The effects of ancymidol,abscisic acid,uniconazole and paclobutrazol on somatic embryogenesis of asparagus

Summary The effects of ancymidol, abscisic acid (ABA), uniconazole, and paclobutrazol on asparagus somatic embryogenesis were evaluated. Calli induced from seedlings of genotype G447 were transferred to embryo induction medium (MS plus 3% sucrose, 0.1 mg L^–1 NAA, 0.5 mg L^–1 kinetin and 3% gelrite), with different concentrations of these compounds. After 8 weeks, the recovered bipolar or globular embryos were placed on germination medium (MS plus 6% sucrose, 0.1 mg L^–1 NAA, 0.1 mg L^–1 kinetin, 0.75 mg L^–1 ancymidol, 40 mg L^–1 adenine sulphate dihydrate, 0.17 mg L^–1 sodium phosphate monobasic and 3% gelrite) for conversion to plantlets. Inclusion of ancymidol, ABA, uniconazole and paclobutrazol in the embryo induction medium did not affect the total number of somatic embryos produced relative to the control without these compounds. However, ancymidol, ABA and uniconazole significantly improved embryo development by increasing the production of bipolar embryos 250–750% and decreasing that of globular embryos 8–35% relative to the control. The bipolar embryos produced with any of the four compounds in the embryo induction medium converted to plantlets at rates 700–1100% greater than the control. None of the globular embryos converted to plantlets. Ancymidol (0.75 mg L^–1) and ABA (0.05 mg L^–1) were the most effective treatments; 61 and 46 bipolar embryos g^–1 callus were produced, and 38% and 37% of the bipolar embryos converted to plantlets, respectively. These results indicated that ancymidol, ABA, uniconazole and paclobutrazol significantly enhanced the production of asparagus somatic embryos and their conversion to plantlets, and ancymidol and ABA were more effective than uniconazole and paclobutrazol.Abbreviations Ancymidol a-cyclopropyl-a(4-methoxyphenyi)-5-pyrimidine methanol - NAA 1-naphthaleneacetic acid - Paclobutrazol I-(4-chlorophenyl)-4,4-dimethyl-2(1H-1,2,4-triazol-1-yl)-pentan-3-ol - Uniconazole (E)-(p-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)-1-pentan-3-ol - ABA abscisic acid - GA gibberellic acid     

Negative Feedbacks by Isoprenoids on a Mevalonate Kinase Expressed in the Corpora Allata of Mosquitoes

Background

Juvenile hormones (JH) regulate development and reproductive maturation in insects. JHs are synthesized through the mevalonate pathway (MVAP), an ancient metabolic pathway present in the three domains of life. Mevalonate kinase (MVK) is a key enzyme in the MVAP. MVK catalyzes the synthesis of phosphomevalonate (PM) by transferring the γ-phosphoryl group from ATP to the C₅ hydroxyl oxygen of mevalonic acid (MA). Despite the importance of MVKs, these enzymes have been poorly characterized in insects.

Results

We functionally characterized an Aedes aegypti MVK (AaMVK) expressed in the corpora allata (CA) of the mosquito. AaMVK displayed its activity in the presence of metal cofactors. Different nucleotides were used by AaMVK as phosphoryl donors. In the presence of Mg²⁺, the enzyme has higher affinity for MA than ATP. The activity of AaMVK was regulated by feedback inhibition from long-chain isoprenoids, such as geranyl diphosphate (GPP) and farnesyl diphosphate (FPP).

Conclusions

AaMVK exhibited efficient inhibition by GPP and FPP (Ki less than 1 μM), and none by isopentenyl pyrophosphate (IPP) and dimethyl allyl pyrophosphate (DPPM). These results suggest that GPP and FPP might act as physiological inhibitors in the synthesis of isoprenoids in the CA of mosquitoes. Changing MVK activity can alter the flux of precursors and therefore regulate juvenile hormone biosynthesis.     

Inhibition of Abscisic Acid Biosynthesis in Cercospora rosicola by Inhibitors of Gibberellin Biosynthesis and Plant Growth Retardants

The fungus Cercospora rosicola produces abscisic acid (ABA) as a secondary metabolite. We developed a convenient system using this fungus to determine the effects of compounds on the biosynthesis of ABA. Inasmuch as ABA and the gibberellins (GAs) both arise via the isoprenoid pathway, it was of interest to determine if inhibitors of GA biosynthesis affect ABA biosynthesis. All five putative inhibitors of GA biosynthesis tested inhibited ABA biosynthesis. Several plant growth retardants with poorly understood actions in plants were also tested; of these, six inhibited ABA biosynthesis to varying degrees and two had no effect. Effects of plant growth retardants on various branches of the isoprenoid biosynthetic pathway may help to explain some of the diverse and unexpected results reported for these compounds. Knowledge that certain inhibitors of GA biosynthesis also have the ability to inhibit ABA biosynthesis in C. rosicola indicates the need for further studies in plants on the mode of action of these compounds.     

Comparative Effects of Substituted Pyrimidines on Growth and Gibberellin Biosynthesis in Gibberella fujikuroi

The fungicide α-(2,4-dichlorophenyl)-α-phenyl-5-pyrimidine methyl alcohol (triarimol) and four other structural analogs of this substance, in which one or more of the substituents were varied, were tested for their comparative effects on growth and gibberellin biosynthesis in the fungus Gibberella fujikuroi. Each of the five analogs tested was capable of inhibiting growth as measured by dry weight in 5-day-old cultures. Three of them [α-(2-chlorophenyl)-α-(4-chlorophenyl)-5-pyrimidine methyl alcohol, fenarimol; α-(2-chlorophenyl)-α-(4-fluorophenyl)-5-pyrimidine methyl alcohol, nuarimol; and triarimol] were effective at appreciably lower concentrations than the other two [α-(4-chlorophenyl)-α-(1-methylethyl)-5-pyrimidine methyl alcohol, experimental compound EL 509; and α-cyclopropyl-α-(4-methoxyphenyl)-5-pyrimidine methyl alcohol, ancymidol].     

Molecular Cloning and Characterisation of Farnesyl Pyrophosphate Synthase from Tripterygium wilfordii

Farnesylpyrophosphate synthase (FPS) catalyzes the biosynthesis of farnesyl pyrophosphate (FPP), which is an important precursor of sesquiterpenoids such as artemisinin and wilfordine. In the present study, we report the molecular cloning and characterization of two full-length cDNAs encoding FPSs from Tripterygium wilfordii (TwFPSs). TwFPSs maintained their capability to synthesise FPP in vitro when purified as recombinant proteins from E. coli. Consistent with the endogenous role of FPS in FPP biosynthesis, TwFPSs were highly expressed in T. wilfordii roots, and were up-regulated upon methyl jasmonate (MeJA) treatment. The global gene expression profiles suggested that the TwFPSs might play an important regulatory role interpenoid biosynthesis in T. wilfordii, laying the groundwork for the future study of the synthetic biology of natural terpene products.     

A Novel Role for Arabidopsis CBL1 in Affecting Plant Responses to Glucose and Gibberellin during Germination and Seedling Development

Glucose and phytohormones such as abscisic acid (ABA), ethylene, and gibberellin (GA) coordinately regulate germination and seedling development. However, there is still inadequate evidence to link their molecular roles in affecting plant responses. Calcium acts as a second messenger in a diverse range of signal transduction pathways. As calcium sensors unique to plants, calcineurin B-like (CBL) proteins are well known to modulate abiotic stress responses. In this study, it was found that CBL1 was induced by glucose in Arabidopsis. Loss-of-function mutant cbl1 exhibited hypersensitivity to glucose and paclobutrazol, a GA biosynthetic inhibitor. Several sugar-responsive and GA biosynthetic gene expressions were altered in the cbl1 mutant. CBL1 protein physically interacted with AKINβ1, the regulatory β subunit of the SnRK1 complex which has a central role in sugar signaling. Our results indicate a novel role for CBL1 in modulating responses to glucose and GA signals.     

Evidence for a gibberellin biosynthetic origin of ceratopteris antheridiogen

The species-specific chemical messenger, antheridiogen A_Ce, mediates the differentiation of male gametophytes in the fern Ceratopteris. In order to investigate the biochemical origin of antheridiogen, the effect of the inhibitors, 2′-isopropyl-4′-(trimethylammoniumchloride)-5′ -methylphenylpiperidine-1-carboxylate (AMO-1618), 2-chloroethyl trimethylammonium chloride (CCC), and α-cyclopropyl-α-(4-methoxyphenyl)-5-pyrimidine methyl alcohol (ancymidol) on gametophytic sex expression was determined in C. richardii. Both AMO-1618 and ancymidol blocked the production of male gametophytes in three genetically defined strains of C. richardii that exhibit different sensitivities to antheridiogen. Antheridiogen supplementation overcame inhibition by AMO-1618 and ancymidol, except in one strain (HaC18) that is insensitive to antheridiogen supplementation. These data suggest that the synthesis of Ceratopteris antheridiogen, a taxon that is insensitive to exogenously supplied gibberellins, occurs via a pathway that may include steps in common with gibberellin biosynthesis or involves similar reactions.     

Monoterpene formation by leucoplasts of Citrofortunella mitis and Citrus unshiu

Leucoplasts of immature calamondin and satsuma fruits were incubated with [1-¹⁴C] isopentenyl pyrophosphate under various conditions. Optimal incorporation of the tracer into geranyl pyrophosphate and monoterpene hydrocarbons occurred in the presence of exogenous dimethylallyl pyrophosphate and Mn²⁺ which was more effective than Mg²⁺. The dependence of dimethylallyl pyrophosphate showed that about 10 moles were required for 1 mole of isopentenyl pyrophosphate for the best recovery in monoterpene hydrocarbon biosynthesis. A time-course incorporation of isopentenyl pyrophosphate revealed that the C₁₀ hydrocarbon elaboration was dependent on the geranyl pyrophosphate production and at no time neryl pyrophosphate was synthesized by leucoplasts. The amount of labelled farnesyl pyrophosphate was rather low whatever the conditions used in the experiments and sesquiterpene hydrocarbon biosynthesis was never observed.Abbreviations DMAPP dimethylallyl pyrophosphate - FPP farnesyl pyrophosphate - GPP geranyl pyrophosphate - IPP isopentenyl pyrophosphate - LPP linalyl pyrophosphate - NPP neryl pyrophosphate     

Biosynthesis of abscisic acid from farnesol derivatives in Cercospora rosicola

(E,E)?[1?¹⁴C]Farnesyl phosphate and (E,E)?[1?¹⁴C]farnesyl pyrophosphate were both converted to abscisic acid by Cercospora rosicola resuspensions. (E,E)?[1?¹⁴C]Farnesol, (E,Z)?[1?¹⁴C]farnesol, (E,Z)?[1?¹⁴C]farnesyl pyrophosphate, (E,E)?[1?¹⁴C]farnesic acid, and (E,Z)?[1?¹⁴C]farnesic acid were not converted to abscisic acid by the fungus. These findings provide information on the sequence of the reactions involved in converting farnesyl pyrophosphate to abscisic acid. Specifically, they suggest that the transformations involving the three terminal carbons in the side chain occur after one or more changes elsewhere in the molecule.     

Inhibition of abscisic acid biosynthesis inCercospora rosicola by triarimol

The fungicide triarimol was tested for its effect on abscisic acid (ABA) accumulation in growing culturesof Cercospora rosicola. ABA accumulation was reduced by approximately 50% with 10^?8 M triarimol. Growth ofC. rosicola, as measured by dry weight accumulation, was inhibited by triarimol concentrations at or greater than 10^?7 M. These results are compared with those obtained with clomazone, ancymidol, and paclobutrazol, which inhibit ABA accumulation by 50% at concentrations of 5 × 10^?5, 5 × 10^?6, and 5 × 10^?7 M, respectively. Triarimol, therefore, is among the most potent inhibitors of ABA biosynthesis reported to date. Feeding studies with [¹⁴C]mevalonic acid confirmed the inhibition of ABA biosynthesis by 5 × 10^?8 M triarimol. These results support previous suggestions that one or more of the steps in the ABA biosynthetic pathway from mevalonic acid is catalyzed by cytochrome P-450. Feeding studies with 1′-deoxy-[²H]-ABA in resuspended cultures ofC. rosicola show that the conversion of this substrate is not inhibited by triarimol.     

Stereochemistry of the biosynthesis of presqualene alcohol

Current hypotheses of the biosynthesis of presqualene pyrophosphate were tested by the examination of presqualene alcohol biosynthesized from [1R,5R,9R-1,5,9-D₃]farnesyl pyrophosphate and from [1-¹⁸O]farnesyl pyrophosphate. Nuclear magnetic resonance spectrometry showed that the octet of the two cyclopropylcarbinyl protons seen in the spectrum of protio-presqualene alcohol, centered at τ 6.35, was replaced by a broad doublet of one proton (τ, 6.23; J, 6.2 Hz), which became sharpened after deuterium decoupling and was reduced to a singlet after deuterium and proton decoupling. Also the doublet of a single olefinic proton adjacent to the cyclopropane ring, seen in the spectrum of protio-presqualene alcohol at τ 5.08 (J, 8.5 Hz), was reduced to a broad singlet. The presqualene alcohol biosynthesized from the [1-¹⁸O]farnesyl pyrophosphate contained the same isotopic concentration as its precursor. The observations, taken together with previous results, are interpreted to mean that the pyrophosphate-bearing group of one farnesyl pyrophosphate molecule appears without chhnge of configuration, and without previous cleavage of the CO bond of farnesyl pyrophosphate, in presqualene pyrophosphate and that the pro-R hydrogen atom at C-1 of the second farnesyl pyrophosphate molecule appears at C-3 of the cyclopropane ring anti to the vinylic substituent. The observations support the view that presqualene pyrophosphate is not an artifact, but a true intermediate in the biosynthesis of squalene.     

Mechanism of cis-prenyltransferase reaction probed by substrate analogues

Undecaprenyl pyrophosphate synthase (UPPS) is a cis-type prenyltransferases which catalyzes condensation reactions of farnesyl diphosphate (FPP) with eight isopentenyl pyrophosphate (IPP) units to generate C₅₅ product. In this study, we used two analogues of FPP, 2-fluoro-FPP and [1,1-²H₂]FPP, to probe the reaction mechanism of Escherichia coli UPPS. The reaction rate of 2-fluoro-FPP with IPP under single-turnover condition is similar to that of FPP, consistent with the mechanism without forming a farnesyl carbocation intermediate. Moreover, the deuterium secondary KIE of 0.985 ± 0.022 measured for UPPS reaction using [1,1-²H₂]FPP supports the associative transition state. Unlike the sequential mechanism used by trans-prenyltransferases, our data demonstrate E. coli UPPS utilizes the concerted mechanism.     

Inhibition of abscisic acid biosynthesis inCercospora rosicola by triarimol

The fungicide triarimol was tested for its effect on abscisic acid (ABA) accumulation in growing culturesof Cercospora rosicola. ABA accumulation was reduced by approximately 50% with 10^–8 M triarimol. Growth ofC. rosicola, as measured by dry weight accumulation, was inhibited by triarimol concentrations at or greater than 10^–7 M. These results are compared with those obtained with clomazone, ancymidol, and paclobutrazol, which inhibit ABA accumulation by 50% at concentrations of 5 × 10^–5, 5 × 10^–6, and 5 × 10^–7 M, respectively. Triarimol, therefore, is among the most potent inhibitors of ABA biosynthesis reported to date. Feeding studies with [¹⁴C]mevalonic acid confirmed the inhibition of ABA biosynthesis by 5 × 10^–8 M triarimol. These results support previous suggestions that one or more of the steps in the ABA biosynthetic pathway from mevalonic acid is catalyzed by cytochrome P-450. Feeding studies with 1-deoxy-[²H]-ABA in resuspended cultures ofC. rosicola show that the conversion of this substrate is not inhibited by triarimol.     

Preparation, Characterization, and Optimization of an In Vitro C30 Carotenoid Pathway

The ispA gene encoding farnesyl pyrophosphate (FPP) synthase from Escherichia coli and the crtM gene encoding 4,4′-diapophytoene (DAP) synthase from Staphylococcus aureus were overexpressed and purified for use in vitro. Steady-state kinetics for FPP synthase and DAP synthase, individually and in sequence, were determined under optimized reaction conditions. For the two-step reaction, the DAP product was unstable in aqueous buffer; however, in situ extraction using an aqueous-organic two-phase system resulted in a 100% conversion of isopentenyl pyrophosphate and dimethylallyl pyrophosphate into DAP. This aqueous-organic two-phase system is the first demonstration of an in vitro carotenoid synthesis pathway performed with in situ extraction, which enables quantitative conversions. This approach, if extended to a wide range of isoprenoid-based pathways, could lead to the synthesis of novel carotenoids and their derivatives.     

Modeling studies with Helicobacter pylori octaprenyl pyrophosphate synthase reveal the enzymatic mechanism of trans-prenyltransferases

Octaprenyl pyrophosphate synthase (OPPs), an enzyme belonging to the trans-prenyltransferases family, is involved in the synthesis of C40 octaprenyl pyrophosphate (OPP) by reacting farnesyl pyrophosphate (FPP) with five isopentenyl pyrophosphates (IPP). It has been reported that OPPs is essential for bacteria's normal growth and is a potential target for novel antibacterial drug design. Here we report the crystal structure of OPPs from Helicobacter pylori, determined by MAD method at 2.8 Å resolution and refined to 2.0 Å resolution. The substrate IPP was docked into HpOPPs structure and residues involved in IPP recognition were identified. The other substrate FPP, the intermediate GGPP and a nitrogen-containing bisphosphonate drug were also modeled into the structure. The resulting model shed some lights on the enzymatic mechanism, including (1) residues Arg87, Lys36 and Arg39 are essential for IPP binding; (2) residues Lys162, Lys224 and Gln197 are involved in FPP binding; (3) the second DDXXD motif may involve in FPP binding by Mg²⁺ mediated interactions; (4) Leu127 is probably involved in product chain length determination in HpOPPs and (5) the intermediate products such as GGPP need a rearrange to occupy the binding site of FPP and then IPP is reloaded. Our results also indicate that the nitrogen-containing bisphosphonate drugs are potential inhibitors of FPPs and other trans-prenyltransferases aiming at blocking the binding of FPP.     

ent-Kaurene Biosynthesis in Germinating Barley (Hordeum vulgare L., cv Himalaya) Caryopses and Its Relation to alpha-Amylase Production

ent-Kaurene biosynthesis as a prerequisite for gibberellin (GA) biosynthesis was studied in germinating Hordeum vulgare L., cv Himalaya caryopses and correlated, in time, with the appearance of α-amylase activity. The rate of ent-kaurene biosynthesis was estimated by inhibiting its further metabolism with plant growth retardants (triapenthenol or tetcyclacis) and measuring its accumulation by isotope dilution using combined gas chromatographymass spectrometry. In the inhibitor-treated caryopses, ent-kaurene accumulation began approximately 24 hours after imbibition and proceeded at a rate of about 1 to 2 picomoles per hour per caryopsis, depending on the batch of seeds. In the absence of inhibitor, ent-kaurene did not accumulate, indicating that it is normally turned over rapidly, presumably to further intermediates of the GA biosynthesis pathway and eventually to GAs. ent-Kaurene accumulation occurred almost exclusively in the shoot, which is, therefore, probably the site of biosynthesis. α-Amylase production began between 30 and 36 hours after imbibition and, thus, correlated well with de novo GA biosynthesis, as estimated from ent-kaurene accumulation. However, inhibition of ent-kaurene oxidation by plant growth retardants did not reduce the α-amylase production significantly, although it did reduce shoot elongation. We conclude that ent-kaurene is produced in the shoot and is continuously converted to GA, which is essential for normal shoot elongation, but not for the production of α-amylase in the aleurone layer.     

Substrate specificity of α‐humulene synthase from Zingiber zerumbet Smith and determination of kinetic constants by a spectrophotometric assay

Terpene synthases are the key enzymes in terpene biosynthesis that provide a structurally complex and highly diverse product spectrum. A suitable and reliable analytical assay is indispensable to measure terpene synthase activity accurately and precisely. In this study, a malachite green assay (MG) was adapted to rapidly assay terpene synthase activity and was validated in comparison to an already established gas chromatography assay. A linear correlation between both assays was observed. Kinetic properties for the previously described sesquiterpene synthase α‐humulene synthase (HUM) from Zingiber zerumbet Smith were investigated for the bioconversion of the monoterpene precursors geranyl pyrophosphate (2E‐GPP) and neryl pyrophosphate (2Z‐NPP) as well as for the sesquiterpene precursor farnesyl pyrophosphate (2E,6E‐FPP). Also, gas chromatography mass spectrometry (GS‐MS) was carried out to identify the products of the bioconversion of (2E)‐GPP and (2Z)‐NPP.     

Presqualene alcohol,squalene, and sterol biosynthesis from bifarnesol

To cast light on the overall biosynthetic conversion of farnesol pyrophosphate to presqualene alcohol pyrophosphate (PSA), the biochemical precursor of squalene as well as all sterols, radiolabeled bifarnesol (1) was prepared and fed toGibberella fujikuroi. The diol (1), acting as a surrogate for a previously suggested phosphorylated version of1, was converted to radiolabeled presqualene alcohol and squalene, as well as various sterols, including lanosterol and24-β-methylcholesta-5,7,9(11),22-tetraen-3β-ol, previously isolated from the same fungus. The results are interpreted to imply that a phosphorylated version of1 may act as a bone fide intermediate in the biosynthesis of PSA, thereby rendering unlikely any type of concerted farnesyl/presqualene pyrophosphate change.     

Abscisic acid physiology and biosynthesis in higher plants

Abscisic acid (ABA) has been postulated to modulate several aspects of plant growth and development. While it is tempting to attribute changes in growth and development to a specific hormone such as ABA, the reality is that these processes are complex and poorly understood. Since there is so little known about basic biochemical events that occur during growth and development, it is difficult ot unambiguously assign a role for ABA in any process. Becuse of this, many of the cited effects of ABA on growth and development have not been conclusively demonstrated. Howver, it is clear that ABA has a function in ameliorating water-stress and preventing vivipary. The roles of ABA in bud dormancy and growth still remain unclear. With the use of biosynthesis inhibitors and mutants which block ABA accumulation, it has been shown that ABA does not play a role in gravitropism.
Knowledge of how the levels of any particular growth regulator are modulated is essential for the understanding of its physiology. The use of mutants, inhibitors and heavy isotopes suggests that ABA may be derived from a carotenoid rather than directly from farnesyl pyrophosphate (FPP), and that the cleavage of a carotenoid is the rate limiting step. However, the relative contribution of each pathway (and the role of xanthoxin) in ABA biosynthesis remain unknown.