Extra-Large G Proteins Expand the Repertoire of Subunits in Arabidopsis Heterotrimeric G Protein Signaling

Heterotrimeric G proteins, consisting of Gα, Gβ, and Gγ subunits, are a conserved signal transduction mechanism in eukaryotes. However, G protein subunit numbers in diploid plant genomes are greatly reduced as compared with animals and do not correlate with the diversity of functions and phenotypes in which heterotrimeric G proteins have been implicated. In addition to GPA1, the sole canonical Arabidopsis (Arabidopsis thaliana) Gα subunit, Arabidopsis has three related proteins: the extra-large GTP-binding proteins XLG1, XLG2, and XLG3. We demonstrate that the XLGs can bind Gβγ dimers (AGB1 plus a Gγ subunit: AGG1, AGG2, or AGG3) with differing specificity in yeast (Saccharomyces cerevisiae) three-hybrid assays. Our in silico structural analysis shows that XLG3 aligns closely to the crystal structure of GPA1, and XLG3 also competes with GPA1 for Gβγ binding in yeast. We observed interaction of the XLGs with all three Gβγ dimers at the plasma membrane in planta by bimolecular fluorescence complementation. Bioinformatic and localization studies identified and confirmed nuclear localization signals in XLG2 and XLG3 and a nuclear export signal in XLG3, which may facilitate intracellular shuttling. We found that tunicamycin, salt, and glucose hypersensitivity and increased stomatal density are agb1-specific phenotypes that are not observed in gpa1 mutants but are recapitulated in xlg mutants. Thus, XLG-Gβγ heterotrimers provide additional signaling modalities for tuning plant G protein responses and increase the repertoire of G protein heterotrimer combinations from three to 12. The potential for signal partitioning and competition between the XLGs and GPA1 is a new paradigm for plant-specific cell signaling.The classical heterotrimeric G protein consists of a GDP/GTP-binding Gα subunit with GTPase activity bound to an obligate dimer formed by Gβ and Gγ subunits. In the signaling paradigm largely elucidated from mammalian systems, the plasma membrane-associated heterotrimer contains Gα in its GDP-bound form. Upon receiving a molecular signal, typically transduced by a transmembrane protein (e.g. a G protein-coupled receptor), Gα exchanges GDP for GTP and dissociates from the Gβγ dimer. Both Gα and Gβγ interact with intracellular effectors to initiate downstream signaling cascades. The intrinsic GTPase activity of Gα restores Gα to the GDP-bound form, which binds Gβγ, thereby reconstituting the heterotrimer (McCudden et al., 2005; Oldham and Hamm, 2008).Signal transduction through a heterotrimeric G protein complex is an evolutionarily conserved eukaryotic mechanism common to metazoa and plants, although there are distinct differences in the functional intricacies between the evolutionary branches (Jones et al., 2011a, 2011b; Bradford et al., 2013). The numbers of each subunit encoded within genomes, and therefore the potential for combinatorial complexity within the heterotrimer, is one of the most striking differences between plants and animals. For example, the human genome encodes 23 Gα (encoded by 16 genes), five Gβ, and 12 Gγ subunits (Hurowitz et al., 2000; McCudden et al., 2005; Birnbaumer, 2007). The Arabidopsis (Arabidopsis thaliana) genome, however, only encodes one canonical Gα (GPA1; Ma et al., 1990), one Gβ (AGB1; Weiss et al., 1994), and three Gγ (AGG1, AGG2, and AGG3) subunits (Mason and Botella, 2000, 2001; Chakravorty et al., 2011), while the rice (Oryza sativa) genome encodes one Gα (Ishikawa et al., 1995), one Gβ (Ishikawa et al., 1996), and either four or five Gγ subunits (Kato et al., 2004; Chakravorty et al., 2011; Botella, 2012). As expected, genomes of polyploid plants have more copies due to genome duplication, with the soybean (Glycine max) genome encoding four Gα, four Gβ (Bisht et al., 2011), and 10 Gγ subunits (Choudhury et al., 2011). However, Arabidopsis heterotrimeric G proteins have been implicated in a surprisingly large number of phenotypes, which is seemingly contradictory given the relative scarcity of subunits. Arabidopsis G proteins have been implicated in cell division (Ullah et al., 2001; Chen et al., 2006) and morphological development in various tissues, including hypocotyls (Ullah et al., 2001, 2003), roots (Ullah et al., 2003; Chen et al., 2006; Li et al., 2012), leaves (Lease et al., 2001; Ullah et al., 2001), inflorescences (Ullah et al., 2003), and flowers and siliques (Lease et al., 2001), as well as in pathogen responses (Llorente et al., 2005; Trusov et al., 2006; Cheng et al., 2015), regulation of stomatal movement (Wang et al., 2001; Coursol et al., 2003; Fan et al., 2008) and development (Zhang et al., 2008; Nilson and Assmann, 2010), cell wall composition (Delgado-Cerezo et al., 2012), responses to various light stimuli (Warpeha et al., 2007; Botto et al., 2009), responses to multiple abiotic stimuli (Huang et al., 2006; Pandey et al., 2006; Trusov et al., 2007; Zhang et al., 2008; Colaneri et al., 2014), responses to various hormones during germination (Ullah et al., 2002), and postgermination development (Ullah et al., 2002; Pandey et al., 2006; Trusov et al., 2007). Since the Gγ subunit appeared to be the only subunit that provides diversity in heterotrimer composition in Arabidopsis, it was proposed that all functional specificity in heterotrimeric G protein signaling was provided by the Gγ subunit (Trusov et al., 2007; Chakravorty et al., 2011; Thung et al., 2012, 2013). This allowed for only three heterotrimer combinations to account for the wide range of G protein-associated phenotypes.In addition to the above typical G protein subunits, the plant kingdom contains a conserved protein family of extra-large GTP-binding proteins (XLGs). XLGs differ from typical Gα subunits in that they possess a long N-terminal extension of unknown function, but they are similar in that they all have a typical C-terminal Gα-like region, with five semiconserved G-box (G1–G5) motifs. The XLGs also possess the two sequence features that differentiate heterotrimeric G protein Gα subunits from monomeric G proteins: a helical region between the G1 and G2 motifs and an Asp/Glu-rich loop between the G3 and G4 motifs (Lee and Assmann, 1999; Ding et al., 2008; Heo et al., 2012). The Arabidopsis XLG family comprises XLG1, XLG2, and XLG3, and all three have demonstrated GTP-binding and GTPase activities, although they differ from GPA1 in exhibiting a much slower rate of GTP hydrolysis, with a Ca²⁺ cofactor requirement instead of an Mg²⁺ requirement, as for canonical Gα proteins (Heo et al., 2012). All three Arabidopsis XLGs were observed to be nuclear localized (Ding et al., 2008). Although much less is known about XLGs than canonical Gα subunits, XLG2 positively regulates resistance to the bacterial pathogen Pseudomonas syringae and was immunoprecipitated with AGB1 from tissue infected with P. syringae (Zhu et al., 2009). xlg3 mutants, like agb1 mutants, are impaired in root-waving and root-skewing responses (Pandey et al., 2008). During the preparation of this report, Maruta et al. (2015) further investigated XLG2, particularly focusing on the link between XLG2 and Gβγ in pathogen responses. Based on symptom progression in xlg mutants, they found that XLG2 is a positive regulator of resistance to both bacterial and fungal pathogens, with a minor contribution from XLG3 in resistance to Fusarium oxysporum. XLG2 and XLG3 are also positive regulators of reactive oxygen species (ROS) production in response to pathogen-associated molecular pattern elicitors. The resistance and pathogen-associated molecular pattern-induced ROS phenotypes of the agg1 agg2 and xlg2 xlg3 double mutants were not additive in an agg1 agg2 xlg2 xlg3 quadruple mutant, indicating that these two XLGs and the two Gγ subunits function in the same, rather than parallel, pathways. Unfortunately, the close proximity of XLG2 and AGB1 on chromosome 4 precluded the generation of an agb1 xlg2 double mutant; therefore, direct genetic evidence of XLG2 and AGB1 interaction is still lacking, but physical interactions between XLG2 and the Gβγ dimers were shown by yeast (Saccharomyces cerevisiae) three-hybrid and bimolecular fluorescence complementation (BiFC) assays (Maruta et al., 2015). Localization of all three XLGs was also reexamined, indicating that XLGs are capable of localizing to the plasma membrane in addition to the nucleus (Maruta et al., 2015).Interestingly, several other plant G protein-related phenotypes, in addition to pathogen resistance, have been observed only in Gβ and Gγ mutants, with opposite phenotypes observed in Gα (gpa1) mutants. Traditionally, the observation of opposite phenotypes in Gα versus Gβγ mutants in plants and other organisms has mechanistically been attributed to signaling mediated by free Gβγ, which increases in abundance in the absence of Gα. However, an intriguing alternative is that XLG proteins fulfill a Gα-like role in forming heterotrimeric complexes with Gβγ and function in non-GPA1-based G protein signaling processes. If XLGs function like Gα subunits, the corresponding increase in subunit diversity could potentially account for the diversity of G protein phenotypes. In light of this possibility, we assessed the heterotrimerization potential of all possible XLG and Gβγ dimer combinations, XLG localization and its regulation by Gβγ, and the effect of xlg mutation on selected known phenotypes associated with heterotrimeric G proteins. Our results provide compelling evidence for the formation of XLG-Gβγ heterotrimers and reveal that plant G protein signaling is substantially more complex than previously thought.     

The K-Segment of Maize DHN1 Mediates Binding to Anionic Phospholipid Vesicles and Concomitant Structural Changes

Dehydrins (DHNs; late embryogenesis abundant D11 family) are a family of intrinsically unstructured plant proteins that accumulate in the late stages of seed development and in vegetative tissues subjected to water deficit, salinity, low temperature, or abscisic acid treatment. We demonstrated previously that maize (Zea mays) DHNs bind preferentially to anionic phospholipid vesicles; this binding is accompanied by an increase in α-helicity of the protein, and adoption of α-helicity can be induced by sodium dodecyl sulfate. All DHNs contain at least one “K-segment,” a lysine-rich 15-amino acid consensus sequence. The K-segment is predicted to form a class A2 amphipathic α-helix, a structural element known to interact with membranes and proteins. Here, three K-segment deletion proteins of maize DHN1 were produced. Lipid vesicle-binding assays revealed that the K-segment is required for binding to anionic phospholipid vesicles, and adoption of α-helicity of the K-segment accounts for most of the conformational change of DHNs upon binding to anionic phospholipid vesicles or sodium dodecyl sulfate. The adoption of structure may help stabilize cellular components, including membranes, under stress conditions.When plants encounter environmental stresses such as drought or low temperature, various responses take place to adapt to these conditions. Typical responses include increased expression of chaperones, signal transduction pathway and late embryogenesis abundant (LEA) proteins, osmotic adjustment, and induction of degradation and repair systems (Ingram and Bartels, 1996).Dehydrins (DHNs; LEA D11 family) are a subfamily of group 2 LEA proteins that accumulate to high levels during late stages of seed development and in vegetative tissues subjected to water deficit, salinity, low temperature, or abscisic acid (ABA) treatment (Svensson et al., 2002). Some DHNs are expressed constitutively during normal growth (Nylander et al., 2001; Rorat et al., 2004, 2006; Rodriguez et al., 2005). DHNs exist in a wide range of photosynthetic organisms, including angiosperms, gymnosperms, algae, and mosses (Svensson et al., 2002). DHNs are encoded by a dispersed multigene family and are differentially regulated, at least in higher plants. For example, 13 Dhn genes have been identified in barley (Hordeum vulgare), dispersed over seven genetic map locations (Choi et al., 1999; Svensson et al., 2002) and regulated variably by drought, low temperature, and embryo development (Tommasini et al., 2008). DHNs are localized in various subcellular compartments, including cytosol (Roberts et al., 1993), nucleus (Houde et al., 1995), chloroplast (Artus et al., 1996), vacuole (Heyen et al., 2002), and proximal to the plasma membrane and protein bodies (Asghar et al., 1994; Egerton-Warburton et al., 1997; Puhakainen et al., 2004). Elevated expression of Dhn genes generally has been correlated with the acquisition of tolerance to abiotic stresses such as drought (Whitsitt et al., 1997), salt (Godoy et al., 1994; Jayaprakash et al., 1998), chilling (Ismail et al., 1999a), or freezing (Houde et al., 1995; Danyluk et al., 1998; Fowler et al., 2001). The differences in expression and tissue location suggest that individual members of the Dhn multigene family have somewhat distinct biological functions (Close, 1997; Zhu et al., 2000; Nylander et al., 2001). Many studies have observed a positive correlation between the accumulation of DHNs and tolerance to abiotic stresses (Svensson et al., 2002). However, overexpression of a single DHN protein has not, in general, been sufficient to confer stress tolerance (Puhakainen et al., 2004).DHNs are subclassified by sequence motifs referred to as the K-segment (Lys-rich consensus sequence), the Y-segment (N-terminal conserved sequence), the S-segment (a tract of Ser residues), and the φ-segment (Close, 1996). Because of high hydrophilicity, high content of Gly (>20%), and the lack of a defined three-dimensional structure in the pure form (Lisse et al., 1996), DHNs have been categorized as “intrinsically disordered/unstructured proteins” or “hydrophilins” (Wright and Dyson, 1999; Garay-Arroyo et al., 2000; Tompa, 2005; Kovacs et al., 2008). On the basis of compositional and biophysical properties and their link to abiotic stresses, several functions of DHNs have been proposed, including ion sequestration (Roberts et al., 1993), water retention (McCubbin et al., 1985), and stabilization of membranes or proteins (Close, 1996, 1997). Observations from in vitro experiments include DHN binding to lipid vesicles (Koag et al., 2003; Kovacs et al., 2008) or metals (Svensson et al., 2000; Heyen et al., 2002; Kruger et al., 2002; Alsheikh et al., 2003; Hara et al., 2005), protection of membrane lipid against peroxidation (Hara et al., 2003), retention of hydration or ion sequestration (Bokor et al., 2005; Tompa et al., 2006), and chaperone activity against the heat-induced inactivation and aggregation of various proteins (Kovacs et al., 2008).Intrinsically disordered/unstructured proteins that lack a well-defined three-dimensional structure have recently been recognized to be prevalent in prokaryotes and eukaryotes (Oldfield et al., 2005). They fulfill important functions in signal transduction, gene expression, and binding to targets such as protein, RNA, ions, and membranes (Wright and Dyson, 1999; Tompa, 2002; Dyson and Wright, 2005). The disorder confers structural flexibility and malleability to adapt to changes in the protein environment, including water potential, pH, ionic strength, and temperature, and to undergo structural transition when complexed with ligands such as other proteins, DNA, RNA, or membranes (Prestrelski et al., 1993; Uversky, 2002). Structural changes from disorder to ordered functional structure also can be induced by the folding of a partner protein (Wright and Dyson, 1999; Tompa, 2002; Mouillon et al., 2008).The idea that DHNs interact with membranes is consistent with many immunolocalization studies, which have shown that DHNs accumulate near the plasma membrane or membrane-rich areas surrounding lipid and protein bodies (Asghar et al., 1994; Egerton-Warburton et al., 1997; Danyluk et al., 1998; Puhakainen et al., 2004). The K-segment is predicted to form a class A2 amphipathic α-helix, in which hydrophilic and hydrophobic residues are arranged on opposite faces (Close, 1996). The amphipathic α-helix is a structural element known to interact with membranes and proteins (Epand et al., 1995). Also, in the presence of helical inducers such as SDS and trifluoroethanol (Dalal and Pio, 2006), DHNs take on α-helicity (Lisse et al., 1996; Ismail et al., 1999b). We previously examined the binding of DHN1 to liposomes and found that DHNs bind preferentially to anionic phospholipids and that this binding is accompanied by an increase in α-helicity of the protein (Koag et al., 2003). Similarly, a mitochondrial LEA protein, one of the group III LEA proteins, recently has been shown to interact with and protect membranes subjected to desiccation, coupled with the adoption of amphipathic α-helices (Tolleter et al., 2007).Here, we explore the basis of DHN-vesicle interaction using K-segment deletion proteins. This study reveals that the K-segment is necessary and sufficient for binding to anionic phospholipid vesicles and that the adoption of α-helicity of DHN proteins can be attributed mainly to the K-segment.     

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.     

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.     

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

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

Focus on Ethylene: Ethylene and the Regulation of Physiological and Morphological Responses to Nutrient Deficiencies

To cope with nutrient deficiencies, plants develop both morphological and physiological responses. The regulation of these responses is not totally understood, but some hormones and signaling substances have been implicated. It was suggested several years ago that ethylene participates in the regulation of responses to iron and phosphorous deficiency. More recently, its role has been extended to other deficiencies, such as potassium, sulfur, and others. The role of ethylene in so many deficiencies suggests that, to confer specificity to the different responses, it should act through different transduction pathways and/or in conjunction with other signals. In this update, the data supporting a role for ethylene in the regulation of responses to different nutrient deficiencies will be reviewed. In addition, the results suggesting the action of ethylene through different transduction pathways and its interaction with other hormones and signaling substances will be discussed.When plants suffer from a mineral nutrient deficiency, they develop morphological and physiological responses (mainly in their roots) aimed to facilitate the uptake and mobilization of the limiting nutrient. After the nutrient has been acquired in enough quantity, these responses need to be switched off to avoid toxicity and conserve energy. In recent years, different plant hormones (e.g. ethylene, auxin, cytokinins, jasmonic acid, abscisic acid, brassinosteroids, GAs, and strigolactones) have been implicated in the regulation of these responses (Romera et al., 2007, 2011, 2015; Liu et al., 2009; Rubio et al., 2009; Kapulnik et al., 2011; Kiba et al., 2011; Iqbal et al., 2013; Zhang et al., 2014).Before the 1990s, there were several publications relating ethylene and nutrient deficiencies (cited in Lynch and Brown [1997] and Romera et al. [1999]) without establishing a direct implication of ethylene in the regulation of nutrient deficiency responses. In 1994, Romera and Alcántara (1994) published an article in Plant Physiology suggesting a role for ethylene in the regulation of Fe deficiency responses. In 1999, Borch et al. (1999) showed the participation of ethylene in the regulation of P deficiency responses. Since then, evidence has been accumulating in support of a role for ethylene in the regulation of both Fe (Romera et al., 1999, 2015; Waters and Blevins, 2000; Lucena et al., 2006; Waters et al., 2007; García et al., 2010, 2011, 2013, 2014; Yang et al., 2014) and P deficiency responses (Kim et al., 2008; Lei et al., 2011; Li et al., 2011; Nagarajan and Smith, 2012; Wang et al., 2012, 2014c). Both Fe and P may be poorly available in most soils, and plants develop similar responses under their deficiencies (Romera and Alcántara, 2004; Zhang et al., 2014). More recently, a role for ethylene has been extended to other deficiencies, such as K (Shin and Schachtman, 2004; Jung et al., 2009; Kim et al., 2012), S (Maruyama-Nakashita et al., 2006; Wawrzyńska et al., 2010; Moniuszko et al., 2013), and B (Martín-Rejano et al., 2011). Ethylene has also been implicated in both N deficiency and excess (Tian et al., 2009; Mohd-Radzman et al., 2013; Zheng et al., 2013), and its participation in Mg deficiency has been suggested (Hermans et al., 2010).In this update, we will review the information supporting a role for ethylene in the regulation of different nutrient deficiency responses. For information relating ethylene to other aspects of plant mineral nutrition, such as N₂ fixation and responses to excess of nitrate or essential heavy metals, the reader is referred to other reviews (for review, see Maksymiec, 2007; Mohd-Radzman et al., 2013; Steffens, 2014).     

Homomeric Interaction of AtVSR1 Is Essential for Its Function as a Vacuolar Sorting Receptor

Vacuolar sorting receptors, BP80/VSRs, play a critical role in vacuolar trafficking of soluble proteins in plant cells. However, the mechanism of action of BP80 is not well understood. Here, we investigate the action mechanism of AtVSR1, a member of BP80 proteins in Arabidopsis (Arabidopsis thaliana), in vacuolar trafficking. AtVSR1 exists as multiple forms, including a high molecular mass homomeric complex in vivo. Both the transmembrane and carboxyl-terminal cytoplasmic domains of AtVSR1 are necessary for the homomeric interaction. The carboxyl-terminal cytoplasmic domain contains specific sequence information, whereas the transmembrane domain has a structural role in the homomeric interaction. In protoplasts, an AtVSR1 mutant, C2A, that contained alanine substitution of the region involved in the homomeric interaction, was defective in trafficking to the prevacuolar compartment and localized primarily to the trans-Golgi network. In addition, overexpression of C2A, but not wild-type AtVSR1, inhibited trafficking of soluble proteins to the vacuole and caused their secretion into the medium. Furthermore, C2A:hemagglutinin in transgenic plants interfered with the homomeric interaction of endogenous AtVSR1 and inhibited vacuolar trafficking of sporamin:green fluorescent protein. These data suggest that homomeric interaction of AtVSR1 is critical for its function as a vacuolar sorting receptor.Newly synthesized organellar proteins are delivered to their respective organelles by a complex mechanism of transport. Vacuolar or secretory proteins are initially sorted and translocated into the endoplasmic reticulum (ER) cotranslationally (Crowley et al., 1994; Rapoport et al., 1996). After correct folding into a mature protein and assembly into complexes in the ER, these proteins are transported to the Golgi complex by COPII vesicles (Lee et al., 2004; Tang et al., 2005). Proteins that arrive nondiscriminantly to the Golgi complex are subject to sorting primarily at the trans-Golgi network (TGN), and depending on their final destination, they are transported to the prelysosomal or prevacuolar compartment (PVC; Harasaki et al., 2005; Traub, 2005). Lysosomal/vacuolar cargo-sorting receptors play a critical role in the sorting of cargoes at this step (Marcusson et al., 1994; Hadlington and Denecke, 2000; Gu et al., 2001; Tse et al., 2009).In plant cells, the search for vacuolar sorting receptors led to the identification of an 80-kD protein called BP80 (Kirsch et al., 1994, 1996; Paris and Neuhaus, 2002). BP80 is a type I membrane protein and a member of a highly conserved family of proteins in plants termed vacuolar sorting receptors (VSRs; Kirsch et al., 1994, 1996; Ahmed et al., 1997). BP80/VSRs localize primarily to the PVC, with a minor portion located in the TGN (Sanderfoot et al., 1998; Li et al., 2002; Tse et al., 2004). Thus, it has been proposed that BP80/VSRs shuttle between the PVC and the TGN. In the TGN, they are involved in sorting of vacuolar proteins containing a vacuolar sorting motif, NPIR, for packaging into clathrin-coated vesicles (CCVs). In support of this theory, it was shown that in vitro, BP80/VSR binds to the N-terminal propeptide-sorting signal, the NPIR motif (Kirsch et al., 1994, 1996; Ahmed et al., 1997, 2000). In addition, overexpression of the ER-localized luminal domain of PV72, a seed-specific vacuolar sorting receptor, interferes with the transport of an NPIR-containing proteinase in Arabidopsis (Arabidopsis thaliana) leaves (Watanabe et al., 2004). The biological role of BP80/VSRs was demonstrated in protoplasts. Expression of a mutant form of BP80/VSR, in which the luminal domain was replaced with GFP, resulted in secretion of a soluble vacuolar protein, indicating that BP80/VSR functions in protein trafficking to the lytic vacuole (daSilva et al., 2005). In addition, recently it has been demonstrated that AtVSR1 plays a role in trafficking of protein storage vacuoles in plant seed cells (Shimada et al., 2003). In the atvsr1 mutant, storage proteins were secreted into the apoplastic space of Arabidopsis seeds. In this case, the sorting signal recognized by AtVSR1 may be different from the NPIR motif found in proteins destined to the central vacuole.Although there is mounting evidence that BP80/VSR functions as a vacuolar sorting receptor in plant cells (daSilva et al., 2005; Oliviusson et al., 2006), the detailed mechanism of its action remains poorly understood. Man-6-P receptors and Vps10p, the sorting receptors for soluble lysosomal and vacuolar hydrolases in animal and yeast, respectively, recruit adaptor proteins such as adaptor protein complex 1 (AP-1) and Golgi-localized, γ-ear-containing Arf-binding proteins using the C-terminal cytoplasmic domain (CCD; Johnson and Kornfeld, 1992; Dintzis et al., 1994; Honing et al., 1997; Seaman et al., 1997; Nothwehr et al., 2000; Puertollano et al., 2001; Dennes et al., 2002; Doray et al., 2002; Nakatsu and Ohno, 2003). Similarly, the CCD of BP80/VSR may also recruit accessory proteins for CCV formation at the TGN. Indeed, AtVSR1 interacts with EpsinR1 (formally EPSIN1), one of the epsin homologs in Arabidopsis (Song et al., 2006). Since EpsinR1 interacts with clathrin directly, this interaction may play a role in CCV formation. In addition, the CCD of BP80 contains a highly conserved sequence motif, YMPL, which conforms to the consensus sequence motif YXXΦ (where X is any amino acid and Φ is an amino acid with a bulky hydrophobic side chain) for binding to AP complexes. A peptide containing the YMPL motif binds in vitro to Arabidopsis μA, a close homolog of AP μ-adaptin in animal cells. The importance of the YXXΦ motif has also been confirmed by a recent study showing that mutation of the YXXΦ motif of BP80 caused its mistargeting in tobacco (Nicotiana tabacum) cells (daSilva et al., 2006). However, the exact role of the YXXΦ motif has not been addressed in trafficking of vacuolar proteins in vivo.In an effort to understand the action mechanism of BP80/VSRs in plant cells, we examined the interaction of AtVSR1 with its binding partners. Here, we demonstrate that AtVSR1 undergoes homomeric interaction through the transmembrane domain (TMD) and CCD and that the homomeric interaction is critical for its function as sorting receptor of vacuolar proteins.     

Surveying Rubisco Diversity and Temperature Response to Improve Crop Photosynthetic Efficiency

The threat to global food security of stagnating yields and population growth makes increasing crop productivity a critical goal over the coming decades. One key target for improving crop productivity and yields is increasing the efficiency of photosynthesis. Central to photosynthesis is Rubisco, which is a critical but often rate-limiting component. Here, we present full Rubisco catalytic properties measured at three temperatures for 75 plants species representing both crops and undomesticated plants from diverse climates. Some newly characterized Rubiscos were naturally “better” compared to crop enzymes and have the potential to improve crop photosynthetic efficiency. The temperature response of the various catalytic parameters was largely consistent across the diverse range of species, though absolute values showed significant variation in Rubisco catalysis, even between closely related species. An analysis of residue differences among the species characterized identified a number of candidate amino acid substitutions that will aid in advancing engineering of improved Rubisco in crop systems. This study provides new insights on the range of Rubisco catalysis and temperature response present in nature, and provides new information to include in models from leaf to canopy and ecosystem scale.In a changing climate and under pressure from a population set to hit nine billion by 2050, global food security will require massive changes to the way food is produced, distributed, and consumed (Ort et al., 2015). To match rising demand, agricultural production must increase by 50 to 70% in the next 35 years, and yet the gains in crop yields initiated by the green revolution are slowing, and in some cases, stagnating (Long and Ort, 2010; Ray et al., 2012). Among a number of areas being pursued to increase crop productivity and food production, improving photosynthetic efficiency is a clear target, offering great promise (Parry et al., 2007; von Caemmerer et al., 2012; Price et al., 2013; Ort et al., 2015). As the gatekeeper of carbon entry into the biosphere and often acting as the rate-limiting step of photosynthesis, Rubisco, the most abundant enzyme on the planet (Ellis, 1979), is an obvious and important target for improving crop photosynthetic efficiency.Rubisco is considered to exhibit comparatively poor catalysis, in terms of catalytic rate, specificity, and CO₂ affinity (Tcherkez et al., 2006; Andersson, 2008), leading to the suggestion that even small increases in catalytic efficiency may result in substantial improvements to carbon assimilation across a growing season (Zhu et al., 2004; Parry et al., 2013; Galmés et al., 2014a; Carmo-Silva et al., 2015). If combined with complimentary changes such as optimizing other components of the Calvin Benson or photorespiratory cycles (Raines, 2011; Peterhansel et al., 2013; Simkin et al., 2015), optimized canopy architecture (Drewry et al., 2014), or introducing elements of a carbon concentrating mechanism (Furbank et al., 2009; Lin et al., 2014a; Hanson et al., 2016; Long et al., 2016), Rubisco improvement presents an opportunity to dramatically increase the photosynthetic efficiency of crop plants (McGrath and Long, 2014; Long et al., 2015; Betti et al., 2016). A combination of the available strategies is essential for devising tailored solutions to meet the varied requirements of different crops and the diverse conditions under which they are typically grown around the world.Efforts to engineer an improved Rubisco have not yet produced a “super Rubisco” (Parry et al., 2007; Ort et al., 2015). However, advances in engineering precise changes in model systems continue to provide important developments that are increasing our understanding of Rubisco catalysis (Spreitzer et al., 2005; Whitney et al., 2011a, 2011b; Morita et al., 2014; Wilson et al., 2016), regulation (Andralojc et al., 2012; Carmo-Silva and Salvucci, 2013; Bracher et al., 2015), and biogenesis (Saschenbrecker et al., 2007; Whitney and Sharwood, 2008; Lin et al., 2014b; Hauser et al., 2015; Whitney et al., 2015).A complementary approach is to understand and exploit Rubisco natural diversity. Previous characterization of Rubisco from a limited number of species has not only demonstrated significant differences in the underlying catalytic parameters, but also suggests that further undiscovered diversity exists in nature and that the properties of some of these enzymes could be beneficial if present in crop plants (Carmo-Silva et al., 2015). Recent studies clearly illustrate the variation possible among even closely related species (Galmés et al., 2005, 2014b, 2014c; Kubien et al., 2008; Andralojc et al., 2014; Prins et al., 2016).Until recently, there have been relatively few attempts to characterize the consistency, or lack thereof, of temperature effects on in vitro Rubisco catalysis (Sharwood and Whitney, 2014), and often studies only consider a subset of Rubisco catalytic properties. This type of characterization is particularly important for future engineering efforts, enabling specific temperature effects to be factored into any attempts to modify crops for a future climate. In addition, the ability to coanalyze catalytic properties and DNA or amino acid sequence provides the opportunity to correlate sequence and biochemistry to inform engineering studies (Christin et al., 2008; Kapralov et al., 2011; Rosnow et al., 2015). While the amount of gene sequence information available grows rapidly with improving technology, knowledge of the corresponding biochemical variation resulting has yet to be determined (Cousins et al., 2010; Carmo-Silva et al., 2015; Sharwood and Whitney, 2014; Nunes-Nesi et al., 2016).This study aimed to characterize the catalytic properties of Rubisco from diverse species, comprising a broad range of monocots and dicots from diverse environments. The temperature dependence of Rubisco catalysis was evaluated to tailor Rubisco engineering for crop improvement in specific environments. Catalytic diversity was analyzed alongside the sequence of the Rubisco large subunit gene, rbcL, to identify potential catalytic switches for improving photosynthesis and productivity. In vitro results were compared to the average temperature of the warmest quarter in the regions where each species grows to investigate the role of temperature in modulating Rubisco catalysis.     

Genetic Interactions between PEROXIN12 and Other Peroxisome-Associated Ubiquitination Components

Most eukaryotic cells require peroxisomes, organelles housing fatty acid β-oxidation and other critical metabolic reactions. Peroxisomal matrix proteins carry peroxisome-targeting signals that are recognized by one of two receptors, PEX5 or PEX7, in the cytosol. After delivering the matrix proteins to the organelle, these receptors are removed from the peroxisomal membrane or matrix. Receptor retrotranslocation not only facilitates further rounds of matrix protein import but also prevents deleterious PEX5 retention in the membrane. Three peroxisome-associated ubiquitin-protein ligases in the Really Interesting New Gene (RING) family, PEX2, PEX10, and PEX12, facilitate PEX5 retrotranslocation. However, the detailed mechanism of receptor retrotranslocation remains unclear in plants. We identified an Arabidopsis (Arabidopsis thaliana) pex12 Glu-to-Lys missense allele that conferred severe peroxisomal defects, including impaired β-oxidation, inefficient matrix protein import, and decreased growth. We compared this pex12-1 mutant to other peroxisome-associated ubiquitination-related mutants and found that RING peroxin mutants displayed elevated PEX5 and PEX7 levels, supporting the involvement of RING peroxins in receptor ubiquitination in Arabidopsis. Also, we observed that disruption of any Arabidopsis RING peroxin led to decreased PEX10 levels, as seen in yeast and mammals. Peroxisomal defects were exacerbated in RING peroxin double mutants, suggesting distinct roles of individual RING peroxins. Finally, reducing function of the peroxisome-associated ubiquitin-conjugating enzyme PEX4 restored PEX10 levels and partially ameliorated the other molecular and physiological defects of the pex12-1 mutant. Future biochemical analyses will be needed to determine whether destabilization of the RING peroxin complex observed in pex12-1 stems from PEX4-dependent ubiquitination on the pex12-1 ectopic Lys residue.Oilseed plants obtain energy for germination and early development by utilizing stored fatty acids (Graham, 2008). This β-oxidation of fatty acids to acetyl-CoA occurs in peroxisomes, organelles that also house other important metabolic reactions, including the glyoxylate cycle, several steps in photorespiration, and phytohormone production (Hu et al., 2012). For example, indole-3-butyric acid (IBA) is β-oxidized into the active auxin indole-3-acetic acid (IAA) in peroxisomes (Zolman et al., 2000, 2007, 2008; Strader et al., 2010; Strader and Bartel, 2011). Many peroxisomal metabolic pathways generate reactive oxygen species (Inestrosa et al., 1979; Hu et al., 2012), and peroxisomes also house antioxidative enzymes, like catalase and ascorbate peroxidase, to detoxify hydrogen peroxide (Wang et al., 1999; Mhamdi et al., 2012).Peroxisomes can divide by fission or be synthesized de novo from the endoplasmic reticulum (ER). Preperoxisomes with peroxisomal membrane proteins bud from the ER and fuse, allowing matrix proteins to be imported to form mature peroxisomes (van der Zand et al., 2012; Mayerhofer, 2016). Peroxin (PEX) proteins facilitate peroxisome biogenesis and matrix protein import. Most peroxins are involved in importing proteins destined for the peroxisome matrix, which are imported after recognition of a type 1 or type 2 peroxisome-targeting signal (PTS). The PTS1 is a tripeptide located at the C terminus of most peroxisome-bound proteins (Gould et al., 1989; Chowdhary et al., 2012). The less common PTS2 is a nonapeptide usually located near the N terminus (Swinkels et al., 1991; Reumann, 2004). PTS1 proteins are recognized by PEX5 (van der Leij et al., 1993; Zolman et al., 2000), PTS2 proteins are recognized by PEX7 (Marzioch et al., 1994; Braverman et al., 1997; Woodward and Bartel, 2005), and PEX7 binds to PEX5 to allow matrix protein delivery in plants and mammals (Otera et al., 1998; Hayashi et al., 2005; Woodward and Bartel, 2005). The cargo-receptor complex docks with the membrane peroxins PEX13 and PEX14 (Urquhart et al., 2000; Otera et al., 2002; Woodward et al., 2014), and PEX5 assists cargo translocation into the peroxisomal matrix (Meinecke et al., 2010) before dissociating from its cargo (Freitas et al., 2011).After cargo delivery, PEX5 is recycled to enable further rounds of cargo recruitment (Thoms and Erdmann, 2006). This process requires a set of peroxins that is implicated in ubiquitinating PEX5 so that it can be retrotranslocated back to the cytosol. PEX5 ubiquitination is best understood in yeast. In Saccharomyces cerevisiae, Pex5 is monoubiquitinated through the action of the peroxisome-tethered ubiquitin-conjugating enzyme Pex4 and the peroxisomal ubiquitin-protein ligase Pex12 (Platta et al., 2009) and returned to the cytosol with the assistance of a peroxisome-tethered ATPase complex containing Pex1 and Pex6 (Grimm et al., 2012). S. cerevisiae Pex5 also can be polyubiquitinated and targeted for proteasomal degradation (Kiel et al., 2005). The cytosolic ubiquitin-conjugating enzyme Ubc4 cooperates with the peroxisomal ubiquitin-protein ligase Pex2 to polyubiquitinate Pex5 (Platta et al., 2009). Pex10 has ubiquitin-protein ligase activity (Williams et al., 2008; Platta et al., 2009; El Magraoui et al., 2012), but whether Pex10 directly ubiquitinates Pex5 is controversial. Pex10 promotes Ubc4-dependent Pex5 polyubiquitination when Pex4 is absent (Williams et al., 2008); however, Pex10 is not essential for Pex5 mono- or polyubiquitination (Platta et al., 2009), but rather enhances both Pex4/Pex12- and Ubc4/Pex2-mediated ubiquitination (El Magraoui et al., 2012). Recycling of the PTS2 receptor PEX7 is less understood, although the Pex5 recycling pathways are implicated in shuttling and degrading Pex7 in Pichia pastoris (Hagstrom et al., 2014).Although PEX5 ubiquitination has not been directly demonstrated in plants, the implicated peroxins are conserved in Arabidopsis, and several have been connected to PEX5 retrotranslocation. The PEX4 ubiquitin-conjugating enzyme binds to PEX22, which is predicted to be a peroxisomal membrane protein based on ability to restore peroxisome function to yeast mutants (Zolman et al., 2005). The pex4-1 mutant displays increased membrane-associated PEX5 (Ratzel et al., 2011; Kao and Bartel, 2015), suggesting that ubiquitin supplied by PEX4 promotes PEX5 retrotranslocation. PEX1 and PEX6 are members of the ATPases associated with diverse cellular activities (AAA) family and are tethered to peroxisomes by the peroxisomal membrane protein PEX26 (Goto et al., 2011; Li et al., 2014). The pex6-1 mutant displays PTS1 import defects and decreased PEX5 levels (Zolman and Bartel, 2004), suggesting that impaired PEX5 recycling can lead to increased PEX5 degradation. Indeed, pex4-1 restores PEX5 levels in the pex6-1 mutant (Ratzel et al., 2011), suggesting that Arabidopsis PEX4 also is involved in PEX5 ubiquitination and degradation when retrotranslocation is impeded.In addition to allowing for further rounds of PTS1 cargo import, several lines of evidence suggest that in the absence of efficient retrotranslocation, PEX5 retention in the peroxisomal membrane impairs peroxisome function. Slightly reducing levels of the PEX13 docking peroxin ameliorates the physiological defects of pex4-1 without restoring matrix protein import (Ratzel et al., 2011), presumably because decreasing PEX5 docking reduces its accumulation in the peroxisomal membrane. In addition, overexpressing PEX5 exacerbates rather than ameliorates the peroxisomal defects of pex4-1 (Kao and Bartel, 2015), suggesting that pex4-1 defects are linked to excessive PEX5 lingering in the peroxisome membrane rather than a lack of PEX5 available for import.The three Really Interesting New Gene (RING) peroxins (PEX2, PEX10, and PEX12) from Arabidopsis each possesses in vitro ubiquitin-protein ligase activity (Kaur et al., 2013). Null mutations in the RING peroxin genes confer embryo lethality in Arabidopsis (Hu et al., 2002; Schumann et al., 2003; Sparkes et al., 2003; Fan et al., 2005; Prestele et al., 2010), necessitating other approaches to study the in vivo functions of these peroxins. Expressing RING peroxins with mutations in the C-terminal zinc-binding RING domains (ΔZn) confers matrix protein import defects for PEX2-ΔZn and photorespiration defects for PEX10-ΔZn but no apparent defects for PEX12-ΔZn (Prestele et al., 2010). Targeting individual RING peroxins using RNAi confers β-oxidation deficiencies and impairs PTS1 cargo import (Fan et al., 2005; Nito et al., 2007). A screen for delayed matrix protein degradation (Burkhart et al., 2013) uncovered a missense pex2-1 mutant and a splicing pex10-2 mutant that both display PTS1 import defects (Burkhart et al., 2014), suggesting roles in regulating the PTS1 receptor, PEX5. A missense pex12 mutant (aberrant peroxisome morphology 4, apm4) has defects in β-oxidation and PTS1 import and increased membrane-associated PEX5 (Mano et al., 2006). These findings highlight the essential roles of the RING peroxins in Arabidopsis development and peroxisomal functions, but the RING peroxin interactions and the individual roles of the RING peroxins in PEX5 retrotranslocation remain incompletely understood.In this study, we describe a missense pex12-1 mutant recovered from a forward genetic screen for β-oxidation deficient mutants. The pex12-1 mutant displayed severe peroxisomal defects, including reduced growth, β-oxidation deficiencies, matrix protein import defects, and inefficient processing of PTS2 proteins. Comparing single and double mutants with impaired RING peroxins revealed that each RING peroxin contributes to complex stability and influences PEX5 accumulation. Furthermore, decreasing PEX4 function ameliorated pex12-1 defects, suggesting that the Glu-to-Lys substitution in pex12-1 lures ubiquitination, perhaps by pex12-1 itself, leading to PEX4-dependent degradation of the mutant protein.