Differential Sodium and Potassium Transport Selectivities of the Rice OsHKT2;1 and OsHKT2;2 Transporters in Plant Cells

Na⁺ and K⁺ homeostasis are crucial for plant growth and development. Two HKT transporter/channel classes have been characterized that mediate either Na⁺ transport or Na⁺ and K⁺ transport when expressed in Xenopus laevis oocytes and yeast. However, the Na⁺/K⁺ selectivities of the K⁺-permeable HKT transporters have not yet been studied in plant cells. One study expressing 5′ untranslated region-modified HKT constructs in yeast has questioned the relevance of cation selectivities found in heterologous systems for selectivity predictions in plant cells. Therefore, here we analyze two highly homologous rice (Oryza sativa) HKT transporters in plant cells, OsHKT2;1 and OsHKT2;2, that show differential K⁺ permeabilities in heterologous systems. Upon stable expression in cultured tobacco (Nicotiana tabacum) Bright-Yellow 2 cells, OsHKT2;1 mediated Na⁺ uptake, but little Rb⁺ uptake, consistent with earlier studies and new findings presented here in oocytes. In contrast, OsHKT2;2 mediated Na⁺-K⁺ cotransport in plant cells such that extracellular K⁺ stimulated OsHKT2;2-mediated Na⁺ influx and vice versa. Furthermore, at millimolar Na⁺ concentrations, OsHKT2;2 mediated Na⁺ influx into plant cells without adding extracellular K⁺. This study shows that the Na⁺/K⁺ selectivities of these HKT transporters in plant cells coincide closely with the selectivities in oocytes and yeast. In addition, the presence of external K⁺ and Ca²⁺ down-regulated OsHKT2;1-mediated Na⁺ influx in two plant systems, Bright-Yellow 2 cells and intact rice roots, and also in Xenopus oocytes. Moreover, OsHKT transporter selectivities in plant cells are shown to depend on the imposed cationic conditions, supporting the model that HKT transporters are multi-ion pores.Intracellular Na⁺ and K⁺ homeostasis play vital roles in growth and development of higher plants (Clarkson and Hanson, 1980). Low cytosolic Na⁺ and high K⁺/Na⁺ ratios aid in maintaining an osmotic and biochemical equilibrium in plant cells. Na⁺ and K⁺ influx and efflux across membranes require the function of transmembrane Na⁺ and K⁺ transporters/channels. Several Na⁺-permeable transporters have been characterized in plants (Zhu, 2001; Horie and Schroeder, 2004; Apse and Blumwald, 2007). Na⁺/H⁺ antiporters mediate sequestration of Na⁺ into vacuoles under salt stress conditions in plants (Blumwald and Poole, 1985, 1987; Sze et al., 1999). Na⁺ (cation)/H⁺ antiporters are encoded by six AtNHX genes in Arabidopsis (Arabidopsis thaliana; Apse et al., 1999; Gaxiola et al., 1999; Yokoi et al., 2002; Aharon et al., 2003). A distinct Na⁺/H⁺ antiporter, Salt Overly Sensitive1, mediates Na⁺/H⁺ exchange at the plasma membrane and mediates cellular Na⁺ extrusion (Shi et al., 2000, 2002; Zhu, 2001; Ward et al., 2003). Electrophysiological analyses reveal that voltage-independent channels, also named nonselective cation channels, mediate Na⁺ influx into roots under high external Na⁺ concentrations (Amtmann et al., 1997; Tyerman et al., 1997; Buschmann et al., 2000; Davenport and Tester, 2000); however, the underlying genes remain unknown.Potassium is the most abundant cation in plants and an essential nutrient for plant growth. The Arabidopsis genome includes 13 genes encoding KUP/HAK/KT transporters (Quintero and Blatt, 1997; Santa-María et al., 1997; Fu and Luan, 1998; Kim et al., 1998), and 17 genes have been identified encoding this family of transporters in rice (Oryza sativa ‘Nipponbare’; Bañuelos et al., 2002). Several KUP/HAK/KT transporters have been characterized as mediating K⁺ uptake across the plasma membrane of plant cells (Rigas et al., 2001; Bañuelos et al., 2002; Gierth et al., 2005).Ionic balance, especially the Na⁺/K⁺ ratio, is a key factor of salt tolerance in plants (Niu et al., 1995; Maathuis and Amtmann, 1999; Shabala, 2000; Mäser et al., 2002a; Tester and Davenport, 2003; Horie et al., 2006; Apse and Blumwald, 2007; Chen et al., 2007; Gierth and Mäser, 2007). Salinity stress is a major problem for agricultural productivity of crops worldwide (Greenway and Munns, 1980; Zhu, 2001). The Arabidopsis AtHKT1;1 transporter plays a key role in salt tolerance of plants by mediating Na⁺ exclusion from leaves (Mäser et al., 2002a; Berthomieu et al., 2003; Gong et al., 2004; Sunarpi et al., 2005; Rus et al., 2006; Davenport et al., 2007; Horie et al., 2009). athkt1;1 mutations cause leaf chlorosis and elevated Na⁺ accumulation in leaves under salt stress conditions in Arabidopsis (Mäser et al., 2002a; Berthomieu et al., 2003; Gong et al., 2004; Sunarpi et al., 2005). AtHKT1;1 and its homolog in rice, OsHKT1;5 (SKC1), mediate leaf Na⁺ exclusion by removing Na⁺ from the xylem sap to protect plants from salinity stress (Ren et al., 2005; Sunarpi et al., 2005; Horie et al., 2006, 2009; Davenport et al., 2007).The land plant HKT gene family is divided into two classes based on their nucleic acid sequences and protein structures (Mäser et al., 2002b; Platten et al., 2006). Class 1 HKT transporters have a Ser residue at a selectivity filter position in the first pore loop, which is replaced by a Gly in all but one known class 2 HKT transporter (Horie et al., 2001; Mäser et al., 2002b; Garciadeblás et al., 2003). While the Arabidopsis genome includes only one HKT gene, AtHKT1;1 (Uozumi et al., 2000), seven full-length OsHKT genes were found in the japonica rice cv Nipponbare genome (Garciadeblás et al., 2003). Members of class 1 HKT transporters, AtHKT1;1 and SKC1/OsHKT1;5, have a relatively higher Na⁺-to-K⁺ selectivity in Xenopus laevis oocytes and yeast than class 2 HKT transporters (Uozumi et al., 2000; Horie et al., 2001; Mäser et al., 2002b; Ren et al., 2005). The first identified plant HKT transporter, TaHKT2;1 from wheat (Triticum aestivum), is a class 2 HKT transporter (Schachtman and Schroeder, 1994). TaHKT2;1 was found to mediate Na⁺-K⁺ cotransport and Na⁺ influx at high Na⁺ concentrations in heterologous expression systems (Rubio et al., 1995, 1999; Gassmann et al., 1996; Mäser et al., 2002b). Thus, class 1 HKT transporters have been characterized as Na⁺-preferring transporters with a smaller K⁺ permeability (Fairbairn et al., 2000; Uozumi et al., 2000; Su et al., 2003; Jabnoune et al., 2009), whereas class 2 HKT transporters function as Na⁺-K⁺ cotransporters or channels (Gassmann et al., 1996; Corratgé et al., 2007). In addition, at millimolar Na⁺ concentrations, class 2 HKT transporters were found to mediate Na⁺ influx, without adding external K⁺ in Xenopus oocytes and yeast (Rubio et al., 1995, 1999; Gassmann et al., 1996; Horie et al., 2001). However, the differential cation transport selectivities of the two types of HKT transporters have not yet been analyzed and compared in plant cells.A study of the barley (Hordeum vulgare) and wheat class 2 transporters has suggested that the transport properties of HvHKT2;1 and TaHKT2;1 expressed in yeast are variable, depending on the constructs from which the transporter is expressed, and have led to questioning of the K⁺ transport activity of HKT transporters characterized in Xenopus oocytes and yeast (Haro et al., 2005). It was further proposed that the 5′ translation initiation of HKT proteins in yeast at nonconventional (non-ATG) sites affects the transporter selectivities of HKT transporters (Haro et al., 2005), although direct evidence for this has not yet been presented. However, recent research has shown a K⁺ permeability of OsHKT2;1 but not of OsHKT1;1 and OsHKT1;3 in Xenopus oocytes. These three OsHKT transporters show overlapping and also distinctive expression patterns in rice (Jabnoune et al., 2009).The report of Haro et al. (2005) has opened a central question addressed in this study: are the Na⁺/K⁺ transport selectivities of plant HKT transporters characterized in heterologous systems of physiological relevance in plant cells, or do they exhibit strong differences in the cation transport selectivities in these nonplant versus plant systems? To address this question, we analyzed the Na⁺/K⁺ transport selectivities of the OsHKT2;1 and OsHKT2;2 transporters expressed in cultured tobacco (Nicotiana tabacum ‘Bright-Yellow 2’ [BY2]) cells. OsHKT2;1 and OsHKT2;2 are two highly homologous HKT transporters from indica rice cv Pokkali, sharing 91% amino acid and 93% cDNA sequence identity (Horie et al., 2001). OsHKT2;1 mediates mainly Na⁺ uptake, which correlates with the presence of a Ser residue in the first pore loop of OsHKT2;1 (Horie et al., 2001, 2007; Mäser et al., 2002b; Garciadeblás et al., 2003). In contrast, OsHKT2;2 mediates Na⁺-K⁺ cotransport in Xenopus oocytes and yeast (Horie et al., 2001). Furthermore, at millimolar Na⁺ concentrations, OsHKT2;2 mediates Na⁺ influx in the absence of added K⁺ (Horie et al., 2001). Recent research on oshkt2;1 loss-of-function mutant alleles has revealed that OsHKT2;1 from japonica rice mediates a large Na⁺ influx component into K⁺-starved roots, thus compensating for lack of K⁺ availability (Horie et al., 2007). But the detailed Na⁺/K⁺ selectivities of Gly-containing, predicted K⁺-transporting class 2 HKT transporters have not yet been analyzed in plant cells.Here, we have generated stable OsHKT2;1- and OsHKT2;2-expressing tobacco BY2 cell lines and characterized the cell lines by ion content measurements and tracer influx studies to directly analyze unidirectional fluxes (Epstein et al., 1963). These analyses showed that OsHKT2;1 exhibits Na⁺ uptake activity in plant BY2 cells in the absence of added K⁺, but little K⁺ (Rb⁺), influx activity. In contrast, OsHKT2;2 was found to function as a Na⁺-K⁺ cotransporter/channel in plant BY2 cells, showing K⁺-stimulated Na⁺ influx and Na⁺-stimulated K⁺ (Rb⁺) influx. The differential K⁺ selectivities of the two OsHKT2 transporters were consistently reproduced by voltage clamp experiments using Xenopus oocytes here, as reported previously (Horie et al., 2001). OsHKT2;2 was also found to mediate K⁺-independent Na⁺ influx at millimolar external Na⁺ concentrations. These findings demonstrate that the cation selectivities of OsHKT2;1 and OsHKT2;2 in plant cells are consistent with past findings obtained from heterologous expression analyses under similar ionic conditions (Horie et al., 2001; Garciadeblás et al., 2003; Tholema et al., 2005). Furthermore, the shift in OsHKT2;2 Na⁺-K⁺ selectivity depending on ionic editions is consistent with the model that HKT transporters/channels are multi-ion pores (Gassmann et al., 1996; Corratgé et al., 2007). Classical studies of ion channels have shown that ion channels, in which multiple ions can occupy the pore at the same time, can change their relative selectivities depending on the ionic conditions (Hille, 2001). Moreover, the presence of external K⁺ and Ca²⁺ was found here to down-regulate OsHKT2;1-mediated Na⁺ influx both in tobacco BY2 cells and in rice roots. The inhibitory effect of external K⁺ on OsHKT2;1-mediated Na⁺ influx into intact rice roots, however, showed a distinct difference in comparison with that of BY2 cells, which indicates a possible posttranslational regulation of OsHKT2;1 in K⁺-starved rice roots.     

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.     

A Signaling Pathway Linking Nitric Oxide Production to Heterotrimeric G Protein and Hydrogen Peroxide Regulates Extracellular Calmodulin Induction of Stomatal Closure in Arabidopsis

Extracellular calmodulin (ExtCaM) regulates stomatal movement by eliciting a cascade of intracellular signaling events including heterotrimeric G protein, hydrogen peroxide (H₂O₂), and Ca²⁺. However, the ExtCaM-mediated guard cell signaling pathway remains poorly understood. In this report, we show that Arabidopsis (Arabidopsis thaliana) NITRIC OXIDE ASSOCIATED1 (AtNOA1)-dependent nitric oxide (NO) accumulation plays a crucial role in ExtCaM-induced stomatal closure. ExtCaM triggered a significant increase in NO levels associated with stomatal closure in the wild type, but both effects were abolished in the Atnoa1 mutant. Furthermore, we found that ExtCaM-mediated NO generation is regulated by GPA1, the Gα-subunit of heterotrimeric G protein. The ExtCaM-dependent NO accumulation was nullified in gpa1 knockout mutants but enhanced by overexpression of a constitutively active form of GPA1 (cGα). In addition, cGα Atnoa1 and gpa1-2 Atnoa1 double mutants exhibited a similar response as did Atnoa1. The defect in gpa1 was rescued by overexpression of AtNOA1. Finally, we demonstrated that G protein activation of NO production depends on H₂O₂. Reduced H₂O₂ levels in guard cells blocked the stomatal response of cGα lines, whereas exogenously applied H₂O₂ rescued the defect in ExtCaM-mediated stomatal closure in gpa1 mutants. Moreover, the atrbohD/F mutant, which lacks the NADPH oxidase activity in guard cells, had impaired NO generation in response to ExtCaM, and H₂O₂-induced stomatal closure and NO accumulation were greatly impaired in Atnoa1. These findings have established a signaling pathway leading to ExtCaM-induced stomatal closure, which involves GPA1-dependent activation of H₂O₂ production and subsequent AtNOA1-dependent NO accumulation.Plant guard cells control opening and closure of the stomata in response to phytohormones (e.g. abscisic acid [ABA]) and various environmental signals such as light and temperature, thereby regulating gas exchange for photosynthesis and water status via transpiration (Schroeder et al., 2001). Cytosolic calcium ([Ca²⁺]_i) has been shown to be a key second messenger that changes in response to multiple stimuli in guard cells (McAinsh et al., 1995; Grabov and Blatt, 1998; Wood et al., 2000). A large proportion of Ca²⁺ is localized in extracellular space. It has been shown that external Ca²⁺ concentration ([Ca²⁺]_o) promotes stomatal closure and induces oscillation in [Ca²⁺]_i in guard cells (MacRobbie, 1992; McAinsh et al., 1995; Allen et al., 2001). However, how the guard cells perceive [Ca²⁺]_o concentration and convert [Ca²⁺]_o changes into [Ca²⁺]_i changes was not understood until a calcium-sensing receptor (CAS) in the plasma membrane of guard cells in Arabidopsis (Arabidopsis thaliana) was identified (Han et al., 2003). The external Ca²⁺ (Ca²⁺_o)-induced [Ca²⁺]_i increase is abolished in CAS antisense lines (Han et al., 2003). Both [Ca²⁺]_o and [Ca²⁺]_i show diurnal oscillation that is determined by stomatal conductance, whereas the amplitude of [Ca²⁺]_i oscillation is reduced in CAS antisense lines (Tang et al., 2007). The reduced amplitude of [Ca²⁺]_i diurnal oscillation in response to Ca²⁺_o treatment suggests the potential existence of other [Ca²⁺]_o sensor(s) that may transmit [Ca²⁺]_o information into the [Ca²⁺]_i response in coordination with CAS. Extracellular calmodulin (ExtCaM) could be such an additional [Ca²⁺]_o sensor.Calmodulin is a well-known Ca²⁺ sensor that is activated upon binding of Ca²⁺. It has been shown that calmodulin exists not only intracellularly but also extracellularly in many plant species (Biro et al., 1984; Sun et al., 1994, 1995; Cui et al., 2005). ExtCaM has been implicated in several important biological functions, such as the promotion of cell proliferation, pollen germination, and tube growth (Sun et al., 1994, 1995; Ma and Sun, 1997; Ma et al., 1999; Cui et al., 2005; Shang et al., 2005). ExtCaM is found in the cell wall of guard cells in Vicia faba and in the epidermis of Arabidopsis by immunogold labeling/electron microscopy and western-blot analyses, respectively, and the endogenous CaM in the extracellular space has been shown to regulate stomatal movements (Chen et al., 2003; Xiao et al., 2004). Under natural conditions, once the activity of ExtCaM has been inhibited by its membrane-impermeable antagonist W7-agrose or CaM antibody, stomatal opening under light is enhanced and stomatal closure in darkness is inhibited in V. faba and Arabidopsis (Chen et al., 2003; Xiao et al., 2004). [Ca²⁺]_i and cytosolic hydrogen peroxide (H₂O₂) changes, two events involved in ExtCaM-regulated stomatal movement (Chen et al., 2004), are likely regulated by light/darkness (Chen and Gallie, 2004; Tang et al., 2007), suggesting that ExtCaM plays an important physiological role in the regulation of stomatal diurnal rhythm. Calmodulin-binding proteins have been found in the protoplast of suspension-cultured Arabidopsis cells, supporting the idea that ExtCaM functions as a peptide-signaling molecule (Cui et al., 2005). Furthermore, ExtCaM triggers [Ca²⁺]_i elevation in guard cells of V. faba and Arabidopsis and in lily (Lilium daviddi) pollen (Chen et al., 2004; Xiao et al., 2004; Shang et al., 2005). These observations support the notion that ExtCaM could be a potential [Ca²⁺]_o sensor for external calcium, and this external calcium sensing could subsequently regulate the [Ca²⁺]_i level through a signaling cascade.It is interesting that ExtCaM and ABA induce some parallel changes in second messengers in guard cell signaling. Our previous studies show that ExtCaM induces [Ca²⁺]_i increase and H₂O₂ generation through the Gα-subunit (GPA1) of a heterotrimeric G protein, and increased H₂O₂ further elevates [Ca²⁺]_i (Chen et al., 2004). G protein, Ca²⁺, and H₂O₂ are well-known second messengers in ABA-induced guard cell signaling (McAinsh et al., 1995; Grabov and Blatt, 1998; Pei et al., 2000; Wang et al., 2001; Zhang et al., 2001; Liu et al., 2007). However, the signaling cascade triggered by ExtCaM in guard cells is poorly understood. New ABA signaling components in guard cells could provide a clue in the study of the molecular mechanism of ExtCaM guard cell signaling.Recently, nitric oxide (NO) has been shown to serve as an important signal molecule involved in many aspects of developmental processes, including floral transition, root growth, root gravitropism, adventitious root formation, xylogenesis, seed germination, and orientation of pollen tube growth (Beligni and Lamattina, 2000; Pagnussat et al., 2002; He et al., 2004; Prado et al., 2004; Gabaldón et al., 2005; Stohr and Stremlau, 2006). Increasing evidence points to a role for NO as an essential component in ABA signaling in guard cells (Garcia-Mata and Lamattina, 2001, 2002; Neill et al., 2002). It has been shown that nitrate reductase (NR) reduces nitrite to NO, and the nia1, nia2 NR-deficient mutant in Arabidopsis showed reduced ABA induction of stomatal closure (Desikan et al., 2002; Bright et al., 2006). Although animal nitric oxide synthase (NOS) activity has been detected in plants and inhibitors of mammalian NOS impair NO production in plants (Barroso et al., 1999; Corpas et al., 2001), the gene(s) encoding NOS in plants is still not clear. AtNOS1 in Arabidopsis was initially reported to encode a protein containing NOS activity (Guo et al., 2003). However, recent studies have raised critical questions regarding the nature of AtNOS1 and suggested that AtNOS1 appears not to encode a NOS (Crawford et al., 2006; Zemojtel et al., 2006). However, the originally described Atnos1 mutant is deficient in NO accumulation (Crawford et al., 2006). Consequently, AtNOS1 was renamed AtNOA1 (for NITRIC OXIDE ASSOCIATED1; Crawford et al., 2006). Therefore, the Atnoa1 mutant provides a useful tool for dissecting the function of NO in plants. At present, the molecules that regulate NO generation in ABA-mediated guard cell signaling are not clear. Evidence suggests that H₂O₂, a second messenger important for the regulation of many developmental processes and stomatal movement (Pei et al., 2000; Zhang et al., 2001; Coelho et al., 2002; Demidchik et al., 2003; Kwak et al., 2003), regulates NO generation in guard cells (Lum et al., 2002; He et al., 2005; Bright et al., 2006).Given the parallel signaling events induced by ABA and ExtCaM, we investigated whether NO is involved in the regulation of ExtCaM-induced stomatal closure in Arabidopsis and whether it is linked to G protein and H₂O₂, two key regulators of both ExtCaM and ABA regulation of stomatal movements. Using Arabidopsis mutants (e.g. GPA1 null mutants, the NO-producing mutant Atnoa1, and the guard cell H₂O₂ synthetic enzymatic mutant atrbohD/F) combined with pharmacological analysis, we present compelling evidence to establish a linear functional relationship between Gα, H₂O₂, and NO in ExtCaM guard cell signaling.     

Phytochelatin Synthesis Is Essential for the Detoxification of Excess Zinc and Contributes Significantly to the Accumulation of Zinc

The synthesis of phytochelatins (PCs) is essential for the detoxification of nonessential metals and metalloids such as cadmium and arsenic in plants and a variety of other organisms. To our knowledge, no direct evidence for a role of PCs in essential metal homeostasis has been reported to date. Prompted by observations in Schizosaccharomyces pombe and Saccharomyces cerevisiae indicating a contribution of PC synthase expression to Zn²⁺ sequestration, we investigated a known PC-deficient Arabidopsis (Arabidopsis thaliana) mutant, cad1-3, and a newly isolated second strong allele, cad1-6, with respect to zinc (Zn) homeostasis. We found that in a medium with low cation content PC-deficient mutants show pronounced Zn²⁺ hypersensitivity. This phenotype is of comparable strength to the well-documented Cd²⁺ hypersensitivity of cad1 mutants. PC deficiency also results in significant reduction in root Zn accumulation. To be able to sensitively measure PC accumulation, we established an assay using capillary liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry of derivatized extracts. Plants grown under control conditions consistently showed PC2 accumulation. Analysis of plants treated with same-effect concentrations revealed that Zn²⁺-elicited PC2 accumulation in roots reached about 30% of the level of Cd²⁺-elicited PC2 accumulation. We conclude from these data that PC formation is essential for Zn²⁺ tolerance and provides driving force for the accumulation of Zn. This function might also help explain the mysterious occurrence of PC synthase genes throughout the plant kingdom and in a wide range of other organisms.Both essential and nonessential metal ions can be toxic when present in excess. Zinc (Zn) ions, for instance, are used in biological systems as catalytic or structural components in a myriad of proteins (Frausto da Silva and Williams, 2001). In humans, about 10% of genes encode Zn-dependent proteins (Andreini et al., 2006) and it is reasonable to postulate similar numbers for plants. When the Zn-buffering capacity of a cell is exceeded, however, aberrant binding of Zn ions to thiols or other functional groups can occur, which disrupts the function of proteins. Also, Zn ions can displace other essential metal ions from their binding sites (Krämer and Clemens, 2005). Toxicity thresholds for Zn were found to range between 100 and 300 μg g⁻¹ dry weight depending on plant species and physiological state (Marschner, 1995). Ions of the nonessential metal cadmium (Cd) have a high affinity for various functional groups in biological molecules, in particular thiols. When taken up into a cell they can inactivate proteins by uncontrolled binding or cause oxidative stress by depleting glutathione pools (Clemens, 2006a).Because of the potential toxicity of metal ions, all living systems possess mechanisms to tightly regulate the distribution of metal ions and to minimize damage under conditions of excess metal supply (Eide, 2006; Grotz and Guerinot, 2006). Principally, detoxification of excess metal ions is assumed to be achieved by efflux, sequestration, and chelation. For instance, in the past few years evidence has been reported for a contribution of AtMTP1 (At2g46800) to vacuolar sequestration of Zn²⁺ (Kobae et al., 2004; Desbrosses-Fonrouge et al., 2005) and of AtHMA4 (At2g19110) to effluxing Zn²⁺ (Mills et al., 2005). Furthermore, the Arabidopsis halleri ortholog of HMA4 is essential for Zn and Cd hypertolerance (Hanikenne et al., 2008). Loss of ZIF1, a transporter of the major facilitator superfamily, results in Zn²⁺ hypersensitivity in Arabidopsis (Arabidopsis thaliana; Haydon and Cobbett, 2007).The synthesis of phytochelatins (PCs), glutathione-derived metal-binding peptides, represents a major detoxification mechanism for Cd and arsenic (As) ions in various species. More recently, PCs have also been implicated in long-distance transport of Cd in the phloem (Mendoza-Cozatl et al., 2008). Formation of PCs is catalyzed by PC synthases (PCS) and genes encoding this enzyme have been cloned from plants, fungi, and nematodes (Clemens et al., 1999, 2001; Ha et al., 1999; Vatamaniuk et al., 1999, 2001). Mutant lines of Arabidopsis, Schizosaccharomyces pombe, or Caenorhabditis elegans that are deficient in PC synthesis show a severe loss of Cd and As tolerance (Clemens et al., 1999; Ha et al., 1999; Vatamaniuk et al., 2001). For other metal ions only minor effects have been reported (Cobbett and Goldsbrough, 2002). Arabidopsis cad1-3 mutant plants, which are defective in AtPCS1, showed about a 2-fold increase in copper (Cu) and mercury sensitivity and no significant increase in Zn sensitivity (Ha et al., 1999). S. pombe PCS-deficient mutants are slightly more Cu²⁺ sensitive than wild-type cells (Clemens et al., 1999). PC-metal complexes have been detected in plant cells only with Cd, silver, Cu, and As (Maitani et al., 1996; Schmöger et al., 2000) even though synthesis of PCs is activated by a wide range of metal ions both in vivo and in vitro (Grill et al., 1987; Vatamaniuk et al., 2000; Oven et al., 2002). Thus, the role of PC synthesis in metal detoxification has so far been seen as being confined to Cd and As (Cobbett and Goldsbrough, 2002). This, however, leaves the question as to why PCS genes are so widespread and why the enzyme is expressed constitutively throughout the plant (Rea et al., 2004). It is not clear how the sporadic need to sequester excess Cd or As ions could have provided the selective pressure to maintain PCS expression throughout the plant kingdom and beyond (Clemens, 2006b). One explanation could be the second enzymatic function of PCS, i.e. breakdown of glutathione conjugates (GS conjugates) to the corresponding β-glutamyl-Cys conjugates (Blum et al., 2007). The catalytic activity of bacterial PCS-like proteins is similar (Harada et al., 2004; Tsuji et al., 2004; Vivares et al., 2005). Another possibility is involvement of PC synthesis in essential metal homeostasis, which has been discussed occasionally (Steffens et al., 1986; Rauser, 1990; Steffens, 1990). As early as 1986, Steffens et al. suggested this based on the observation that small amounts of PCs were detectable in tomato (Solanum lycopersicum) cell cultures in the absence of excess metals. Similarly, Grill et al. reported the induction of PC synthesis by Cu and Zn ions after transfer of cultured cells into fresh medium. The amount of detectable PCs was correlated linearly with the Zn²⁺ concentration in the culture medium and an involvement of PC synthesis in metal homeostasis was proposed (Grill et al., 1987, 1988). However, no genetic evidence for such a role of PCs has been reported to date. Here we present evidence that PC formation indeed contributes significantly to Zn²⁺ detoxification in Arabidopsis and enhances Zn accumulation.     

Disentangling the Complexity of Mitogen-Activated Protein Kinases and Reactive Oxygen Species Signaling

We report here on the identification of the major plasma membrane (PM) ascorbate-reducible b-type cytochrome of bean (Phaseolus vulgaris) and soybean (Glycine max) hypocotyls as orthologs of Arabidopsis (Arabidopsis thaliana) AIR12 (for auxin induced in root cultures). Soybean AIR12, which is glycosylated and glycosylphosphatidylinositol-anchored to the external side of the PM in vivo, was expressed in Pichia pastoris in a recombinant form, lacking the glycosylphosphatidylinositol modification signal and purified from the culture medium. Recombinant AIR12 is a soluble protein predicted to fold into a β-sandwich domain and belonging to the DOMON (for dopamine β-monooxygenase N terminus) domain superfamily. It is shown to be a b-type cytochrome with a symmetrical α-band at 561 nm, fully reduced by ascorbate, and fully oxidized by monodehydroascorbate radical. AIR12 is a high-potential cytochrome b showing a wide bimodal dependence from the redox potential between +80 mV and +300 mV. Optical absorption and electron paramagnetic resonance analysis indicate that AIR12 binds a single, highly axial low-spin heme, likely coordinated by methionine-91 and histidine-76, which are strongly conserved in AIR12 sequences. Phylogenetic analyses reveal that the auxin-responsive genes AIR12 represent a new family of PM b-type cytochromes specific to flowering plants. Circumstantial evidence suggests that AIR12 may interact with other redox partners within the PM to constitute a redox link between cytoplasm and apoplast.Complex interactions between plant cells and the environment are mediated by the apoplast. The apoplastic liquid phase permeating the cell wall contains relatively low concentrations of solutes (Dietz, 1997). Its composition, although largely determined by the protoplast, is easily perturbed by environmental challenges that can thus be perceived by the apoplast and translated into signals that trigger cell responses (Pignocchi and Foyer, 2003; Foyer and Noctor, 2005). Environmental challenges affecting the apoplast commonly result in an oxidative load, caused, for instance, by pollutants (e.g. ozone; Sandermann, 2008) or by endogenously generated reactive oxygen species (ROS). Several enzymatic and nonenzymatic systems are able to generate ROS in the apoplast (Fry, 1998; Apel and Hirt, 2004), an event that is not restricted to biotic or abiotic stresses (Torres and Dangl, 2005), but also involved in diverse physiological conditions, including stomata closure and cell growth (Foreman et al., 2003; Mori and Schroeder, 2004; Gapper and Dolan, 2006; Schopfer and Liszkay, 2006).Apoplastic reductants not only act as an antioxidant barrier, but they could also modulate oxidative signals, thus actively contributing to plant adaptation to the environment. Ascorbate occurs at 10⁻⁴ to 10⁻³ m concentrations in the apoplast, where it represents the major pool of low-molecular-mass antioxidants (Dietz, 1997; Pignocchi and Foyer, 2003; Padu et al., 2005). Maintenance of the apoplastic ascorbate pool depends on transport systems of the plasma membrane (PM; Horemans et al., 2000). The redox state of the ascorbate in the apoplast is relatively flexible and typically more oxidized than in the symplast (Cordoba-Pedregosa et al., 2003, 2005; de Pinto and De Gara, 2004; Padu et al., 2005; Pignocchi et al., 2006). Ascorbate oxidation can be effected enzymatically by ascorbate oxidase or ascorbate peroxidase, and nonenzymatically by direct interaction with ROS (including ozone; Sandermann, 2008), transition metals (e.g. iron, copper; Fry, 1998), or phenolic radicals (Takahama, 1993). Oxidation of ascorbate gives rise to the monodehydroascorbate (MDA) radical, which can disproportionate into ascorbate and fully oxidized dehydroascorbate. In addition, the apoplastic MDA radical can be reduced back to ascorbate by a trans-PM redox system that uses cytosolic ascorbate as a reductant and involves a high-potential cytochrome b (Horemans et al., 1994). The latter has escaped molecular identification thus far (Trost et al., 2000; Bérczi et al., 2003; Griesen et al., 2004; Preger et al., 2005).It was suggested (Asard et al., 2001) that the trans-PM electron transfer from cytosolic ascorbate to apoplastic MDA may be effected by a cytochrome b561, in analogy to the electron transfer of animal chromaffin vesicles (Kelley and Njus, 1986). Cytochromes b561 are high-potential, transmembrane redox proteins of about 25 kD made of six membrane-spanning α-helices, which bind two hemes b. One heme is predicted to be close to an ascorbate binding site facing the cytosol, whereas the second heme faces the opposite side of the membrane and can be oxidized by either MDA or ferrichelates (Tsubaki et al., 1997; McKie et al., 2001; Bérczi et al., 2005; Kamensky et al., 2007). Plants contain several orthologous genes to animal cytochrome b561 (Asard et al., 2000; Bashtovyy et al., 2003). Arabidopsis (Arabidopsis thaliana) contains four genes belonging to this family (Tsubaki et al., 2005) and one of these (At4g25570, CYBASC1), expressed in recombinant form, showed similar biochemical properties to animal cytochrome b561 (Bérczi et al., 2007). However, the localization in vivo of plant cytochrome b561 is controversial. Arabidopsis CYBASC1 was found to be associated with the tonoplast membrane (Griesen et al., 2004) and annotated in proteomic studies as either a tonoplast protein (Carter et al., 2004; Shimaoka et al., 2004) or a chloroplast protein (Zybailov et al., 2008). Tonoplast localization was also reported for bean (Phaseolus vulgaris) CYBASC1 in etiolated hypocotyls (Preger et al., 2005), whereas a GFP construct of CYBASC1 from wild watermelon (Citrullus lanatus) was shown to be targeted to the PM in transformed onion (Allium cepa) epidermal cells (Nanasato et al., 2005). No data are available for any other isoform of cytochrome b561 in plants.An ascorbate-reducible cytochrome b from enriched PM preparations was purified as a glycosylated protein of 55 to 63 kD (bean hypocotyls; Trost et al., 2000) or 120 kD (Arabidopsis; Bérczi et al., 2003) in SDS-PAGE. The association to the PM of the bean hypocotyl cytochrome was confirmed by analytical Suc gradient centrifugation (Preger et al., 2005). Based on potentiometric redox titrations, both bean and Arabidopsis cytochrome b preparations were suggested to bind two hemes with distant redox potentials (E_m7 +135 and +180/+200 mV). However, the nature of this high-potential cytochrome b of plant PM remained elusive, although clearly different from tonoplast cytochrome b561 (Preger et al., 2005).In this article, we report on the purification, molecular identification, cloning, and biochemical characterization of the major ascorbate-reducible cytochrome b associated with the PM of soybean (Glycine max) etiolated hypocotyls. The coding gene, known as AIR12 (for auxin induced in root cultures), is early expressed during auxin-induced lateral root formation in Arabidopsis (Laskowski et al., 2006). We demonstrate that AIR12 is a member of a new family of ascorbate-reducible cytochromes b specific to flowering plant species. The protein is glycosylated and hydrophilic and predicted to be associated in vivo with the external face of the PM by means of a glycosylphosphatidylinositol (GPI) anchor (Borner et al., 2003). AIR12 has been found to be associated with lipid rafts together with other redox proteins (Lefebvre et al., 2007), which may act as its partners in a possible electron link between apoplast and symplast.     

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.     

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.     

NaCl-Induced Alternations of Cellular and Tissue Ion Fluxes in Roots of Salt-Resistant and Salt-Sensitive Poplar Species

Using the scanning ion-selective electrode technique, fluxes of H⁺, Na⁺, and Cl⁻ were investigated in roots and derived protoplasts of salt-tolerant Populus euphratica and salt-sensitive Populus popularis 35-44 (P. popularis). Compared to P. popularis, P. euphratica roots exhibited a higher capacity to extrude Na⁺ after a short-term exposure to 50 mm NaCl (24 h) and a long term in a saline environment of 100 mm NaCl (15 d). Root protoplasts, isolated from the long-term-stressed P. euphratica roots, had an enhanced Na⁺ efflux and a correspondingly increased H⁺ influx, especially at an acidic pH of 5.5. However, the NaCl-induced Na⁺/H⁺ exchange in root tissues and cells was inhibited by amiloride (a Na⁺/H⁺ antiporter inhibitor) or sodium orthovanadate (a plasma membrane H⁺-ATPase inhibitor). These results indicate that the Na⁺ extrusion in stressed P. euphratica roots is the result of an active Na⁺/H⁺ antiport across the plasma membrane. In comparison, the Na⁺/H⁺ antiport system in salt-stressed P. popularis roots was insufficient to exclude Na⁺ at both the tissue and cellular levels. Moreover, salt-treated P. euphratica roots retained a higher capacity for Cl⁻ exclusion than P. popularis, especially during a long term in high salinity. The pattern of NaCl-induced fluxes of H⁺, Na⁺, and Cl⁻ differs from that caused by isomotic mannitol in P. euphratica roots, suggesting that NaCl-induced alternations of root ion fluxes are mainly the result of ion-specific effects.Soil salinity causes increasingly agricultural and environmental problems on a worldwide scale, especially in arid areas. When plant roots are subjected to saline environments with high NaCl content, external Na⁺ and Cl⁻ establish a large electrochemical gradient favoring the passive entry of salt ions through a variety of cation and anion channels and/or transporters in the plasma membrane (PM; Blumwald et al., 2000; Hasegawa et al., 2000; White and Broadley, 2001; Roberts, 2006; Demidchik and Maathuis, 2007). The entry and accumulation of toxic ions lead to disruption of ion homeostasis and finally cause secondary stress, e.g. oxidative bursts (Zhu, 2001, 2003). Accordingly, the maintenance of low salt concentration in the cytosol is of great importance for salt adaptation of plants (Greenway and Munns, 1980; Munns and Tester, 2008).Active Na⁺ extrusion to the apoplast or external environment is essential for sustaining Na⁺ homeostasis in the cytosol (Blumwald et al., 2000; Tester and Davenport, 2003; Zhu, 2003; Apse and Blumwald, 2007). PM Na⁺/H⁺ antiporters have been widely considered to play a crucial role in active Na⁺ extrusion under saline conditions (Shi et al., 2000, 2002; Qiu et al., 2002; Martínez-Atienza et al., 2007). NaCl-induced activity of PM Na⁺/H⁺ antiporter has been reported in crop species, tomato (Solanum lycopersicum; Wilson and Shannon, 1995), Arabidopsis (Arabidopsis thaliana; Qiu et al., 2002, 2003), and rice (Oryza sativa; Martínez-Atienza et al., 2007). Furthermore, overexpression of the Na⁺/H⁺ antiporter gene AtSOS1 decreases the accumulation of Na⁺ in transgenic Arabidopsis under NaCl stress (Shi et al., 2003). These PM Na⁺/H⁺ antiporters depend on electrochemical H⁺ gradients, which are generated by PM H⁺-ATPase (Blumwald et al., 2000; Zhu, 2003). Using an ion-selective microelectrode, Shabala and a colleague suggested the involvement of PM H⁺-ATPase in the Na⁺/H⁺ antiport according to H⁺ kinetics on salt shock (Shabala, 2000; Shabala and Newman, 2000). Therefore, the NaCl-induced H⁺ pumping is fundamental to Na⁺/H⁺ exchange and salinity tolerance (Ayala et al., 1996; Vitart et al., 2001; Chen et al., 2007; Gévaudant et al., 2007). However, the active Na⁺/H⁺ antiport across PM and the contribution to salt exclusion have been rarely investigated in tree species.Munns and Tester (2008) claimed that Cl⁻ toxicity is more important than Na⁺ toxicity in some woody species, e.g. citrus. Similarly, we have noticed that the inability to restrict Cl⁻ uptake contributes to the NaCl-induced salt damage in salt-sensitive poplar (Populus spp.) species, in addition to toxicity of excess Na⁺ (Chen et al., 2001, 2002, 2003). The differences in Cl⁻ tolerance exhibited by plants are usually related to the ability to restrict Cl⁻ transport to the aerial part (Greenway and Munns, 1980; White and Broadley, 2001). Excluding Cl⁻ from the xylem seems to be an effective mechanism for lotus to cope with the interactive effect of salt and water logging (Teakle et al., 2007). An influx of Cl⁻, immediately after addition of NaCl, was observed in bean (Vicia faba) mesophyll tissue (Shabala, 2000). The Cl⁻ flux response to salt shock is helpful to reveal the rapid adjustments of plants to salinity. However, Cl⁻ fluxes in salt-adapted roots, which are necessary to clarify plant adaptations to long durations of salinity, have not been examined.Populus euphratica has been widely considered as a model plant to elucidate physiological and molecular mechanisms of salt tolerance in woody species (Chen et al., 2001, 2002, 2003; Gu et al., 2004; Ottow et al., 2005a, 2005b; Junghans et al., 2006; Wang et al., 2007, 2008; Wu et al., 2007; Zhang et al., 2007). Comparative studies have shown that salt-stressed P. euphratica seedlings accumulate less Na⁺ and Cl⁻ in root and shoot tissues than salt-sensitive poplar species (Chen et al., 2001, 2002). It is suggested that the greater capacity to exclude NaCl in P. euphratica is likely the result of salt uptake and transport restriction in roots (Chen et al., 2002, 2003). However, this needs further investigations, e.g. by electrophysiology, to clarify.In this study, we used a noninvasive ion flux technique to measure the tissue and cellular fluxes of H⁺, Na⁺, and Cl⁻ in roots of the salt-tolerant P. euphratica and salt-sensitive P. popularis 35-44. The aim was to compare the NaCl-induced alternations of ion fluxes in poplar species differing in salt tolerance. Furthermore, we examined the effects of pH, salt shock, and PM transport inhibitors on Na⁺ and H⁺ fluxes in root-derived protoplasts of the salt-tolerant species, P. euphratica, which exhibited an evident Na⁺ exclusion under saline conditions.     

Development of a Novel Aluminum Tolerance Phenotyping Platform Used for Comparisons of Cereal Aluminum Tolerance and Investigations into Rice Aluminum Tolerance Mechanisms

The genetic and physiological mechanisms of aluminum (Al) tolerance have been well studied in certain cereal crops, and Al tolerance genes have been identified in sorghum (Sorghum bicolor) and wheat (Triticum aestivum). Rice (Oryza sativa) has been reported to be highly Al tolerant; however, a direct comparison of rice and other cereals has not been reported, and the mechanisms of rice Al tolerance are poorly understood. To facilitate Al tolerance phenotyping in rice, a high-throughput imaging system and root quantification computer program was developed, permitting quantification of the entire root system, rather than just the longest root. Additionally, a novel hydroponic solution was developed and optimized for Al tolerance screening in rice and compared with the Yoshida''s rice solution commonly used for rice Al tolerance studies. To gain a better understanding of Al tolerance in cereals, comparisons of Al tolerance across cereal species were conducted at four Al concentrations using seven to nine genetically diverse genotypes of wheat, maize (Zea mays), sorghum, and rice. Rice was significantly more tolerant than maize, wheat, and sorghum at all Al concentrations, with the mean Al tolerance level for rice found to be 2- to 6-fold greater than that in maize, wheat, and sorghum. Physiological experiments were conducted on a genetically diverse panel of more than 20 rice genotypes spanning the range of rice Al tolerance and compared with two maize genotypes to determine if rice utilizes the well-described Al tolerance mechanism of root tip Al exclusion mediated by organic acid exudation. These results clearly demonstrate that the extremely high levels of rice Al tolerance are mediated by a novel mechanism, which is independent of root tip Al exclusion.Aluminum (Al) is the most abundant metal in the earth''s crust, constituting approximately 7% of the soil (Wolt, 1994). Al is predominately found as a key component of soil clays; however, under highly acidic soil conditions (pH < 5.0), Al³⁺ is solubilized into the soil solution and is highly phytotoxic. Al³⁺ causes a rapid inhibition of root growth that leads to a reduced and stunted root system, thus having a direct effect on the ability of a plant to acquire both water and nutrients. Approximately 30% of the world''s total land area and over 50% of potentially arable lands are acidic, with the majority (60%) found in the tropics and subtropics (von Uexkull and Mutert, 1995). Thus, acidic soils are a major limitation to crop production, particularly in the developing world.As a whole, cereal crops (Poaceae) provide an excellent model for studying Al tolerance because of their abundant genetic resources, large, active research communities, and importance to agriculture. In addition, work in one cereal species can rapidly translate into impact throughout the family. Previous research has focused on understanding the genetic and physiological mechanisms of Al tolerance in maize (Zea mays), sorghum (Sorghum bicolor), and wheat (Triticum aestivum). The most recognized physiological mechanism conferring Al tolerance in plants involves exclusion of Al from the root tip (Miyasaka et al., 1991; Delhaize and Ryan, 1995; Kochian, 1995; Kochian et al., 2004a, 2004b). The exclusion mechanism is primarily mediated by Al-activated exudation of organic acids such as malate, citrate, or oxalate from the root apex, the site of Al toxicity (Ryan et al., 1993, 2001; Ma et al., 2001). These organic acids chelate Al in the rhizosphere, reducing the concentration and toxicity of Al at the growing root tip (Ma et al., 2001). Phosphate has also been identified as a class of root exudates involved in cation chelation and therefore can be considered a potential exudate involved in Al exclusion from the root tip (Pellet et al., 1996).Al-activated malate and citrate anion efflux transporters have been cloned from wheat (ALMT1; Sasaki et al., 2004) and sorghum (SbMATE; Magalhaes et al., 2007), and root citrate efflux transporters have been implicated in Al tolerance in maize (Piñeros and Kochian, 2001; Zhang et al., 2001). Recently, a maize homolog of sorghum SbMATE was shown to be the root citrate efflux transporter that plays a role in maize Al tolerance (Maron et al., 2010). Although organic acids have been shown to play a major role in Al tolerance in these species, another exclusion mechanism has been identified in an Arabidopsis (Arabidopsis thaliana) mutant, where a root-mediated increase in rhizosphere pH lowers the Al³⁺ activity and thus participates in Al exclusion from the root apex (Degenhardt et al., 1998). Furthermore, there is clear evidence that tolerance in maize cannot be fully explained by organic acid release (Piñeros et al., 2005). These types of findings strongly suggest that multiple Al tolerance mechanisms exist in plants.Rice (Oryza sativa) has been reported to be the most Al-tolerant cereal crop under field conditions, capable of withstanding significantly higher concentrations of Al than other major cereals (Foy, 1988). Despite this fact, very little is known about the physiological mechanisms of Al tolerance in rice. Two independent studies have identified increased Al accumulation in the root apex in susceptible compared with Al-tolerant rice varieties, but no differences were observed in organic acid exudation or rhizosphere pH (Ma et al., 2002; Yang et al., 2008). These studies suggest that rice may contain novel physiological and/or genetic mechanisms that confer significantly higher levels of Al tolerance than those found in other cereals. A more thorough analysis is required to clarify the mechanism of Al tolerance in rice.Cultivated rice is characterized by deep genetic divergence between the two major varietal groups: Indica and Japonica (Dally and Second, 1990; Garris et al., 2005; Hu et al., 2006; Londo et al., 2006). Extensive selection pressure over the last 10,000 years has resulted in the formation of five genetically distinct subpopulations: indica and aus within the Indica varietal group, and temperate japonica, tropical japonica, and aromatic/groupV within the Japonica varietal group (Garris et al., 2005; Caicedo et al., 2007; K. Zhao and S. McCouch, personal communication). (Note: When referring to varietal groups, the first letter will be capitalized, while lowercase letters will be used to refer to the subpopulation groups.) Subpopulation differences in trait performance are often significant, particularly with respect to biotic and abiotic stress (Champoux et al., 1995; Lilley et al., 1996; Garris et al. 2003; Xu et al., 2009). This can lead to confusion because trait or performance differences may be confounded with subpopulation structure, leading to false positives (type 1 error; Devlin and Roeder, 1999; Pritchard and Donnelly, 2001; Yu et al., 2006; Zhao et al., 2007). Therefore, it is important to consider the subpopulation origin of genotypes being compared when studying the genetics and physiology of Al tolerance in rice.Al tolerance screening is typically conducted by comparing root growth of seedlings grown in hydroponic solutions, with and without Al (Piñeros and Kochian, 2001; Magalhaes et al., 2004; Sasaki et al., 2004). Sorghum and maize are often screened for Al tolerance in Magnavaca''s nutrient solution (Piñeros and Kochian, 2001; Magalhaes et al., 2004; Piñeros et al., 2005), while rice seedlings are typically grown in Yoshida''s solution (Yoshida et al., 1976). Furthermore, Al concentrations used to screen for Al tolerance in maize (222 μm), sorghum (148 μm), and wheat (100 μm) are significantly lower than those used for screening Al tolerance in rice (1,112–1,482 μm; Wu et al., 2000; Nguyen et al., 2001, 2002, 2003). These differences in chemical composition of the nutrient solutions make it difficult to directly compare plant response to Al across these cereals. In rice, the high Al concentrations required to observe significant differences in root growth between susceptible and resistant varieties also complicate Al tolerance screening due to the precipitation of Al along with other elements. The result is that control (−Al) and treatment (+Al) solutions may differ with regard to essential mineral nutrients that react with Al, leading to differences in growth not directly attributable to Al. Additionally, because the active form of Al that is toxic to root growth is Al³⁺, any Al that precipitates out of solution has no effect on root growth (Kochian et al., 2004a). In a hydroponic solution, Al may be found in one of four forms: (1) as free Al³⁺, where it actively inhibits root growth; (2) precipitated with other elements and essentially unavailable to inhibit plant growth; (3) different hydroxyl monomers of Al, which are not believed to be toxic to roots (Parker et al., 1988); or (4) complexed with other elements in an equilibrium between its active and inactive states. The degree to which Al inhibits root growth is primarily dependent upon the activity of free Al³⁺ in solution (Kochian et al., 2004a).The objectives of this study were to (1) develop and optimize a suitable nutrient solution and high-throughput Al tolerance screening method for rice; (2) quantify and compare differences in Al tolerance between maize, sorghum, wheat, and rice; and (3) use the developed screening methods to determine if rice utilizes the organic acid-mediated Al exclusion mechanism that is observed in maize, sorghum, and wheat.