular movement of P6 from cytoplasm to nucleus and the disparate cytoplasmic functions of this protein, factors controlling intracellular transport of P6 remain unknown. The cytoskeleton has been implicated in the intracellular trafficking of a number of plant viral proteins. For example, proteins encoded by several viruses have been found to colocalize with actin microfilaments, including the TGBp2 movement protein from Potato virus X (PVX), TGBp2 and TGBp3 from Potato mop-top virus, the Hsp70 homolog from Beet yellows virus, as well as both the movement (MP) and 126-kD proteins from Tobacco mosaic virus (TMV; McLean et al., 1995; Haupt et al., 2005; Ju et al., 2005; Liu et al., 2005; Prokhnevsky et al., 2005) In addition, inhibitor studies recently demonstrated that the intracellular trafficking of potato leafroll virus MP to the plasmodesmata (PD) is dependent upon an intact actin cytoskeleton (Vogel et al., 2007). Together, these studies suggest that the trafficking of viral proteins along actin filaments is a mechanism utilized by highly divergent RNA viruses.The only documented example of a plant viral protein found to colocalize with both microfilaments and microtubules in cells is the TMV MP (McLean et al., 1995; reviewed in Beachy and Heinlein, 2000; Lucas, 2006), which has been shown to associate with and stabilize microtubules and contains a motif thought to mimic the region of tubulin responsible for lateral junctions between microtubules (Boyko et al., 2000; Ashby et al., 2006). Interestingly, the CaMV gene II product (P2), an aphid transmission factor, was previously shown by immunoelectron microscopy to associate with microtubules in both insect and plant cells, although the significance of this interaction remains unclear (Blanc et al., 1996). In addition to these two viral proteins found to colocalize with microtubules in planta, the Hsp70 homolog from Beet yellows virus and the coat protein from PVX have both been shown to interact with microtubules in vitro (Karasev et al., 1992; Serazev et al., 2003). Evidence that the intracellular localization of grapevine fanleaf virus MP is disturbed by oryzalin, as well as the finding that the geminivirus replication protein AL1 interacts with a kinesin by yeast two-hybrid assay, may also indicate a potential association of these proteins with microtubules (Kong and Hanley-Bowdoin, 2002; Laporte et al., 2003).In this study, we utilize a fusion between the C terminus of P6 and GFP to visualize P6 inclusions in live cells. We demonstrate that the fusion of P6 with GFP does not interfere with its ability to act as a TAV. We further demonstrate that P6-GFP inclusion bodies move intracellularly and are associated with microtubules, actin microfilaments, and the endoplasmic reticulum (ER). Although P6-GFP inclusion bodies associated with microtubules appear stationary, we show that P6-GFP bodies can traffic along microfilaments and that this movement is severely reduced by treatment with the actin inhibitor latrunculin B (LatB). LatB treatment of N. edwardsonii leaves inhibits the formation of local lesions by CaMV, indicating the potential that P6 trafficking on microfilaments is necessary for CaMV cell-to-cell movement. Additionally, the association of P6-GFP inclusion bodies with microtubules prevents the disruption of microtubules by oryzalin, denoting a tight association between these two proteins. We discuss the potential role of P6 movement and cytoskeletal association in CaMV infection.     

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.     

SUGAR-INSENSITIVE3, a RING E3 Ligase,Is a New Player in Plant Sugar Response

Sugars, such as sucrose and glucose, have been implicated in the regulation of diverse developmental events in plants and other organisms. We isolated an Arabidopsis (Arabidopsis thaliana) mutant, sugar-insensitive3 (sis3), that is resistant to the inhibitory effects of high concentrations of exogenous glucose and sucrose on early seedling development. In contrast to wild-type plants, sis3 mutants develop green, expanded cotyledons and true leaves when sown on medium containing high concentrations (e.g. 270 mm) of sucrose. Unlike some other sugar response mutants, sis3 exhibits wild-type responses to the inhibitory effects of abscisic acid and paclobutrazol, a gibberellic acid biosynthesis inhibitor, on seed germination. Map-based cloning revealed that SIS3 encodes a RING finger protein. Complementation of the sis3-2 mutant with a genomic SIS3 clone restored sugar sensitivity of sis3-2, confirming the identity of the SIS3 gene. Biochemical analyses demonstrated that SIS3 is functional in an in vitro ubiquitination assay and that the RING motif is sufficient for its activity. Our results indicate that SIS3 encodes a ubiquitin E3 ligase that is a positive regulator of sugar signaling during early seedling development.Almost all living organisms rely on the products of plant photosynthesis for sustenance, either directly or indirectly. Carbohydrates, the major photosynthates, provide both energy and carbon skeletons for fungi, plants, and animals. In addition, sugars, such as Suc and Glc, function as signaling molecules to regulate plant growth, development, gene expression, and metabolic processes. Sugar response pathways are integrated with other signaling pathways, such as those for light, phytohormones, stress, and nitrogen (Dijkwel et al., 1997; Zhou et al., 1998; Roitsch, 1999; Arenas-Huertero et al., 2000; Huijser et al., 2000; Laby et al., 2000; Coruzzi and Zhou, 2001; Rook et al., 2001; Rolland et al., 2006).Several components of plant sugar response pathways have been identified based on the conservation of sugar-sensing mechanisms among eukaryotic cells (Rolland et al., 2001, 2006) or by mutant screens. Yeast HEXOKINASE2 functions in the Glc-mediated catabolite repression pathway (Entian, 1980). In Arabidopsis (Arabidopsis thaliana), mutations in HEXOKINASE1 (HXK1) cause a Glc-insensitive phenotype, and HXK1 demonstrates dual functions in Glc sensing and metabolism (Moore et al., 2003; Cho et al., 2006). Recent studies revealed the involvement of G-protein-coupled receptor systems in sugar response in yeast and Arabidopsis (Chen et al., 2003; Lemaire et al., 2004). Arabidopsis regulator of G-protein signaling1 (rgs1) mutant seedlings are insensitive to 6% Glc (Chen and Jones, 2004), whereas G-protein α-subunit (gpa1) null mutant seedlings are hypersensitive to Glc (Chen et al., 2003). The SNF1/AMPK/SnRK1 protein kinases are postulated to be global regulators of energy control (Polge and Thomas, 2007). Studies conducted on two members of the Arabidopsis SnRK1 (for SNF1-Related Protein Kinases1) family, AKIN10 and AKIN11, have revealed their pivotal roles in stress and sugar signaling (Baena-González et al., 2007). A genetic screen for reduced seedling growth on 175 mm Suc identified the pleiotropic regulatory locus1 (prl1) mutant, which encodes a nuclear WD protein. Further analyses revealed that PRL1 functions in Glc and phytohormone responses (Németh et al., 1998). Interestingly, PRL1 negatively regulates the Arabidopsis SnRK1s AKIN10 and AKIN11 in vitro (Bhalerao et al., 1999).Isolation of additional mutants defective in sugar response has revealed cross talk between sugar and phytohormone response pathways. For example, abscisic acid (ABA) biosynthesis and signaling mutants have been isolated by several genetic screens for seedlings with reduced responses to the inhibitory effects of high levels of Suc or Glc on seedling development. These mutants include abscisic acid-deficient1 (aba1), aba2, aba3, salt-tolerant1/nine-cis-epoxycarotenoid dioxygenase3, abscisic acid-insensitive3 (abi3), and abi4 (Arenas-Huertero et al., 2000; Huijser et al., 2000; Laby et al., 2000; Rook et al., 2001; Cheng et al., 2002; Rolland et al., 2002; Huang et al., 2008), indicating interplay between ABA- and sugar-mediated signaling. Ethylene also exhibits interactions with sugars in controlling seedling development. Both the ethylene overproduction mutant eto1 and the constitutive ethylene response mutant ctr1 exhibit Glc (Zhou et al., 1998) and Suc (Gibson et al., 2001) insensitivity, whereas the ethylene-insensitive mutants etr1, ein2, and ein4 show sugar hypersensitivity (Zhou et al., 1998; Gibson et al., 2001; Cheng et al., 2002).Further characterization of sugar response factors has suggested that ubiquitin-mediated protein degradation may play a role in sugar response. In particular, the PRL1-binding domains of SnRK1s have been shown to recruit SKP1/ASK1, a conserved SCF ubiquitin ligase subunit, as well as the α4/PAD1 proteasomal subunit, indicating a role for SnRK1s in mediating proteasomal binding of SCF ubiquitin ligases (Farrás et al., 2001). In addition, recent studies indicate that PRL1 is part of a CUL4-based E3 ligase and that AKIN10 exhibits decreased rates of degradation in prl1 than in wild-type extracts (Lee et al., 2008). The ubiquitin/26S proteasome pathway plays important roles in many cellular processes and signal transduction pathways in yeast, animals, and plants (Hochstrasser, 1996; Hershko and Ciechanover, 1998; Smalle and Vierstra, 2004). The key task of the pathway is to selectively ubiquitinate substrate proteins and target them for degradation by the 26S proteasome. In short, the multistep ubiquitination process starts with the formation of a thiol-ester linkage between ubiquitin and a ubiquitin-activating enzyme (E1). The activated ubiquitin is then transferred to a ubiquitin-conjugating enzyme (E2), and a ubiquitin protein ligase (E3) then mediates the covalent attachment of ubiquitin to the substrate protein. The specificity of the pathway is largely realized by the E3s, which recognize the substrates that should be ubiquitinated. In Arabidopsis, more than 1,300 genes encode putative E3 subunits and the E3 ligases can be grouped into defined families based upon the presence of HECT (for Homology to E6-AP C Terminus), RING (for Really Interesting New Gene), or U-box domains (Smalle and Vierstra, 2004). The RING-type E3s can be subdivided into single-subunit E3s, which contain the substrate recognition and RING finger domains on the same protein, and multisubunit E3s, which include the SCF (for Skp1-Cullin-F-box), CUL3-BTB (for Broad-complex, Tramtrack, Bric-a-Brac), and APC (for Anaphase-Promoting Complex) complexes (Weissman, 2001; Moon et al., 2004).The Cys-rich RING finger was first described in the early 1990s (Freemont et al., 1991). It is defined as a linear series of conserved Cys and His residues (C3HC/HC3) that bind two zinc atoms in a cross-brace arrangement. RING fingers can be divided into two types, C3HC4 (RING-HC) and C3H2C3 (RING-H2), depending on the presence of either a Cys or a His residue in the fifth position of the motif (Lovering et al., 1993; Freemont, 2000). A recent study of the RING finger ubiquitin ligase family encoded by the Arabidopsis genome resulted in the identification of 469 predicted proteins containing one or more RING domains (Stone et al., 2005). However, the in vivo biological functions of all but a few of the RING proteins remain unknown. Recent studies have implicated several Arabidopsis RING proteins in a variety biological processes, including COP1 and CIP8 (photomorphogenesis; Hardtke et al., 2002; Seo et al., 2004), SINAT5 (auxin signaling; Xie et al., 2002), ATL2 (defense signaling; Serrano and Guzman, 2004), BRH1 (brassinosteroid response; Molnár et al., 2002), RIE1 (seed development; Xu and Li, 2003), NLA (nitrogen limitation adaptation; Peng et al., 2007), HOS1 (cold response; Dong et al., 2006), AIP2 (ABA signaling; Zhang et al., 2005), KEG (ABA signaling; Stone et al., 2006), and SDIR1 (ABA signaling; Zhang et al., 2007).Here, we report the isolation, identification, and characterization of an Arabidopsis mutant, sugar-insensitive3 (sis3), which is resistant to the early seedling developmental arrest caused by high exogenous sugar levels. The responsible locus, SIS3, was identified through a map-based cloning approach and confirmed with additional T-DNA insertional mutants and complementation tests. The SIS3 gene encodes a protein with a RING-H2 domain and three putative transmembrane domains. Glutathione S-transferase (GST)-SIS3 recombinant proteins exhibit in vitro ubiquitin E3 ligase activity. Together, these results indicate that a ubiquitination pathway involving the SIS3 RING protein is required to mediate the sugar response during early seedling development.     

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.