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.     

Plant growth regulation with triazoles of the dioxanyl type

The plant growth retarding activities of several dioxanylalkyl and dioxanylalkenyl triazoles were determined in seedlings of barley, rice, and oilseed rape. Out of these groups some substances proved to be among the most efficient growth retardants known. The compound 1-(4-trifluormethyl)-2-(1,2,4-triazolyl-(1))-3-(5-methyl-1,3-dioxan-5-yl)-propen-3-ol was investigated more closely. Shoot growth is reduced more intensively than root growth by this compound. At lower dosages root growth may even be stimulated. The action of this retardant can be antagonized by gibberellin A₃ and byent-kaurenoic acid. It is suggested that its main biochemical action is to block the reactions that lead froment-kaurene toent-kaurenoic acid in the course of gibberellin biosynthesis.     

Identification and Functional Characterization of Monofunctional ent-Copalyl Diphosphate and ent-Kaurene Synthases in White Spruce Reveal Different Patterns for Diterpene Synthase Evolution for Primary and Secondary Metabolism in Gymnosperms

The biosynthesis of the tetracyclic diterpene ent-kaurene is a critical step in the general (primary) metabolism of gibberellin hormones. ent-Kaurene is formed by a two-step cyclization of geranylgeranyl diphosphate via the intermediate ent-copalyl diphosphate. In a lower land plant, the moss Physcomitrella patens, a single bifunctional diterpene synthase (diTPS) catalyzes both steps. In contrast, in angiosperms, the two consecutive cyclizations are catalyzed by two distinct monofunctional enzymes, ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS). The enzyme, or enzymes, responsible for ent-kaurene biosynthesis in gymnosperms has been elusive. However, several bifunctional diTPS of specialized (secondary) metabolism have previously been characterized in gymnosperms, and all known diTPSs for resin acid biosynthesis in conifers are bifunctional. To further understand the evolution of ent-kaurene biosynthesis as well as the evolution of general and specialized diterpenoid metabolisms in gymnosperms, we set out to determine whether conifers use a single bifunctional diTPS or two monofunctional diTPSs in the ent-kaurene pathway. Using a combination of expressed sequence tag, full-length cDNA, genomic DNA, and targeted bacterial artificial chromosome sequencing, we identified two candidate CPS and KS genes from white spruce (Picea glauca) and their orthologs in Sitka spruce (Picea sitchensis). Functional characterization of the recombinant enzymes established that ent-kaurene biosynthesis in white spruce is catalyzed by two monofunctional diTPSs, PgCPS and PgKS. Comparative analysis of gene structures and enzyme functions highlights the molecular evolution of these diTPSs as conserved between gymnosperms and angiosperms. In contrast, diTPSs for specialized metabolism have evolved differently in angiosperms and gymnosperms.Conifers (Coniferophyta) are well known for producing an abundant and diverse assortment of oleoresin diterpenoids, predominantly in the form of diterpene resin acids from specialized (or secondary) metabolism, that play roles in conifer defense (Trapp and Croteau, 2001a; Keeling and Bohlmann, 2006a; Bohlmann, 2008) and are an important source of biomaterials (Bohlmann and Keeling, 2008). Several conifer diterpene synthases (diTPSs) that biosynthesize these compounds have been functionally characterized (Stofer Vogel et al., 1996; Peters et al., 2000; Martin et al., 2004; Keeling and Bohlmann, 2006b; Ro and Bohlmann, 2006). The formation of diterpene resin acids of conifer specialized metabolism parallels the formation of ent-kaurenoic acid in the biosynthesis of the gibberellin diterpenoid phytohormones (Fig. 1; Keeling and Bohlmann, 2006a; Yamaguchi, 2008). In gibberellin biosynthesis, geranylgeranyl diphosphate (GGPP) is cyclized by diTPS activity to ent-copalyl diphosphate (ent-CPP), and the ent-CPP is further cyclized by diTPS activity to ent-kaurene. A cytochrome P450 (P450)-dependent monooxygenase (CYP701) oxidizes ent-kaurene to ent-kaurenoic acid (Davidson et al., 2006), paralleling the activity of a P450 (CYP720B1) that oxidizes abietadiene to abietic acid in conifer diterpene resin acid biosynthesis (Ro et al., 2005). Other P450s further functionalize ent-kaurenoic acid to form the biologically active gibberellins. Surprisingly, no conifer diTPS involved in the general (or primary) metabolism of gibberellins has been reported to date, while metabolite profiles of gibberellins have been well characterized in conifers for their role in flowering (Moritz et al., 1990).Open in a separate windowFigure 1.Comparison of the biosynthesis of gibberellins, as it is known in angiosperm and lower plants, with the biosynthesis of diterpene resin acids in conifers, a large group of gymnosperm trees. In conifers, the formation of diterpene resin acids involves bifunctional diTPS (e.g. abietadiene synthase) for the stepwise cyclization of GGPP into diterpenes such as abietadiene via a copalyl diphosphate intermediate that moves between the two active sites of the bifunctional diTPS (Peters et al., 2001). The products of the diTPS are subsequently oxidized by P450 to the resin acids. In contrast, gibberellin biosynthesis in angiosperms requires two monofunctional diTPSs to convert GGPP into ent-kaurene, which is subsequently modified by P450s. The two monofunctional diTPSs in angiosperm gibberellin biosynthesis are CPS and KS. In the lower plant P. patens, the CPS and KS activities are combined in a bifunctional diTPS similar to the bifunctional diTPS in conifer diterpene resin acid biosynthesis. Prior to this work, to our knowledge, it was not known if the formation of gibberellins in a gymnosperm involves two monofunctional diTPSs, as in angiosperms, or a bifunctional diTPS, as in gymnosperm diterpene resin acid biosynthesis and in P. patens gibberellin biosynthesis. (Figure adapted from Keeling and Bohlmann [2006a].)In the fungi Gibberella fujikuroi (Toyomasu et al., 2000) and Phaeosphaeria species L487 (Kawaide et al., 1997) and in the primitive land plant Physcomitrella patens (Bryophyta; Hayashi et al., 2006; Anterola and Shanle, 2008), the formation of ent-kaurene from GGPP is catalyzed by bifunctional diTPS enzymes. These enzymes contain two active sites. The N-terminal active site domain harbors a conserved DXDD motif and catalyzes the protonation-initiated cyclization of GGPP to ent-CPP (Prisic et al., 2007). In the C-terminal active site domain, a conserved DDXXD motif is essential for the diphosphate ionization-initiated cyclization of ent-CPP to ent-kaurene (Christianson, 2006). The presence of two active sites with their characteristic DXDD and DDXXD motifs resembles the structure of conifer bifunctional diTPSs in specialized metabolism of diterpene resin acid biosynthesis (Fig. 1), such as the grand fir (Abies grandis) abietadiene synthase (AgAS) and Norway spruce (Picea abies) levopimaradiene/abietadiene synthases (PaLAS; Peters et al., 2001; Martin et al., 2004; Keeling and Bohlmann, 2006a). In contrast, the formation of ent-kaurene from GGPP in angiosperms is catalyzed by two separate monofunctional enzymes, one with only the DXDD motif and having ent-copalyl diphosphate synthase (ent-CPS) activity and the other with only the DDXXD motif and having ent-kaurene synthase (ent-KS) activity (Yamaguchi, 2008).A previously published model for the evolution of plant diTPS (Trapp and Croteau, 2001b) suggests that genes encoding the monofunctional CPS and KS enzymes known in angiosperms originated by gene duplication and subfunctionalization (Lynch and Force, 2000) of an ancestral bifunctional CPS/KS gene that may have been similar to the gene for the CPS/KS enzyme of the moss P. patens. The same model also suggests that genes for diTPSs of gymnosperm specialized diterpene resin acid metabolism arose from duplication and subsequent neofunctionalization of an ancestral bifunctional diTPS of the gibberellin pathway (Trapp and Croteau, 2001b). The pathways to specialized oleoresin diterpenes existed in ancient plants prior to the differentiation of gymnosperms and angiosperms (Bray and Anderson, 2009). Vascular plants split from nonvascular plants approximately 500 million years ago, and angiosperms split from gymnosperms approximately 300 million years ago (Palmer et al., 2004). As there has been no report to date of genes involved in gibberellin biosynthesis in gymnosperms, it remains unresolved and cannot be predicted whether conifers have a bifunctional CPS/KS for the formation of ent-kaurene similar to the primitive land plant P. patens and paralleling the diTPSs for conifer specialized diterpene resin acid biosynthesis or whether they have separate monofunctional CPS and KS enzymes, as is the case in angiosperms.In this study, we made use of the extensive EST resources for spruce species (Pavy et al., 2005; Ralph et al., 2008), combined with isolation and sequencing of full-length cDNAs, genomic (g)DNA, and targeted bacterial artificial chromosome (BAC) clones, as well as enzyme assays with recombinant proteins to search for, and functionally characterize, possible monofunctional or bifunctional diTPS for ent-kaurene biosynthesis in a gymnosperm. In summary, we successfully isolated and characterized monofunctional ent-CPS (PgCPS) and ent-KS (PgKS) from white spruce (Picea glauca) and isolated orthologous cDNAs from Sitka spruce (Picea sitchensis). Comparison of enzyme functions and gene structures support common ancestry but different routes of evolution of monofunctional and bifunctional diTPS in conifer general and specialized metabolism, respectively.     

CYP701A26 is characterized as an <Emphasis Type="Italic">ent</Emphasis>-kaurene oxidase with putative involvement in maize gibberellin biosynthesis

Objectives

To characterize the ent-kaurene oxidase (KO) involved in maize (Zea mays) gibberellin (GA) biosynthesis.

Results

Two putative KO genes were identified in maize based on the homologous alignment. Biochemical characterization indicated that one of them encoded a cytochrome P450 monooxygenase (P450) CYP701A26, which reacted with ent-kaurene to form ent-kaurenoic acid, the key intermediate of GA biosynthesis. CYP701A26 showed constitutive expression in active growing tissues and no inducible expression, which led to putative designation of CYP701A26 as the ZmKO. CYP701A26 exhibited substrate promiscuity to catalyze oxidation of other labdane related diterpenes. Another maize KO homologue, CYP701A43 did not show any catalytic activities on ent-kaurene or other tested diterpenes. It exhibited inducible gene expression and might accept unknown substrates to play roles in specialized metabolism for stress response.

Conclusions

CYP701A26 was characterized to exhibit ent-kaurene oxidase activity with substrate promiscuity and might be involved in maize GA biosynthesis, and its homologue CYP701A43 did not show such function and might play roles in stress response.

     

Arabidopsis ent-Kaurene Oxidase Catalyzes Three Steps of Gibberellin Biosynthesis

The Arabidopsis GA3 cDNA was expressed in yeast (Saccharomyces cerevisiae) and the ability of the transformed yeast cells to metabolize ent-kaurene was tested. We show by full-scan gas chromatography-mass spectrometry that the transformed cells produce ent-kaurenoic acid, and demonstrate that the single enzyme GA3 (ent-kaurene oxidase) catalyzes the three steps of gibberellin biosynthesis from ent-kaurene to ent-kaurenoic acid.     

The P450-4 Gene of Gibberella fujikuroi Encodes ent-Kaurene Oxidase in the Gibberellin Biosynthesis Pathway

At least five genes of the gibberellin (GA) biosynthesis pathway are clustered on chromosome 4 of Gibberella fujikuroi; these genes encode the bifunctional ent-copalyl diphosphate synthase/ent-kaurene synthase, a GA-specific geranylgeranyl diphosphate synthase, and three cytochrome P450 monooxygenases. We now describe a fourth cytochrome P450 monooxygenase gene (P450-4). Gas chromatography-mass spectrometry analysis of extracts of mycelia and culture fluid of a P450-4 knockout mutant identified ent-kaurene as the only intermediate of the GA pathway. Incubations with radiolabeled precursors showed that the metabolism of ent-kaurene, ent-kaurenol, and ent-kaurenal was blocked in the transformants, whereas ent-kaurenoic acid was metabolized efficiently to GA₄. The GA-deficient mutant strain SG139, which lacks the 30-kb GA biosynthesis gene cluster, converted ent-kaurene to ent-kaurenoic acid after transformation with P450-4. The B1-41a mutant, described as blocked between ent-kaurenal and ent-kaurenoic acid, was fully complemented by P450-4. There is a single nucleotide difference between the sequence of the B1-41a and wild-type P450-4 alleles at the 3′ consensus sequence of intron 2 in the mutant, resulting in reduced levels of active protein due to a splicing defect in the mutant. These data suggest that P450-4 encodes a multifunctional ent-kaurene oxidase catalyzing all three oxidation steps between ent-kaurene and ent-kaurenoic acid.     

Presence of endogenous ent-kaurene in a microsomal preparation from Cucurbita maxima L. Endosperm and implications for kinetic studies of ent-kaurene oxidase

Microsomal and soluble cell-free extracts prepared from liquid endosperm of Cucurbita maxima L. were found to contain high concentrations of endogenous ent-kaurene and ent-kaurenol by gas chromatography-mass spectrometry-chemical ionization with deuterated internal standards. Increases in the levels of ent-kaurenol, ent-kaurenoic acid, and ent-7-hydroxykaurenoic acid are correlated with a decline in the amount of endogenous ent-kaurene following a 10 min incubation of microsomes with NADPH and FAD. The rate of oxidation of radiolabeled ent-kaurene by the microsomal fraction was determined, and the need to account for endogenous substrate is shown. Endogenous ent-kaurene present in soluble extracts had the effect of diluting the [¹⁴C]ent-kaurene synthesized from [¹⁴C]mevalonic acid, resulting in reduced specific radioactivity of the product. The dilution of [¹⁴C]ent-kaurene was more pronounced in extracts with higher endogenous ent-kaurene levels or when the reactions were run in the presence of O₂ and NADPH. Evidence is presented that suggests differential metabolism of endogenous ent-kaurene and radiolabeled ent-kaurene in both microsomal and soluble extracts.Abbreviations Kaurene ent-kaur-16-ene - MVA mevalonic acid - kaurenol ent-kaur-16-en-19-ol - kaurenoic acid ent-kaur-16-en-19-oic acid - EtOAc ethyl acetate - MeOH methanol - GC-MS-CI gas chromatography-mass spectrometry-chemical ionization - 13-OH KA ent-13-hydroxykaur-16-en-19-oic acid - 7-OH kaurenoic acid ent-7-hydroxykaur-16-en-19-oic acid - kaurenal ent-kaur-16-en-19-al - Me(x) methyl ester of x - TMS(x) trimethylsilyl ether or ester of x - GA(x) gibberellin A(x)     

A Step in the Biosynthesis of Gibberellins that Is Controlled by the Mutation in the Semi-Dwarf Rice Cultivar Tan-Ginbozu

Genetic analysis and a comparison of endogenous levels of gibberellinsbetween the semi-dwarf rice cultivar Tan-ginbozu and the correspondingnormal cultivar Ginbozu have confirmed that Tan-ginbozu is agibberellin deficient mutant and that the semi-dwarfism of Tan-ginbozuis controlled by a single recessive gene. A step in the biosynthesisof gibberellins that is blocked by the mutation in Tan-ginbozuhad been considered to be the synthesis of ent-kaurene or anearlier step. However, the rate of production of ent-kaureneby Tan-ginbozu was almost the same as that by Ginbozu. By contrast,accumulation of only a small amount of ent-kaurene was detectedin Tan-ginbozu, and the amount that accumulated was similarto that in Ginbozu that had been treated with 6.9 x 10^-8 M uniconazole-P(an effective inhibitor of three oxidative steps in the pathwayfrom ent-kaurene to ent-kaurenoic acid via entkaurenol and ent-kaurenal).The height of the treated Ginbozu plants was reduced to thesame as that of Tanginbozu plants. Resembling Tan-ginbozu plants,Ginbozu plants that had been treated with uniconazole-P respondedwell to ent-kaurenoic acid and slightly to ent-kaurene and ent-kaurenol.Since the growth-promoting activity of enf-kaurenal in Tan-ginbozuwas similar to that of ent-kaurene, our results suggest thatthe mutation in Tan-ginbozu blocks the three oxidative stepswhereby ent-kaurene is converted to ent-kaurenoic acid. (Received June 9, 1995; Accepted February 15, 1996)     

Molecular cloning and functional analysis of a blue light receptor gene MdCRY2 from apple (Malus domestica)

Key message

MdCRY2 was isolated from apple fruit skin, and its function was analyzed in MdCRY2 transgenic Arabidopsis. The interaction between MdCRY2 and AtCOP1 was found by yeast two-hybrid and BiFC assays.

Abstract

Cryptochromes are blue/ultraviolet-A (UV-A) light receptors involved in regulating various aspects of plant growth and development. Investigations of the structure and functions of cryptochromes in plants have largely focused on Arabidopsis (Arabidopsis thaliana), tomato (Solanum lycopersicum), pea (Pisum sativum), and rice (Oryza sativa). However, no data on the function of CRY2 are available in woody plants. In this study, we isolated a cryptochrome gene, MdCRY2, from apple (Malus domestica). The deduced amino acid sequences of MdCRY2 contain the conserved N-terminal photolyase-related domain and the flavin adenine dinucleotide (FAD) binding domain, as well as the C-terminal DQXVP-acidic-STAES (DAS) domain. Relationship analysis indicates that MdCRY2 shows the highest similarity to the strawberry FvCRY protein. The expression of MdCRY2 is induced by blue/UV-A light, which represents a 48-h circadian rhythm. To investigate the function of MdCRY2, we overexpressed the MdCRY2 gene in a cry2 mutant and wild type (WT) Arabidopsis, assessed the phenotypes of the resulting transgenic plants, and found that MdCRY2 functions to regulate hypocotyl elongation, root growth, flower initiation, and anthocyanin accumulation. Furthermore, we examined the interaction between MdCRY2 and AtCOP1 using a yeast two-hybrid assay and a bimolecular fluorescence complementation assay. These data provide functional evidence for a role of blue/UV-A light-induced MdCRY2 in controlling photomorphogenesis in apple.     

Biotransformation of Methylphenylacetonitriles by Brazilian Marine Fungal Strain Aspergillus sydowii CBMAI 934: Eco-friendly Reactions

This study reports the biotransformation of methylphenylacetonitriles by Brazilian marine filamentous fungus Aspergillus sydowii CBMAI 934 under eco-friendly reaction conditions. The phenylacetonitrile 1, 2-methylphenylacetonitrile 2, 3-methylphenylacetonitrile 3, and 4-methylphenylacetonitrile 4 were quantitatively biotransformed into 2-hydroxyphenylacetic 1a, 2-methylphenylacetic acid 2a, 3-methylphenylacetic acid 3a, and 4-methylphenylacetic acid 4a by enzymatic processes using whole cell as biocatalyst. The marine fungus A. sydowii CBMAI 934 is thus a promising biocatalyst for the preparation of important carboxylic acids under mild conditions (pH 7.5 and 32 °C) from nitrile compounds.     

The effect of growth retardants on phytochrome-induced lettuce seed germination

Experiments were carried out to explore the involvement of the plant hormone gibberellin (GA) in the light-induced germination of lettuce seeds. Three growth retardants known to be inhibitors of GA biosynthesis were tested for their effect on red-light-induced germination. Chlormequat chloride (CCC) and AMO-1618 had no effect, but ancymidol was strongly inhibitory. Moreover, the inhibition caused by ancymidol was completely overcome by GA₃. CCC and AMO-1618 inhibit the formation ofent-kaurene, while ancymidol blocks the oxidation ofent-kaurene toent-kaurenoic acid. Ancymidol also was found to inhibit GA-induced dark germination of lettuce seeds, and this inhibition was partially reversed by higher levels of GA. Therefore, the results suggest two possibilities for the relationship between phytochrome and GA in this system: first, the rate-limiting step in the germination of light-sensitive lettuce seeds, that which is regulated by phytochrome, is the oxidation ofent-kaurene toent-kaurenoic acid. Alternatively, red-light treatment may result in the release of active GAlike substances which, in turn, induce germination. In either case the results presented here support the view that phytochrome exerts its effect on lettuce seed germination by means of GA rather than via an independent pathway.     

Density functional studies on photophysical properties and chemical reactivities of the triarylboranes: effect of the constraint of planarity

The geometric and electronic structures, absorption spectra, transporting properties, chemical reactivity indices and electrostatic potentials of the planar three-coordinate organoboron compounds 1-2 and twisted reference compound Mes ₃ B, have been investigated by employing density functional theory (DFT) and conceptual DFT methods to shed light on the planarity effects on the photophysical properties and the chemical reactivity. The results show that the planar compounds 1-2 exhibit significantly lower HOMO level than Mes ₃ B, owing to the stronger electronic induction effect of boron centers. This feature conspicuously induces a blue shifted absorption for 1, although 1 seemingly possesses more extended conjugation framework than Mes ₃ B. Importantly, the reactivity strength of the boron atoms in 1-2 is much lower than that in Mes ₃ B, despite the fact that the tri-coordinate boron centers of 1-2 are completely naked. The interesting and abnormal phenomenon is caused by the strong p-π electronic interactions, that is, the empty p-orbital of boron center is partly filled by π-electron of the neighbor carbon atoms in 1-2, which are confirmed by the analysis of Laplacian of the electron density and natural bond orbitals. Furthermore, the negative electrostatic potentials of the boron centers in 1-2 also interpret that they are not the most preferred sites for incoming nucleophiles. Moreover, it is also found that the planar compounds 1-2 can act as promising electron transporting materials since the internal reorganization energies for electron are really small.

Kaurenolide biosynthesis in a cell-free system from Cucurbita maxima seeds

A new product obtained by incubation of [2-¹⁴C ]-mevalonic acid with a cell-free system from Cucurbita maxima endosperm was identified by GC-MS as ent-kaura-6,16-dien-19-oic acid. When this compound was reincubated with the microsomal fraction it was converted to 7β-hydroxykaurenolide and hence to 7β,12α-dihydroxykaurenolide. The dienoic acid was also obtained by incubation of ent-kaurene, ent1-kaurenol, ent-kaurenal and ent-kaurenoic acid, but not ent-7α-hydroxykaurenoic acid, with the microsomal fraction. Thus, in the C. maxima cell-free system, the kaurenolides are formed by a pathway which branches from the GA pathway at ent-kaurenoic acid and proceeds via the dienoic acid.     

Photophysics of novel coumarin-labeled depsipeptides in solution: sensing interactions with SDS micelle via TICT model

N-Acylbenzotriazoles enable the synthesis (69–92 % yield) of blue to green fluorescent coumarin-labeled depsidipeptides 8a–f (quantum yields 0.004–0.97) and depsitripeptides 12a–d (quantum yields 0.02–0.96). Detailed photophysical studies of fluorescent coumarin-labeled depsipeptides 8a–f and 12a–d are reported for both polar protic and polar aprotic solvents. 7-Methoxy and 7-diethylaminocoumarin-3-ylcarbonyl depsipeptides 8c,f and 12d are highly solvent sensitive. These highly fluorescent compounds could be useful for peptide assays. Further photophysical studies of 7-diethylaminocoumarin-labeled depsipeptides 8c,12d within the micellar microenvironment of SDS reflect their ability to bind with the biological membrane, suggesting potential applications in the fields of bio- and medicinal chemistry.     

Synthesis of Pyrrolo[2′,3′:4,5]Furo[3,2-c]Pyridine-2-Carboxylic Acids

1H-Pyrrolo[2′,3′:4,5]furo[3,2-c]pyridine-2-carboxylic acid (6a) and its 1-methyl (6b) and 1-benzyl (6c) derivatives were synthesized. 3-(5-Methoxycarbonyl-4H-furo[3,2-b]-pyrrole-2-yl)propenoic acid (1) was converted to the corresponding azide 2, which in turn was cyclized to give 3 by heating in diphenylether. The pyridone 3 obtained was aromatized with phosphorus oxychloride, then reduced with zinc in acetic acid to give methyl 1H-pyrrolo[2′,3′:4,5]furo[3,2-c]pyridine-2-carboxylate (5), which by hydrolysis gave the corresponding carboxylic acid 6a.     

Cytotoxic metabolites from the cultures of endophytic fungi from Panax ginseng

Two strains of endophytic fungi, Penicillium melinii Yuan-25 and Penicillium janthinellum Yuan-27, with strong anti-Pyricularia oryzae activity, were obtained from the roots of Panax ginseng. Based on bioactivity-oriented isolation, a new benzaldehyde derivative, ginsenocin (1), together with six known compounds, methyl 2,4-dihydroxy-3,5,6-trimethylbenzoate (2), 3,4,5-trimethyl-1,2-benzenediol (3), penicillic acid (4), mannitol (5), ergosterol (6), and ergosterol peroxide (7), were separated from the EtOAc extract of Yuan-25 culture, while brefeldin A (8) was isolated as the major constituent from the EtOAc extract of Yuan-27 culture. The chemical structures were determined based on spectroscopic methods. All the isolated compounds 1–8 were evaluated for their cytotoxicity against six human cancer cell lines. Brefeldin A (8) was the most cytotoxic constituent against all the tested cell lines with IC₅₀ values <0.12 μg/ml, while ginsenocin (1) and penicillic acid (4) also exhibited potent cytotoxicity with IC₅₀ values ranging from 0.49 to 7.46 μg/ml. Our results suggest that endophytic fungi isolated from P. ginseng are a promising natural source of potential anticancer agents.