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.     

Uptake of Gibberellin Methyl Esters by Spores and Induction of Dark Germination in Lygodium japonicum

In the fern Lygodium japonicum, the effect of the exogenousapplication of two gibberellin methyl esters, gibberellin A₄methyl ester (GA₄Me) and gibberellin A₂₀ methyl ester (GA₂₀Me)on spore germination in the dark and uptake of GA₄Me and GA₂₀Meby spores was investigated. Tritiated GA₄Me and GA₂₀Me wereprepared and used as radioactive tracers. The activity of GA₄Mewas more than 100-fold that of GA₂₀Me for the induction of sporegermination. When treated for 24 h, the activity for inducingspore germination remained after removal of the gibberellinmethyl esters from the medium. The amount of GA₄Me taken upby spores was more than three times that of GA₂₀Me throughoutthe 24 h time course of treatment. The uptake of both gibberellinmethyl esters was proportional to the external concentrationfor the range of concentrations between 10^–9 M and 10^–6M. When treated with the tritiated gibberellin methyl estersat 10^–6 M and 10^–7 M for 24 h, most of the gibberellinmethyl esters taken up by the spores were not metabolized. Althoughthe uptake of the two gibberellin methyl esters differed by3- to 5- fold, their abilities to induce spore germination differedby more than 100-fold. Therefore, the difference in the activityof the two gibberellin methyl esters regarding the inductionof spore germination could not be explained solely by the differencein their uptake. (Received January 11, 1988; Accepted May 26, 1988)     

海金沙的化学成分和药理活性研究进展

海金沙是海金沙科海金沙属多年生蕨类植物,在我国广东、海南、江苏、浙江等地广泛分布。海金沙含有黄酮类化合物、酚酸及其糖苷类化合物及三萜类化合物等多种生物活性成分,具有利胆、防治结石、抗氧化、抗茵等多种药理活性。对海金沙的化学成分和药理活性做了综述,以便更好地对其进行开发利用。     

Effects of Gibberellins and Their Methyl Esters on Dark Germination and Antheridium Formation in Lygodium japonicum and Anemia phyllitidis

The effects of GA₃, GA₄ and GA₉ and their methyl esters on darkspore germination and antheridium formation of the ferns Lygodiumjaponicum and Anemia phyllitidis were investigated. Althoughall induced both germination and antheridium formation in Lygodium,only the gibberellins induced these effects inAnemia. (Received August 28, 1986; Revision received November 14, 1986. )     

海金沙配子体发育及卵发生的显微观察

用光镜观察海金沙(Lygodium japonicum)配子体发育和卵发生。海金沙孢子为四面体形,具三裂缝,孢子萌发方式为密穗蕨型(Anemia-type);配子体的发育形态多样,通常丝状体长至3~5个细胞时通过顶细胞纵分裂发育为片状体,进而发育为心形原叶体,在心形原叶体上可产生精子器和颈卵器。但在培养过程中也可产生10个细胞以上的丝状体,这种丝状体发育成的片状体和原叶体形态通常不规则,只产生精子器,不产生颈卵器。原叶体发育是铁线蕨型(Adiantum-type),性器官是薄囊蕨型(Leptosporangiate-type)。切片观察海金沙颈卵器产生于生长点下方表面细胞,经两次分裂形成了顶细胞、初生细胞和基细胞。其中初生细胞再经两次不等分裂产生卵细胞、腹沟细胞和颈沟细胞,此时三个细胞紧密相连,随发育,颈沟细胞和和腹沟细胞退化,卵周围形成了分离腔,光镜观察显示成熟卵细胞上无典型卵膜形成,未观察到受精孔的结构。     

海金沙配子体形态学特征的新观察

海金沙[Lygodium japonicum(Thunb)Sw.]是常见的药用蕨类之一。在人工培养条件下,利用光学显微镜详细观察其配子体发育的过程。在前人工作的基础上,补充了丝状体较短,2~3细胞长,片状体和幼原叶体发育多样,成熟原叶体心形,具单细胞或两细胞的毛状体,单细胞的假根,内含叶绿体或根尖膨大等,从海金沙配子体特征看说明其为系统进化的中间类群。孢子人工培养得到大量的海金沙幼孢子体,为保护性开发利用该资源奠定基础。     

Synthesis and Biological Activities of Novel (Z)-/(E)-Anisaldehyde-Based Oxime Ester Compounds

In search of novel natural product-based bioactive molecules, twenty (ten pairs) novel (Z)-/(E)-anisaldehyde-based oxime ester compounds were designed and synthesized by using anisaldehyde as starting material. Structural characterization of the target compounds was carried out by NMR, FT-IR, ESI-MS, and elemental analysis. Their herbicidal and antifungal activities were preliminarily tested. As a result, at 50 μg/mL, compound (E)- 5b exhibited excellent to good inhibition rates of 92.3 %, 79.2 %, and 73.9 %, against Rhizoctonia solani, Fusarium oxysporum f. sp. cucumerinum, and Bipolaris maydis, respectively, better than or comparable to that of the positive control chlorothalonil. In addition, at 100 μg/mL, compounds (E)- 5b , (E)- 5f , (Z)- 5f and (E)- 5d exhibited excellent to good inhibition rates of 85.8 %, 82.9 %, 78.6 % and 64.2 %, respectively, against the root-growth of rape (B. campestris), much better than that of the positive control flumioxazin. The bioassay result also showed that the synthesized compounds had obvious differences in antifungal and herbicidal activities between (Z)- and (E)-isomers. Preliminary structure–activity relationship was also discussed by theoretical calculation.     

Interaction between Biological Activities on Rice Seedlings of Dehydroabietic Acid Derivatives and Gibberellin A3

Effects of hydrofluorene and hydrophenanthrene compounds derived from dehydroabietic acid on the second leaf sheath growth of rice seedlings were examined in the presence and absence of gibberellin A₃(GA₃). In the absence of GA₃, nineteen compounds at 100 ppm inhibited more than 20% the growth of normal rice seedlings. In the presence of GA₃ (1.5 ppm) with dwarf rice seedlings, nine compounds at 500 ppm suppressed the elongation caused by the hormone, and a compound was slightly promotive. Then, three compounds were selected and subjected to the bioassay under various conditions.     

Synthesis and Biological Activity of Nitrilic Derivatives of the Methyl Ester of Maleopimaric Acid

An effective method of a modification of the anhydride ring of the maleopimaric acid methyl ester by means of the cyanoethylation reaction was developed. A primary screening of a cytotoxic activity in vitro demonstrated an ability of the cyanoethyl derivative of the maleopimaric acid methyl ester to induce the death of the PC-3 cells of prostate cancer.     

Relationship between Germination and PFR Level in Spores of the Fern Lygodium japonicum

The relationship between germination and P_FR level in sporesof the fern Lygodium japonicum was investigated. Percent P_FRestimated from direct spectrophotometric measurement of sporesincreased with the logarithm of total fluence of 660 nm-light.The transformation from P_R to P_FR was saturated by giving ca.200 Jm^–2 of 660 nm-light and half-saturated by ca. 55J^–2 of 660 nm-light. Clear positive correlation was observedbetween % P_FR levels and germination rates in spores irradiatedwith 660 nm and/or 730 nm-light, or with 686 or 700 nm-light.The P_FR percentage in spores was raised to 16–34% by blue(415 nm) light irradiation. This P_FR level was enough to causesome germination when produced by monochromatic light of redto far-red region, but blue light did not cause any germination. After 660 nm-light irradiation, the P_FR level decreased graduallyin darkness (25±1°C) and P_FR completely disappearedin 8 h, but 730 nm-light given even 16 h after 660 nm-lightirradiation inhibited germination. ⁴Present address: Tropical Botanic Garden and Research Institute,Navaranga Road, Trivandrum 695 011, India. (Received March 15, 1983; Accepted June 4, 1983)     

海金沙提取物体外抑菌性能研究

用M／C和纸片法考查了海金沙提取物对藤黄球菌、枯草芽孢杆菌、金黄色葡萄球菌和乙型溶血性链球菌的抑菌性能。结果表明：海金沙对4种受试菌株都有抑菌活性;37℃时,醇提物对乙型溶血性链球菌的抑菌效果最好,最大抑菌圈为8．5mm;42℃时,对藤黄球菌、金黄色葡萄球菌和枯草杆菌的抑菌效果最好,最大抑菌圈分别为21、13．2和6．5mm。pH值为7．6时,海金沙醇提物在对藤黄球菌、金黄色葡萄球菌、枯草杆菌和乙型溶血性链球菌的最大抑菌圈分别为9．1、8．2、9mm和11．3mm。海金沙水提物和醇提物对藤黄球菌、乙型溶血性链球菌、枯草芽孢杆菌、金黄色葡萄球菌的最低抑菌质量分数分别为25％、12．5％、12．5％、25％和3．12％、1．56％、6．25％、3．12％．     

Changes in Phytochrome Content during Imbibition in Spores of the Fern Lygodium japonicum

The phytochrome content was determined in intact fern sporesof Lygodium japonicum (Thunb.) Sw. by difference spectrophotometry.The spectral characteristics thus estimated in spores whichhad been imbibed for 9 days in darkness were: far-red maximumat 730?2.5 nm, red maximum at 662?1.5 nm and isosbestic pointat 684.5?1.4 nm. A detectable amount of phytochrome first appearedafter 3 days of dark imbibition, and the level then increasedduring the rest of the imbibition period. On the 7th day, thephytochrome content leveled off. During the dark imbibitionperiod, the phytochrome was revealed to be in the P_R form. (Received February 22, 1982; Accepted July 9, 1982)     

Identification of gibberellin A9 methyl ester as a natural substance regulating formation of reproductive organs in Lygodium japonicum

Prothallia of Lygodium japonicum (Thunb.) Sw. were aseptically cultured under white light in a mineral solution. Solvent fractionation of the resultant culture medium and subsequent preparative thinlayer chromatography yielded a fraction that induced antheridium formation and inhibited archegonium formation. Combined gas chromatography-selected ion monitoring analysis of this fraction confirmed the presence of gibberellin A₉ methyl ester (GA₉-me) as an antheridiogen and an inhibitor of archegonium formation. Exogenously applied [³H]GA₉ was rapidly converted to [³H]GA₉-me in the prothallial tissue. Authentic GA₉-me was active to 10^-10M in antheridium formation and to 10^-9M in the inhibition of archegonium formation.Abbreviations GAs gibberellins - GA_n gibberellin A_n - GA_n me, gibberellin A_n methyl ester - TLC thin-layer chromatography - GCSIM Combined gas chromatography-selected ion monitoring     

Spore germination and phytochrome biosynthesis in the fern Lygodium japonicum as affected by gabaculine and cycloheximide

We investigated whether the gradual increase in phytochrome content in the fern Lygodium japonicum (Thunb.) Sw. during dark imbibition results from hydration or from biosynthesis of phytochrome. Addition of gabaculine or cycloheximide to the culture medium caused inhibitions of both red light-induced spore germination and of the appearance of phytochrome in the spores. Fifty percent inhibition of both red light-induced germination and of the appearance of phytochrome in the spores occurred at ca 10⁻7 M cycloheximide. Red light-induced germination and phytochrome appearance were markedly inhibited by 10⁻4 M and completely by 10⁻3 M gabaculine, but germination induced by gibberellic acid was unaffected. Phytochrome was not detected in spores after forced hydration. These results suggest that the increase in phytochrome during imbibition was mainly due to de novo synthesis of the phytochrome apoprotein and to synthesis of the chromophore and/or proteins required for phytochrome formation, rather than to hydration of preexisting phytochrome molecules.     

海金沙胚胎发育的研究

利用石蜡切片法研究了海金沙胚胎发育过程。合子的第一次分裂面垂直于颈卵器的长轴,产生两个相等的子细胞,靠近颈卵器颈部的营养器官原始细胞和远离颈部的基足原始细胞。前者发育成子代孢子体的营养器官,后者发育成基足。胚胎在32细胞阶段后,第一叶顶端细胞与第一根顶端细胞几乎同时发生。第一叶突出帽状体之后,由第一叶基部保留下来的茎干顶端细胞产生第二叶。据营养器官的形态结构判断,在海金沙胚胎发育中最早出现的营养器官是叶和根。     

Division and gametophytic tissue formation from protoplasts of young sporophytes in fern Lygodium japonicum

Protoplasts isolated enzymatically from juvenile leaflets of sporophytes in fern Lyqodium japonicum were inoculated in a MS medium containing NAA 2.7 m, BAP 2.2 m, 0.6 M mannitol, and 0.05 M sucrose. Cell division took place within 8 days of culture initiation, and after 30 days cell-clusters with 10–15 cells were observed. When the cell-clusters were transferred into fresh hormone-free medium containing no mannitol, they developed rhizoids and protonema-like regenerants. About 2 months later, cordate prothallia developed bearing both antheridia and archegonia.Abbreviations MS Murashige and Skoog (1962) - NAA naphthalene acetic acid - BAP 6-benzylaminopurine     

Isolation and identification of antheridiogens in the ferns, Lygodium microphyllum and Lygodium reticulatum.

Antheridiogens in culture media of 6-week-old prothallia of two species of Schizaeaceous ferns, Lygodium microphyllum and Lygodium reticulatum, were analyzed by gas chromatography-mass spectrometry. In both species, the gibberellin A73 methyl ester (GA73-Me) was identified as the most abundant antheridiogen, and the methyl esters of GA9 and of several monohydroxy-GA73 derivatives were also detected. Since both species produced antheridiogens at a high level, they were classified into high-antheridiogen-producing ferns. The response to GA73-Me of gametophytes of both species is also discussed.     

An Ultrastructural Study of the Development of Blepharoplast and the Origin of Multilayered Structure in the Sperm of Lygodium japonicum

The blepharoplast, an elliptical, compact spherical body ranging 0.5～0.6 /μm in diameter, appears in the cytoplasm of the sperm mother cell during the sperm development of Lygodium japonicum (Thunb.) Sw. It consists of cartwheel tubular subunits arranged in radiant pattern and amorphous material. Numerous microtubules extended from the blepharoplast into cytoplasm. In the process of sperm cell development, the blepharoplast became loose, and the centrioles originated from the subunits separated from each other and dispersed towards the surface, with the amorphous material in the center. Accompanying the differentiation of the centrioles, muhilayered structure with the spline ( i. e. microtubular band) and lamella strata came into existence, with one end attaching to the amorphous material in the center. Then the muhilayered structure (MIS) associated with a mitochondrion and moved towards the nucleus, and a dent facing the MIS is formed in the nucleus. A cotton-like structure consisting of amorphous material and dardyed small tubular structure appeard in late stage of spermatogenesis. Microtubules were seen protruding from the surface of the cotton-like structure. It is suggested that this structure might be relevant to the origin of the blepharoplast.     

Accumulation of Gibberellin A1 and the Metabolism of Gibberellin A9 to Gibberellin A1 in a Phaeosphaeria sp. L487 Culture

Gibberellin A₁ (GA₁), which was identified as the major GA from the GA-producing fungus Phaeosphaeria sp. L487, was accumulated in the culture with a maltose-yeast extract medium, its amount in the culture filtrate being about 50 mg per liter after a 3-week culture. The new fungal biosynthetic pathway to GA₁ from GA₉ via GA₄ was elucidated by feeding experiments with synthetic [17-²H₂]GA₉ and [17-²H₂]GA₄.