The Calcium Ion Is a Second Messenger in the Nitrate Signaling Pathway of Arabidopsis

Understanding how plants sense and respond to changes in nitrogen availability is the first step toward developing strategies for biotechnological applications, such as improvement of nitrogen use efficiency. However, components involved in nitrogen signaling pathways remain poorly characterized. Calcium is a second messenger in signal transduction pathways in plants, and it has been indirectly implicated in nitrate responses. Using aequorin reporter plants, we show that nitrate treatments transiently increase cytoplasmic Ca²⁺ concentration. We found that nitrate also induces cytoplasmic concentration of inositol 1,4,5-trisphosphate. Increases in inositol 1,4,5-trisphosphate and cytoplasmic Ca²⁺ levels in response to nitrate treatments were blocked by {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122, a pharmacological inhibitor of phospholipase C, but not by the nonfunctional phospholipase C inhibitor analog {"type":"entrez-nucleotide","attrs":{"text":"U73343","term_id":"1688125","term_text":"U73343"}}U73343. In addition, increase in cytoplasmic Ca²⁺ levels in response to nitrate treatments was abolished in mutants of the nitrate transceptor NITRATE TRANSPORTER1.1/Arabidopsis (Arabidopsis thaliana) NITRATE TRANSPORTER1 PEPTIDE TRANSPORTER FAMILY6.3. Gene expression of nitrate-responsive genes was severely affected by pretreatments with Ca²⁺ channel blockers or phospholipase C inhibitors. These results indicate that Ca²⁺ acts as a second messenger in the nitrate signaling pathway of Arabidopsis. Our results suggest a model where NRT1.1/AtNPF6.3 and a phospholipase C activity mediate the increase of Ca²⁺ in response to nitrate required for changes in expression of prototypical nitrate-responsive genes.Plants are sessile organisms that evolved sophisticated sensing and response mechanisms to adapt to changing environmental conditions. Calcium, a ubiquitous second messenger in all eukaryotes, has been implicated in plant signaling pathways (Harper et al., 2004; Hetherington and Brownlee, 2004; Reddy and Reddy, 2004; Hepler, 2005). Multiple abiotic and biotic cues elicit specific and distinct spatiotemporal patterns of change in the concentration of cytosolic Ca²⁺ ([Ca2+]_cyt) in plants (Sanders et al., 2002; Hetherington and Brownlee, 2004; Reddy and Reddy, 2004; Hepler, 2005). Abscisic acid and heat shock treatments cause a rapid intracellular Ca²⁺ increase that is preceded by a transient increase in the level of inositol 1,4,5-trisphosphate (IP₃; Sanchez and Chua, 2001; Zheng et al., 2012). Ca²⁺ signatures are detected, decoded, and transmitted to downstream responses by a set of Ca²⁺ binding proteins that functions as Ca²⁺ sensors (White and Broadley, 2003; Dodd et al., 2010).Nitrate is the main source of N in agriculture and a potent signal that regulates the expression of hundreds of genes (Wang et al., 2004; Vidal and Gutiérrez, 2008; Ho and Tsay, 2010). Despite progress in identifying genome-wide responses, only a handful of molecular components involved in nitrate signaling has been identified. Several pieces of evidence indicate that NITRATE TRANSPORTER1.1 (NRT1.1)/Arabidopsis (Arabidopsis thaliana) NITRATE TRANSPORTER1 PEPTIDE TRANSPORTER FAMILY6.3 (AtNPF6.3) is a nitrate sensor in Arabidopsis (Ho et al., 2009; Gojon et al., 2011; Bouguyon et al., 2015). NRT1.1/AtNPF6.3 is required for normal expression of more than 100 genes in response to nitrate in Arabidopsis roots (Wang et al., 2009). Downstream of NRT1.1/AtNPF6.3, CALCINEURIN B-LIKE INTERACTING SER/THR-PROTEINE KINASE8 (CIPK8) is required for normal nitrate-induced expression of primary nitrate response genes, and the CIPK23 kinase is able to control the switch from low to high affinity of NRT1.1/AtNPF6.3 (Ho et al., 2009; Hu et al., 2009; Ho and Tsay, 2010; Castaings et al., 2011). CIPKs act in concert with CALCINEURIN B-LIKE proteins, plant-specific calcium binding proteins that activate CIPKs to phosphorylate downstream targets (Albrecht et al., 2001). Early experiments using maize (Zea mays) and barley (Hordeum vulgare) detached leaves showed that nitrate induction of two nitrate primary response genes was altered by pretreating leaves with the calcium chelator EGTA or the calcium channel blocker LaCl₃ (Sakakibara et al., 1997; Sueyoshi et al., 1999), suggesting an interplay between nitrate response and calcium-related signaling pathways. However, the role of calcium as a second messenger in the nitrate signaling pathway has not been directly addressed.We show that nitrate treatments cause a rapid increase of IP₃ and [Ca2+]_cyt levels and that blocking phospholipase C (PLC) activity inhibits both IP₃ and [Ca2+]_cyt increases after nitrate treatments. We provide evidence that NRT1.1/AtNPF6.3 is required for increasing both IP₃ and [Ca2+]_cyt in response to nitrate treatments. Altering [Ca2+]_cyt or blocking PLC activity hinders regulation of gene expression of nitrate-responsive genes. Our results indicate that Ca²⁺ is a second messenger in the nitrate signaling pathway of Arabidopsis.     

Nitrogen Use Efficiency Is Mediated by Vacuolar Nitrate Sequestration Capacity in Roots of Brassica napus

Enhancing nitrogen use efficiency (NUE) in crop plants is an important breeding target to reduce excessive use of chemical fertilizers, with substantial benefits to farmers and the environment. In Arabidopsis (Arabidopsis thaliana), allocation of more NO₃⁻ to shoots was associated with higher NUE; however, the commonality of this process across plant species have not been sufficiently studied. Two Brassica napus genotypes were identified with high and low NUE. We found that activities of V-ATPase and V-PPase, the two tonoplast proton-pumps, were significantly lower in roots of the high-NUE genotype (Xiangyou15) than in the low-NUE genotype (814); and consequently, less vacuolar NO₃⁻ was retained in roots of Xiangyou15. Moreover, NO₃⁻ concentration in xylem sap, [¹⁵N] shoot:root (S:R) and [NO₃⁻] S:R ratios were significantly higher in Xiangyou15. BnNRT1.5 expression was higher in roots of Xiangyou15 compared with 814, while BnNRT1.8 expression was lower. In both B. napus treated with proton pump inhibitors or Arabidopsis mutants impaired in proton pump activity, vacuolar sequestration capacity (VSC) of NO₃⁻ in roots substantially decreased. Expression of NRT1.5 was up-regulated, but NRT1.8 was down-regulated, driving greater NO₃⁻ long-distance transport from roots to shoots. NUE in Arabidopsis mutants impaired in proton pumps was also significantly higher than in the wild type col-0. Taken together, these data suggest that decrease in VSC of NO₃⁻ in roots will enhance transport to shoot and essentially contribute to higher NUE by promoting NO₃⁻ allocation to aerial parts, likely through coordinated regulation of NRT1.5 and NRT1.8.China is the largest consumer of nitrogen (N) fertilizer in the world; however, the average N use efficiency (NUE) in fertilizer is only around 35%, suggesting considerable potential for improvements (Shen et al., 2003; Wang et al., 2014). With the high amounts of N-fertilizer being used, crop yields are declining in some areas, where application is exceeding the optimum required for local field crops (Shen et al., 2003; Miller and Smith, 2008; Xu et al., 2012). The extremely low NUE results in waste of resources and environmental contamination, and also presents serious hazards for human health (Xu et al., 2012; Chen et al., 2014). Consequently, exploiting the maximum potential for improving NUE in crop plants will have practical significance for agriculture production and the environment (Zhang et al., 2010; Schroeder et al., 2013; Wang et al., 2014). Elucidating the genetic and physiological regulatory mechanisms governing NUE in plants will allow breeding crops and varieties with higher NUE.Ammonium (NH₄⁺) and nitrate (NO₃⁻) are the main N species absorbed and utilized by crops, and NO₃⁻ accumulation and utilization are of major emphasis for N nutrient studies in dry land crops, such as Brassica napus. Several studies revealed the close relationship between NO₃⁻ content and NUE in plant tissues (Shen et al., 2003; Zhang et al., 2012; Tang et al., 2013; Han et al., 2015a). When plants are sufficiently illuminated, NO₃⁻ assimilation efficiency significantly increase in shoots compared with roots (Smirnoff and Stewart, 1985; Tang et al., 2013). Consequently, under daytime with optimal illumination, higher proportion of NO₃⁻ in plant tissue is transported from root to shoot, as an advantageous physiological adaptation that reduces the cost of energy for metabolism (Tang et al., 2013). NO₃⁻ assimilation in plant shoots can therefore take advantage of solar energy while improving NUE (Smirnoff and Stewart, 1985; Andrews, 1986; Tang et al., 2012, 2013).The NO₃⁻ long-distance transport and distribution between root and shoot is regulated by two genes encoding long transport mechanisms. NRT1.5 is responsible for xylem NO₃⁻ loading, while NRT1.8 is responsible for xylem NO₃⁻ unloading (Lin et al., 2008; Li et al., 2010). Expression of the two genes is influenced by NO₃⁻ concentration. NRT1.5 is strongly induced by NO₃⁻ (Lin et al., 2008), while NRT1.8 expression is extremely up-regulated in nrt1.5 mutants (Chen et al., 2012). A negative correlation between the extents of expression of the two genes was observed when plants are subjected to abiotic stresses (Chen et al., 2012). Moreover, expression of NRT1.5 is strongly inhibited by 1-aminocyclopropane-1-carboxylic acid (ACC) and methyl jasmonate (MeJA), whereas the expression of NRT1.8 is significantly up-regulated (Zhang et al., 2014). Based on these studies, we argue that the expression and functioning of NO₃⁻ long-distance transport genes NRT1.5 and NRT1.8 are regulated by cytosolic NO₃⁻ concentration. In addition, the vacuolar and cytosolic NO₃⁻ distribution is likely regulated by proton pumps located within the tonoplast (V-ATPase and V-PPase; Granstedt and Huffaker, 1982; Glass et al., 2002; Krebs et al., 2010). Therefore, NO₃⁻ use efficiency must be affected by NO₃⁻ long-distant transport (between shoot and root) and short-distant transport (between vacuole and cytosol). However, the physiological mechanisms controlling this regulation are still obscure.Previous studies showed that the chloride channel protein (CLCa) is mainly responsible for vacuole NO₃⁻ short-distance transport, as it is the main channel for NO₃⁻ movement between the vacuoles and cytosol (De Angeli et al., 2006; Wege et al., 2014). The vacuole proton-pumps (V-ATPase and V-PPase) located in the tonoplast supply energy for active transport of NO₃⁻ and accumulation within the vacuole (Gaxiola et al., 2001; Brüx et al., 2008; Krebs et al., 2010). Despite the fact about 90% of the volume of mature plant cells is occupied by vacuoles, vacuolar NO₃⁻ cannot be efficiently assimilated because the enzyme nitrate reductase (NR) is cytosolic (Shen et al., 2003; Han et al., 2015a). However, retranslocation of NO₃⁻ from the vacuole to the cytosol will permit its immediate assimilation and utilization.Generally, NO₃⁻ concentrations in plant cell vacuoles and the cytoplasm are in the range of 30–50 mol m⁻³ and 3–5 mol m⁻³, respectively (Martinoia et al., 1981, 2000). Because vacuoles are obviously the organelle for high NO₃⁻ accumulation and storage in plant tissues, their function in NO₃⁻ use efficiency cannot be ignored (Martinoia et al., 1981; Zhang et al., 2012; Han et al., 2015b). NO₃⁻ assimilatory system in the cytoplasm is sufficient for its assimilation when it is transported out of the vacuoles. Therefore, NO₃⁻ use efficiency could in part be dependent on vacuolar-cytosolic NO₃⁻ short-distance transport in plant tissues (Martinoia et al., 1981; Shen et al., 2003; Zhang et al., 2012; Han et al., 2015a).Evidently, NO₃⁻ use efficiency is regulated by both NO₃⁻ long-distance transport from root to shoot and short-distance transport and distribution between vacuoles and cytoplasm within cells (Glass et al., 2002; Dechorgnat et al., 2011; Han et al., 2015a). Although vacuoles compartment excess NO₃⁻ that accumulates in plant cells (Granstedt and Huffaker, 1982; Krebs et al., 2010), neither NO₃⁻ inducible NR genes (NIA1 and NIA2; Fan et al., 2007; Han et al., 2015a) nor the NO₃⁻ long-distance transport gene NRT1.5 (Lin et al., 2008) are regulated by vacuolar NO₃⁻, even though they are essential for NO₃⁻ assimilation. Only NO₃⁻ transported from the vacuole to the cytosol can play a role in regulating NO₃⁻ inducible genes. Consequently, we argue that both NO₃⁻ assimilation in cells and its long-distance transport from root to shoot are regulated by cytosolic NO₃⁻ concentration. However, this hypothesis needs to be substantiated. The mechanisms underlying both NO₃⁻ short-distance (Gaxiola et al., 2001; De Angeli et al., 2006; Brüx et al., 2008; Krebs et al., 2010) and long-distance transport (Lin et al., 2008; Li et al., 2010) have been previously investigated, yet the underlying mechanisms regulating the flux of NO₃⁻ and the obvious relationship between the two transport pathways, as well as their relation to NUE, are not well understood.The NRT family of genes play a partial role in vacuolar NO₃⁻ accumulation in petioles (Chiu et al., 2004) and seed tissues (Chopin et al., 2007), whereas the proton pumps and CLCa system in the tonoplast play a major role in accumulating NO₃⁻ in vacuoles (Gaxiola et al., 2001; De Angeli et al., 2006; Brüx et al., 2008; Krebs et al., 2010). The vacuolar NO₃⁻ short-distance transport system is spread throughout the plant tissues and is the principal means by which vacuolar NO₃⁻ short-distance transport and distribution is controlled (De Angeli et al., 2006; Krebs et al., 2010).The NRT genes seem to work synergistically to control NO₃⁻ long-distance transport between roots and shoots. NRT1.9 is responsible for NO₃⁻ loading into the phloem (Wang and Tsay, 2011), whereas NO₃⁻ loading and unloading into xylem are regulated by NRT1.5 and NRT1.8, respectively (Lin et al., 2008; Li et al.; 2010). Phloem transport mainly involves organic N; the inorganic-N (NO₃⁻) concentrations in the phloem sap are typically very low, ranging from one-tenth to one-hundredth of that of the inorganic-N in xylem sap (Lin et al., 2008; Fan et al., 2009). Therefore, this study focused on NO₃⁻ short-distance transport mediated through the tonoplast proton pumps and the CLCa system and the long-distant transport mechanisms responsible for xylem NO₃⁻ loading and unloading via NRT1.5 and NRT1.8, respectively.Questions related to how long- and short-distance transport of NO₃⁻ are coupled in plant tissues and their role in determining NUE were addressed using a pair of high- and low-NUE B. napus genotypes and Arabidopsis (Arabidopsis thaliana). Application of proton pump inhibitors and ACC in the former, and use of mutants with defective proton pumps in the latter, allowed experimental distinction of the physiological mechanisms regulating these processes. Data presented here provide strong evidence from both model plants supporting this linkage and strongly suggest that cytosolic NO₃⁻ concentration in roots regulates NO₃⁻ long-distance transport from roots to shoots. We also investigated how NO₃⁻ concentration in plant tissues would be affected by NO₃⁻ long-distance transport, vacuolar NO₃⁻ sequestration, and the ensuing relationship with NO₃⁻ use efficiency. We also proposed the physiological mechanisms likely to be important for enhancing NO₃⁻ use efficiency in plants. These findings will provide scientific rationales for improving NUE in important industrial and food crops.     

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.     

Functional Characterization of Proanthocyanidin Pathway Enzymes from Tea and Their Application for Metabolic Engineering

A reevaluation of flux data for Arabidopsis mutants reveals that nitrate uptake through AtNRT1.1 conforms to a single low-affinity transport system that makes virtually no contribution to high-affinity nitrate uptake.In papers by Wang et al. (1998), Liu et al. (1999), and Liu and Tsay (2003), it was proposed that Arabidopsis thaliana Nitrate Transporter1.1 (AtNRT1.1; CHL1) encodes a dual-affinity nitrate transporter that “plays a major role in high-affinity nitrate uptake.” Here, we evaluate this concept by reexamining the uptake kinetics of Arabidopsis (Arabidopsis thaliana) mutant lines defective in NRT1.1 or other nitrate transporters.The uptake of inorganic ions by plant roots conforms to a pattern of biphasic kinetics. At low external ion concentration, ions are absorbed by saturable high-affinity transport systems (HATS), while at high concentrations, nonsaturating low-affinity transport systems (LATS) operate. Such is the case for K⁺, NH₄⁺, NO₃⁻, and ClO₃⁻ (a NO₃⁻ analog; Kochian and Lucas, 1982; Ullrich et al., 1984; Pace and McClure, 1986; Guy et al., 1988; Siddiqi et al., 1990; Aslam et al., 1992). The LATS for ³⁶ClO₃⁻ uptake was linear at [ClO₃⁻] down to 200 μm in tobacco (Nicotiana tabacum; Guy et al., 1988) and for nitrate uptake by barley (Hordeum vulgare) down to 100 μm NO₃⁻ (Aslam et al., 1992). These concentrations were the lowest examined by the latter authors. In the studies by Pace and McClure (1986), Guy et al. (1988), Siddiqi et al. (1990), and Aslam et al. (1992), LATS fluxes were extremely small at low external [NO₃⁻] and linear at both low and high [NO₃⁻].In barley, both constitutive HATS (CHATS) and inducible HATS (IHATS) were demonstrated at low [NO₃⁻], while a constitutive LATS (CLATS) failed to saturate even at 50 mm NO₃⁻ (Siddiqi et al., 1990). Likewise, CHATS and IHATS for nitrate have been demonstrated in Arabidopsis, as well as CLATS and inducible LATS (ILATS; Tsay et al., 1993; Huang et al., 1999).Doddema and Telkamp (1979) isolated an Arabidopsis B1 mutant that was defective in the LATS for nitrate (but not the HATS) by screening for survival on ClO₃⁻. Tsay et al. (1993) isolated the nitrate-inducible AtNRT1.1 gene that encodes the ILATS. Interestingly, Touraine and Glass (1997) were unable to detect reduced LATS or HATS influxes in AtNRT1.1 mutants grown on KNO₃, while Muños et al. (2004) reported increased HATS influx in AtNRT1.1 mutants. Likewise, Remans et al. (2006) failed to detect reduced uptake rates at low (0.5 mm) or high (10 mm) nitrate in AtNRT1.1 mutants.Among eukaryotes, genes encoding IHATS for nitrate were first isolated from Aspergillus nidulans (Unkles et al., 1991) and subsequently from Chlamydomonas reinhardtii (Quesada et al., 1994) and several higher plants (Glass, 2009), and based on the correlations between AtNRT2.1 expression and IHATS influx, it became accepted that IHATS was encoded by AtNRT2.1. This conclusion was supported by the demonstration that transfer DNA mutants disrupted in both AtNRT2.1 and AtNRT2.2 exhibited 67% reduction of HATS but no reduction in LATS function (Filleur et al., 2001). A gene encoding CHATS has not yet been identified, although a mutant with defective CHATS has been isolated (Wang and Crawford, 1996). In summary, it was held that in Arabidopsis, AtNRT2.1 was responsible for IHATS, while AtNRT1.1 and AtNRT1.2 encoded ILATS and CLATS, respectively (Forde, 2000; Li et al., 2007).In papers by Wang et al. (1998), Liu et al. (1999), and Liu and Tsay (2003), it was demonstrated that AtNRT1.1 mutants of Arabidopsis exhibited reduced nitrate uptake even at 10 μm nitrate. The authors concluded that AtNRT1.1 fluxes exhibited saturation kinetics in planta and in Xenopus laevis oocytes and proposed that NRT1.1 encodes a dual-affinity nitrate transporter that “plays a major role in high-affinity nitrate uptake” (Wang et al., 1998). Liu and Tsay (2003) demonstrated that the AtNRT1.1 protein was capable of switching between high- and low-affinity states by phosphorylation of Thr residue 101; under low-nitrogen (N) conditions, phosphorylation mediated via the activation of protein kinase CIPK23 generated a high-affinity transporter (Ho et al., 2009), whereas high-N favored the dephosphorylated low-affinity configuration.