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.     

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.     

Calcineurin B-Like Protein-Interacting Protein Kinase CIPK21 Regulates Osmotic and Salt Stress Responses in Arabidopsis

The role of calcium-mediated signaling has been extensively studied in plant responses to abiotic stress signals. Calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs) constitute a complex signaling network acting in diverse plant stress responses. Osmotic stress imposed by soil salinity and drought is a major abiotic stress that impedes plant growth and development and involves calcium-signaling processes. In this study, we report the functional analysis of CIPK21, an Arabidopsis (Arabidopsis thaliana) CBL-interacting protein kinase, ubiquitously expressed in plant tissues and up-regulated under multiple abiotic stress conditions. The growth of a loss-of-function mutant of CIPK21, cipk21, was hypersensitive to high salt and osmotic stress conditions. The calcium sensors CBL2 and CBL3 were found to physically interact with CIPK21 and target this kinase to the tonoplast. Moreover, preferential localization of CIPK21 to the tonoplast was detected under salt stress condition when coexpressed with CBL2 or CBL3. These findings suggest that CIPK21 mediates responses to salt stress condition in Arabidopsis, at least in part, by regulating ion and water homeostasis across the vacuolar membranes.Drought and salinity cause osmotic stress in plants and severely affect crop productivity throughout the world. Plants respond to osmotic stress by changing a number of cellular processes (Xiong et al., 1999; Xiong and Zhu, 2002; Bartels and Sunkar, 2005; Boudsocq and Lauriére, 2005). Some of these changes include activation of stress-responsive genes, regulation of membrane transport at both plasma membrane (PM) and vacuolar membrane (tonoplast) to maintain water and ionic homeostasis, and metabolic changes to produce compatible osmolytes such as Pro (Stewart and Lee, 1974; Krasensky and Jonak, 2012). It has been well established that a specific calcium (Ca²⁺) signature is generated in response to a particular environmental stimulus (Trewavas and Malhó, 1998; Scrase-Field and Knight, 2003; Luan, 2009; Kudla et al., 2010). The Ca²⁺ changes are primarily perceived by several Ca²⁺ sensors such as calmodulin (Reddy, 2001; Luan et al., 2002), Ca²⁺-dependent protein kinases (Harper and Harmon, 2005), calcineurin B-like proteins (CBLs; Luan et al., 2002; Batistič and Kudla, 2004; Pandey, 2008; Luan, 2009; Sanyal et al., 2015), and other Ca²⁺-binding proteins (Reddy, 2001; Shao et al., 2008) to initiate various cellular responses.Plant CBL-type Ca²⁺ sensors interact with and activate CBL-interacting protein kinases (CIPKs) that phosphorylate downstream components to transduce Ca²⁺ signals (Liu et al., 2000; Luan et al., 2002; Batistič and Kudla, 2004; Luan, 2009). In several plant species, multiple members have been identified in the CBL and CIPK family (Luan et al., 2002; Kolukisaoglu et al., 2004; Pandey, 2008; Batistič and Kudla, 2009; Weinl and Kudla, 2009; Pandey et al., 2014). Involvement of specific CBL-CIPK pair to decode a particular type of signal entails the alternative and selective complex formation leading to stimulus-response coupling (D’Angelo et al., 2006; Batistič et al., 2010).Several CBL and CIPK family members have been implicated in plant responses to drought, salinity, and osmotic stress based on genetic analysis of Arabidopsis (Arabidopsis thaliana) mutants (Zhu, 2002; Cheong et al., 2003, 2007; Kim et al., 2003; Pandey et al., 2004, 2008; D’Angelo et al., 2006; Qin et al., 2008; Tripathi et al., 2009; Held et al., 2011; Tang et al., 2012; Drerup et al., 2013; Eckert et al., 2014). A few CIPKs have also been functionally characterized by gain-of-function approach in crop plants such as rice (Oryza sativa), pea (Pisum sativum), and maize (Zea mays) and were found to be involved in osmotic stress responses (Mahajan et al., 2006; Xiang et al., 2007; Yang et al., 2008; Tripathi et al., 2009; Zhao et al., 2009; Cuéllar et al., 2010).In this report, we examined the role of the Arabidopsis CIPK21 gene in osmotic stress response by reverse genetic analysis. The loss-of-function mutant plants became hypersensitive to salt and mannitol stress conditions, suggesting that CIPK21 is involved in the regulation of osmotic stress response in Arabidopsis. These findings are further supported by an enhanced tonoplast targeting of the cytoplasmic CIPK21 through interaction with the vacuolar Ca²⁺ sensors CBL2 and CBL3 under salt stress condition.     

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

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

Disruption of Fumarylacetoacetate Hydrolase Causes Spontaneous Cell Death under Short-Day Conditions in Arabidopsis

Fumarylacetoacetate hydrolase (FAH) hydrolyzes fumarylacetoacetate to fumarate and acetoacetate, the final step in the tyrosine (Tyr) degradation pathway that is essential to animals. Deficiency of FAH in animals results in an inborn lethal disorder. However, the role for the Tyr degradation pathway in plants remains to be elucidated. In this study, we isolated an Arabidopsis (Arabidopsis thaliana) short-day sensitive cell death1 (sscd1) mutant that displays a spontaneous cell death phenotype under short-day conditions. The SSCD1 gene was cloned via a map-based cloning approach and found to encode an Arabidopsis putative FAH. The spontaneous cell death phenotype of the sscd1 mutant was completely eliminated by further knockout of the gene encoding the putative homogentisate dioxygenase, which catalyzes homogentisate into maleylacetoacetate (the antepenultimate step) in the Tyr degradation pathway. Furthermore, treatment of Arabidopsis wild-type seedlings with succinylacetone, an abnormal metabolite caused by loss of FAH in the Tyr degradation pathway, mimicked the sscd1 cell death phenotype. These results demonstrate that disruption of FAH leads to cell death in Arabidopsis and suggest that the Tyr degradation pathway is essential for plant survival under short-day conditions.Programmed cell death (PCD) has been defined as a sequence of genetically regulated events that lead to the elimination of specific cells, tissues, or whole organs (Lockshin and Zakeri, 2004). In plants, PCD is essential for developmental processes and defense responses (Dangl et al., 1996; Greenberg, 1996; Durrant et al., 2007). One well-characterized example of plant PCD is the hypersensitive response occurring during incompatible plant-pathogen interactions (Lam, 2004), which results in cell death to form visible lesions at the site of infection by an avirulent pathogen and consequently limits the pathogen spread (Morel and Dangl, 1997).To date, a large number of mutants that display spontaneous cell death lesions have been identified in barley (Hordeum vulgare), maize (Zea mays), rice (Oryza sativa), and Arabidopsis (Arabidopsis thaliana; Marchetti et al., 1983; Wolter et al., 1993; Dietrich et al., 1994; Gray et al., 1997). Because lesions form in the absence of pathogen infection, these mutants have been collectively termed as lesion-mimic mutants. Many genes with regulatory roles in PCD and defense responses, including LESION SIMULATING DISEASE1, ACCELERATED CELL DEATH11, and VASCULAR ASSOCIATED DEATH1, have been cloned and characterized (Dietrich et al., 1997; Brodersen et al., 2002; Lorrain et al., 2004).The appearance of spontaneous cell death lesions in some lesion-mimic mutants is dependent on photoperiod. For example, the Arabidopsis mutant lesion simulating disease1 and myoinositol-1-phosphate synthase1 show lesions under long days (LD; Dietrich et al., 1994; Meng et al., 2009), whereas the lesion simulating disease2, lesion initiation1, enhancing RPW8-mediated HR-like cell death1, and lag one homolog1 display lesions under short days (SD; Dietrich et al., 1994; Ishikawa et al., 2003; Wang et al., 2008; Ternes et al., 2011).Blockage of some metabolic pathways in plants may cause cell death and result in lesion formation. For example, the lesion-mimic phenotypes in the Arabidopsis mutants lesion initiation2 and accelerated cell death2 and the maize mutant lesion mimic22 result from an impairment of porphyrin metabolism (Hu et al., 1998; Ishikawa et al., 2001; Mach et al., 2001). Deficiency in fatty acid, sphingolipid, and myoinositol metabolism also causes cell death in Arabidopsis (Mou et al., 2000; Liang et al., 2003; Wang et al., 2008; Meng et al., 2009; Donahue et al., 2010; Berkey et al., 2012).Tyr degradation is an essential five-step pathway in animals (Lindblad et al., 1977). First, Tyr aminotransferase catalyzes the conversion of Tyr into 4-hydroxyphenylpyruvate, which is further transformed into homogentisate by 4-hydroxyphenylpyruvate dioxygenase. Through the sequential action of homogentisate dioxygenase (HGO), maleylacetoacetate isomerase (MAAI), and fumarylacetoacetate hydrolase (FAH), homogentisate is catalyzed to generate fumarate and acetoacetate (Lindblad et al., 1977). Blockage of this pathway in animals results in metabolic disorder diseases (Lindblad et al., 1977; Ruppert et al., 1992; Grompe et al., 1993). For example, human FAH deficiency causes hereditary tyrosinemia type I (HT1), an inborn lethal disease (St-Louis and Tanguay, 1997). Although the homologous genes putatively encoding these enzymes exist in plants (Dixon et al., 2000; Lopukhina et al., 2001; Dixon and Edwards, 2006), it is unclear whether this pathway is essential for plant growth and development.In this study, we report the isolation and characterization of a recessive short-day sensitive cell death1 (sscd1) mutant in Arabidopsis. Map-based cloning of the corresponding gene revealed that SSCD1 encodes the Arabidopsis putative FAH. Further knockout of the gene encoding the Arabidopsis putative HGO completely eliminated the spontaneous cell death phenotype in the sscd1 mutant. Furthermore, we found that treatment of Arabidopsis wild-type seedlings with succinylacetone, an abnormal metabolite caused by loss of FAH in the Tyr degradation pathway (Lindblad et al., 1977), is able to mimic the sscd1 cell death phenotype. These results demonstrate that disruption of FAH leads to cell death in Arabidopsis and suggest that the Tyr degradation pathway is essential for plant survival under SD.