Endogenous Diterpenes Derived from ent-Kaurene,a Common Gibberellin Precursor,Regulate Protonema Differentiation of the Moss Physcomitrella patens

Gibberellins (GAs) are a group of diterpene-type plant hormones biosynthesized from ent-kaurene via ent-kaurenoic acid. GAs are ubiquitously present in seed plants. The GA signal is perceived and transduced by the GID1 GA receptor/DELLA repressor pathway. The lycopod Selaginella moellendorffii biosynthesizes GA and has functional GID1-DELLA signaling components. In contrast, no GAs or functionally orthologous GID1-DELLA components have been found in the moss Physcomitrella patens. However, P. patens produces ent-kaurene, a common precursor for GAs, and possesses a functional ent-kaurene synthase, PpCPS/KS. To assess the biological role of ent-kaurene in P. patens, we generated a PpCPS/KS disruption mutant that does not accumulate ent-kaurene. Phenotypic analysis demonstrates that the mutant has a defect in the protonemal differentiation of the chloronemata to caulonemata. Gas chromatography-mass spectrometry analysis shows that P. patens produces ent-kaurenoic acid, an ent-kaurene metabolite in the GA biosynthesis pathway. The phenotypic defect of the disruptant was recovered by the application of ent-kaurene or ent-kaurenoic acid, suggesting that ent-kaurenoic acid, or a downstream metabolite, is involved in protonemal differentiation. Treatment with uniconazole, an inhibitor of ent-kaurene oxidase in GA biosynthesis, mimics the protonemal phenotypes of the PpCPS/KS mutant, which were also restored by ent-kaurenoic acid treatment. Interestingly, the GA₉ methyl ester, a fern antheridiogen, rescued the protonemal defect of the disruption mutant, while GA₃ and GA₄, both of which are active GAs in angiosperms, did not. Our results suggest that the moss P. patens utilizes a diterpene metabolite from ent-kaurene as an endogenous developmental regulator and provide insights into the evolution of GA functions in land plants.GAs are a large family of tetracyclic diterpenoids, and bioactive GAs play crucial roles in aspects of plant growth and development, including seed germination, stem elongation, leaf expansion, trichome development, and flower and fruit development (Olszewski et al., 2002). GAs are biosynthesized from ent-kaurene, the key intermediate of the GA biosynthetic pathway (Olszewski et al., 2002; Yamaguchi, 2008; Fig. 1). ent-Kaurene is synthesized via sequential cyclization steps of geranylgeranyl diphosphate (GGDP) by ent-copalyl diphosphate synthase (CPS; Sun and Kamiya, 1994) and ent-kaurene synthase (KS; Yamaguchi et al., 1996, 1998). The bioactive GAs (GA₁ and GA₄) are synthesized through a series of oxidation reactions of ent-kaurene by two types of oxidases. Both ent-kaurene oxidase and ent-kaurenoic acid oxidase are cytochrome P450 monooxygenases that successively convert ent-kaurene to GA₁₂. GA₁₂ is further converted to bioactive GAs by two 2-oxoglutarate-dependent dioxygenases, GA 20-oxidase and GA 3-oxidase (Phillips et al., 1995; Olszewski et al., 2002; Yamaguchi, 2008; Fig. 1). GA 2-oxidase is another member of the 2-oxoglutarate-dependent dioxygenase family and is responsible for GA inactivation (Fig. 1). The active GAs can bind to the soluble GA receptor, GID1, and promote the interaction of GID1 with DELLA repressors, which are negative regulators of GA signaling (Ueguchi-Tanaka et al., 2005; Nakajima et al., 2006). This GA-promoted GID1-DELLA interaction triggers the degradation of DELLA repressors via the SCF^GID2/SLY1 proteasome pathway and consequently activates GA signaling (Ueguchi-Tanaka et al., 2007).Open in a separate windowFigure 1.The biosynthetic pathway of GA. The enzyme names are shown in boldface below or to the right of each arrow. AMO-1618 is an angiosperm inhibitor of CPS. Uniconazole, a GA biosynthesis inhibitor, blocks ent-kaurene oxidase activity. GA₁ and GA₄ are the bioactive GAs, and GA₈ and GA₃₄ are their inactive catabolites, respectively. KAO, ent-Kaurenoic acid oxidase.In nonseed land vascular plants, auxin, cytokinin, and abscisic acid function as regulators of plant growth and development (Chopra and Kumra, 1988; Raghavan, 1989). Various physiological responses to these phytohormones are investigated in nonseed land plants, especially in the model moss Physcomitrella patens (Cove et al., 2006). Auxin and cytokinin function in developmental phase changes of chloronemata, caulonemata, and gametophores as well as in cellular growth regulation in P. patens (Imaizumi et al., 2002; Sakakibara et al., 2003; Decker et al., 2006). Abscisic acid mediates the establishment of tolerance to dehydration, cold temperature, and osmotic stresses in P. patens as in angiosperms (Decker et al., 2006; Cho et al., 2009; Khandelwal et al., 2010). In contrast to these hormones, there are only a few studies on the physiological activity of GA in mosses (Von Maltzahn and Macquarrie, 1958; Chopra and Mehta, 1987; Vandenbussche et al., 2007), and the GA function and signaling pathways are still unclear.Recent progress in plant molecular biology and chemical analysis of GA revealed the biosynthesis, perception, and signaling of GA in P. patens and the lycopod Selaginella moellendorffii (Hirano et al., 2007; Vandenbussche et al., 2007; Yasumura et al., 2007). Genome sequence for these organisms has enabled the identification of genes orthologous to flowering plant genes encoding GA biosynthetic enzymes and GA signaling components involved in the GID1-DELLA pathway (Hirano et al., 2007; Vandenbussche et al., 2007). Recently, two reports demonstrated that GID1-DELLA-mediated signaling is functionally conserved in the fern Selaginella and in angiosperms (Hirano et al., 2007; Yasumura et al., 2007). GA-dependent protein-protein interactions were observed between SmGID1 and SmDELLA proteins, the S. moellendorffii proteins orthologous to the rice (Oryza sativa) GID1 and DELLA proteins, respectively. The introduction of either the SmGID1a or SmGID1b gene rescued the rice Osgid1-3 mutant, and the overproduction of SmDELLA1 suppressed GA action in the wild-type background. These reports indicate that the GID1 and DELLA proteins function similarly in S. moellendorffii and in angiosperms. Additionally, S. moellendorffii has functional GA biosynthetic enzymes similar to the angiosperm GA 20- and GA 3-oxidases and endogenous active GA₄ detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. However, endogenous GAs were not detected in P. patens by LC-MS/MS analysis, and the putative P. patens GA oxidases did not show any enzymatic activity on the known substrate for the orthologous angiosperm GA oxidases (Hirano et al., 2007). Furthermore, the PpGID1-like and PpDELLA-like proteins did not interact in the presence of active GA in yeast cells, and the PpDELLA-like protein did not complement the rice DELLA function. These findings suggest that GID1-DELLA-mediated GA signaling evolved in the vascular plant lineage after bryophyte divergence (Hirano et al., 2008).GA₁ and GA₄ are recognized as major biologically active GAs in angiosperms. S. moellendorffii biosynthesizes GA₄ as an active GA. Additionally, the Schizaeaceae family of ferns utilize GA methyl esters (methyl esters of GA₉, GA₂₀, and GA₇₃) as regulators of antheridium development, whereas these GA methyl esters are inactive in angiosperms (Yamauchi et al., 1996, 1997; Kurumatani et al., 2001). The biologically active GAs present in angiosperms were not detected in P. patens (Hirano et al., 2007). Although diverse GA metabolites have been found in plants and fungi, all the GA metabolites are thought to be derived from ent-kaurene, a common intermediate in early GA biosynthetic steps in both land plants and fungi (Kawaide, 2006). In angiosperms, two separate enzymes (CPS and KS) are involved in ent-kaurene synthesis from GGDP via ent-copalyl diphosphate as a reaction intermediate (Fig. 1). We have reported that PpCPS/KS, catalyzing the direct cyclization of GGDP to ent-kaurene, was a bifunctional diterpene cyclase with both CPS and KS activities in a single polypeptide (Hayashi et al., 2006). This type of bifunctional ent-kaurene synthase was also found in GA-producing fungi but was not identified in angiosperms (Kawaide et al., 1997; Toyomasu et al., 2000). The P. patens genome contains a single CPS/KS homolog, and no diterpene cyclase gene was found on the basis of sequence similarity in this organism. Anterola et al. (2009) reported that AMO-1618, an inhibitor of CPS, suppressed spore germination in P. patens; the suppression was recovered by exogenous ent-kaurene application. These results led the authors to hypothesize a role for ent-kaurene in regulating spore germination (Anterola et al., 2009). However, the hypothesis should be examined because the AMO-1618 inhibitory effect was not fully recovered by ent-kaurene application, probably because of the unspecific inhibitory effect of AMO-1618 on spore germination (Anterola et al., 2009).To assess the biological role of ent-kaurene and its metabolites in P. patens, we performed an insertional knockout of the ent-kaurene synthase gene, CPS/KS, in P. patens; the loss of ent-kaurene production was confirmed by gas chromatography-mass spectrometry (GC-MS) analysis. We also determined the abundance of all possible GAs and their precursors in P. patens by LC-MS/MS analysis. The PpCPS/KS disruption mutant (Ppcps/ks KO) lines have a defect in protonemal development. The differentiation of chloronemata to caulonemata was suppressed in the Ppcps/ks KO mutants, and the defect was recovered by the exogenous application of ent-kaurene or ent-kaurenoic acid. Furthermore, the GA₉ methyl ester, an antheridiogen of schizaeaceous ferns, rescued the protonemal defect of the mutants, but GA₃ and GA₄, the representative active GAs for angiosperm, did not. Our results demonstrate that P. patens utilizes GA-type diterpenes synthesized from ent-kaurene as an endogenous growth regulator in protonemal development.     

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.