Leaf Senescence Signaling: The Ca2+-Conducting Arabidopsis Cyclic Nucleotide Gated Channel2 Acts through Nitric Oxide to Repress Senescence Programming

Ca²⁺ and nitric oxide (NO) are essential components involved in plant senescence signaling cascades. In other signaling pathways, NO generation can be dependent on cytosolic Ca²⁺. The Arabidopsis (Arabidopsis thaliana) mutant dnd1 lacks a plasma membrane-localized cation channel (CNGC2). We recently demonstrated that this channel affects plant response to pathogens through a signaling cascade involving Ca²⁺ modulation of NO generation; the pathogen response phenotype of dnd1 can be complemented by application of a NO donor. At present, the interrelationship between Ca²⁺ and NO generation in plant cells during leaf senescence remains unclear. Here, we use dnd1 plants to present genetic evidence consistent with the hypothesis that Ca²⁺ uptake and NO production play pivotal roles in plant leaf senescence. Leaf Ca²⁺ accumulation is reduced in dnd1 leaves compared to the wild type. Early senescence-associated phenotypes (such as loss of chlorophyll, expression level of senescence-associated genes, H₂O₂ generation, lipid peroxidation, tissue necrosis, and increased salicylic acid levels) were more prominent in dnd1 leaves compared to the wild type. Application of a Ca²⁺ channel blocker hastened senescence of detached wild-type leaves maintained in the dark, increasing the rate of chlorophyll loss, expression of a senescence-associated gene, and lipid peroxidation. Pharmacological manipulation of Ca²⁺ signaling provides evidence consistent with genetic studies of the relationship between Ca²⁺ signaling and senescence with the dnd1 mutant. Basal levels of NO in dnd1 leaf tissue were lower than that in leaves of wild-type plants. Application of a NO donor effectively rescues many dnd1 senescence-related phenotypes. Our work demonstrates that the CNGC2 channel is involved in Ca²⁺ uptake during plant development beyond its role in pathogen defense response signaling. Work presented here suggests that this function of CNGC2 may impact downstream basal NO production in addition to its role (also linked to NO signaling) in pathogen defense responses and that this NO generation acts as a negative regulator during plant leaf senescence signaling.Senescence can be considered as the final stage of a plant’s development. During this process, nutrients will be reallocated from older to younger parts of the plant, such as developing leaves and seeds. Leaf senescence has been characterized as a type of programmed cell death (PCD; Gan and Amasino, 1997; Quirino et al., 2000; Lim et al., 2003). During senescence, organelles such as chloroplasts will break down first. Biochemical changes will also occur in the peroxisome during this process. When the chloroplast disassembles, it is easily observed as a loss of chlorophyll. Mitochondria, the source of energy for cells, will be the last cell organelles to undergo changes during the senescence process (Quirino et al., 2000). At the same time, other catabolic events (e.g. protein and lipid breakdown, etc.) are occurring (Quirino et al., 2000). Hormones may also contribute to this process (Gepstein, 2004). From this information we can infer that leaf senescence is regulated by many signals.Darkness treatment can induce senescence in detached leaves (Poovaiah and Leopold, 1973; Chou and Kao, 1992; Weaver and Amasino, 2001; Chrost et al., 2004; Guo and Crawford, 2005; Ülker et al., 2007). Ca²⁺ can delay the senescence of detached leaves (Poovaiah and Leopold, 1973) and leaf senescence induced by methyl jasmonate (Chou and Kao, 1992); the molecular events that mediate this effect of Ca²⁺ are not well characterized at present.Nitric oxide (NO) is a critical signaling molecule involved in many plant physiological processes. Recently, published evidence supports NO acting as a negative regulator during leaf senescence (Guo and Crawford, 2005; Mishina et al., 2007). Abolishing NO generation in either loss-of-function mutants (Guo and Crawford, 2005) or transgenic Arabidopsis (Arabidopsis thaliana) plants expressing NO degrading dioxygenase (NOD; Mishina et al., 2007) leads to an early senescence phenotype in these plants compared to the wild type. Corpas et al. (2004) showed that endogenous NO is mainly accumulated in vascular tissues of pea (Pisum sativum) leaves. This accumulation is significantly reduced in senescing leaves (Corpas et al., 2004). Corpas et al. (2004) also provided evidence that NO synthase (NOS)-like activity (i.e. generation of NO from l-Arg) is greatly reduced in senescing leaves. Plant NOS activity is regulated by Ca²⁺/calmodulin (CaM; Delledonne et al., 1998; Corpas et al., 2004, 2009; del Río et al., 2004; Valderrama et al., 2007; Ma et al., 2008). These studies suggest a link between Ca²⁺ and NO that could be operating during senescence.In animal cells, all three NOS isoforms require Ca²⁺/CaM as a cofactor (Nathan and Xie, 1994; Stuehr, 1999; Alderton et al., 2001). Notably, animal NOS contains a CaM binding domain (Stuehr, 1999). It is unclear whether Ca²⁺/CaM can directly modulate plant NOS or if Ca²⁺/CaM impacts plant leaf development/senescence through (either direct or indirect) effects on NO generation. However, recent studies from our lab suggest that Ca²⁺/CaM acts as an activator of NOS activity in plant innate immune response signaling (Ali et al., 2007; Ma et al., 2008).Although Arabidopsis NO ASSOCIATED PROTEIN1 (AtNOA1; formerly named AtNOS1) was thought to encode a NOS enzyme, no NOS-encoding gene has yet been identified in plants (Guo et al., 2003; Crawford et al., 2006; Zemojtel et al., 2006). However, the AtNOA1 loss-of-function mutant does display reduced levels of NO generation, and several groups have used the NO donor sodium nitroprusside (SNP) to reverse some low-NO related phenotypes in Atnoa1 plants (Guo et al., 2003; Bright et al., 2006; Zhao et al., 2007). Importantly, plant endogenous NO deficiency (Guo and Crawford, 2005; Mishina et al., 2007) or abscisic acid/methyl jasmonate (Hung and Kao, 2003, 2004) induced early senescence can be successfully rescued by application of exogenous NO. Addition of NO donor can delay GA-elicited PCD in barley (Hordeum vulgare) aleurone layers as well (Beligni et al., 2002).It has been suggested that salicylic acid (SA), a critical pathogen defense metabolite, can be increased in natural (Morris et al., 2000; Mishina et al., 2007) and transgenic NOD-induced senescent Arabidopsis leaves (Mishina et al., 2007). Pathogenesis related gene1 (PR1) expression is up-regulated in transgenic Arabidopsis expressing NOD (Mishina et al., 2007) and in leaves of an early senescence mutant (Ülker et al., 2007).Plant cyclic nucleotide gated channels (CNGCs) have been proposed as candidates to conduct extracellular Ca²⁺ into the cytosol (Sunkar et al., 2000; Talke et al., 2003; Lemtiri-Chlieh and Berkowitz, 2004; Ali et al., 2007; Demidchik and Maathuis, 2007; Frietsch et al., 2007; Kaplan et al., 2007; Ma and Berkowitz, 2007; Urquhart et al., 2007; Ma et al., 2009a, 2009b). Arabidopsis “defense, no death” (dnd1) mutant plants have a null mutation in the gene encoding the plasma membrane-localized Ca²⁺-conducting CNGC2 channel. This mutant also displays no hypersensitive response to infection by some pathogens (Clough et al., 2000; Ali et al., 2007). In addition to involvement in pathogen-mediated Ca²⁺ signaling, CNGC2 has been suggested to participate in the process of leaf development/senescence (Köhler et al., 2001). dnd1 mutant plants have high levels of SA and expression of PR1 (Yu et al., 1998), and spontaneous necrotic lesions appear conditionally in dnd1 leaves (Clough et al., 2000; Jirage et al., 2001). Endogenous H₂O₂ levels in dnd1 mutants are increased from wild-type levels (Mateo et al., 2006). Reactive oxygen species molecules, such as H₂O₂, are critical to the PCD/senescence processes of plants (Navabpour et al., 2003; Overmyer et al., 2003; Hung and Kao, 2004; Guo and Crawford, 2005; Zimmermann et al., 2006). Here, we use the dnd1 mutant to evaluate the relationship between leaf Ca²⁺ uptake during plant growth and leaf senescence. Our results identify NO, as affected by leaf Ca²⁺ level, to be an important negative regulator of leaf senescence initiation. Ca²⁺-mediated NO production during leaf development could control senescence-associated gene (SAG) expression and the production of molecules (such as SA and H₂O₂) that act as signals during the initiation of leaf senescence programs.     

CPK13, a Noncanonical Ca2+-Dependent Protein Kinase,Specifically Inhibits KAT2 and KAT1 Shaker K+ Channels and Reduces Stomatal Opening

Ca²⁺-dependent protein kinases (CPKs) form a large family of 34 genes in Arabidopsis (Arabidopsis thaliana). Based on their dependence on Ca²⁺, CPKs can be sorted into three types: strictly Ca²⁺-dependent CPKs, Ca²⁺-stimulated CPKs (with a significant basal activity in the absence of Ca²⁺), and essentially calcium-insensitive CPKs. Here, we report on the third type of CPK, CPK13, which is expressed in guard cells but whose role is still unknown. We confirm the expression of CPK13 in Arabidopsis guard cells, and we show that its overexpression inhibits light-induced stomatal opening. We combine several approaches to identify a guard cell-expressed target. We provide evidence that CPK13 (1) specifically phosphorylates peptide arrays featuring Arabidopsis K⁺ Channel KAT2 and KAT1 polypeptides, (2) inhibits KAT2 and/or KAT1 when expressed in Xenopus laevis oocytes, and (3) closely interacts in plant cells with KAT2 channels (Förster resonance energy transfer-fluorescence lifetime imaging microscopy). We propose that CPK13 reduces stomatal aperture through its inhibition of the guard cell-expressed KAT2 and KAT1 channels.Stomata are microscopic organs at the leaf surface, each made of two so-called guard cells forming a pore. Opening or closing these pores is the way through which plants control their gas exchanges with the atmosphere (i.e. carbon dioxide uptake to feed the photosynthetic process and transpirational loss of water vapor). Stomatal movements result from osmotically driven fluxes of water, which follow massive exchanges of solutes, including K⁺ ions, between the guard cells and the surrounding tissues (Hetherington, 2001; Nilson and Assmann, 2007).Both Ca²⁺-dependent and Ca²⁺-independent signaling pathways are known to control stomatal movements (MacRobbie, 1993, 1998; Blatt, 2000; Webb et al., 2001; Mustilli et al., 2002; Israelsson et al., 2006; Marten et al., 2007; Laanemets et al., 2013). In particular, Ca²⁺ signals have been reported to promote stomatal closure through the inhibition of inward K⁺ channels and the activation of anion channels (Blatt, 1991, 1992, 2000; Thiel et al., 1992; Grabov and Blatt, 1999; Schroeder et al., 2001; Hetherington and Brownlee, 2004; Mori et al., 2006; Marten et al., 2007; Geiger et al., 2010; Brandt et al., 2012; Scherzer et al., 2012). However, little is known about the molecular identity of the links between Ca²⁺ events and Shaker K⁺ channel activity. Several kinases and phosphatases are believed to be involved in both the Ca²⁺-dependent and Ca²⁺-independent signaling pathways. Plants express two large kinase families whose activity is related to Ca²⁺ signaling. Firstly, CBL-interacting protein kinases (CIPKs; 25 genes in Arabidopsis [Arabidopsis thaliana]) are indirectly controlled by their interaction with a set of calcium sensors, the calcineurin B-like proteins (CBLs; 10 genes in Arabidopsis). This complex forms a fascinating network of potential Ca²⁺ signaling decoders (Luan, 2009; Weinl and Kudla, 2009), which have been addressed in numerous reports (Xu et al., 2006; Hu et al., 2009; Batistic et al., 2010; Held et al., 2011; Chen et al., 2013). In particular, some CBL-CIPK pairs have been shown to regulate Shaker channels such as Arabidopsis K⁺ Transporter1 (AKT1; Xu et al., 2006; Lan et al., 2011) or AKT2 (Held et al., 2011). Second, Ca²⁺-dependent protein kinases (CPKs) form an even larger family (34 genes in Arabidopsis) of proteins combining a kinase domain with the ability to bind Ca²⁺, thanks to the so-called EF hands (Harmon et al., 2000; Harper et al., 2004). CPKs, which, interestingly, are not found in animal cells, exhibit different calcium dependencies (Boudsocq et al., 2012). With respect to this, three types of CPKs can be considered: strictly Ca²⁺-dependent CPKs, Ca²⁺-stimulated CPKs (with a significant basal activity in the absence of Ca²⁺), and essentially Ca²⁺-insensitive CPKs (however, structurally close to kinases of groups 1 and 2).Pioneering work by Luan et al. (1993) demonstrated in Vicia faba guard cells that inward K⁺ channels were regulated by some Ca²⁺-dependent kinases. Then, such a Ca²⁺-dependent kinase was purified from guard cell protoplasts of V. faba and shown to actually phosphorylate the in vitro-translated KAT1 protein, a Shaker channel subunit natively expressed in Arabidopsis guard cells (Li et al., 1998). KAT1 regulation by CPK was shown by the inhibition of KAT1 currents after the coexpression of KAT1 and CDPK from soybean (Glycine max) in oocytes (Berkowitz et al., 2000). Since then, several cpk mutant lines of Arabidopsis have been shown to be impaired in stomatal movements, for example cpk10 (Ca²⁺ insensitive), cpk4/cpk11 (Ca²⁺ dependent), and cpk3/cpk6/cpk23 (Ca²⁺ dependent; Mori et al., 2006; Geiger et al., 2010; Munemasa et al., 2011; Hubbard et al., 2012).Of the nine genes encoding voltage-dependent K⁺ channels (Shaker) in Arabidopsis (Véry and Sentenac, 2002, 2003; Lebaudy et al., 2007; Hedrich, 2012), six are expressed in guard cells and play a role in stomatal movements: the Gated Outwardly-Rectifying K⁺ (GORK) gene, encoding an outward K⁺ channel subunit, and the AKT1, AKT2, Arabidopsis K⁺ Rectifying Channel1 (AtKC1), KAT1, and KAT2 genes, encoding inward K⁺ channel subunits (Pilot et al., 2001; Szyroki et al., 2001; Hosy et al., 2003; Pandey et al., 2007; Lebaudy et al., 2008a). Shaker channels result from the assembly of four subunits, and it has been shown that inward subunits tend to heterotetramerize, thus potentially widening the functional and regulatory scope of inward K⁺ conductance in guard cells (Xicluna et al., 2007; Jeanguenin et al., 2008; Lebaudy et al., 2008a, 2010). Inhibition of inward K⁺ channels has been shown to reduce stomatal opening (Liu et al., 2000; Kwak et al., 2001). This has grounded a strategy for disrupting inward K⁺ channel conductance in guard cells by expressing a nonfunctional KAT2 subunit (dominant negative mutation) in a kat2 knockout Arabidopsis line. The resulting Arabidopsis lines, named kincless, have no functional inward K⁺ channels and exhibit delayed stomatal opening (Lebaudy et al., 2008b) with, in the long term, a biomass reduction compared with the Arabidopsis wild-type line.Among the CPKs presumably expressed in Arabidopsis guard cells (Leonhardt et al., 2004), we looked for CPK13, which belongs to the atypical Ca²⁺-insensitive type of CPKs (Kanchiswamy et al., 2010; Boudsocq et al., 2012; Liese and Romeis, 2013) and whose role remains unknown in stomatal movements. Here, we confirm first that CPK13 kinase activity is independent of Ca²⁺ and show that CPK13 expression is predominant in Arabidopsis guard cells using CPK13-GUS lines. We then report that overexpression of CPK13 in Arabidopsis induces a dramatic default in stomatal aperture. Based on the previously reported kincless phenotype (Lebaudy et al., 2008b), we propose that CPK13 could reduce the activity of inward K⁺ channels in guard cells, particularly that of KAT2. We confirm this hypothesis by voltage-clamp experiments and show an inhibition of KAT2 and KAT1 activity by CPK13 (but not that of AKT2). In addition, we present peptide array phosphorylation assays showing that CPK13 targets, with some specificity, several KAT2 and KAT1 polypeptides. Finally, we demonstrate that KAT2 and CPK13 interact in planta using Förster resonance energy transfer (FRET)-fluorescence lifetime imaging microscopy (FLIM).     

Focus Issue on Calcium Signaling: Identification of Cyclic GMP-Activated Nonselective Ca2+-Permeable Cation Channels and Associated CNGC5 and CNGC6 Genes in Arabidopsis Guard Cells

Cytosolic Ca²⁺ in guard cells plays an important role in stomatal movement responses to environmental stimuli. These cytosolic Ca²⁺ increases result from Ca²⁺ influx through Ca²⁺-permeable channels in the plasma membrane and Ca²⁺ release from intracellular organelles in guard cells. However, the genes encoding defined plasma membrane Ca²⁺-permeable channel activity remain unknown in guard cells and, with some exceptions, largely unknown in higher plant cells. Here, we report the identification of two Arabidopsis (Arabidopsis thaliana) cation channel genes, CNGC5 and CNGC6, that are highly expressed in guard cells. Cytosolic application of cyclic GMP (cGMP) and extracellularly applied membrane-permeable 8-Bromoguanosine 3′,5′-cyclic monophosphate-cGMP both activated hyperpolarization-induced inward-conducting currents in wild-type guard cells using Mg²⁺ as the main charge carrier. The cGMP-activated currents were strongly blocked by lanthanum and gadolinium and also conducted Ba²⁺, Ca²⁺, and Na⁺ ions. cngc5 cngc6 double mutant guard cells exhibited dramatically impaired cGMP-activated currents. In contrast, mutations in CNGC1, CNGC2, and CNGC20 did not disrupt these cGMP-activated currents. The yellow fluorescent protein-CNGC5 and yellow fluorescent protein-CNGC6 proteins localize in the cell periphery. Cyclic AMP activated modest inward currents in both wild-type and cngc5cngc6 mutant guard cells. Moreover, cngc5 cngc6 double mutant guard cells exhibited functional abscisic acid (ABA)-activated hyperpolarization-dependent Ca²⁺-permeable cation channel currents, intact ABA-induced stomatal closing responses, and whole-plant stomatal conductance responses to darkness and changes in CO₂ concentration. Furthermore, cGMP-activated currents remained intact in the growth controlled by abscisic acid2 and abscisic acid insensitive1 mutants. This research demonstrates that the CNGC5 and CNGC6 genes encode unique cGMP-activated nonselective Ca²⁺-permeable cation channels in the plasma membrane of Arabidopsis guard cells.Plants lose water via transpiration and take in CO₂ for photosynthesis through stomatal pores. Each stomatal pore is surrounded by two guard cells, and stomatal movements are driven by the change of turgor pressure in guard cells. The intracellular second messenger Ca²⁺ functions in guard cell signal transduction (Schroeder and Hagiwara, 1989; McAinsh et al., 1990; Webb et al., 1996; Grabov and Blatt, 1998; Allen et al., 1999; MacRobbie, 2000; Mori et al., 2006; Young et al., 2006; Siegel et al., 2009; Chen et al., 2010; Hubbard et al., 2012). Plasma membrane ion channel activity and gene expression in guard cells are finely regulated by the intracellular free calcium concentration ([Ca2+]_cyt; Schroeder and Hagiwara, 1989; Webb et al., 2001; Allen et al., 2002; Siegel et al., 2009; Kim et al., 2010; Stange et al., 2010). Ca²⁺-dependent protein kinases (CPKs) function as targets of the cytosolic Ca²⁺ signal, and several members of the CPK family have been shown to function in stimulus-induced stomatal closing, including the Arabidopsis (Arabidopsis thaliana) CPK3, CPK4, CPK6, CPK10, and CPK11 proteins (Mori et al., 2006; Zhu et al., 2007; Zou et al., 2010; Brandt et al., 2012; Hubbard et al., 2012). Further research found that several CPKs could activate the S-type anion channel SLAC1 in Xenopus laevis oocytes, including CPK21, CPK23, and CPK6 (Geiger et al., 2010; Brandt et al., 2012). At the same time, the Ca²⁺-independent protein kinase Open Stomata1 mediates stomatal closing and activates the S-type anion channel SLAC1 (Mustilli et al., 2002; Yoshida et al., 2002; Geiger et al., 2009; Lee et al., 2009; Xue et al., 2011), indicating that both Ca²⁺-dependent and Ca²⁺-independent pathways function in guard cells.Multiple essential factors of guard cell abscisic acid (ABA) signal transduction function in the regulation of Ca²⁺-permeable channels and [Ca2+]_cyt elevations, including Abscisic Acid Insensitive1 (ABI1), ABI2, Enhanced Response to Abscisic Acid1 (ERA1), the NADPH oxidases AtrbohD and AtrbohF, the Guard Cell Hydrogen Peroxide-Resistant1 (GHR1) receptor kinase, as well as the Ca²⁺-activated CPK6 protein kinase (Pei et al., 1998; Allen et al., 1999, 2002; Kwak et al., 2003; Miao et al., 2006; Mori et al., 2006; Hua et al., 2012). [Ca2+]_cyt increases result from both Ca²⁺ release from intracellular Ca²⁺ stores (McAinsh et al., 1992) and Ca²⁺ influx across the plasma membrane (Hamilton et al., 2000; Pei et al., 2000; Murata et al., 2001; Kwak et al., 2003; Hua et al., 2012). Electrophysiological analyses have characterized nonselective Ca²⁺-permeable channel activity in the plasma membrane of guard cells (Schroeder and Hagiwara, 1990; Hamilton et al., 2000; Pei et al., 2000; Murata et al., 2001; Köhler and Blatt, 2002; Miao et al., 2006; Mori et al., 2006; Suh et al., 2007; Vahisalu et al., 2008; Hua et al., 2012). However, the genetic identities of Ca²⁺-permeable channels in the plasma membrane of guard cells have remained unknown despite over two decades of research on these channel activities.The Arabidopsis genome includes 20 genes encoding cyclic nucleotide-gated channel (CNGC) homologs and 20 genes encoding homologs to animal Glu receptor channels (Lacombe et al., 2001; Kaplan et al., 2007; Ward et al., 2009), which have been proposed to function in plant cells as cation channels (Schuurink et al., 1998; Arazi et al., 1999; Köhler et al., 1999). Recent research has demonstrated functions of specific Glu receptor channels in mediating Ca²⁺ channel activity (Michard et al., 2011; Vincill et al., 2012). Previous studies have shown cAMP activation of nonselective cation currents in guard cells (Lemtiri-Chlieh and Berkowitz, 2004; Ali et al., 2007). However, only a few studies have shown the disappearance of a defined plasma membrane Ca²⁺ channel activity in plants upon mutation of candidate Ca²⁺ channel genes (Ali et al., 2007; Michard et al., 2011; Laohavisit et al., 2012; Vincill et al., 2012). Some CNGCs have been found to be involved in cation nutrient intake, including monovalent cation intake (Guo et al., 2010; Caballero et al., 2012), salt tolerance (Guo et al., 2008; Kugler et al., 2009), programmed cell death and pathogen responses (Clough et al., 2000; Balagué et al., 2003; Urquhart et al., 2007; Abdel-Hamid et al., 2013), thermal sensing (Finka et al., 2012; Gao et al., 2012), and pollen tube growth (Chang et al., 2007; Frietsch et al., 2007; Tunc-Ozdemir et al., 2013a, 2013b). Direct in vivo disappearance of Ca²⁺ channel activity in cngc disruption mutants has been demonstrated in only a few cases thus far (Ali et al., 2007; Gao et al., 2012). In this research, we show that CNGC5 and CNGC6 are required for a cyclic GMP (cGMP)-activated nonselective Ca²⁺-permeable cation channel activity in the plasma membrane of Arabidopsis guard cells.     

Enhanced Abscisic Acid-Mediated Responses in nia1nia2noa1-2 Triple Mutant Impaired in NIA/NR- and AtNOA1-Dependent Nitric Oxide Biosynthesis in Arabidopsis

Nitric oxide (NO) regulates a wide range of plant processes from development to environmental adaptation. Despite its reported regulatory functions, it remains unclear how NO is synthesized in plants. We have generated a triple nia1nia2noa1-2 mutant that is impaired in nitrate reductase (NIA/NR)- and Nitric Oxide-Associated1 (AtNOA1)-mediated NO biosynthetic pathways. NO content in roots of nia1nia2 and noa1-2 plants was lower than in wild-type plants and below the detection limit in nia1nia2noa1-2 plants. NIA/NR- and AtNOA1-mediated biosynthesis of NO were thus active and responsible for most of the NO production in Arabidopsis (Arabidopsis thaliana). The nia1nia2noa1-2 plants displayed reduced size, fertility, and seed germination potential but increased dormancy and resistance to water deficit. The increasing deficiency in NO of nia1nia2, noa1-2, and nia1nia2noa1-2 plants correlated with increased seed dormancy, hypersensitivity to abscisic acid (ABA) in seed germination and establishment, as well as dehydration resistance. In nia1nia2noa1-2 plants, enhanced drought tolerance was due to a very efficient stomata closure and inhibition of opening by ABA, thus uncoupling NO from ABA-triggered responses in NO-deficient guard cells. The NO-deficient mutants in NIA/NR- and AtNOA1-mediated pathways in combination with the triple mutant will be useful tools to functionally characterize the role of NO and the contribution of both biosynthetic pathways in regulating plant development and defense.Nitric oxide (NO) is a small ubiquitous molecule derived from nitrogen-containing precursors that is one of the earliest and most widespread signaling molecules in living organisms from metazoans to mammals (Torreilles, 2001). The regulatory functions of NO have been extensively studied in mammals, where it is synthesized from Arg through the activity of NO synthases (Knowles and Moncada, 1994). By contrast, the biosynthesis and function of this molecule in plants are largely unknown. During the last 10 years, NO biosynthesis in plants has been one of the most controversial topics in plant biology (Durner and Klessig, 1999; Wendehenne et al., 2001; del Río et al., 2004; Zeier et al., 2004; Lamotte et al., 2005; Meyer et al., 2005; Modolo et al., 2005; Crawford, 2006; Crawford et al., 2006; Zemojtel et al., 2006a). Despite the controversy about its biosynthesis, it is now clear that NO regulates many physiological processes of plants, including seed germination, cell death, defense responses against pathogens, stomata function, senescence, and flowering (Beligni and Lamattina, 2000; Pedroso et al., 2000; Neill et al., 2002; Lamattina et al., 2003; He et al., 2004; Romero-Puertas et al., 2004; Wendehenne et al., 2004; Delledonne, 2005; Guo and Crawford, 2005; Simpson, 2005; Grün et al., 2006; Melotto et al., 2006; Planchet et al., 2006; Ali et al., 2007; Mishina et al., 2007).The molecular mechanisms underlying the control of seed dormancy and germination are still poorly characterized. Genetic data support a central role of abscisic acid (ABA) in regulating seed dormancy, whereas gibberellins promote germination (Finkelstein et al., 2008; Holdsworth et al., 2008). In addition, NO has been lately characterized as a new component in the signaling pathway leading to dormancy breakage. NO-releasing compounds reduce dormancy in a NO-dependent manner in Arabidopsis (Arabidopsis thaliana), some warm-season grasses, and certain barley (Hordeum vulgare) cultivars (Bethke et al., 2004; Sarath et al., 2006). More recently, the aleurone layer cells have been characterized as responsive to NO, gibberellins, and ABA, thus becoming a primary determinant of seed dormancy in Arabidopsis (Bethke et al., 2007).Two main enzyme-based pathways have been proposed to be functional for NO biosynthesis in plants. One is based on the activity of nitrate reductases (Meyer et al., 2005; Modolo et al., 2005), and another one, yet undefined, is based on the direct or indirect function of the Nitric Oxide-Associated1/Resistant to Inhibition by Fosfidomycin1 (AtNOA1/RIF1) protein. It has been also reported that NO synthesis from nitrite occurs in mitochondria associated with mitochondrial electron transport (Planchet et al., 2005) and also that this pathway is mainly functioning in roots under anoxia (Gupta et al., 2005). Moreover, the balance between mitochondrial nitrite reduction and superoxide-dependent NO degradation seems to be derived from factors controlling NO levels in Arabidopsis (Wulff et al., 2009). It has been recently reported that the synthesis of NO in floral organs requires nitrate reductase activity (Seligman et al., 2008) and also that homologues of AtNOA1 participate in NO biosynthesis in diatoms (Vardi et al., 2008), mammals (Zemojtel et al., 2006b; Parihar et al., 2008a, 2008b), and Nicotiana benthamiana (Kato et al., 2008). Recently, the identification of the rif1 mutant, carrying a null mutation in the AtNOA1 locus (At3g47450), allowed uncovering of a function for AtNOA1/RIF1 in the expression of plastome-encoded proteins (Flores-Pérez et al., 2008). Moreover, another recent report claims that AtNOA1 is not a NO synthase but a cGTPase (Moreau et al., 2008), likely playing a role in ribosome assembly and subsequent mRNA translation to proteins in the chloroplasts.To date, it is not clear if both pathways coexist in plants and, if so, the corresponding contributions of each pathway to NO biosynthesis. In this work, we have addressed the functions of both pathways in Arabidopsis by generating a triple mutant in both nitrate reductases and AtNOA1 that is severely impaired in NO production. Further characterization of NO-deficient plants allowed us to identify a functional cross talk between NO and ABA in controlling seed germination and dormancy as well as plant resistance to water deficit.     

Focus Issue on Calcium Signaling: Calcium-Dependent and -Independent Stomatal Signaling Network and Compensatory Feedback Control of Stomatal Opening via Ca2+ Sensitivity Priming

Guard cells use compensatory feedback controls to adapt to conditions that produce excessively open stomata.In the past 15 years or more, many mutants that are impaired in stimulus-induced stomatal closing and opening have been identified and functionally characterized in Arabidopsis (Arabidopsis thaliana), leading to a mechanistic understanding of the guard cell signal transduction network. However, evidence has only recently emerged that mutations impairing stomatal closure, in particular those in slow anion channel SLOW ANION CHANNEL-ASSOCIATED1 (SLAC1), unexpectedly also exhibit slowed stomatal opening responses. Results suggest that this compensatory slowing of stomatal opening can be attributed to a calcium-dependent posttranslational down-regulation of stomatal opening mechanisms, including down-regulation of inward K⁺ channel activity. Here, we discuss this newly emerging stomatal compensatory feedback control model mediated via constitutive enhancement (priming) of intracellular Ca²⁺ sensitivity of ion channel activity. The CALCIUM-DEPENDENT PROTEIN KINASE6 (CPK6) is strongly activated by physiological Ca²⁺ elevations and a model is discussed and open questions are raised for cross talk among Ca²⁺-dependent and Ca²⁺-independent guard cell signal transduction pathways and Ca²⁺ sensitivity priming mechanisms.Stomatal pores formed by two guard cells enable CO₂ uptake from the atmosphere, but also ensure leaf cooling and provide a pulling force for nutrient uptake from the soil via transpiration. These vitally important processes are inevitably accompanied by water loss through stomata. Stomatal opening and closure is caused by the uptake and release of osmotically active substances and is tightly regulated by signaling pathways that lead to the activation or inactivation of guard cell ion channels and pumps. Potassium ions enter guard cells through the inward-rectifying K⁺ channels (K+_in) during stomatal opening and are released via outward-rectifying K⁺ channels during stomatal closure (Schroeder et al., 1987; Hosy et al., 2003; Roelfsema and Hedrich 2005). Cytosolic Ca²⁺, an important second messenger in plants, mediates ion channel regulation, particularly down-regulation of inward-conducting K+_in channels and activation of S-type anion channels, thus mediating stomatal closure and inhibiting stomatal opening (Schroeder and Hagiwara, 1989; Dodd et al., 2010; Kim et al., 2010). Stomatal closure is initiated by anion efflux via the slow S-type anion channel SLAC1 (Negi et al., 2008; Vahisalu et al., 2008; Kollist et al., 2011) and the voltage-dependent rapid R-type anion channel QUICK-ACTIVATING ANION CHANNEL1 (Meyer et al. 2010; Sasaki et al., 2010).In recent years, advances have been made toward understanding mechanisms mediating abscisic acid (ABA)-induced stomatal closure (Cutler et al., 2010; Kim et al., 2010; Raghavendra et al., 2010). The core ABA signaling module, consisting of PYR/RCAR (for pyrabactin resistance 1/regulatory components of ABA receptors) receptors, clade A protein phosphatases (PP2Cs), SNF-related protein kinase OPEN STOMATA1 (OST1), and downstream targets, is Ca²⁺-independent (Ma et al., 2009; Park et al., 2009; Hubbard et al., 2010). However, ABA-induced stomatal closure was reduced to only 30% of the normal stomatal closure response under conditions that inhibited intracellular cytosolic free calcium ([Ca2+]_cyt) elevations in Arabidopsis (Siegel et al., 2009), consistent with previous findings in other plants (De Silva et al., 1985; Schwartz, 1985; McAinsh et al., 1991; MacRobbie, 2000). Together these and other studies show the importance of [Ca2+]_cyt for a robust ABA-induced stomatal closure. Here, we discuss Ca²⁺-dependent and Ca²⁺-independent signaling pathways in guard cells and open questions on how these may work together.Plants carrying mutations in the SLAC1 anion channel have innately more open stomata, and exhibit clear impairments in ABA-, elevated CO₂-, Ca²⁺-, ozone-, air humidity-, darkness-, and hydrogen peroxide-induced stomatal closure (Negi et al., 2008; Vahisalu et al., 2008; Merilo et al., 2013). Recent research, however, unexpectedly revealed that mutations in SLAC1 also down-regulate stomatal opening mechanisms and slow down stomatal opening (Laanemets et al., 2013).     

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.     

Phosphorylation of the C Terminus of RHD3 Has a Critical Role in Homotypic ER Membrane Fusion in Arabidopsis

The endoplasmic reticulum (ER) consists of dynamically changing tubules and cisternae. In animals and yeast, homotypic ER membrane fusion is mediated by fusogens (atlastin and Sey1p, respectively) that are membrane-associated dynamin-like GTPases. In Arabidopsis (Arabidopsis thaliana), another dynamin-like GTPase, ROOT HAIR DEFECTIVE3 (RHD3), has been proposed as an ER membrane fusogen, but direct evidence is lacking. Here, we show that RHD3 has an ER membrane fusion activity that is enhanced by phosphorylation of its C terminus. The ER network was RHD3-dependently reconstituted from the cytosol and microsome fraction of tobacco (Nicotiana tabacum) cultured cells by exogenously adding GTP, ATP, and F-actin. We next established an in vitro assay system of ER tubule formation with Arabidopsis ER vesicles, in which addition of GTP caused ER sac formation from the ER vesicles. Subsequent application of a shearing force to this system triggered the formation of tubules from the ER sacs in an RHD-dependent manner. Unexpectedly, in the absence of a shearing force, Ser/Thr kinase treatment triggered RHD3-dependent tubule formation. Mass spectrometry showed that RHD3 was phosphorylated at multiple Ser and Thr residues in the C terminus. An antibody against the RHD3 C-terminal peptide abolished kinase-triggered tubule formation. When the Ser cluster was deleted or when the Ser residues were replaced with Ala residues, kinase treatment had no effect on tubule formation. Kinase treatment induced the oligomerization of RHD3. Neither phosphorylation-dependent modulation of membrane fusion nor oligomerization has been reported for atlastin or Sey1p. Taken together, we propose that phosphorylation-stimulated oligomerization of RHD3 enhances ER membrane fusion to form the ER network.In eukaryotic cells, the endoplasmic reticulum (ER) is the organelle with the largest membrane area. The ER consists of an elaborate network of interconnected membrane tubules and cisternae that is continually moving and being remodeled (Friedman and Voeltz, 2011). In plant cells, ER movement and remodeling is primarily driven by the actin-myosin XI cytoskeleton (Sparkes et al., 2009; Ueda et al., 2010; Yokota et al., 2011; Griffing et al., 2014) and secondarily by the microtubule cytoskeleton (Hamada et al., 2014). Several factors involved in creating the ER architecture have been also identified (Anwar et al., 2012; Chen et al., 2012; Goyal and Blackstone, 2013; Sackmann, 2014; Stefano et al., 2014a; Westrate et al., 2015). Among them, ER membrane-bound GTPases, animal atlastins and yeast Sey1p (Synthetic Enhancement of Yop1), function as ER fusogens to form the interconnected tubular network (Hu et al., 2009; Orso et al., 2009; Anwar et al., 2012). Atlastin molecules on the two opposed membranes have been proposed to transiently dimerize to attract the two membranes to each other (Bian et al., 2011; Byrnes and Sondermann, 2011; Morin-Leisk et al., 2011; Moss et al., 2011; Lin et al., 2012; Byrnes et al., 2013). Closely attracted lipid bilayers are supposed to be destabilized by an amphipathic helical domain at the atlastin C terminus to facilitate membrane fusion (Bian et al., 2011; Liu et al., 2012; Faust et al., 2015). Knockdown of atlastins leads to fragmentation of the ER and unbranched ER tubules, while overexpression of atlastins enhances ER membrane fusion, which enlarges the ER profiles (Hu et al., 2009; Orso et al., 2009).An Arabidopsis (Arabidopsis thaliana) protein, ROOT HAIR DEFECTIVE3 (RHD3), has been proposed as a fusogen because (1) when it is disrupted, the ER network is modified into large cable-like strands of poorly branched membranes (Zheng et al., 2004; Chen et al., 2011; Stefano et al., 2012), (2) it shares sequence similarity with the above-mentioned fusogen Sey1p (Hu et al., 2009), and (3) it has structural similarity to atlastin and Sey1p, with a functional GTPase domain at the N-terminal cytosolic domain (Stefano et al., 2012) followed by two transmembrane domains and a cytosolic tail. RHD3 has a longer cytosolic C-terminal tail than do atlastin and Sey1p (Stefano and Brandizzi, 2014). It contains not only an amphipathic region but also a Ser/Thr-rich C terminus.Arabidopsis has two RHD3 isoforms called RHD3-Like 1 and RHD3-Like 2. Fluorescently tagged RHD3 and RHD3-Like 2 localize to the ER (Chen et al., 2011; Stefano et al., 2012; Lee et al., 2013). RHD3 and the two RHD3-Like proteins likely have redundant roles in ER membrane fusion (Zhang et al., 2013). Overexpression of either RHD3 or RHD3-Like 2 with a defective GTPase domain phenocopies the aberrant ER morphology in rhd3-deficient mutants (Chen et al., 2011; Lee et al., 2013).In this study, we show that the Ser/Thr-rich C terminus enhances ER membrane fusion following phosphorylation of its C terminus. We propose a model in which phosphorylation and oligomerization of RHD3 is required for efficient ER membrane fusion. Our findings clarify the mechanisms that regulate RHD3 and consequently the homeostasis of membrane fusion in the ER.     

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.