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.     

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.     

Patterns of Metabolite Changes Identified from Large-Scale Gene Perturbations in Arabidopsis Using a Genome-Scale Metabolic Network

Metabolomics enables quantitative evaluation of metabolic changes caused by genetic or environmental perturbations. However, little is known about how perturbing a single gene changes the metabolic system as a whole and which network and functional properties are involved in this response. To answer this question, we investigated the metabolite profiles from 136 mutants with single gene perturbations of functionally diverse Arabidopsis (Arabidopsis thaliana) genes. Fewer than 10 metabolites were changed significantly relative to the wild type in most of the mutants, indicating that the metabolic network was robust to perturbations of single metabolic genes. These changed metabolites were closer to each other in a genome-scale metabolic network than expected by chance, supporting the notion that the genetic perturbations changed the network more locally than globally. Surprisingly, the changed metabolites were close to the perturbed reactions in only 30% of the mutants of the well-characterized genes. To determine the factors that contributed to the distance between the observed metabolic changes and the perturbation site in the network, we examined nine network and functional properties of the perturbed genes. Only the isozyme number affected the distance between the perturbed reactions and changed metabolites. This study revealed patterns of metabolic changes from large-scale gene perturbations and relationships between characteristics of the perturbed genes and metabolic changes.Rational and quantitative assessment of metabolic changes in response to genetic modification (GM) is an open question and in need of innovative solutions. Nontargeted metabolite profiling can detect thousands of compounds, but it is not easy to understand the significance of the changed metabolites in the biochemical and biological context of the organism. To better assess the changes in metabolites from nontargeted metabolomics studies, it is important to examine the changed metabolites in the context of the genome-scale metabolic network of the organism.Metabolomics is a technique that aims to quantify all the metabolites in a biological system (Nikolau and Wurtele, 2007; Nicholson and Lindon, 2008; Roessner and Bowne, 2009). It has been used widely in studies ranging from disease diagnosis (Holmes et al., 2008; DeBerardinis and Thompson, 2012) and drug discovery (Cascante et al., 2002; Kell, 2006) to metabolic reconstruction (Feist et al., 2009; Kim et al., 2012) and metabolic engineering (Keasling, 2010; Lee et al., 2011). Metabolomic studies have demonstrated the possibility of identifying gene functions from changes in the relative concentrations of metabolites (metabotypes or metabolic signatures; Ebbels et al., 2004) in various species including yeast (Saccharomyces cerevisiae; Raamsdonk et al., 2001; Allen et al., 2003), Arabidopsis (Arabidopsis thaliana; Brotman et al., 2011), tomato (Solanum lycopersicum; Schauer et al., 2006), and maize (Zea mays; Riedelsheimer et al., 2012). Metabolomics has also been used to better understand how plants interact with their environments (Field and Lake, 2011), including their responses to biotic and abiotic stresses (Dixon et al., 2006; Arbona et al., 2013), and to predict important agronomic traits (Riedelsheimer et al., 2012). Metabolite profiling has been performed on many plant species, including angiosperms such as Arabidopsis, poplar (Populus trichocarpa), and Catharanthus roseus (Sumner et al., 2003; Rischer et al., 2006), basal land plants such as Selaginella moellendorffii and Physcomitrella patens (Erxleben et al., 2012; Yobi et al., 2012), and Chlamydomonas reinhardtii (Fernie et al., 2012; Davis et al., 2013). With the availability of whole genome sequences of various species, metabolomics has the potential to become a useful tool for elucidating the functions of genes using large-scale systematic analyses (Fiehn et al., 2000; Saito and Matsuda, 2010; Hur et al., 2013).Although metabolomics data have the potential for identifying the roles of genes that are associated with metabolic phenotypes, the biochemical mechanisms that link functions of genes with metabolic phenotypes are still poorly characterized. For example, we do not yet know the principles behind how perturbing the expression of a single gene changes the metabolic system as a whole. Large-scale metabolomics data have provided useful resources for linking phenotypes to genotypes (Fiehn et al., 2000; Roessner et al., 2001; Tikunov et al., 2005; Schauer et al., 2006; Lu et al., 2011; Fukushima et al., 2014). For example, Lu et al. (2011) compared morphological and metabolic phenotypes from more than 5,000 Arabidopsis chloroplast mutants using gas chromatography (GC)- and liquid chromatography (LC)-mass spectrometry (MS). Fukushima et al. (2014) generated metabolite profiles from various characterized and uncharacterized mutant plants and clustered the mutants with similar metabolic phenotypes by conducting multidimensional scaling with quantified metabolic phenotypes. Nonetheless, representation and analysis of such a large amount of data remains a challenge for scientific discovery (Lu et al., 2011). In addition, these studies do not examine the topological and functional characteristics of metabolic changes in the context of a genome-scale metabolic network. To understand the relationship between genotype and metabolic phenotype, we need to investigate the metabolic changes caused by perturbing the expression of a gene in a genome-scale metabolic network perspective, because metabolic pathways are not independent biochemical factories but are components of a complex network (Berg et al., 2002; Merico et al., 2009).Much progress has been made in the last 2 decades to represent metabolism at a genome scale (Terzer et al., 2009). The advances in genome sequencing and emerging fields such as biocuration and bioinformatics enabled the representation of genome-scale metabolic network reconstructions for model organisms (Bassel et al., 2012). Genome-scale metabolic models have been built and applied broadly from microbes to plants. The first step toward modeling a genome-scale metabolism in a plant species started with developing a genome-scale metabolic pathway database for Arabidopsis (AraCyc; Mueller et al., 2003) from reference pathway databases (Kanehisa and Goto, 2000; Karp et al., 2002; Zhang et al., 2010). Genome-scale metabolic pathway databases have been built for several plant species (Mueller et al., 2005; Zhang et al., 2005, 2010; Urbanczyk-Wochniak and Sumner, 2007; May et al., 2009; Dharmawardhana et al., 2013; Monaco et al., 2013, 2014; Van Moerkercke et al., 2013; Chae et al., 2014; Jung et al., 2014). Efforts have been made to develop predictive genome-scale metabolic models using enzyme kinetics and stoichiometric flux-balance approaches (Sweetlove et al., 2008). de Oliveira Dal’Molin et al. (2010) developed a genome-scale metabolic model for Arabidopsis and successfully validated the model by predicting the classical photorespiratory cycle as well as known key differences between redox metabolism in photosynthetic and nonphotosynthetic plant cells. Other genome-scale models have been developed for Arabidopsis (Poolman et al., 2009; Radrich et al., 2010; Mintz-Oron et al., 2012), C. reinhardtii (Chang et al., 2011; Dal’Molin et al., 2011), maize (Dal’Molin et al., 2010; Saha et al., 2011), sorghum (Sorghum bicolor; Dal’Molin et al., 2010), and sugarcane (Saccharum officinarum; Dal’Molin et al., 2010). These predictive models have the potential to be applied broadly in fields such as metabolic engineering, drug target discovery, identification of gene function, study of evolutionary processes, risk assessment of genetically modified crops, and interpretations of mutant phenotypes (Feist and Palsson, 2008; Ricroch et al., 2011).Here, we interrogate the metabotypes caused by 136 single gene perturbations of Arabidopsis by analyzing the relative concentration changes of 1,348 chemically identified metabolites using a reconstructed genome-scale metabolic network. We examine the characteristics of the changed metabolites (the metabolites whose relative concentrations were significantly different in mutants relative to the wild type) in the metabolic network to uncover biological and topological consequences of the perturbed genes.     

AtGEN1 and AtSEND1, Two Paralogs in Arabidopsis,Possess Holliday Junction Resolvase Activity

Holliday junctions (HJs) are physical links between homologous DNA molecules that arise as central intermediary structures during homologous recombination and repair in meiotic and somatic cells. It is necessary for these structures to be resolved to ensure correct chromosome segregation and other functions. In eukaryotes, including plants, homologs of a gene called XPG-like endonuclease1 (GEN1) have been identified that process HJs in a manner analogous to the HJ resolvases of phages, archaea, and bacteria. Here, we report that Arabidopsis (Arabidopsis thaliana), a eukaryotic organism, has two functional GEN1 homologs instead of one. Like all known eukaryotic resolvases, AtGEN1 and Arabidopsis single-strand DNA endonuclease1 both belong to class IV of the Rad2/XPG family of nucleases. Their resolvase activity shares the characteristics of the Escherichia coli radiation and UV sensitive C paradigm for resolvases, which involves resolving HJs by symmetrically oriented incisions in two opposing strands. This leads to ligatable products without the need for further processing. The observation that the sequence context influences the cleavage by the enzymes can be interpreted as a hint for the existence of sequence specificity. The two Arabidopsis paralogs differ in their preferred sequences. The precise cleavage positions observed for the resolution of mobile nicked HJs suggest that these cleavage positions are determined by both the substrate structure and the sequence context at the junction point.To counter the effects of endogenous and exogenous factors that threaten the genome integrity, efficient mechanisms have evolved to ensure the faithful transmission of genetic information (Tuteja et al., 2001). Double-strand breaks, induced by conditions such as ionizing radiation or replication fork (RF) stalling, are among the most deleterious lesions (Jackson and Bartek, 2009). To protect the genome from consequences of these lesions, the cells have ancient double-strand break repair mechanisms, including the homologous recombination (HR) pathway. The HR mechanism is also of great importance in the intentional genetic recombination during sexual reproduction. A key intermediate in HR is the so-called Holliday junction (HJ), a structure that was first suggested in the context of a gene conversion model in fungi (Holliday, 1964) and later shown to arise in somatic and meiotic cells (Szostak et al., 1983; Schwacha and Kleckner, 1995; Cromie et al., 2006; Bzymek et al., 2010).HJs are structures consisting of four DNA strands of two homologous DNA helices (e.g. homologous chromosomes or sister chromatids). They arise through invasion of one single strand from each of two helices into the other double strand. This results in two continuous strands (one per helix) and two strands that cross from one helix into the other. Schematics often depict the HJs with a parallel orientation of the helices, in which the crossing strands cross each other as was originally postulated (Holliday, 1964). However, HJs based on oligonucleotides have been shown to adopt an antiparallel conformation (for review, see Lilley, 2000). In this configuration, the junction resembles the letter H in a lateral view, and the crossing strands actually perform U turns. The crossing strands represent physical links between the two DNA strands involved. If a RF is restored by HR-mediated repair during mitosis, the resulting HJ usually involves the two sister chromatids of one chromosome (Li and Heyer, 2008). In meiosis, the physical links in the shape of HJs arise because of meiotic crossover between homologous chromosomes. In either case, these links must be resolved to ensure unperturbed cell survival.The importance of resolving the HJs for the survival of cells and organisms is highlighted by the phenotypes described for mutants defective for the known pathways of HJ resolution. One of these pathways is the resolution by canonical HJ resolvases, enzymes that cleave the two opposing strands of a HJ in perfectly symmetric positions relative to the junction point, which results in readily ligatable nicked duplex (nD) products (Svendsen and Harper, 2010). This property distinguishes the canonical HJ resolvases from the noncanonical resolvases (see below).The main resolvase of Escherichia coli is radiation and UV sensitive C (RuvC), which is part of the E. coli resolvasome (RuvABC complex; Otsuji et al., 1974; Sharples et al., 1990, 1999). In this complex, a HJ is sandwiched between two RuvA tetramers (Panyutin and Hsieh, 1994). Two RuvB complexes form ATP-dependent motors of branch migration, with two opposing helical arms of the junction threaded through their central openings. For the resolution of the HJ, one RuvA tetramer is replaced by a RuvC homodimer. This homodimer positions two active sites at the center of the junction that are poised to cleave the junction point if a preferred consensus sequence of the form 5′-(A/T)TT^↓(G/C)-3′ is encountered. The requirement for this correct sequence is quite strict; even a single base change can lead to a drastic reduction of the cleavage efficiency (Shah et al., 1994). Isolated EcRuvC is also active in vitro and binds only HJ structures with high specificity. This binding is independent of the sequence context, but the cleavage depends on the specific sequence (Iwasaki et al., 1991; Benson and West, 1994; Dunderdale et al., 1994). The exact cleavage position has been determined to be either one nucleotide 3′ or 5′ from the junction or at the junction point (Bennett and West, 1996; Shida et al., 1996; Osman et al., 2009). The well-characterized EcRuvC is often referred to as a paradigm of canonical HJ resolution.Eukaryotes have evolved a more complex interplay of different HJ resolution pathways (Schwartz and Heyer, 2011; Zakharyevich et al., 2012). A defined complex, consisting of a recombination deficiency Q (RecQ) helicase (AtRECQ4A in Arabidopsis [Arabidopsis thaliana], Bloom syndrome protein in human, and Slow growth suppression1 (Sgs1) in yeast [Saccharomyces cerevisiae]), a type IA topoisomerase (DNA topoisomerase 3-alpha [TOP3A] in Arabidopsis, HsTOPOIIIα in human, and ScTop3 in yeast), and the structural protein RecQ-mediated genome instability1 (AtRMI1 in Arabidopsis, HsRMI1 in human, and ScRmi1 in yeast; RTR complex), mediates the so-called dissolution pathway. The crossing points of a double HJ are brought together by branch migration catalyzed by the helicase followed by decatenation catalyzed by the topoisomerase (Wu and Hickson, 2003; Hartung et al., 2007a, 2008; Mankouri and Hickson, 2007; Yang et al., 2010). In addition to the catalytic activities, a functional RTR complex also requires structural functions based on protein-protein interactions, for which RMI1 plays an essential role (Mullen et al., 2005; Chen and Brill, 2007; Bonnet et al., 2013; Schröpfer et al., 2014). Dissolution leads to noncross-over products and therefore, is a major mechanism in somatic yeast cells (Gangloff et al., 1994; Ira et al., 2003; Matos et al., 2011). In Arabidopsis, the loss of RTR component function leads to elevated rates of HR as well as sensitivity to UV light and methylmethane sulfonate (MMS; Bagherieh-Najjar et al., 2005; Hartung et al., 2007a; Bonnet et al., 2013). Mutants of AtRMI1 and AtTOP3A exhibit severe and unique meiotic phenotypes (Chelysheva et al., 2008; Hartung et al., 2008). This meiosis I arrest is dependent on HR, but the exact nature of the recombination intermediates that are involved remains unclear (Li et al., 2004; Hartung et al., 2007b; Knoll et al., 2014).Dissolution acts in parallel with a second pathway mediated by the structure-specific endonuclease MMS and UV-sensitive protein81 (MUS81) as shown by the fact that the additional mutation of ScSgs1/AtRECQ4A leads to synthetic lethality (Mullen et al., 2001; Hartung et al., 2006; Mannuss et al., 2010). Single mutants of MUS81 in yeast, human, Drosophila melanogaster, and Arabidopsis are sensitive to DNA-damaging agents that perturb RFs and show reduced HR after induction of double-strand breaks (Boddy et al., 2001; Hanada et al., 2006; Hartung et al., 2006). The MUS81 homologs form heterodimers with the noncatalytic subunit essential meiotic endonuclease1 (EME1; ScMms4 in S. cerevisiae). SpMus81-Eme1 was, to our knowledge, the first nuclear endonuclease reported to be capable of resolving HJs (Boddy et al., 2001). The Arabidopsis complexes can be formed with the two different subunits: AtEME1A or AtEME1B (Geuting et al., 2009). AtMUS81-EME1A/B, like the fission yeast ortholog, preferentially cleaves nicked Holliday junctions (nHJs) and 3′-flaps but also shows weaker activity on intact HJs in vitro (Boddy et al., 2001; Osman et al., 2003; Geuting et al., 2009; Schwartz and Heyer, 2011). MUS81 homologs are key players in meiotic cross-over generation (Osman et al., 2003; Berchowitz et al., 2007; Higgins et al., 2008). Although cross-over formation is solely dependent on SpMus81 in fission yeast, this function was shown to be shared with ScYen1 in budding yeast (Osman et al., 2003; Blanco et al., 2010; Ho et al., 2010; Tay and Wu, 2010). Tightly regulated by cell division cycle5-dependent hyperphosphorylation at the end of prophase I, the main activity of ScMus81-Mms4 is timed to coordinate with the formation of chiasmata and HJs that link the homologous chromosomes. This role in meiosis I is shown by the failure of chromosome segregation at the end of meiosis I in ScMus81 mutants (Matos et al., 2011). Interestingly, the chromosomes could be segregated at the end of meiosis II because of the presence of ScYen1. In contrast to canonical HJ resolvases, the hallmark of the MUS81-EME1 cleavage mechanism is the asymmetry of the second incision relative to either a first incision or a preexisting nick. This difference classifies MUS81-EME1 as a noncanonical resolvase. Its products need additional processing by gap-filling or flap-cleaving enzymes to allow religation (Boddy et al., 2001; Geuting et al., 2009).In very recent studies, HsMUS81-EME1 was found to constitute an essential canonical HJ resolvase with HsSLX1-SLX4 (SLX for synthetic lethal of unknown function), in which a first incision is made by HsSLX1-SLX4 followed by the enhanced action of the HsMUS81-EME1 subunits on the resulting nHJ (Garner et al., 2013; Wyatt et al., 2013). HsSLX1-SLX4 had previously been described as a canonical resolvase, albeit producing only a low level of symmetrically cut ligatable products (Fekairi et al., 2009).In addition to the mechanisms described above, an activity resembling that of EcRuvC had long been known to be present in mammalian cell-free extracts. In 2008, the group of Steven C. West succeeded in identifying, to their knowledge, the first nuclear proteins analogous to the EcRuvC paradigm: ScYen1 and Homo sapiens XPG-like endonuclease1 (HsGEN1; Ip et al., 2008). These proteins are members of the large and well-characterized Rad2/XPG family of nucleases. The Rad2/XPG family consists of the Xeroderma pigmentosum group G-complementing protein (XPG) endonucleases of the nucleotide excision repair (class I), the flap endonuclease1 (FEN1) replication-associated flap endonucleases (class II), the exodeoxyribonuclease1 (EXO1) exonucleases of recombination and repair (class III), and class IV (containing the [putative] eukaryotic HJ resolvases). This last class was introduced after the identification of the rice (Oryza sativa) single-strand DNA endonuclease1 (OsSEND-1) based on sequence homology. The class IV members show a domain composition homologous to FEN1 and EXO1, with no spacer region between their N-terminal XPG (XPG-N) and internal XPG (XPG-I) domains, whereas the primary structure of these domains is more similar to the sequence of the nuclease domain of XPG (Furukawa et al., 2003).Although all Rad2/XPG homologs share a common cleavage mechanism as observed for the typical 5′-flap substrate (Tsutakawa et al., 2011; Tsutakawa and Tainer, 2012), the striking evolutionary difference between classes I, II, and III on the one hand and the HJ resolvases (class IV) on the other hand is the ability of class IV members to form homodimers in vitro at their preferred substrate, the HJs (Rass et al., 2010). The homodimer configuration ensures the presence of two active sites positioned on the opposing strands of the HJ, which is necessary for resolution. The mode of eukaryotic HJ resolution is largely similar to the bacterial paradigm: (1) cleavage occurs one nucleotide in the 3′ direction of a static junction point (equivalent to the main cleavage site on 5′-flaps), (2) the incisions occur with almost perfect point symmetry, (3) the incisions result in readily ligatable nDs, and (4) certain sites within a migratable HJ core are preferred, providing evidence for a (yet to be determined) sequence specificity (Ip et al., 2008; Bailly et al., 2010; Rass et al., 2010; Yang et al., 2012).In the absence of MUS81-EME1/Mms4, the proteins HsGEN1, ScYen1, and CeGEN-1 have been shown to play a role in response to replication-associated perturbations, such as MMS- and UV-induced DNA damage (Bailly et al., 2010; Blanco et al., 2010; Tay and Wu, 2010; Gao et al., 2012; Muñoz-Galván et al., 2012). It is also likely that these proteins provide a backup mechanism in mitosis and meiosis, ensuring proper chromosome segregation after a failure of other mechanisms, including MUS81-EME1/Mms4 (Blanco et al., 2010; Matos et al., 2011).Although canonical HJ resolvases in animals and fungi are a current topic of great interest, very little is known about these proteins in plants. In rice, two members of the Rad2/XPG class IV have been described: OsSEND-1 (the founding member) and OsGEN-like (OsGEN-L). OsSEND-1 was shown to digest single-stranded circular DNA, and its expression is induced on MMS-induced genotoxic stress, whereas OsGEN-L is implicated in late spore development (Furukawa et al., 2003; Moritoh et al., 2005). Both studies (Furukawa et al., 2003; Moritoh et al., 2005) proposed putative homologs in other plants, and the gene locus At1g01880 of Arabidopsis, coding for the protein AtGEN1, is considered the ortholog of HsGEN1 and ScYen1 (Ip et al., 2008). However, currently, only OsGEN-L has been further investigated and described to possess in vitro properties similar to both Rad2/XPG nucleases and EcRuvC. This protein shows a well-defined 5′-flap activity as well as a poorly characterized ability, similar to that of EcRuvC, to resolve mobile HJs (Yang et al., 2012).Thus, of two members of Rad2/XPG class IV of plants, only one member has so far been analyzed with respect to a possible HJ resolvase activity. However, Arabidopsis expression data show that both proteins are expressed in plants and do not reveal marked differences (Laubinger et al., 2008). In this study, the goal was, therefore, to characterize the in vitro activities of not only AtGEN1 but also, AtSEND1, focusing on the idea that Arabidopsis and (seed) plants in general might encode not one but actually two HJ resolvases with functional homology to EcRuvC.     

Nontransgenic Genome Modification in Plant Cells

Zinc finger nucleases (ZFNs) are a powerful tool for genome editing in eukaryotic cells. ZFNs have been used for targeted mutagenesis in model and crop species. In animal and human cells, transient ZFN expression is often achieved by direct gene transfer into the target cells. Stable transformation, however, is the preferred method for gene expression in plant species, and ZFN-expressing transgenic plants have been used for recovery of mutants that are likely to be classified as transgenic due to the use of direct gene-transfer methods into the target cells. Here we present an alternative, nontransgenic approach for ZFN delivery and production of mutant plants using a novel Tobacco rattle virus (TRV)-based expression system for indirect transient delivery of ZFNs into a variety of tissues and cells of intact plants. TRV systemically infected its hosts and virus ZFN-mediated targeted mutagenesis could be clearly observed in newly developed infected tissues as measured by activation of a mutated reporter transgene in tobacco (Nicotiana tabacum) and petunia (Petunia hybrida) plants. The ability of TRV to move to developing buds and regenerating tissues enabled recovery of mutated tobacco and petunia plants. Sequence analysis and transmission of the mutations to the next generation confirmed the stability of the ZFN-induced genetic changes. Because TRV is an RNA virus that can infect a wide range of plant species, it provides a viable alternative to the production of ZFN-mediated mutants while avoiding the use of direct plant-transformation methods.Methods for genome editing in plant cells have fallen behind the remarkable progress made in whole-genome sequencing projects. The availability of reliable and efficient methods for genome editing would foster gene discovery and functional gene analyses in model plants and the introduction of novel traits in agriculturally important species (Puchta, 2002; Hanin and Paszkowski, 2003; Reiss, 2003; Porteus, 2009). Genome editing in various species is typically achieved by integrating foreign DNA molecules into the target genome by homologous recombination (HR). Genome editing by HR is routine in yeast (Saccharomyces cerevisiae) cells (Scherer and Davis, 1979) and has been adapted for other species, including Drosophila, human cell lines, various fungal species, and mouse embryonic stem cells (Baribault and Kemler, 1989; Venken and Bellen, 2005; Porteus, 2007; Hall et al., 2009; Laible and Alonso-González, 2009; Tenzen et al., 2009). In plants, however, foreign DNA molecules, which are typically delivered by direct gene-transfer methods (e.g. Agrobacterium and microbombardment of plasmid DNA), often integrate into the target cell genome via nonhomologous end joining (NHEJ) and not HR (Ray and Langer, 2002; Britt and May, 2003).Various methods have been developed to indentify and select for rare site-specific foreign DNA integration events or to enhance the rate of HR-mediated DNA integration in plant cells. Novel T-DNA molecules designed to support strong positive- and negative-selection schemes (e.g. Thykjaer et al., 1997; Terada et al., 2002), altering the plant DNA-repair machinery by expressing yeast chromatin remodeling protein (Shaked et al., 2005), and PCR screening of large numbers of transgenic plants (Kempin et al., 1997; Hanin et al., 2001) are just a few of the experimental approaches used to achieve HR-mediated gene targeting in plant species. While successful, these approaches, and others, have resulted in only a limited number of reports describing the successful implementation of HR-mediated gene targeting of native and transgenic sequences in plant cells (for review, see Puchta, 2002; Hanin and Paszkowski, 2003; Reiss, 2003; Porteus, 2009; Weinthal et al., 2010).HR-mediated gene targeting can potentially be enhanced by the induction of genomic double-strand breaks (DSBs). In their pioneering studies, Puchta et al. (1993, 1996) showed that DSB induction by the naturally occurring rare-cutting restriction enzyme I-SceI leads to enhanced HR-mediated DNA repair in plants. Expression of I-SceI and another rare-cutting restriction enzyme (I-CeuI) also led to efficient NHEJ-mediated site-specific mutagenesis and integration of foreign DNA molecules in plants (Salomon and Puchta, 1998; Chilton and Que, 2003; Tzfira et al., 2003). Naturally occurring rare-cutting restriction enzymes thus hold great promise as a tool for genome editing in plant cells (Carroll, 2004; Pâques and Duchateau, 2007). However, their wide application is hindered by the tedious and next to impossible reengineering of such enzymes for novel DNA-target specificities (Pâques and Duchateau, 2007).A viable alternative to the use of rare-cutting restriction enzymes is the zinc finger nucleases (ZFNs), which have been used for genome editing in a wide range of eukaryotic species, including plants (e.g. Bibikova et al., 2001; Porteus and Baltimore, 2003; Lloyd et al., 2005; Urnov et al., 2005; Wright et al., 2005; Beumer et al., 2006; Moehle et al., 2007; Santiago et al., 2008; Shukla et al., 2009; Tovkach et al., 2009; Townsend et al., 2009; Osakabe et al., 2010; Petolino et al., 2010; Zhang et al., 2010). Here too, ZFNs have been used to enhance DNA integration via HR (e.g. Shukla et al., 2009; Townsend et al., 2009) and as an efficient tool for the induction of site-specific mutagenesis (e.g. Lloyd et al., 2005; Zhang et al., 2010) in plant species. The latter is more efficient and simpler to implement in plants as it does not require codelivery of both ZFN-expressing and donor DNA molecules and it relies on NHEJ—the dominant DNA-repair machinery in most plant species (Ray and Langer, 2002; Britt and May, 2003).ZFNs are artificial restriction enzymes composed of a fusion between an artificial Cys₂His₂ zinc-finger protein DNA-binding domain and the cleavage domain of the FokI endonuclease. The DNA-binding domain of ZFNs can be engineered to recognize a variety of DNA sequences (for review, see Durai et al., 2005; Porteus and Carroll, 2005; Carroll et al., 2006). The FokI endonuclease domain functions as a dimer, and digestion of the target DNA requires proper alignment of two ZFN monomers at the target site (Durai et al., 2005; Porteus and Carroll, 2005; Carroll et al., 2006). Efficient and coordinated expression of both monomers is thus required for the production of DSBs in living cells. Transient ZFN expression, by direct gene delivery, is the method of choice for targeted mutagenesis in human and animal cells (e.g. Urnov et al., 2005; Beumer et al., 2006; Meng et al., 2008). Among the different methods used for high and efficient transient ZFN delivery in animal and human cell lines are plasmid injection (Morton et al., 2006; Foley et al., 2009), direct plasmid transfer (Urnov et al., 2005), the use of integrase-defective lentiviral vectors (Lombardo et al., 2007), and mRNA injection (Takasu et al., 2010).In plant species, however, efficient and strong gene expression is often achieved by stable gene transformation. Both transient and stable ZFN expression have been used in gene-targeting experiments in plants (Lloyd et al., 2005; Wright et al., 2005; Maeder et al., 2008; Cai et al., 2009; de Pater et al., 2009; Shukla et al., 2009; Tovkach et al., 2009; Townsend et al., 2009; Osakabe et al., 2010; Petolino et al., 2010; Zhang et al., 2010). In all cases, direct gene-transformation methods, using polyethylene glycol, silicon carbide whiskers, or Agrobacterium, were deployed. Thus, while mutant plants and tissues could be recovered, potentially without any detectable traces of foreign DNA, such plants were generated using a transgenic approach and are therefore still likely to be classified as transgenic. Furthermore, the recovery of mutants in many cases is also dependent on the ability to regenerate plants from protoplasts, a procedure that has only been successfully applied in a limited number of plant species. Therefore, while ZFN technology is a powerful tool for site-specific mutagenesis, its wider implementation for plant improvement may be somewhat limited, both by its restriction to certain plant species and by legislative restrictions imposed on transgenic plants.Here we describe an alternative to direct gene transfer for ZFN delivery and for the production of mutated plants. Our approach is based on the use of a novel Tobacco rattle virus (TRV)-based expression system, which is capable of systemically infecting its host and spreading into a variety of tissues and cells of intact plants, including developing buds and regenerating tissues. We traced the indirect ZFN delivery in infected plants by activation of a mutated reporter gene and we demonstrate that this approach can be used to recover mutated plants.     

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.