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.     

Focus on Ethylene: Ethylene Biosynthesis Is Promoted by Very-Long-Chain Fatty Acids during Lysigenous Aerenchyma Formation in Rice Roots

In rice (Oryza sativa) roots, lysigenous aerenchyma, which is created by programmed cell death and lysis of cortical cells, is constitutively formed under aerobic conditions, and its formation is further induced under oxygen-deficient conditions. Ethylene is involved in the induction of aerenchyma formation. reduced culm number1 (rcn1) is a rice mutant in which the gene encoding the ATP-binding cassette transporter RCN1/OsABCG5 is defective. Here, we report that the induction of aerenchyma formation was reduced in roots of rcn1 grown in stagnant deoxygenated nutrient solution (i.e. under stagnant conditions, which mimic oxygen-deficient conditions in waterlogged soils). 1-Aminocyclopropane-1-carboxylic acid synthase (ACS) is a key enzyme in ethylene biosynthesis. Stagnant conditions hardly induced the expression of ACS1 in rcn1 roots, resulting in low ethylene production in the roots. Accumulation of saturated very-long-chain fatty acids (VLCFAs) of 24, 26, and 28 carbons was reduced in rcn1 roots. Exogenously supplied VLCFA (26 carbons) increased the expression level of ACS1 and induced aerenchyma formation in rcn1 roots. Moreover, in rice lines in which the gene encoding a fatty acid elongase, CUT1-LIKE (CUT1L; a homolog of the gene encoding Arabidopsis CUT1, which is required for cuticular wax production), was silenced, both ACS1 expression and aerenchyma formation were reduced. Interestingly, the expression of ACS1, CUT1L, and RCN1/OsABCG5 was induced predominantly in the outer part of roots under stagnant conditions. These results suggest that, in rice under oxygen-deficient conditions, VLCFAs increase ethylene production by promoting 1-aminocyclopropane-1-carboxylic acid biosynthesis in the outer part of roots, which, in turn, induces aerenchyma formation in the root cortex.Aerenchyma formation is a morphological adaptation of plants to complete submergence and waterlogging of the soil, and facilitates internal gas diffusion (Armstrong, 1979; Jackson and Armstrong, 1999; Colmer, 2003; Voesenek et al., 2006; Bailey-Serres and Voesenek, 2008; Licausi and Perata, 2009; Sauter, 2013; Voesenek and Bailey-Serres, 2015). To adapt to waterlogging in soil, rice (Oryza sativa) develops lysigenous aerenchyma in shoots (Matsukura et al., 2000; Colmer and Pedersen, 2008; Steffens et al., 2011) and roots (Jackson et al., 1985b; Justin and Armstrong, 1991; Kawai et al., 1998), which is formed by programmed cell death and subsequent lysis of some cortical cells (Jackson and Armstrong, 1999; Evans, 2004; Yamauchi et al., 2013). In rice roots, lysigenous aerenchyma is constitutively formed under aerobic conditions (Jackson et al., 1985b), and its formation is further induced under oxygen-deficient conditions (Colmer et al., 2006; Shiono et al., 2011). The former and latter are designated constitutive and inducible lysigenous aerenchyma formation, respectively (Colmer and Voesenek, 2009). The gaseous plant hormone ethylene regulates adaptive growth responses of plants to submergence (Voesenek and Blom, 1989; Voesenek et al., 1993; Visser et al., 1996a,b; Lorbiecke and Sauter, 1999; Hattori et al., 2009; Steffens and Sauter, 2009; van Veen et al., 2013). Ethylene also induces lysigenous aerenchyma formation in roots of some gramineous plants (Drew et al., 2000; Shiono et al., 2008). The treatment of roots with ethylene or its precursor (1-aminocyclopropane-1-carboxylic acid [ACC]) stimulates aerenchyma formation in rice (Justin and Armstrong, 1991; Colmer et al., 2006; Yukiyoshi and Karahara, 2014), maize (Zea mays; Drew et al., 1981; Jackson et al., 1985a; Takahashi et al., 2015), and wheat (Triticum aestivum; Yamauchi et al., 2014a,b). Moreover, treatment of roots with inhibitors of ethylene action or ethylene biosynthesis effectively blocks aerenchyma formation under hypoxic conditions in maize (Drew et al., 1981; Konings, 1982; Jackson et al., 1985a; Rajhi et al., 2011).Ethylene biosynthesis is accomplished by two main successive enzymatic reactions: conversion of S-adenosyl-Met to ACC by 1-aminocyclopropane-1-carboxylic acid synthase (ACS), and conversion of ACC to ethylene by 1-aminocyclopropane-1-carboxylic acid oxidase (ACO; Yang and Hoffman, 1984). The activities of both enzymes are enhanced during aerenchyma formation under hypoxic conditions in maize root (He et al., 1996). Since the ACC content in roots of maize is increased by oxygen deficiency and is strongly correlated with ethylene production (Atwell et al., 1988), ACC biosynthesis is essential for ethylene production during aerenchyma formation in roots. In fact, exogenously supplied ACC induced ethylene production in roots of maize (Drew et al., 1979; Konings, 1982; Atwell et al., 1988) and wheat (Yamauchi et al., 2014b), even under aerobic conditions. Ethylene production in plants is inversely related to oxygen concentration (Yang and Hoffman, 1984). Under anoxic conditions, the oxidation of ACC to ethylene by ACO, which requires oxygen, is almost completely repressed (Yip et al., 1988; Tonutti and Ramina, 1991). Indeed, anoxic conditions stimulate neither ethylene production nor aerenchyma formation in maize adventitious roots (Drew et al., 1979). Therefore, it is unlikely that the root tissues forming inducible aerenchyma are anoxic, and that the ACO-mediated step is repressed. Moreover, aerenchyma is constitutively formed in rice roots even under aerobic conditions (Jackson et al., 1985b), and thus, after the onset of waterlogging, oxygen can be immediately supplied to the apical regions of roots through the constitutively formed aerenchyma.Very-long-chain fatty acids (VLCFAs; ≥20 carbons) are major constituents of sphingolipids, cuticular waxes, and suberin in plants (Franke and Schreiber, 2007; Kunst and Samuels, 2009). In addition to their structural functions, VLCFAs directly or indirectly participate in several physiological processes (Zheng et al., 2005; Reina-Pinto et al., 2009; Roudier et al., 2010; Ito et al., 2011; Nobusawa et al., 2013; Tsuda et al., 2013), including the regulation of ethylene biosynthesis (Qin et al., 2007). During fiber cell elongation in cotton ovules, ethylene biosynthesis is enhanced by treatment with saturated VLCFAs, especially 24-carbon fatty acids, and is suppressed by an inhibitor of VLCFA biosynthesis (Qin et al., 2007). The first rate-limiting step in VLCFA biosynthesis is condensation of acyl-CoA with malonyl-CoA by β-ketoacyl-CoA synthase (KCS; Joubès et al., 2008). KCS enzymes are thought to determine the substrate and tissue specificities of fatty acid elongation (Joubès et al., 2008). The Arabidopsis (Arabidopsis thaliana) genome has 21 KCS genes (Joubès et al., 2008). In the Arabidopsis cut1 mutant, which has a defect in the gene encoding CUT1 that is required for cuticular wax production (i.e. one of the KCS genes), the expression of AtACO genes and growth of root cells were reduced when compared with the wild type (Qin et al., 2007). Furthermore, expression of the AtACO genes was rescued by exogenously supplied saturated VLCFAs (Qin et al., 2007). These observations imply that VLCFAs or their derivatives work as regulatory factors for gene expression during some physiological processes in plants.reduced culm number1 (rcn1) was first identified as a rice mutant with a low tillering rate in a paddy field (Takamure and Kinoshita, 1985; Yasuno et al., 2007). The rcn1 (rcn1-2) mutant has a single nucleotide substitution in the gene encoding a member of the ATP-binding cassette (ABC) transporter subfamily G, RCN1/OsABCG5, causing an Ala-684Pro substitution (Yasuno et al., 2009). The mutation results in several mutant phenotypes, although the substrates of RCN1/OsABCG5 have not been determined (Ureshi et al., 2012; Funabiki et al., 2013; Matsuda et al., 2014). We previously found that the rcn1 mutant has abnormal root morphology, such as shorter root length and brownish appearance of roots, under stagnant (deoxygenated) conditions (which mimics oxygen-deficient conditions in waterlogged soils). We also found that the rcn1 mutant accumulates less of the major suberin monomers originating from VLCFAs in the outer part of adventitious roots, and this results in a reduction of a functional apoplastic barrier in the root hypodermis (Shiono et al., 2014a).The objective of this study was to elucidate the molecular basis of inducible aerenchyma formation. To this end, we examined lysigenous aerenchyma formation and ACC, ethylene, and VLCFA accumulation and their biosyntheses in rcn1 roots. Based on the results of these studies, we propose that VLCFAs are involved in inducible aerenchyma formation through the enhancement of ethylene biosynthesis in rice roots.     

Integrating Image-Based Phenomics and Association Analysis to Dissect the Genetic Architecture of Temporal Salinity Responses in Rice

Salinity affects a significant portion of arable land and is particularly detrimental for irrigated agriculture, which provides one-third of the global food supply. Rice (Oryza sativa), the most important food crop, is salt sensitive. The genetic resources for salt tolerance in rice germplasm exist but are underutilized due to the difficulty in capturing the dynamic nature of physiological responses to salt stress. The genetic basis of these physiological responses is predicted to be polygenic. In an effort to address this challenge, we generated temporal imaging data from 378 diverse rice genotypes across 14 d of 90 mm NaCl stress and developed a statistical model to assess the genetic architecture of dynamic salinity-induced growth responses in rice germplasm. A genomic region on chromosome 3 was strongly associated with the early growth response and was captured using visible range imaging. Fluorescence imaging identified four genomic regions linked to salinity-induced fluorescence responses. A region on chromosome 1 regulates both the fluorescence shift indicative of the longer term ionic stress and the early growth rate decline during salinity stress. We present, to our knowledge, a new approach to capture the dynamic plant responses to its environment and elucidate the genetic basis of these responses using a longitudinal genome-wide association model.Nearly one-third of the 54 million ha of the highly saline soils in the world are located in South and Southeast Asia. Rice (Oryza sativa), which is the primary source of calories and protein for these two regions, is very sensitive to salinity stress, with even moderate salinity levels known to decrease yields by 50% (Zeng et al., 2002). Projected sea level rise due to climate change is expected to increase saltwater ingress in coastal rice-growing regions of South and Southeast Asia. Therefore, development of salt-tolerant rice cultivars is essential to maintain rice productivity in the salinity-affected regions globally.Salt tolerance, defined as the ability to maintain growth and productivity in saline conditions, is a complex polygenic trait that may be influenced by distinct physiological mechanisms (Munns et al., 1982; Munns and Termaat, 1986; Cheeseman, 1988; Munns and Tester, 2008; Horie et al., 2012; for a comprehensive review of genes involved in salinity tolerance in rice, see Negrão et al., 2011) At the cellular level, plants respond to saline conditions in two phases, namely an osmotic (shoot ion independent) and an ionic stress phase, which can occur in an overlapping manner with varying intensity during the course of salinity stress (Munns and Termaat, 1986; Munns, 2002; Munns and James, 2003; Munns and Tester, 2008; Horie et al., 2012). During the osmotic stress phase, which occurs soon after the onset of salinity, the reduction in external osmotic potential disrupts water uptake and impedes cell expansion, which, at the whole plant level, leads to reduced growth rate (Matsuda and Riazi, 1981; Munns and Passioura, 1984; Rawson and Munns, 1984; Azaizeh and Steudle, 1991; Fricke and Peters, 2002; Fricke, 2004; Boursiac et al., 2005). As salinity stress persists over several days and weeks, sodium ions (Na⁺) accumulate to toxic levels, resulting in cell death and precocious leaf senescence (Lutts and Bouharmont, 1996; Munns, 2002; Munns and James, 2003; Ghanem et al., 2008). This is typically observed during the ionic phase of the salinity response (Munns, 2002; Munns and James, 2003; Munns and Tester, 2008). Plants possess distinct mechanisms to adapt to these osmotic and ionic stresses that are controlled by a suite of genes (Apse et al., 1999; Carvajal et al., 1999; Halfter et al., 2000; Ishitani et al., 2000; Shi et al., 2000; Zeng and Shannon, 2000; Rus et al., 2001; Berthomieu et al., 2003; Martínez-Ballesta et al., 2003; Boursiac et al., 2005, 2008; Ren et al., 2005; Huang et al., 2006; Davenport et al., 2007; Obata et al., 2007; Székely et al., 2008; Horie et al., 2011; Rivandi et al., 2011; Asano et al., 2012; Munns et al., 2012; Latz et al., 2013; Schmidt et al., 2013; Campo et al., 2014; Choi et al., 2014; Liu et al., 2014). The genetic basis of temporal adaptive responses to salinity stress remains to be explored in rice and other crops. This is primarily due to challenges in capturing the dynamic physiological responses to salinity for a large number of genotypes in a nondestructive manner. Manual phenotyping to detect incremental changes in growth rate during the osmotic stress and slight shifts in leaf color due to ionic stress is difficult to quantify for a large number of genotypes.In rice, at least one major quantitative trait loci (QTL; saltol) for salinity tolerance has been characterized based on end point measurements of biomass, senescence/injury, and Na⁺ and K⁺ concentrations (Bonilla et al., 2002; Lin et al., 2004; Thomson et al., 2010). SHOOT K⁺ CONTENT1 (SKC1) is the causative gene underlying the saltol region. SKC1 encodes a Na⁺-selective high-affinity potassium transporter that regulates Na⁺/K⁺ homeostasis during salinity stress (Ren et al., 2005). High levels of Na⁺ displace cellular K⁺, an essential element for several enzymatic reactions and physiological processes (Gierth and Mäser, 2007). The ability to maintain cellular K⁺ levels during salinity through the action of Na⁺-selective potassium transporters or Na⁺/H⁺ antiporters is a well-characterized tolerance mechanism in cereals including rice (Ren et al., 2005; Sunarpi et al., 2005; Huang et al., 2006; Møller et al., 2009; Mian et al., 2011; Munns et al., 2012).Numerous studies have utilized conventional linkage mapping to identify QTL for morphological and physiological responses to salinity in rice using discrete end point measurements (Bonilla et al., 2002; Lin et al., 2004; Ren et al., 2005; Negrão et al., 2011; Wang et al., 2012). However, the physiological adaptation to saline conditions is a complex continuous process that develops over time. While some accessions will exhibit similar end point phenotypic values, the genetic and physiological mechanisms giving rise to the similar phenotypes may be very different and the growth trajectories throughout the experiment may be distinct. Although single time point studies have yielded important information regarding the genetic basis of salinity tolerance, such approaches are too simple to reveal the genetic architecture of stress adaptation. With the advent of high-throughput image-based phenotyping platforms, it is now feasible to quantify dynamic responses during the stress treatment for a large number of genotypes (Berger et al., 2010; Golzarian et al., 2011; Chen et al., 2014; Honsdorf et al., 2014).Image-based phenotyping has been combined with genome-wide association studies (GWAS) and linkage mapping to examine the genetic basis of complex developmental processes (Busemeyer et al., 2013; Moore et al., 2013; Topp et al., 2013; Slovak et al., 2014; Würschum et al., 2014; Yang et al., 2014; Bac-Molenaar et al., 2015). Moreover, the introduction of the time axis provides a better understanding of the physiological processes underlying complex stress and developmental responses compared with single end point measurements (Zhang et al., 2012; Moore et al., 2013; Brown et al., 2014; Chen et al., 2014; Slovak et al., 2014; Bac-Molenaar et al., 2015). However, to date, no studies have implemented an association mapping approach using image-derived phenotypes to address the genetic basis of dynamic stress responses in plants. Image-based phenotyping offers several advantages over conventional phenotyping: (1) quantitative measurements can be recorded over discrete time points to capture morphological and physiological responses in a nondestructive manner, and (2) the use of various types of spectral imaging address phenotypes that are not detectable to the human eye such as chlorophyll fluorescence and leaf water content. Integrating dynamic phenotypic data and association mapping has the potential to query genetic diversity across hundreds of accessions for complex traits and provides much higher resolution compared with conventional linkage mapping. Here, we explored the dynamic growth and chlorophyll responses to salinity of a diverse set of rice accessions using high-throughput visible and fluorescence imaging. To assess the genetic basis of plant growth in saline conditions, a logistic model was used to describe the temporal growth responses and was incorporated into the statistical framework necessary for association mapping. Coupled with temporal fluorescence imaging, we present, to our knowledge, new insights into the genetic architecture of osmotic and ionic responses during salinity stress in rice.     

Enhanced Nodulation and Nitrogen Fixation in the Abscisic Acid Low-Sensitive Mutant enhanced nitrogen fixation1 of Lotus japonicus

The phytohormone abscisic acid (ABA) is known to be a negative regulator of legume root nodule formation. By screening Lotus japonicus seedlings for survival on an agar medium containing 70 μm ABA, we obtained mutants that not only showed increased root nodule number but also enhanced nitrogen fixation. The mutant was designated enhanced nitrogen fixation1 (enf1) and was confirmed to be monogenic and incompletely dominant. The low sensitivity to ABA phenotype was thought to result from either a decrease in the concentration of the plant''s endogenous ABA or from a disruption in ABA signaling. We determined that the endogenous ABA concentration of enf1 was lower than that of wild-type seedlings, and furthermore, when wild-type plants were treated with abamine, a specific inhibitor of 9-cis-epoxycarotenoid dioxygenase, which results in reduced ABA content, the nitrogen fixation activity of abamine-treated plants was elevated to the same levels as enf1. We also determined that production of nitric oxide in enf1 nodules was decreased. We conclude that endogenous ABA concentration not only regulates nodulation but also nitrogen fixation activity by decreasing nitric oxide production in nodules.Many legumes establish nitrogen-fixing root nodules following reciprocal signal exchange between the plant and rhizobia (Hayashi et al., 2000; Hirsch et al., 2003). The host plant produces chemical compounds, frequently flavonoids, which induce rhizobial nod genes, whose products are involved in the synthesis and secretion of Nod factor. Perception of this chitolipooligosaccharide by the host plant results in the triggering of a signal transduction cascade that leads to root hair deformation and curling and subsequent cortical cell divisions, which establish the nodule primordium. The rhizobia enter the curled root hair cell and nodule primordial cells through an infection thread. Eventually, the rhizobia are released into nodule cells, enclosed within a membrane, and differentiate into nitrogen-fixing bacteroids that reduce atmospheric nitrogen into ammonia. In return, the host plant supplies photosynthetic products, to be used as carbon sources, to the rhizobia (Zuanazzi et al., 1998; Hayashi et al., 2000).The host plant is known to be important for regulating the number of nodules established on its roots. For example, hypernodulating mutants such as nitrate-tolerant symbiotic1 (nts1; Glycine max), hypernodulation aberrant root formation1 (har1; Lotus japonicus), super numeric nodules (sunn; Medicago truncatula), and symbiosis29 (sym29; Pisum sativum) disrupt the balance between supply and demand by developing excessive root nodules (Oka-Kira and Kawaguchi, 2006). Grafting experiments demonstrated that leaf tissue is a principal source of the systemic signals contributing to the autoregulation of nodulation (Pierce and Bauer, 1983; Kosslak and Bohlool, 1984; Krusell et al., 2002; Nishimura et al., 2002b; van Brussel et al., 2002; Searle et al., 2003; Schnabel et al., 2005). The Nts1, Har1, Sunn, and Sym29 genes encode a receptor-like kinase similar to CLAVATA1, which regulates meristem cell number and differentiation (Krusell et al., 2002; Nishimura et al., 2002a; Searle et al., 2003; Schnabel et al., 2005).Phytohormones are also known to regulate nodulation (Hirsch and Fang, 1994). For example, ethylene is a well-known negative regulator of nodulation, influencing the earliest stages from the perception of Nod factor to the growth of infection threads (Nukui et al., 2000; Oldroyd et al., 2001; Ma et al., 2003). The ethylene-insensitive mutant sickle1 (skl1) of M. truncatula has a hypernodulating phenotype (Penmetsa and Cook, 1997). Skl1 is homologous to Ethylene insensitive2 of Arabidopsis (Arabidopsis thaliana), which is part of the ethylene-signaling pathway (Alonso et al., 1999; Penmetsa et al., 2008). In contrast, cytokinin is a positive regulator of nodulation. The cytokinin-insensitive mutant hyperinfected1 (loss of function) of L. japonicus and the spontaneous nodule formation2 (gain of function) mutants of M. truncatula provide genetic evidence demonstrating that cytokinin plays a critical role in the activation of nodule primordia (Gonzalez-Rizzo et al., 2006; Murray et al., 2007; Tirichine et al., 2007).Abscisic acid (ABA), added at concentrations that do not affect plant growth, also negatively regulates nodulation in some legumes (Phillips, 1971; Cho and Harper, 1993; Bano et al., 2002; Bano and Harper, 2002; Suzuki et al., 2004; Nakatsukasa-Akune et al., 2005; Liang et al., 2007). Recently, M. truncatula overexpressing abscisic acid insensitive1-1, a gene that encodes a mutated protein phosphatase of the type IIC class derived from Arabidopsis and that suppresses the ABA-signaling pathway (Leung et al., 1994; Hagenbeek et al., 2000; Gampala et al., 2001; Wu et al., 2003), was shown to exhibit ABA insensitivity as well as a hypernodulating phenotype (Ding et al., 2008).In this study, we isolated a L. japonicus (Miyakojima MG20) mutant that showed an increased root nodule phenotype and proceeded to carry out its characterization. This mutant, named enhanced nitrogen fixation1 (enf1), exhibits enhanced symbiotic nitrogen fixation activity. Most legume nitrogen fixation activity mutants, such as ineffective greenish nodules1 (ign1), stationary endosymbiont nodule1, and symbiotic sulfate transporter1 (sst1), are Fix⁻ (Suganuma et al., 2003; Krusell et al., 2005; Kumagai et al., 2007).