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.     

ent-Kaurene Biosynthesis in Cell-Free Extracts of Excised Parts of Tall and Dwarf Pea Seedlings

Investigations on the sites of ent-kaur-16-ene (ent-kaurene) biosynthesis were conducted with cell-free extracts from several excised parts of 10-, 13-, and 16-d-old tall and dwarf pea (Pisum sativum L.) seedlings. [¹⁴C]Mevalonic acid was incorporated into ent-kaurene in cell-free extracts from young developing leaves and elongating internodes of tall (`Alaska') and dwarf (`Progress No.9') pea seedlings at all three stages of development. ent-Kaurene biosynthesis also occurred readily in cell-free extracts from shoot tips, petioles, and stipules near the young elongating internodes. The ent-kaurene-synthesizing activity found in young developing tissues declined as tissues matured. Little or no activity was detectable in enzyme extracts from cotyledons and root tips at different stages. In light grown tall pea internodes ent-kaurene-synthesizing activity was low as they began to elongate, reached a maximum when the internodes reached about 2 cm in length and declined as they matured. Activity in extracts of dwarf shoot tips and internodes was generally lower than in equivalent tall plants, but the activity in dwarf leaves and stipules was somewhat higher than in tall plants. With the exception of root tips, there is a strong correlation between growth potential of a tissue and the rate of ent-kaurene biosynthesis in extracts from that tissue.     

Concentrations ofent-kaurene and squalene in vegetative rice shoots

Ent-kaurene (ent-kaur-16-ene) and squalene were analyzed in extracts of the shoots of three cultivars of rice (Oryza sativa L.) of 14 and 28 days of age by gas chromatography-mass spectrometry (GS-MS) and GC-selected ion monitoring (GS-SIM).Ent-kaurene occurred at approximate concentrations of <1 to 13 ng/g f.w. in 14-day-old plants and 26 to 147 ng/g f.w. in 28-day-old plants. Shoots of the dwarf cultivar Waito-C contained much lessent-kaurene than the other two cultivars at both developmental stages. The level ofent-kaurene in the dwarf cultivar Tan-ginbozu was similar to that in the normal cultivar Nihonbare at 14 days but was lower than in Nihonbare at 28 days. Trace amounts ofent-isokaurene (ent-kaur-15-ene) were also found in the extracts of all three cultivars of shoots at 28 days. Squalene occurred in approximate concentrations from as low as 19 ng/g f.w. in 28-day-old Waito-C shoots to as much as 626 ng/g f.w. in 14-day-old Nihonbare shoots. Both Tan-ginbozu and Waito-C shoots contained less squalene than Nihonbare shoots at both developmental stages.     

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.     

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.     

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.     

Apparent endogenous circadian rhythm inent-kaurene biosynthesis

Net synthesis of [¹⁴C]ent-kaurene from [¹⁴C]2-mevalonic acid was assayed in cell-free enzyme extracts prepared from Alaska pea (Pisum sativum L.) seedlings throughout 44 h of a regimen consisting of a 16-h day and an 8-h night. Activities generally followed an upward trend during the dark period and a downward trend during the photoperiod. Activity was also assayed in enzyme extracts prepared at intervals during a 12-h photoperiod and a following, continuous 36-h dark period after entrainment of plants to a regimen of 12-h days and 12-h nights.Ent-kaurene synthesis activity again followed an upward trend in enzyme extracts prepared during what would have been the entrainment dark period, and a downward trend during the entrainment photoperiod. The apparent endogenous rhythm ofent-kaurene biosynthesis may have implications for the regulation 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.     

Inhibition ofEnt-kaurene oxidation by cytokinins

Cytokinins, which have some structural similarities to ancymidol, a plant growth retardant, were tested for their effects on the cell-free oxidation ofent-kaurene. Results indicate that several cytokinins inhibit this reaction in microsomal extracts of liquid endosperm from immature wild cucumber seeds. N⁶-cyclohexanemethyladenine was the most active (inhibiting 50% of the controlent-kaurene oxidation at 2×10^?6 M). N⁶-isoamyladenine, N⁶-benzyladenine, N⁶-(Δ²-isopentenyl)adenine and dihydrozeatin were active at successively higher concentrations. Zeatin, kinetin, adenine, N⁶-benzyladenosine, and N⁶-(isopentenyl)adenosine were inactive in this system. The basis for the inhibition ofent-kaurene oxidation by cytokinins may be similar to that of ancymidol: interaction with cytochrome P-450. A binding spectrum similar to that of ancymidol with cytochrome P-450 from wild cucumber endosperm microsomes was obtained with four active cytokinins. The cytokinin binding properties of this protein are currently under investigation. No metabolism of N⁶-benzyladenine could be detected under conditions in which the cytokinin inhibited the oxidation ofent-kaurene toent-kaurenol.     

Apparent endogenous circadian rhythm inent-kaurene biosynthesis

Net synthesis of [¹⁴C]ent-kaurene from [¹⁴C]2-mevalonic acid was assayed in cell-free enzyme extracts prepared from Alaska pea (Pisum sativum L.) seedlings throughout 44 h of a regimen consisting of a 16-h day and an 8-h night. Activities generally followed an upward trend during the dark period and a downward trend during the photoperiod. Activity was also assayed in enzyme extracts prepared at intervals during a 12-h photoperiod and a following, continuous 36-h dark period after entrainment of plants to a regimen of 12-h days and 12-h nights.Ent-kaurene synthesis activity again followed an upward trend in enzyme extracts prepared during what would have been the entrainment dark period, and a downward trend during the entrainment photoperiod. The apparent endogenous rhythm ofent-kaurene biosynthesis may have implications for the regulation of gibberellin biosynthesis.     

Gibberellin biosynthesis in bacteria: Separate ent-copalyl diphosphate and ent-kaurene synthases in Bradyrhizobium japonicum

Gibberellins are ent-kaurene-derived diterpenoid phytohormones produced by plants, fungi, and bacteria. The distinct gibberellin biosynthetic pathways in plants and fungi are known, but not that in bacteria. Plants typically use two diterpene synthases to form ent-kaurene, while fungi use only a single bifunctional diterpene synthase. We demonstrate here that Bradyrhizobium japonicum encodes separate ent-copalyl diphosphate and ent-kaurene synthases. These are found in an operon whose enzymatic composition indicates that gibberellin biosynthesis in bacteria represents a third independently assembled pathway relative to plants and fungi. Nevertheless, sequence comparisons also suggest potential homology between diterpene synthases from bacteria, plants, and fungi.     

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.     

Separation of Light-Induced [C]ent-Kaurene Metabolism and Light-Induced Germination in Grand Rapids Lettuce Seeds

The effect of light on the metabolism of [¹⁴C]kaurene in light-requiring lettuce seeds (Lactuca sativa L. cv Grand Rapids) was investigated. Seeds were soaked in a solution of [¹⁴C]ent-kaurene in methylene chloride with 0.01% Tween-20, dried, and incubated in 20% polyethylene glycol (PEG) to prevent seedling development. Labeled metabolites were extracted and analyzed by high performance liquid chromatography and gas chromatography-radio counting. [¹⁴C]ent-Kaurenol and [¹⁴C]ent-kaurenal were identified in seeds incubated in constant white light, while no ethyl acetate-soluble metabolites were found in seeds incubated in the dark. In time course experiments using acid scarified seeds, metabolism began after 18 hours of incubation and greatly increased after 24 hours of incubation in 20% PEG. By 48 hours, several unidentified, more polar metabolites were found. Germination was induced in seeds imbibed in 20% PEG by 4 hours of red or 4 hours of white light following 20 hours in the dark, and was fully reversed by 2 hours of far red light. However, in metabolism experiments, [¹⁴C]ent-kaurene oxidation was observed only with constant white light. These results indicate that although ent-kaurene oxidation is a light sensitive step in the biosynthesis of gibberellins in Grand Rapids lettuce seeds, ent-kaurene metabolism is not required for light-induced germination.     

Sites of gibberellin biosynthesis in pea seedlings

Potential sites of gibberellin biosynthesis in 10-day-old `Alaska' pea (Pisum sativum L.) seedlings were investigated using a cell-free ezyme system capable of incorporating [¹⁴C]-mevalonic acid into ent-kaurene. In peas, ent-kaurene is assumed to be a committed intermediate in the gibberellin biosynthetic pathway. Comparative results from enzyme assays using extracts from shoot tips, leaf blades, internodes, and root tips indicate that the highest capacity for ent-kaurene (and presumably gibberellin) synthesis is in those tissues with the greatest potential for growth. The highest rates were obtained with extracts prepared from the fifth (youngest) internode, the fourth (youngest) expanded leaf, and the shoot tip itself. This report represents the first direct evidence that the enzymes responsible for early stages in gibberellin biosynthesis occur in internode tissues with potential for rapid elongation.     

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 ofEnt-kaurene synthetase A and B activities in cell-free extracts from young tomato fruits of wild-type andgib-1, gib-2, andgib-3 tomato plants

The nonallelicgib-1 andgib-3 tomato (Lycopersion esculentum Mill.) mutants are gibberellin deficient and exhibit a dwarfed growth habit. Previous work has shown that this dwarfed growth pattern can be reversed by the application of a number of gibberellins and their precursors, includingent-kaurene (ent-kaur-16-ene). This indicates that they are blocked in gibberellin biosynthesis at a step prior toent-kaurene metabolism. The normal accumulation of carotenoids observed in these mutants suggests a functionally normal isoprenoid pathway.Ent-kaurene is synthesized from geranylgeranyl pyrophosphate in a two-step process with copalyl pyrophosphate as an intermediate.In vitro assays using young fruit extracts from wild-type andgib-2 plants resulted in the conversion of geranylgeranyl pyrophosphate to copalyl pyrophosphate, and the conversion of copalyl pyrophosphate toentkaurene. Similar assays usinggib-1 plants indicated a reduced ability for synthesis of copalyl pyrophosphate from geranylgeranyl pyrophosphate, and thus a reducedent-kaurene synthetase A activity. Furthermore,gib-3 extracts demonstrated a reduced ability to synthesizeent-kaurene from copalyl pyrophosphate, and thus a reducedent-kaurene synthetase B activity. These results establish the enzymatic conversion of geranylgeranyl pyrophosphate to copalyl pyrophosphate, and copalyl pyrophosphate toent-kaurene, as the sites of the mutations ingib-1 andgib-3 tomatoes, respectively. We also note that tomato fruit extracts contain components which are inhibitory toent-kaurene synthesis.     

Biosynthesis of ent-kaurene in cell-free extracts of Pisum sativum shoot tips

Soluble enzyme preparations from pea shoot tips incorporated mevalonic acid-2-¹⁴C into ent-kaurene-¹⁴C, squalene-¹⁴C and other products. The assay for either ent-kaurene or squalene is quite direct; both products can be obtained apparently free of radioactive contaminants by TLC on silica gel G in hexane. The enzyme system is dependent upon added ATP and Mn²⁺ or Mg²⁺, with Mn²⁺ being a more effective activator than Mg²⁺ under the experimental conditions. Reduced pyridine nucleotide had no effect on ent-kaurene production but stimulated squalene synthesis. The accumulation of both ent-kaurene and squalene was stimulated by dithiothreitol and carbon monoxide and was reduced by the addition of particulate cell components. AMO-1618 inhibited ent-kaurene production and had no effect on the synthesis of squalene. Enzyme extracts from shoot tips are much less active in ent-kaurene synthesis than extracts from the cotyledons of immature seeds on either a fresh weight or protein basis.     

Conversion of geranylgeranyl pyrophosphate to ent-kaurene in enzyme extracts of sonicated chloroplasts

Farnesyl pyrophosphate-[¹⁴C] and geranylgeranyl pyrophosphate-[¹⁴C] were biosynthesized from mevalonic acid-[2-¹⁴C] by cell-free enzyme extracts of pea (Pisum sativum) cotyledons containing MgCl₂, MnCl₂, ATP and AMO-1618. Maximum yields of farnesyl pyrophosphate were obtained after 30 min incubation while geranylgeranyl pyrophosphate was the primary product after 180 min. Biosynthesized geranylgeranyl pyrophosphate-[¹⁴C] served as an efficient substrate for ent-kaurene biosynthesis in reaction mixtures containing cotyledon enzymes when AMO-1618 was omitted. Enzyme extracts from green pea shoot tips and chloroplasts also converted geranylgeranyl pyrophosphate to ent-kaurene in very low yields. Ent-kaurene production from mevalonic acid-[2-¹⁴C] in extracts of pea shoot tips was also enhanced by addition of chloroplast enzymes. This evidence indicates that kaurene synthetase is present in pea chloroplasts and adds to the possibility that some gibberellin biosynthesis may be compartmentalized in those organelles.     

Gibberellin precursor is involved in spore germination in the moss Physcomitrella patens

Gibberellins are ent-kaurene derived phytohormones that are involved in seed germination, stem elongation, and flower induction in seed plants, as well as in antheridia formation and spore germination in ferns. Although ubiquitous in vascular plants, the occurrence and potential function(s) of gibberellins in bryophytes have not yet been resolved. To determine the potential role of gibberellin and/or gibberellin-like compounds in mosses, the effect of AMO-1618 on spores of Physcomitrella patens (Hedw.) B.S.G. was tested. AMO-1618, which inhibited ent-kaurene and gibberellin biosynthesis in angiosperms, also inhibited the bifunctional copalyl diphosphate synthase (E.C. 5.5.1.13)/ent-kaurene synthase (E.C. 4.2.3.19) of P. patens. AMO-1618 also caused a decrease in spore germination rates of P. patens, and this inhibitory effect was less pronounced in the presence of ent-kaurene. These results suggest that ent-kaurene biosynthesis is required by P. patens spores to germinate, implying the presence of gibberellin-like phytohormones in mosses.     

Ent-kaurene,occurrence and metabolism in Hordeum distichon

Barley grains contain hydrocarbons, including a material indistinguishable from ent-kaurene by GLC, and which after appropriate chemical conversions contain material behaving like ent-kauran-16,17-diol, ent-kaurene norketone and ent-17-nor-kaurane on TLC and GLC. The presence of ent-kaurene was confirmed by conversion to ent-kauran-16-ol and, following formation of acetate-[³H], recrystallization to constant specific activity with unlabelled carrier. In the initial ca. 15 hr of germination, preceding the rise in endogenous gibberellins, the level of ent-kaurene falls. Exogenous ent-kaurene-[¹⁴C] was not metabolized by intact barley grains. ent-Kauran-16,17-epoxide was formed non-enzymically by boiled extracts. Unboiled homogenates also formed ent-kauran-17-ol and ent-kauran-16,17-diol. The diol appeared to be formed from the epoxide, but the ent-kauran-17-ol was not. No recognized gibberellin precursors were detected. Nevertheless, endogenous ent-kaurene may be the stored biosynthetic precursor of gibberellins in germinating barley grains.