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.     

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.     

Position of the metabolic block for gibberellin biosynthesis in mutant b1-41a of Gibberella fujikuroi

Mutant B1-41a, obtained by UV-irradiation of Gibberella fujikuroi strain GF-1a, does not metabolise mevalonic acid lactone (MVL), ent-kaur-16-ene, ent-kaurenol, and ent-kaurenal to gibberellins. ent-Kaur-16-ene-19-oic acid is completely metabolised to give the same gibberellins in similar concentration as unsupplemented cultures of the parent strain. It is concluded that this mutant is blocked for gibberellin synthesis at the step from ent-kaurenal to ent-kaurenoic acid. Comparison of the incorporation of MVL into GA₃ by the mutant and the parent strains indicate that the metabolic block is 97·5% effective. A method of preparing ent-kaur-16-ene, labelled at C-15 and C-17 by [²H] and [³H] is described.     

Inhibition of gibberellin biosynthesis by paclobutrazol in cell-free homogenates ofCucurbita maxima endosperm andMalus pumila embryos

The plant growth retardant paclobutrazol, (PP333) (2RS, 3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)pentan-3-ol, inhibits specifically the three steps in the oxidation of the gibberellin-precursorent-kaurene toent-kaurenoic acid in a cell-free system fromCucurbita maxima endosperm. The KI₅₀ for this inhibition is 2×10^–8 M. The KI₅₀ values for the separated2S, 3S, and2R, 3R enantiomers of paclobutrazol in this system are 2×10^–8 M and 7×10^–7 M, respectively. A cell-free preparation from immatureMalus pumila embryos convertsent-kaurene to gibberellin A₉, whereas no conversion occurs in a similar preparation fromMalus endosperm. The conversion ofent-kaurene by the embryo preparation is inhibited by paclobutrazol with KI₅₀ values for the2S,3S and2R,3R enantiomers of 2×10^–8 M and 6×10^–8 M, respectively.     

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 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.     

Comparison of Biological Activities of Gibberellins and Gibberellin-Precursors Native to Thlaspi arvense L

Field pennycress (Thlaspi arvense L.) is a winter annual weed with a cold requirement for stem elongation and flowering. The relative abilities of several native gibberellins (GAs) and GA-precursors to elicit stem growth were compared. Of the eight compounds tested, gibberellin A₁, (GA₁), GA₉, and GA₂₀ caused stem growth in noninduced (no cold treatment) plants. No stem growth was observed in plants treated with ent-kaurene, ent-kaurenol, ent-kaurenoic acid, GA₅₃, or GA₈. Moreover, of the biologically active compounds, GA₉ was the most active followed closely by GA₁. In thermoinduced plants (4-week cold treatment at 6°C) that were continuously treated with 2-chlorocholine chloride to reduce endogenous GA production, GA₉ was the most biologically active compound. However, the three kaurenoid GA precursors also promoted stem growth in thermoinduced plants, and were almost as active as GA₂₀. No such increase in activity was observed for either GA_[unk] or GA₅₃. The results are discussed in relation to thermoinductive regulation of GA metabolism and its significance to the initiation of stem growth in field pennycress. It is proposed that thermoinduction results in increased conversion of ent-kaurenoic acid to GAs through the C-13 desoxy pathway and that GA₉ is the endogenous mediator of thermoinduced stem growth in field pennycress.     

Gibberellin biosynthesis in a cell-free system from immature seeds of Pisum sativum

A cell-free system from immature pea seeds converts ¹⁴C-labelled $\text{ent}$-kaurene to $\text{ent}$-kaurenol, $\text{ent}$-kaurenal, $\text{ent}$-kaurenoic acid, $\text{ent}$-7α-hydroxykaurenoic acid, and gibberellin A₁₂-aldehyde. The latter becomes converted further to 13-hydroxygibberellin A₁₂, gibberellin A44, gibberellin A₁₂-alcohol, and several unidentified products. Thus the biosynthesis of gibberellins $\text{via ent}$-kaurene is now established for a member of the $\text{Leguminosae}$. It is the first time that 13-hydroxylation of gibberellins has been observed in a cell-free system and that gibberellin A₁₂-alcohol has been obtained in any biological system.     

Inhibition of gibberellin biosynthesis by paclobutrazol in cell-free homogenates ofCucurbita maxima endosperm andMalus pumila embryos

The plant growth retardant paclobutrazol, (PP333) (2RS, 3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)pentan-3-ol, inhibits specifically the three steps in the oxidation of the gibberellin-precursorent-kaurene toent-kaurenoic acid in a cell-free system fromCucurbita maxima endosperm. The KI₅₀ for this inhibition is 2×10^?8 M. The KI₅₀ values for the separated2S, 3S, and2R, 3R enantiomers of paclobutrazol in this system are 2×10^?8 M and 7×10^?7 M, respectively. A cell-free preparation from immatureMalus pumila embryos convertsent-kaurene to gibberellin A₉, whereas no conversion occurs in a similar preparation fromMalus endosperm. The conversion ofent-kaurene by the embryo preparation is inhibited by paclobutrazol with KI₅₀ values for the2S,3S and2R,3R enantiomers of 2×10^?8 M and 6×10^?8 M, respectively.     

Metabolism of ent-Kaurene to Gibberellin A(12)-Aldehyde in Young Shoots of Normal Maize

Young shoots of normal maize (Zea mays L.) were used to determine both the stepwise metabolism of ent-kaurene to gibberellin A₁₂-aldehyde and the endogenous presence of the members in this series. Each of the five steps in the sequence was established by feeds of 17-¹³C, ³H-labeled kauranoids to cubes from the cortex of elongating internodes, to homogenates from the cortex of elongating internodes, and/or to homogenates from dark-grown seedlings. The ¹³C-metabolites were identified by Kovats retention indices (KRI) and full-scan capillary gas chromatography-mass spectrometry (GC-MS). Five substrates and the final product in this sequence were shown to be native by the isotopic dilution of 17-¹³C, ³H-labeled substrates added as internal standards to extracts obtained from elongating internodes. Evidence for the isotopic dilution was obtained by KRI and full-scan capillary GC-MS. Thus, we document the presence in young maize shoots of the metabolic steps, ent-kaurene → ent-kaurenol → ent-kaurenal → ent-kaurenoic acid → ent-7 α-hydroxykaurenoic acid → gibberellin A₁₂-aldehyde.     

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)     

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.     

Blue-light irradiation up-regulates the ent-kaurene synthase gene and affects the avoidance response of protonemal growth in Physcomitrella patens

Main conclusion

We report a novel physiological response to blue light in the moss Physcomitrella patens . Blue light regulates ent -kaurene biosynthesis and avoidance response to protonemal growth.

Abstract

Gibberellins (GAs) are a group of diterpene-type plant hormones biosynthesized from ent-kaurenoic acid via ent-kaurene. While the moss Physcomitrella patens has part of the GA biosynthetic pathway, from geranylgeranyl diphosphate to ent-kaurenoic acid, no GA is found in this species. Caulonemal differentiation in a P. patens mutant with a disrupted bifunctional ent-copalyl diphosphate synthase/ent-kaurene synthase (PpCPS/KS) gene is suppressed under red light, and is recovered by application of ent-kaurene and ent-kaurenoic acid. This indicates that derivatives of ent-kaurenoic acid, not GAs, might act as endogenous developmental regulators. Here, we found unique responses in the protonemal growth of P. patens under unilateral blue light, and these regulators were involved in the responses. When protonemata of the wild type were incubated under blue light, the chloronemal filaments grew in the opposite direction to the light source. Although this avoidance was not observed in the ent-kaurene deficient mutant, chloronemal growth toward a blue-light source in the mutant was suppressed by application of ent-kaurenoic acid, and the growth was rescued to that in the wild type. Expression analysis of the PpCPS/KS gene showed that the mRNA level under blue light was rapidly increased and was five times higher than under red light. These results suggest that regulators derived from ent-kaurenoic acid are strongly involved not only in the growth regulation of caulonemal differentiation under red light, but also in the light avoidance response of chloronemal growth under blue light. In particular, growth under blue light is regulated via the PpCPS/KS gene.     

Metabolism of ent-kaurenol-[17-14C], ent-kaurenal-[17-14C] and ent-kaurenoic acid-[17-14C] by germinating Hordeum distichon grains

Subcellular fractions from germinated barley embryos, chloroplast preparations and whole germinating barley grains are able to carry out the conversions ent-kaurenol → ent-kaurenal → ent-kaurenoic acid → ent-hydroxykaurenoic acid, the initial steps of the biosynthetic pathway to gibberellins. Whole grains, and chloroplasts to a slight extent, incorporate radioactivity from ent-kaurenol-[17-¹⁴C] and ent-kaurenoic acid-[17-¹⁴C] into materials with similar but distinct properties from the gibberellins GA₁, GA₃, GA₄ and GA₇.     

Effects of different sterols on the inhibition of cell culture growth caused by the growth retardant tetcyclacis

Cell division in cell suspension cultures can be completely blocked by the growth retardant tetcyclacis at a concentration of 10^-4 mol l^-1. In rice cells it has been demonstrated that the growth inhibition can be completely overcome by application of cholesterol independent of the duration of pretreatment with tetcyclacis. In suspension cultures of maize and soybean, too, the effect of tetcyclacis on cell division was neutralized by adding cholesterol. Other plant sterols, stigmasterol, campesterol and sitosterol were active in a decreasing order. Modifications in the cholesterol perhydro-cyclopentanophenanthrene-ring system indicate that the hydroxyl group at C-3 and the double bond between C-5 and C-6 in ring B are required for the activity. In contrast, gibberellic acid as well as ent-kaurenoic acid could not compensate retardant effects. Likewise, tetcyclasis did not change the level of gibberellins in rice cells as shown by radioimmunoassay. Thus, it is concluded that in cell suspension cultures sterols play a more important role in cell division than gibberellins.Abbreviation GA_x gibberelin A_x     

The conversion of mevalonic acid into gibberellin A12-aldehyde in a cell-free system from Cucurbita pepo

Summary A cell-free system prepared from immature seed of Cucurbita pepo incorporates the label from mevalonate-2-¹⁴C into ent-kaur-16-en-19-oic acid (I), ent-7-hydroxy-kaur-16-en-19-oic acid (II), and ent-gibberell-16-en-7-al-19-oic acid (III) (gibberellin A₁₂-aldehyde). The products were identified by gas liquid chromatography and by combined gas chromatography-mass spectrometry of the methylated and trimethylsilylated fractions. The radioactivity of the compounds was established by recrystallisation to constant specific radioactivity. Gibberellin A₁₂ (IV), also detected in the system after incubation by combined gas chromatographymass spectrometry may be an artefact, derived from gibberellin A₁₂-aldehyde by a non-enzymatic conversion.With gibberellin A₁₂-aldehyde, the cell-free biosynthesis of an ent-gibberellane has been achieved for the first time.     

Inhibition of growth by ancymidol and tetcyclacis in the gibberellin-deficientdwarf-5 mutant ofZea mays L. and its prevention by exogenous gibberellin

Leaf sheath length and shoot dry matter of the gibberellin-deficientdwarf-5 mutant ofZea mays L. were further reduced by micromolar concentrations of two putative gibberellin biosynthesis inhibitors, ancymidol [-cyclopropyl--(p-methoxyphenyl)-5-pyrimidine methyl alcohol] and tetcyclacis [5-(4-chlorophenyl)-3,4,5,9,10-pentaazatetracyclo-5,4,1,0^2,6,0^8,11-dodeca-3,9-diene]. Growth retardant action was prevented by the subsequent application of gibberellin (GA₄₊₇). Plants treated with both gibberellin and growth retardants were identical in all outward respects to those treated with gibberellin alone. Although thedwarf-5 mutant is blocked in the synthesis ofent-kaurene and does not contain detectable quantities of gibberellin, the above results are consistent with the interpretation that biologically active levels of endogenous gibberellin are present in the dwarf which can be decreased by biosynthesis inhibitors.Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the United States Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.     

Metabolism of kaurenoids by Gibberella fujikuroi in the presence of the plant growth retardant,N,N,N-trimethyl-1-methyl-(2′,6′,6′-trimethylcyclohex-2′-en-1′-yl)prop-2-enylammonium iodide

The plant growth retardant, N,N,N-trimethyl-1-methyl-(2′,6′,6′-trimethylcyclohex-2′-en-1′-yl)prop-2-enylammonium iodide, is shown to block gibberellin biosynthesis in Gibberella fujikuroi between mevalonate and ent-kaur-16-ene, probably by inhibiting ent-kaur-16-ene synthetase A-activity. In the presence of the plant growth retardant, cultures of the fungus incorporate (26.5%) added ent-[¹⁴C]-kaur-16-ene into gibberellin A₃. Under the same conditions kaur-16-ene, 13β-kaur-16-ene, and ent-kaur-15-ene are not metabolised to gibberellin analogues.     

Reversing the inhibitory effect of paclobutrazol on seed germination of Amaranthus paniculatus by GA3, ethephon or ACC

The germination of Amaranthus paniculatus seeds was inhibited by applying paclobutrazol, a specific inhibitor of gibberellin biosynthesis. This inhibition was markedly counteracted by gibberellin A₃ (GA₃), suggesting that endogenous gibberellins are required for germination in this species. The inhibitory effect of paclobutrazol was also overcome by ethephon (2-chloroethylphosphonic acid) or the precursor of ethylene biosynthesis, ACC (1-aminocyclopropane-l-carboxylic acid). Thus the physiological effect of gibberellin can be mimicked by ethylene released from ethephon or synthesised from exogenous ACC. It is suggested, that endogenous gibberellins are involved in germination of Amaranthus paniculatus seeds and that action of GA₃ can be substituted by ethylene.Abbreviations ACC 1-aminocyclopropane-l-carboxylic acid - AMO-1618 (2-isopropyl-5methyl-4-trimethylammoniumchloride)-phenyl-l-piperidinium-carboxylate - ancymidol -cyclopropyl--(4-methoxyphenyl)-5-pyrimidine methanol - chloromequat chloride (2-chloroethyl)trimethylammoniumchloride - ethephon 2-chloroethylphosphonic acid - GA gibberellin A₃ - paclobutrazol (2RS, 3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-lyl)pentan-3-ol - Phosphon D 2,4,dichlorobenzyl-tributhylphosphoniumchloride - tetcyclacis 5,(4-chlorophenyl)-3,4,5,9,10-pentaaza-tetracyclo)5,4,1,0,Z,6,0^8,11 dodeca-3,9-diene     

Metabolism of Steviol and Its Derivatives by Gibberella fujikuroi

Steviol (ent-13-hydroxykaur-16-en-19-oic acid)* is metabolized by Gibberella fujikuroi in the presence of inhibitors of gibberellin biosynthesis, such as quaternary ammonium salt-type growth retardants, to afford 7β-Miydroxy- and 6β,7β-dihydroxysteviol, gibberelhns A₁, A₁₈, A₁₉, A₅₃ and 7β,13-dihydroxykaurenolide. Steviol acetate (ent-13-acetoxykaur-16-en-19-oic acid) is also metabolized to the 6β,7β-dihydroxy-derivative and to the 13-acetyl derivatives of gibberellins A₁₇ and A₂₀ and steviol methyl ester (methyl ent-13-hydroxykaur-16-en-19-oate) into the monohydroxy-, dihydroxy- and hydroxyoxo-derivatives. These results indicate a low substrate specificity of the enzymes in the fungus and provide a useful preparative methodology of several important plant gibberellins carrying the 13-hydroxyl group.