Purification and Properties of ent-Kaurene Synthase B from Immature Seeds of Pumpkin.

ent-Kaurene synthase B (KSB) was purified 291-fold from a crude enzyme preparation from endosperm of pumpkin (Cucurbita maxima L.). Separation of ent-kaurene synthase A and KSB was achieved by hydrophobic interaction chromatography. The fractions containing KSB activity were further purified by diethylaminoethyl, phenyl, and hydroxyapatite column chromatography. Using sodium dodecyl phosphate-polyacrylamide gel electrophoresis, the purest enzyme preparation showed a major band at an apparent molecular mass of 81 kD. The amount of protein in this band was correlated with KSB activity after diethylaminoethyl and hydroxyapatite chromatography. The N terminus of the 81-kD protein was blocked. Therefore, the protein was partially digested with protease and the amino acid sequences of the resulting major peptide fragments were analyzed. A polyclonal antibody was raised against a synthetic peptide based on the longest peptide fragment combined with a keyhole limpet hemocyanin. The antibody recognized only the 81-kD denatured protein and not the native KSB. The properties of KSB were examined using the phenyl-purified enzyme preparation. The Km value for copalyl pyrophosphate was 0.35 [mu]M, and the optimal pH was 6.8 to 7.5. The KSB activity required divalent cations such as Mg2+, Mn2+, and Co2+, whereas Cu2+, Ca2+, and Ba2+ inhibited the activity.

Identification of Antheridiogens in Lygodium circinnatum and Lygodium flexuosum.

Antheridiogens in two species of Schizaeaceous ferns, Lygodium circinnatum and Lygodium flexuosum, were analyzed by gas chromatography-mass spectrometry. In L. circinnatum, gibberellin A73 (GA73) methyl ester (GA73-Me), which had originally been identified in L. japonicum, was identified as a principal antheridiogen, and the methyl esters of five known GAs (GA9, GA20, GA70, GA88, and 3-epi-GA88) were also identified as minor antheridiogens. In addition, four compounds corresponding to isomers of monohydroxy-GA73-Me were detected. One of these was shown to be 12[beta]-hydroxy-GA73-Me, the parent acid of which has been allocated the GA assignment GA96. The other three compounds, tentatively named X1, X2, and X3, have not been fully characterized. In L. flexuosum, GA73-Me was also identified as a major antheridiogen, with X2 being detected as a minor one. The total antheridium-formation activity in the culture medium of 7-week-old prothallia of L. circinnatum and L. flexuosum was more than 1000 times higher than that of L. japonicum. On the other hand, the response of gametophytes of the former two Lygodium ferns to GA73-Me was more than 100 times lower than that of L. japonicum.

Biosynthesis of GA73 Methyl Ester in Lygodium Ferns.

Biosynthesis of GA73 methyl ester (GA73-Me), the principal antheridiogen in Lygodium ferns, was investigated. From the methanol extract of prothallia of Lygodium circinnatum, GA25, GA73, GA73-Me, GA88-Me, and a few unknown GA73 derivatives were detected by GC-MS. Because the presence of GA25 suggests that GA24, a direct precursor of GA25, could also be present in L. circinnatum prothallia, we used feeding experiments to investigate the possibility that GA24 is a precursor of GA73-Me. In L. circinnatum prothallia, [2H2]GA24 was converted into [2H2]GA73-Me and a trace amount of [2H2]GA9-Me, whereas [2H3]GA9 was converted into [2H3]GA9-Me and [2H3]monohydroxy-GA9-Me. Because GA73-Me, GA9-Me, and their monohydroxy derivatives had been identified by GC-MS from the culture medium of L. circinnatum prothallia, our results suggest that GA73-Me is biosynthesized from GA24 via GA73, and that neither GA9 nor GA9-Me is a precursor of GA73-Me. Though the possibility had been suggested that GA73-Me is biosynthesized from 9,15-cyclo-GA9 (GA103), [2H2]GA103 was not converted into [2H2]GA73-Me.

Involvement of Jasmonic Acid in Elicitor-Induced Phytoalexin Production in Suspension-Cultured Rice Cells.

It has been suggested that jasmonic acid (JA) could be an integral part of a general signal transduction system regulating inducible defense genes in plants. It was reported that treatment with an elicitor (N-acetylchitoheptaose) induced production of phytoalexin in suspension-cultured rice (Oryza sativa L.) cells. In this study, the role of JA in the induction of phytoalexin production by N-acetylchitoheptaose was investigated. Exogenously applied ([plus or minus])-JA (10-4 M) clearly induced the production of momilactone A, a major phytoalexin, in suspension-cultured rice cells. On the other hand, in rice cells treated with N-acetylchitoheptaose, endogenous JA was rapidly and transiently accumulated prior to accumulation of momilactone A. Treatment with ibuprofen, an inhibitor of JA biosynthesis, reduced production of momilactone A in the cells treated with N-acetylchitoheptaose, but the addition of ([plus or minus])-JA increased production of momilactone A to levels higher than those in the elicited rice cells. These results strongly suggest that JA functions as a signal transducer in the induction of biosynthesis of momilactone A by N-acetylchitoheptaose in suspension-cultured rice cells.

Isolation and initial characterization of 13(2)-hydroxychlorophyll a induced by cyclohexanedione derivatives in tobacco cell suspension cultures.

This paper reports that a new photobleaching compound, 2-(2-chloro-5-propoxycarbonylphenyl)aminomethylidene-5-5-dim ethyl- cyclohexane-1,3-dione (RWH-21), stimulates accumulation of 13(2)-hydroxychlorophyll a in cultured tobacco cells. This was shown based on isolation of 13(2)-hydroxychlorophyll a from pigment extracts of cultured tobacco cells by diode-array HPLC and subsequent fast atom bombardment mass spectrometry analysis. 13(2)-Hydroxychlorophyll a rapidly accumulates in tobacco cells both in the light and dark in the presence of RWH-21 (50 microM). Analysis of 13(2)-hydroxychlorophyll a formation in tobacco cells indicates that 13(2)-hydroxychlorophyll a is rapidly accumulated within 20 h incubation time both in the dark and light. Although the amount of 13(2)-hydroxychlorophyll a is continuously increased in the dark, the amount of 13(2)-hydroxychlorophyll a decreased remarkably in the light after 20 h incubation. Analysis of 13(2)-hydroxychlorophyll a formation and lipid peroxidation by determination malondialdehyde in tobacco cells suggests that RWH-21-induced 13(2)-hydroxychlorophyll a has the potential to cause a photodynamic action in cultured tobacco cells.

Antiproliferative effects of small fruit juices on several cancer cell lines.

Juices prepared from small fruits, mainly growing in the northern part of Japan, were studied in an attempt to explore the feasibility of an assay that screens cytotoxic properties. Screening of 43 small fruit juices indicated that Actinidia polygama Maxim., Rosa rugosa Thunb., Vaccinium smallii A. Gray and Sorbus sambucifolia Roem, strongly inhibited the proliferation of all cancer cell lines examined and yet these juices were substantially less cytotoxic toward normal human cell lines.

Pharbitis class-1 knotted-like homeobox gene, PKn3, shares similar characteristics to those of class-2 knotted-like genes.

Three novel homeobox genes, PKn1-3 (Pharbitis knotted-like), were isolated from Japanese morning glory (Pharbitis nil Chois, strain Violet). A sequence analysis showed that these genes belong to the knotted class-1 gene family and that PKn3 has a relatively unique sequence. All PKn genes are expressed in shoot apices, stems and roots, but not in cotyledons. Transformed tobacco with PKn1 or PKn2 displayed leaf shrinkage and a dwarf phenotype, while the ectopic expression of PKn3 gave no altered phenotypes. In situ hybridization showed that PKn3 is up-regulated in developing leaf primordia and that this expression becomes restricted in the basal region of young leaf blades, which is reminiscent of the expression pattern of the class-2 knotted gene, NTH23. These data suggest that these Pharbitis homeobox genes participate in the differentiation in shoots and suggest a unique function of PKn3 in developing leaves.

Differentiation-inducing effects of small fruit juices on HL-60 leukemic cells.

Epidemiological studies indicate that high intakes of fruits and vegetables are associated with a reduced risk of cancer, and several plant-derived drugs have been developed in medical oncology. Since only a small part of the flora has been tested for any kind of bioactivity, we chose small fruits as sources of differentiation-inducing activity against HL-60 leukemic cells. We have prepared juices from various small fruits that grow mainly in the northern part of Japan. Screening of 43 samples indicated that juices of Actinidia polygama Maxim., Rosa rugosa Thunb., Vaccinium smallii A. Gray, and Sorbus sambucifolia Roem. strongly induced differentiation of HL-60 cells to monocyte/macrophage characteristics in a concentration-dependent manner as indicated by histochemical and biochemical examinations.

Identification of gibberellins in the rice plant and quantitative changes of gibberellin A19 throughout its life cycle.

The major endogenous gibberellin (GA) in shoots, roots and ears of the rice plant, Oryza sativa L. japonica cv. Nihonbare, was identified as GA19 by combined gas liquid chromatography-mass spectrometry (GC-MS) and GC-selected ion current monitoring (GC-SICM). Another GA present in these tissues in small quantity was tentatively identified as GA1 by GC-SICM, and GA4 may be present in the seeds (kernels) of 3rd-leaf-stage seedlings. Using GC-SICM, the GA19 content was quantified throughout the life cycle of rice plants. It was found to reach high levels (ca. 10-15 µg/kg fresh weight) in 3rd-leaf seedlings, at panicle initiation (shoots), and during heading and anthesis (ears). The levels of GA19 in Oryza sativa indica cv. T-136 underwent changes closely similar to those found in Nihonbare. The growth-promoting activity in rice of exogenous GA19 is generally considerably less than that of GA1. It therefore seems possible that GA19 functions as a "pool GA". The level of active GAs such as GA1 may be regulated by the rate of biosynthesis of GA19 or its metabolic conversions.

Isolation of gibberellins A26 and A 27 and their glucosides from immature seeds of Pharbitis nil.

Two new gibberellins, gibberellins A26 and A27 (GA26 and GA27), and their glucosides have been isolated from immature seeds of Japanese morning-glory (Pharbitis nil), together with GA8 and its glucoside. GA26, GA27 and their glucosides showed only slight growth-promoting activities on seedlings of rice, dwarf maize and cucumber.

Biological evaluation of 5-substituted pyrimidine derivatives as inhibitors of brassinosteroid biosynthesis.

A series of 5-substituted pyrimidine derivatives was synthesized, and their ability to inhibit brassinosteroid biosynthesis was tested. The biological activity of these compounds was evaluated by the cress stem elongation method. Among the synthesized compounds, alpha-(4-chlorophenyl)-alpha-phenyl-5-pyrimidinemethanol (DPPM 4) exhibited potent inhibitory activity for retarding cress stem elongation in the light. This inhibition was reversed by the application of 10 nM brassinolide, but not by 1 microM GA3. DPPM 4 also affected Arabidopsis growth in the dark. DPPM 4-treated Arabidopsis had phenotypes like those of brassinosteroid-deficient mutants, with short hypocotyls and open cotyledons, in the dark. These biological changes were restored by the co-application of 10 nM brassinolide, but not by 1 microM GA3, suggesting that the primary site of action of DPPM 4 was the brassinosteroid biosynthetic pathway.

cDNA cloning and characterization of gibberellin-responsive genes in photoblastic lettuce seeds.

Two cDNA clones, cLRG5 and cLRG11, that respond to gibberellin (GA) were isolated from seeds of photoblastic lettuce (Lectuca sativa L. cv. Grand Rapids) by differential screening. Northern blot analysis indicated that the levels of LRG5 and LRG11 mRNAs were raised to slightly higher levels 10 h after the start of GA treatment and the levels were maintained at least for further 8 h, while those in the control seeds gradually decreased. Red light irradiation had effects similar to GA treatment. The cLRG5 insert encodes a putative polypeptide of 380 amino acids that is highly homologous to alcohol dehydrogenases from several higher plants. With regard to the cLRG11 insert, no homologous gene has been reported.

Structure of malformin B, a phytotoxic metabolite produced by Aspergillus niger.

Malformin B, produced by Aspergillus niger, was separated into six compounds by HPLC. Their structures were determined by amino acid analyses, and by mass spectral and two-dimensional NMR experiments to be cyclic pentapeptides structurally related to malformin A1. Both the NMR and MS/MS experiments suggest cyclo-D-cysteinyl-D-cysteinyl-L-amino acid-D-amino acid-L-amino acid as the essential structure of malformins.

Synthesis of New Abscisic Acid (ABA) Analogs Possessing a Geometrically Rigid Cyclized Side Chain.

In a solution, cis-abscisic acid (ABA) isomerizes into the trans-isomer which is physiologically inactive, and this structural instability is regarded as a reason for insuitability of ABA in agricultural applications. The side chain of ABA, the 2-cis-4-trans-3-methyl-2,4-pentadienoic acid moiety, was replaced with various substituted phenyl groups to examine biological effects of the inflexibility of the part. Construction of the phenyl moiety was achieved by cyclizing the ionone derivatives and subsequent aromatization. Some of those new compounds showed ABA-like activity in both seed germination and transpiration assays.

Effects of 3-Methylgibberellin Analogs on Gibberellin 3ß-Hydroxylases and Plant Growth.

Six 3-methylgibberellin analogs were synthesized, and their effects on the GA 3ß-hydroxylases from immature seeds of Phaseolus vulgaris and Cucurbita maxima, and/or on the growth of dwarf rice (Oryza sativa L. cv. Tan-ginbozu) and cucumber (Cucumis sativus L. cv. Spacemaster) were investigated. 3-Methyl-GA5 and 2, 3-didehydro-3-methyl-GA9· inhibited the conversion of [2, 3-(3)H2]GA9 to [2-(3)H]GA4 by GA 3ß-hydroxylases from both P. vulgaris and C. maxima at 3 µM and higher. Their C/D-ring-rearranged isomers, 2, 3-didehydro-3-methyl-DGC and 16-deoxo-2, 3-didehydro-3-methyl-DGC, inhibited 3ß-hydroxylation by the enzyme from P. vulgaris threefold more strongly than the non-C/D-ring-rearranged compounds, but exhibited no effect on 3ß-hydroxylation by the enzyme from C. maxima. In a dwarf rice seedling assay, 3-methyl-GA5 and 2, 3-didehydro-3-methyl-GA9 promoted shoot elongation at doses of 300 ng/plant and higher, and 3α-methyl-GA1 and 3α-methyl-GA4 at doses of 30 ng/plant and higher. In contrast 2, 3-didehydro-3-methyl-DGC inhibited shoot growth to half that of the control at a dose of 300 ng/plant, and 16-deoxo-2, 3-didehydro-3-methyl-DGC showed no effect on growth. In a cucumber seedling assay, 3α-methyl-GA4 promoted hypocotyl elongation at doses of 300 ng/plant and higher. The other C-3 methyl compounds showed no effect on the hypocotyl elongation of cucumber seedlings.

cDNA cloning and characterization of a gibberellin-responsive gene in hypocotyls of Cucumis sativus L.

A cDNA clone corresponding to a gibberellin-responsive gene (CRG16) was isolated from cucumber hypocotyls. CRG16 was deduced to encode an extremely hydrophobic protein of 65 amino acids. The deduced sequence exhibited no significant homology to other proteins. Levels of CRG16 mRNA reflected the gibberellin-induced elongation of cucumber hypocotyls.

Molecular cloning and characterization of a cDNA encoding the gibberellin biosynthetic enzyme ent-kaurene synthase B from pumpkin (Cucurbita maxima L.).

The first committed step in the formation of diterpenoids leading to gibberellin (GA) biosynthesis is the conversion of geranylgeranyl diphosphate (GGDP) to ent-kaurene. ent-Kaurene synthase A (KSA) catalyzes the conversion of GGDP to copalyl diphosphate (CDP), which is subsequently converted to ent-kaurene by ent-kaurene synthase B (KSB). A full-length KSB cDNA was isolated from developing cotyledons in immature seeds of pumpkin (Cucurbita maxima L.). Degenerate oligonucleotide primers were designed from the amino acid sequences obtained from the purified protein to amplify a cDNA fragment, which was used for library screening. The isolated full-length cDNA was expressed in Escherichia coli as a fusion protein, which demonstrated the KSB activity to cyclize [3H]CDP to [3H]ent-kaurene. The KSB transcript was most abundant in growing tissues, but was detected in every organ in pumpkin seedlings. The deduced amino acid sequence shares significant homology with other terpene cyclases, including the conserved DDXXD motif, a putative divalent metal ion-diphosphate complex binding site. A putative transit peptide sequence that may target the translated product into the plastids is present in the N-terminal region.

Structure of Malformin A, a Phytotoxic Metabolite Produced by Aspergillus niger.

Malformins-produced by Aspergillus niger were separated by HPLC and subjected to structural determination. Amino acid analyses and mass spectra suggested that all of them structurally resembled cyclic pentapeptide malformin A1. Two-dimensional NMR experiments and MS/MS experiments led us to deduce cyclo-D-cysteinyl-D-cysteinyl-L-amino acid-D-amino acid-L-amino acid as being the essential structure of malformins.

Characterization of a protein kinase gene responsive to auxin and gibberellin in cucumber hypocotyls.

By means of the PCR, cDNA clones encoding putative protein kinases have been obtained from cucumber hypocotyls. The abundance of the transcript of one of these genes, which was named CsPK3, increased on treatment with gibberellin (GA4) and/or auxin (IAA). We screened a cucumber cDNA library to clone CsPK3 cDNA. The cDNA clone (cCsPK3) encodes an open reading frame of 1,413 bp (471 amino acids), and its predicted amino acid sequence showed homology with those of serine/threonine protein kinases. Northern blot analysis indicated that IAA was more active than GA4 in increasing the level of CsPK3 mRNA in cucumber hypocotyls and that the increase in the level of CsPK3 mRNA on treatment with IAA was not inhibited by pretreatment with a protein synthesis inhibitor. The level of CsPK3 mRNA was high in hypocotyls of dark-grown cucumber seedlings and decreased to less than 50% of the original level within 15 min of the start of irradiation with white light.