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.     

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.     

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.     

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.     

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)     

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.

     

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.     

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)     

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.     

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.     

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.     

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.     

Biosynthesis of gibberellins A12, A15, A24, A36, and A37 by a cell-free system from Cucurbita maxima

GA₁₂-aldehyde obtained from mevalonate via ent-kaurene, ent-kaurenol, ent-kaurenoic acid and ent-7α-hydroxykaurenoic acid in a cell-free system from immature seeds of Cucurbita maxima was converted to GA₁₂ by the same system. When Mn²⁺ was omitted from the system GA₁₂-aldehyde and GA₁₂ were converted further to several products. Among these GA₁₅, GA₂₄, GA₃₆ and GA₃₇ were conclusively identified by GC-MS. With the exception of GA₃₇ these GAs have not previously been found in higher plants. Another biosynthetic pathway led from ent-7α-hydroxykaurenoic acid to very polar products via what was tentatively identified as ent-6α, 7α-dihydroxykaurenoic acid. An unidentified component with an MS resembling that of a dihydroxykaurenolide was also obtained from incubations with mevalonate.     

Deletions in the Gibberellin Biosynthesis Gene Cluster of Gibberella fujikuroi by Restriction Enzyme-Mediated Integration and Conventional Transformation-Mediated Mutagenesis

We induced mutants of Gibberella fujikuroi deficient in gibberellin (GA) biosynthesis by transformation-mediated mutagenesis with the vector pAN7-1. We recovered 24 GA-defective mutants in one of nine transformation experiments performed without the addition of a restriction enzyme. Each mutant had a similar Southern blot pattern, suggesting the integration of the vector into the same site. The addition of a restriction enzyme by restriction enzyme-mediated integration (REMI) significantly increased the transformation rate and the rate of single-copy integration events. Of 1,600 REMI transformants, two produced no GAs. Both mutants had multiple copies of the vector pAN7-1 and one had a Southern blot pattern similar to those of the 24 conventionally transformed GA-deficient mutants. Biochemical analysis of the two REMI mutants confirmed that they cannot produce ent-kaurene, the first specific intermediate of the GA pathway. Feeding the radioactively labelled precursors ent-kaurene and GA₁₂-aldehyde followed by high-performance liquid chromatography and gas chromatography-mass spectrometry analysis showed that neither of these intermediates was converted to GAs in the mutants. Southern blot analysis and pulsed-field gel electrophoresis of the transformants using the bifunctional ent-copalyl diphosphate/ent-kaurene synthase gene (cps/ks) and the flanking regions as probes revealed a large deletion in the GA-deficient REMI transformants and in the GA-deficient transformants obtained by conventional insertional transformation. We conclude that transformation procedures with and without the addition of restriction enzymes can lead to insertion-mediated mutations and to deletions and chromosome translocations.     

Induction ofent-kaurene biosynthesis by low temperature in dwarf peas

Germinating pea (Pisum sativum L.) seeds of two dwarf cultivars, “Progress No. 9” and “Green Arrow”, and two tall cultivars, “Alaska” and “Alderman”, were treated with low temperature (3–5°C) for 14 days and then transferred to normal growing conditions (19–21°C for 16 h/14.5–16.5°C for 8 h) for an additional 10 days. Biosynthesis of [¹⁴C]ent-kaurene from [¹⁴C]2-mevalonic acid (2-MVA) was assayed in cell-free enzyme extracts prepared from shoot tips 10 days after cold treatment and was compared with activity in enzyme extracts prepared from noncold-treated, 10-day-old control plants. Shoot lengths of cold-treated plants were measured throughout a 35-day period and compared with shoot lengths of plants grown without cold treatment for 25–35 days. Low temperature induced a five-to 10-fold enhancement ofent-kaurene, hence potentially gibberellin (GA), biosynthesis in seedlings of the two dwarf cultivars but not in the tall cultivars. However, the lack of an increase in growth rate in the cold-treated dwarfs indicated that endogenous GA biosynthesis remained blocked at some point beyondent-kaurene in the biosynthetic pathway. Since the late-flowering “Alderman” cultivar did not exhibit enhanced biosynthesis ofent-kaurene, it appears that if vernalization in late-flowering cultivars of peas is correlated with enhanced GA biosynthesis, it is not the early part of the biosynthetic pathway which is affected.     

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.     

Biosynthesis of ent-kauranes: Evidence for a C-17, C-16 hydrogen 1,2-shift

The incorporation of ent-kaurenoic acid into the derived hydroxy acid and dihydroxy acid by Beyeria calycina has been studied. The biosynthesis of the hydroxy acid involves a hydrogen 1,2-shift from the C-17 position of ent-kaurenoic acid.     

Blue Light Inhibition of Tuberization in a Day-Neutral Potato

In tests on the effects of light quality on potato tuberization, continuous blue light was found to consistently inhibit tuberization of tissue-cultured plantlets of Solanum tuberosum ssp. tuberosum cv. ??Norland??. Other tested cultivars, including sports of ??Norland??, formed tubers under continuous blue light. Microarrays identified BL, GA7ox, and Nudix genes as exhibiting altered expression in response to blue light treatment. Quantitative RT-PCR (qRT-PCR) showed that GA7ox RNA increased in ??Norland?? but not in ??Sangre?? plantlets in blue light compared to darkness. RNA levels of genes identified in the literature as having roles in potato tuberization were also measured using qRT-PCR. Levels of GA20o1x, but not GA2ox, RNA increased in response to blue light in ??Norland?? plantlets. BEL5 RNA content was greater under blue light compared to darkness for both ??Norland?? and ??Sangre?? plants. Levels of FT were not significantly different in blue light compared to dark-treated ??Norland?? plants, but were low in blue light-treated compared to dark-treated ??Sangre?? plants. Addition of ancymidol to ??Norland?? plants exposed to blue light overcame blue light inhibition of tuberization. Ancymidol prevents the oxidation of ent-kaurene to ent-kaurenoic acid, thus inhibiting gibberellin biosynthesis. These data suggest that blue light may increase GA accumulation in ??Norland?? plants, as has been shown to occur in Arabidopsis plants. The novel effect of blue light in inhibiting tuberization of ??Norland?? plants suggests that this system could be a useful tool in further elucidating the mechanisms of day-neutral potato tuberization.     

The biosynthesis of the gibberellin precursorent-kaurene in cell-free extracts and the endogenous gibberellins of Japanese morning glory in relation to seed development

The endogenous levels of gibberellins (GAs) determined by a combined HPLC-bioassay procedure and the formation ofent-kaurene, an immediate GA precursor, in cell-free extracts were studied in relation to seed development inPharbitis nil Choisy cv. Violet. Three biologically active GA fractions were obtained, tentatively identified as GA₃, GA₅/ GA₂₀, and a GA fraction, possibly GA₁₉ and/or GA₄₄, which all increased in activity during early seed development and subsequently declined during maturation of the seeds. The total endogenous GA level reached its maximum at 19 days after anthesis, just before the seeds had attained their maximum fresh weight at about 23 days after anthesis. Similarly, theent-kaurene synthesizing capacity showed a rapid increase during the period of rapid growth of the seeds, followed by a decline during maturation. A direct relationship between the endogenous GA levels and theent-kaurene synthesizing capacity of a particular tissue was indicated.