Structure comparison between oxidized and reduced plastocyanin from a fern, Dryopteris crassirhizoma.

The X-ray crystal structures of oxidized and reduced plastocyanin obtained from the fern Dryopteris crassirhizoma have been determined at 1.7 and 1.8 A resolution, respectively. The fern plastocyanin is unique in the longer main chain composed of 102 amino acid residues and in the unusual pH dependence due to the pi-pi stacking interaction around the copper site [Kohzuma, T., et al. (1999) J. Biol. Chem. 274, 11817-11823]. Here we report the structural comparison between the fern plastocyanin and other plastocyanins from cyanobacteria, green algae, and other higher plants, together with the structural changes of fern plastocyanin upon reduction. Glu59 hydrogen bonds to the OH of Tyr83, which is thought to be a possible conduit for electrons, in the oxidized state. However, it moves away from Tyr83 upon reduction like poplar plastocyanin.     

The structure and unusual pH dependence of plastocyanin from the fern Dryopteris crassirhizoma. The protonation of an active site histidine is hindered by pi-pi interactions

Spectroscopic properties, amino acid sequence, electron transfer kinetics, and crystal structures of the oxidized (at 1.7 A resolution) and reduced form (at 1.8 A resolution) of a novel plastocyanin from the fern Dryopteris crassirhizoma are presented. Kinetic studies show that the reduced form of Dryopteris plastocyanin remains redox-active at low pH, under conditions where the oxidation of the reduced form of other plastocyanins is inhibited by the protonation of a solvent-exposed active site residue, His87 (equivalent to His90 in Dryopteris plastocyanin). The x-ray crystal structure analysis of Dryopteris plastocyanin reveals pi-pi stacking between Phe12 and His90, suggesting that the active site is uniquely protected against inactivation. Like higher plant plastocyanins, Dryopteris plastocyanin has an acidic patch, but this patch is located closer to the solvent-exposed active site His residue, and the total number of acidic residues is smaller. In the reactions of Dryopteris plastocyanin with inorganic redox reagents, the acidic patch (the "remote" site) and the hydrophobic patch surrounding His90 (the "adjacent" site) are equally efficient for electron transfer. These results indicate the significance of the lack of protonation at the active site of Dryopteris plastocyanin, the equivalence of the two electron transfer sites in this protein, and a possibility of obtaining a novel insight into the photosynthetic electron transfer system of the first vascular plant fern, including its molecular evolutionary aspects. This is the first report on the characterization of plastocyanin and the first three-dimensional protein structure from fern plant.     

粗茎鳞毛蕨应用的研究

为开发利用粗茎鳞毛蕨(Dryopteris crassirhizoma Nakai)的药用和观赏价值、提高其质量和产量,对其进行了人工有性繁殖和复壮研究.在大批量人工有性育苗的基础上,予以分组复壮、对照栽培.筛选出了经济简便的技术路线,观察了形态发育与栽培条件的相关性.     

绵马鳞毛蕨精母细胞和游动精子超微结构特征的研究

应用电镜技术对蕨类植物绵马鳞毛蕨(RYOPTERIS CRASSIRHIZOMA Nakai)精母细胞和游动精子的超微结构特征进行了研究。精母细胞为多边形,细胞质内含有丰富的线粒体、质体、内质网、高尔基体等常见的细胞器．在细胞质中还可见到一些同心圆膜状结构,位于质膜的附近或精母细胞的角偶。同心圆膜状结构由双层膜环绕构成,外被l层单位膜。精母细胞与精子器的璧细胞之间形成了分离腔。在精母细胞质膜外形成了嗜锇层,这些结构的形成说明精母细胞已经开始与雄配子体逐渐分离,进入独立发育的阶段。尽管精母细胞之间也有嗜锇层的形成,但嗜锇层是不连续的,其上有一些空隙,精母细胞之间可通过空隙进行物质和信息的交流。成熟的精子细胞外被l层透明的薄膜,里面为游动精子。螺旋状。由环状细胞器环绕3～4圈构成．这些环状细胞器包括多层结卡构、微管带、巨大线粒体、鞭毛带和1个长形浓缩的细胞核。游动精子的后端为一些泡囊化的细胞质．其中包括一些残存的线粒体、造粉质体及大的囊泡等。当成熟的精子细胞排出精子器后。其内的游动精子挣脱透明质膜的束缚,摆脱后端的囊泡,成为1条游动精子。本文还对绵马鳞毛蕨和其它蕨类植物精子的超微结构特征进行了比较。     

Phytochemical investigation of the rhizomes of Dryopteris crassirhizoma

A comprehensive phytochemical investigation of the rhizomes of Dryopteris crassirhizoma Nakai resulted in the isolation and identification of one new trimeric phloroglucinol, trisflavaspidic acid ABB (1) together with 12 known compounds including phloroglucinol, flavan, terpene, phenolic and acyclic analogs. Their structures were established by spectroscopic methods including IR, UV, MS and 1D and 2D NMR.     

Ultrastructure of Oogenesis in Dryopteris crassirhizoma Nakai

The ultrastructure of oogenesis in Dryopteris crassirhizoma Nakai has been investigated using transmission electron microscopy. The nucleus in the young egg is rounded with an uneven outline. As it develops, it becomes amoeboid and extends nuclear protrusions that are not only sac-like nuclear evaginations like those often seen in the oogenesis of other ferns, but also mushroom-like and finger-like, with an opening at their end allowing the nucleolus material to flow out from the openings. This has not been observed previously. The nuclear protrusions differ from Dryopteris filix-mas (L.) Schott. in the absence of sheets of nuclear membrane in the form of a closed ring. As the egg matures, the nucleus transforms into a tuber-like structure with a smooth surface, lying transversely in the egg cell. In the immature egg, vesicles almost encircle the nucleus twice and are most remarkable. In the maturing egg, the vesicles are distributed at the periphery, except for at the top of the egg, and affect the formation of the separation cavity and extra egg membrane. Simultaneously, vesicles from the venter canal cell move to the egg and take part in the formation of separation cavity and extra egg membrane. In the mature egg, a large number of small vesicles containing fragments of lamellae or osmiophilic material emerge from the cytoplasm. The origin of these vesicles is obscure. Irregular plastids containing a cylindrical starch grain dedifferentiated progressively.Mitochondria seem to have been undeveloped during the process, but return to normal at later stages of oogenesis. There is a high frequency of ribosomes in the mature egg. Microtubules, rarely seen in the eggs ofD. filix-mas (L.) Schott. and Pteridium aquilinum (L.) Kuhn, have been observed inside the plasmalemma of the maturing egg in D. crassirhizoma.     

Ultrastructure of Oogenesis in Dryopteris crassirhizoma Nakai

The ultrastructure ofoogenesis in Dryopteris crassirhizoma Nakai has been investigated using transmission electron microscopy. The nucleus in the young egg is rounded with an uneven outline. As it develops, it becomes amoeboid and extends nuclear protrusions that are not only sac-like nuclear evaginations like those often seen in the oogenesis of other ferns, but also mushroom-like and finger-like, with an opening at their end allowing the nucleolus material to flow out from the openings. This has not been observed previously. The nuclear protrusions differ from Dryopterisfilix-mas (L.) Schott. in the absence of sheets of nuclear membrane in the form of a closed ring. As the egg matures, the nucleus transforms into a tuber-like structure with a smooth surface, lying transversely in the egg cell. In the immature egg, vesicles almost encircle the nucleus twice and are most remarkable. In the maturing egg, the vesicles are distributed at the periphery, except for at the top of the egg, and affect the formation of the separation cavity and extra egg membrane. Simultaneously, vesicles from the venter canal cell move to the egg and take part in the formation of separation cavity and extra egg membrane. In the mature egg, a large number of small vesicles containing fragments of lamellae or osmiophilic material emerge from the cytoplasm. The origin of these vesicles is obscure. Irregular plastids containing a cylindrical starch grain dedifferentiated progressively.Mitochondria seem to have been undeveloped during the process, but return to normal at later stages of oogenesis. There is a high frequency of ribosomes in the mature egg. Microtubules, rarely seen in the eggs of D.filix-mas (L.) Schott. and Pteridium aquilinum (L.) Kuhn, have been observed inside the plasmalemma of the maturing egg in D. crassirhizoma.     

Inhibitory effects of phloroglucinols from the roots of Dryopteris crassirhizoma on melanogenesis

Two new phloroglucinols (7 and 10), along with 12 known derivatives (1-6, 8-9, and 11-14), were isolated from the roots of Dryopteris crassirhizoma (Aspiadaceae). Their chemical structures were elucidated by various spectroscopic methods (¹H NMR, ¹³C NMR, COSY, HMQC, and HMBC) and high-resolution mass spectrometry. All isolates (1-14) were tested for their inhibitory effects on melanin production in B16F10 murine melanoma cells. Norflavaspidic acid AB (8), norflavaspidic acid PB (9), and flavaspidic acid PB (12) inhibited melanin production with IC₅₀ values of 181.3, 35.7, and 38.9 μM, respectively. Compound 9 was also shown to inhibit tyrosinase activity in a dose-dependent manner and melanogenesis with an IC₅₀ value of 5.9 μM in B16F10 cells stimulated with 3-isobutyl-1-methylxanthine (IBMX).     

Fatty acid synthase inhibitory activity of acylphloroglucinols isolated from Dryopteris crassirhizoma

Fatty acid synthase (FAS) is emerging as a potential therapeutic target to treat cancer and obesity. Bioassay-guided fractionation of a MeOH extract of the rhizomes of Dryopteris crassirhizoma (Dryopteridaceae), using an in vitro FAS inhibitory assay, resulted in the isolation of a series of acylphloroglucinols, as the active principles. The isolates 1-10 inhibited FAS with IC50 values ranging from 23.1+/-1.4 to 71.7+/-3.9 microM. The results of the present study indicate that the acylphloroglucinol derivatives could be considered to be a promising class of FAS inhibitors.     

粗茎鳞毛蕨孢子萌发研究

以经过3年低温储藏的粗茎鳞毛蕨孢子为实验材料,从孢子离心、孢子消毒、培养基种类、光质等4方面对孢子萌发进行研究,结果表明:在离心转数≤14 000 r.min-1、离心时间≤30 min条件下,离心处理对孢子萌发基本无影响;对孢子进行1%NaClO水溶液浸泡处理20～30 min为最佳消毒条件;改良Knop’s培养基为最佳孢子萌发培养基;黑暗条件下孢子不能萌发,但是黑暗处理能够明显提高孢子萌发整齐性;红光比白光能促进孢子提早萌发1 d左右,但对提高萌发率效果不显著。     

Dammaradiene synthase, a squalene cyclase, from Dryopteris crassirhizoma Nakai

Ferns produce a variety of cyclic triterpene hydrocarbons in large amount. Squalene cyclases (SCs) are responsible enzymes for formation of cyclic triterpene hydrocarbon skeletons. Although more than ten bacterial SCs have been cloned and four of them characterized for their enzymatic products, the only example of a fern SC is ACH, from Adiantum capillus-veneris, which produces hydroxyhopane. To obtain a deeper understanding of the molecular evolution of SCs and the origin of the structural diversity of fern triterpenes, further cloning and characterization of SCs have been pursued. In this study, a SC cDNA, DCD, was cloned from Dryopteris crassirhizoma by homology-based RT-PCR. DCD contains a 2058-bp open reading frame that encodes a 685 amino acid polypeptide exhibiting 66% identity to the previously identified fern SC, ACH, and 35-40% identity to bacterial SCs. Heterologous expression of DCD in yeast established it to be a dammaradiene synthase affording dammara-18(28),21-diene, a tetracyclic triterpene hydrocarbon. Although neither this compound nor any derived metabolites have been previously reported from D. crassirhizoma, re-investigation of the leaflets demonstrated the presence of dammara-18(28),21-diene. DCD represents the first SC that produces a tetracyclic triterpene hydrocarbon.     

Functional characterization of the evolutionarily divergent fern plastocyanin.

Plastocyanin (Pc) is a soluble copper protein that transfers electrons from cytochrome b(6)f to photosystem I (PSI), two protein complexes that are localized in the thylakoid membranes in chloroplasts. The surface electrostatic potential distribution of Pc plays a key role in complex formation with the membrane-bound partners. It is practically identical for Pcs from plants and green algae, but is quite different for Pc from ferns. Here we report on a laser flash kinetic analysis of PSI reduction by Pc from various eukaryotic and prokaryotic organisms. The reaction of fern Pc with fern PSI fits a two-step kinetic model, consisting of complex formation and electron transfer, whereas other plant systems exhibit a mechanism that requires an additional intracomplex rearrangement step. The fern Pc interacts inefficiently with spinach PSI, showing no detectable complex formation. This can be explained by assuming that the unusual surface charge distribution of fern Pc impairs the interaction. Fern PSI behaves in a similar way as spinach PSI in reaction with other Pcs. The reactivity of fern Pc towards several soluble c-type cytochromes, including cytochrome f, has been analysed by flavin-photosensitized laser flash photolysis, demonstrating that the specific surface motifs for the interaction with cytochrome f are conserved in fern Pc.     

绵马鳞毛蕨的组织培养与植株再生

1植物名称绵马鳞毛蕨(Dryopteris crassirhizoma Nakai),又称粗茎鳞毛蕨、野鸡翅膀子。2材料类别嫩叶。3培养条件愈伤组织诱导培养基:(1)1/2MS 6-BA0.3mg·L-1(单位下同) IBA0.3 NH4H2PO3200,(2)     

Lectins of Dryopteris Adans fern species

Lectin activity of three ferns species (Dryopteris bushiana Fom., D. laeta (Kom.) C. Chr, D. pseudomas (Wollaston) Holub et Pouzar) has been studied. We used hemagglutination reaction albumin and globulin fractions of fronds and rhizomes extracts with rat erythrocytes. The frond extract of D. pseudomas have shown higher activity as compared with other extracts. The lectin activity of D. laeta leaves was absent. The rhizomes of all three fern species could be characterized as high activity. Based on lectin activity the species were placed as follows order: D. buschiana > D. pseudomas > D. laeta. The dependence with lectin activity and elements (Mn, Fe, Cu, Ni, Zn, Pb, Rb, Sr) content were not found.     

Unusual terpenylated acylphloroglucinols from Dryopteris wallichiana

Four unusual terpenylated acylphloroglucinols were isolated from the diethyl ether extract of the scales and rhizomes of the fern Dryopteris wallichiana together with the known compounds albaspidins AA and AB, and filixic acids ABA and ABB. Structures of the isolated compounds were established by extensive spectroscopic analysis and their absolute configuration at C-14″ was determined by comparing their CD spectra with those simulated for the respective isomers. Pure acylphloroglucinols displayed moderate in vitro nematocidal activity against L4 stage larvae of Nippostrongylus brasiliensis (LD₅₀ = 22–121 μM).     

Overwintering leaves of a forest-floor fern, Dryopteris crassirhizoma (Dryopteridaceae): a small contribution to the resource storage and photosynthetic carbon gain

BACKGROUND AND AIMS: Dryopteris crassirhizoma is a semi-evergreen fern growing on the floor of deciduous forests. The present study aimed to clarify the photosynthetic and storage functions of overwintering leaves in this species. METHODS: A 2-year experiment with defoliation and shading of overwintering leaves was conducted. Photosynthetic light response was measured in early spring (for overwintering leaves) and summer (for current-year leaves). KEY RESULTS: No nitrogen limitation of growth was detected in plants subjected to defoliation. The number of leaves, their size, reproductive activity (production of sori) and total leaf mass were not affected by the treatment. The defoliation of overwintering leaves significantly reduced the bulk density of rhizomes and the root weight. The carbohydrates consumed by the rhizomes were assumed to be translocated for leaf production. Photosynthetic products of overwintering leaves were estimated to be small. CONCLUSION: Overwintering leaves served very little as nutrient-storage and photosynthetic organs. They partly functioned as a carbon-storage organ but by contrast to previous studies, their physiological contribution to growth was found to be modest, probably because this species has a large rhizome system. The small contribution of overwintering leaves during the short-term period of this study may be explained by the significant storage ability of rhizomes in this long-living species. Other ecological functions of overwintering leaves, such as suppression of neighbouring plants in spring, are suggested.     

粗茎鳞毛蕨原叶体细胞有丝分裂过程中微管列阵的变化

粗茎鳞毛蕨原叶体细胞有丝分裂过程中微管列阵的变化 何群 尤瑞麟 姆旺戈     

粗茎鳞毛蕨原叶体细胞有丝分裂过程中微管列阵的变化

应用Steedman‘s wax切片法,间接免疫荧光标记技术和激光共聚焦扫描显微镜技术研究了粗茎鳞毛蕨（Dryopteris crassirhizoma Nakai)原叶体大液泡化细胞和分生组织细胞有丝分裂过程中微管列阵的变化。结果显示：应用高浓度的多聚甲醛（8%）可以很好地保持大液泡化细胞的结构和微管的抗原性。结果也显示Steedman‘s wax切片法和间接免疫荧光标记技术的优点;（1）避免在微管标记过程中酶解细胞壁;（2）在乙醇脱水过程中样品中叶绿素的自发荧光被减到最小;（3）能够详细观察到有丝分裂过程中微管骨架的变化。因此,这种方法可以被广泛用来调查简单植物体和复杂植物体中细胞的有丝分裂过程以及发育过程中微管骨架的变化。     

The nucleotide sequence of chloroplast 4.5S rRNA from a fern, Dryopteris acuminata.

The 4.5S rRNA was isolated from the chloroplast ribosomes from Dryopteris acuminata. The complete nucleotide sequence was determined to be: OHUAAGGUCACGGCAAGACGAGCCGUUUAUCACCACGAUAGGUGCUAAGUGGAGGUGCAGUAAUGUAUGCAGCUGAGGC AUCCUAAUAGACCGAGAGGUUUGAACOH. The 4.5S rRNA is composed of 103 nucleotides and shows strong homology with those from flowering plants.     

The nucleotide sequence of chloroplast 5S ribosomal RNA from a fern, Dryopteris acuminata.

Dryopteris acuminata chloroplasts were found to contain three species of 5S rRNAs with different electrophoretic mobility. The large 5S rRNA species is composed of 122 nucleotides and its sequence is: pUAUUCUGGUGUCCCAGGCGUAGAGGAACCACAC-CGAUCCAUCUCGAACUUGGUGGUGAAACUCUGCCGCGGUAACCA AUACUCGGGGGGGGCCCU-GCGGAAAAAUAGCUCGAUGCCAGGAUAOH. This 5S rRNA shows high sequence homology with those from chloroplasts of flowering plants and from a blue-green alga, Anacystis nidulans.