喜树各器官解剖学及其喜树碱含量测定的研究

运用植物解剖学方法研究了喜树各器官的结构,对喜树不同器官中喜树碱含量进行了HPLC法测定。结果表明:(1)喜树根尖纵切面的结构由根冠、分生区、伸长区和根毛区4个部分组成。根和茎的初生结构都由表皮、皮层和维管柱3部分组成;在根和茎初生结构的皮层和髓部具有被锇酸染成黑色的嗜锇细胞;在次生生长中,根的木栓形成层起源于中柱鞘,茎的木栓形成层起源于皮层细胞;随着次生维管组织的增加,茎的中央形成髓腔。(2)叶片由表皮、叶肉和叶脉组成,叶肉细胞中分布有嗜锇细胞;光学显微镜和扫描电子显微镜观察显示,喜树叶的上表皮没有气孔器分布;下表皮气孔器的保卫细胞呈肾形,没有副卫细胞,被几个表皮细胞不规则的围绕,属于无规则型;在上下表皮均分布着单细胞非腺毛和2种类型的腺毛,其中下表皮的分布相对稠密;上表皮腺毛呈球形,下表皮腺毛大部分为长卵形,其中间杂着一些球形腺毛;观察发现,越幼嫩的叶中,分布的腺毛和非腺毛越多。(3)喜树不同器官中喜树碱含量以幼叶和种子中较高,木质部中较低,髓中含量最少;随着叶的发育成熟,叶中喜树碱的含量逐渐降低,且同一叶的不同部位,喜树碱含量也有差异;喜树幼苗发育过程中喜树碱含量也在不断变化。     

Leaf glandular trichomes in Empetrum nigrum: morphology,histochemistry, ultrastructure and secondary metabolites

The morphology, histochemistry and ultrastructure of the glandular trichomes on Empetrumnigrum leaves have been studied and more than a third of the metabolites were identified. Samples of the leaves were fixed and processed for light and electron microscopy. Glandular trichomes are situated on the inner surface of the rolled leaves. They have a clavate head and a short stalk. Histochemical tests and fluorescent microscopy demonstrate differentiated staining of the various cell types in the glandular trichome for proteins, pectins, lipids, tannins and phenylpropanoids. During secretion, the secretory cells contain rough and smooth endoplasmic reticulum, Golgi stacks with large vesicles, diversiform leucoplasts in contact with a reticular sheath and opaque deposits in the vacuoles. There are ultrastructural and functional differences between the secretory cells in the trichome head: synthesis of hydrophilic substances predominately occurs in the upper and middle secretory cells, whereas synthesis of lipophilic compounds takes place in the middle and lower secretory cells. Gas chromatography–mass spectrometry was used to determine the content of metabolites in the methanol extracts from the leaves. Many phenolic compounds (phenolic acids, bibenzyls, catechins, flavanones and flavan‐3‐ols) as well as several terpenoids were found. Two chalcones (2′,4′‐dimethoxydihydrochalcone and 2′,4′,6′‐trihydroxydihydrochalcone), one bibenzyl (batatasin III), one flavanone (7‐hydroxyflavanone) and 8 terpenoids (including phytol, α‐tocopherol, ß‐sitosterol, α‐amyrin, uvaol, oleanolic acid, ursolic acid and dehydroursolic acid) were identified in E. nigrum leaf extracts. The total yield of phenolic compounds is five to six times higher than the yield of terpenoids. It has been established that chalcones have no hydroxyl groups in ring B whereas bibenzyls have a hydroxyl group in the 3‐position in ring B. On the basis of the histochemistry, fluorescent microscopy, ultrastructure and chemical analysis, it may be concluded that synthesis and accumulation of phenolic substances and terpenoids takes place in the clavate glandular trichomes. Secondary metabolites synthesized in the trichomes protect leaf tissues from viruses, bacteria and pathogenic fungi.     

Glandular trichomes on aerial and underground organs in Chrysolaena species (Vernonieae – Asteraceae): Structure,ultrastructure and chemical composition

Although the occurrence of glandular trichomes is frequently reported for aerial vegetative organs, many questions still remain opened about the presence of such trichomes in underground systems. Here, we present, for the first time, a comparative study concerning the structure, ultrastructure and chemical aspects of both, the aerial and underground glandular trichomes of two different Chrysolaena species, C. obovata and C. platensis. Glandular trichomes (GTs) were examined using LM, SEM, and TEM and also analyzed by GC–MS and HPLC coupled to UV/DAD and HR-ESI-MS (HPLC–UV–MS). In both aerial (leaf and bud) and underground (rhizophore) organs, the GTs are multicellular, biseriate and formed by five pairs of cells: a pair of support cells, a pair of basal cells, and three pairs of secreting cells. These secreting cells have, at the beginning of secretory process, abundance of smooth ER. The same classes of secondary metabolites are biosynthesized and stored in both aerial and underground GTs of C. platensis and C. obovata. These GTs from aerial and underground organs have similar cellular and sub-cellular anatomy, however the belowground trichomes show a higher diversity of compounds when compared to those from the leaves. We also demonstrate by means of HPLC–UV–DAD that the sesquiterpene lactones are located inside the trichomes and that hirsutinolides are not artifacts.     

Volatiles and Nonvolatiles in Flourensia campestris Griseb. (Asteraceae), How Much Do Capitate Glandular Trichomes Matter?

The distribution and ultrastructure of capitate glandular trichomes (GTs) in Flourensia species (Asteraceae) have been recently elucidated, but their metabolic activity and potential biological function remain unexplored. Selective nonvolatile metabolites from isolated GTs were strikingly similar to those found on leaf surfaces. The phytotoxic allelochemical sesquiterpene (–)‐hamanasic acid A ((–)‐HAA) was the major constituent (ca. 40%) in GTs. Although GTs are quaternary ammonium compounds (QACs)‐accumulating species, glycine betaine was not found in GTs; it was only present in the leaf mesophyll. Two (–)‐HAA accompanying surface secreted products: compounds 4‐hydroxyacetophenone (piceol; 1 ) and 2‐hydroxy‐5‐methoxyacetophenone ( 2 ), which were isolated and fully characterized (GC/MS, NMR), were present in the volatiles found in GTs. The essential oils of fresh leaves revealed ca. 33% monoterpenes, 26% hydrocarbon‐ and 30% oxygenated sesquiterpenes, most of them related to cadinene and bisabolene derivatives. Present results suggest a main role of GTs in determining the volatile and nonvolatile composition of F. campestris leaves. Based on the known activities of the compounds identified, it can be suggested that GTs in F. campestris would play key ecological functions in plant‐pathogen and plant‐plant interactions. In addition, the strikingly high contribution of compounds derived from cadinene and bisabolene pathways, highlights the potential of this species as a source of high‐valued bioproducts.     

Glandular trichomes in Connarus suberosus (Connaraceae): distribution, structural organization and probable functions

Connarus suberosus is a typical species of the Brazilian Cerrado biome, and its inflorescences and young vegetative branches are densely covered by dendritic trichomes. The objective of this study was to report the occurrence of a previously undescribed glandular trichome of this species. The localization, origin and structure of these trichomes were investigated under light, transmission and scanning electron microscopy. Collections were made throughout the year, from five adult specimens of Connarus suberosus near Botucatu, S?o Paulo, Brazil, including vegetative and reproductive apices, leaves and fruits in different developmental stages, as well as floral buds and flowers at anthesis. Glandular trichomes (GTs) occurred on vegetative and reproductive organs during their juvenile stages. The GTs consisted of a uniseriate, multicellular peduncle, whose cells contain phenolic compounds, as well as a multicellular glandular portion that accumulates lipids. The glandular cell has thin wall, dense cytoplasm (with many mitochondria, plastids and dictyosomes), and a large nucleus with a visible nucleolus. The starch present in the plastids was hydrolyzed during the synthesis phase, reducing the density of the plastid stroma. Some plastids were fused to vacuoles, and some evidence suggested the conversion of plastids into vacuoles. During the final activity stages of the GTs, a darkening of the protoplasm was observed in some of the glandular cells, as a programmed cell death; afterwards, became caducous. The GTs in C. suberosus had a temporal restriction, being limited to the juvenile phase of the organs. Their presence on the exposed surfaces of developing organs and the chemical nature of the reserve products, suggest that these structures are food bodies. Field observations and detailed studies of plant-environment interactions, as well as chemical analysis of the reserve compounds, are still necessary to confirm the role of these GTs as feeding rewards.     

Integrating genes and phenotype: a wheat–Arabidopsis–rice glycosyltransferase database for candidate gene analyses

Glycosyltransferases (GTs) constitute a very large multi-gene superfamily, containing several thousand members identified in sequenced organisms especially in plants. GTs are key enzymes involved in various biological processes such as cell wall formation, storage polysaccharides biosynthesis, and glycosylation of various metabolites. GTs have been identified in rice (Oryza sativa) and Arabidopsis thaliana, but their precise function has been demonstrated biochemically for only a few. In this work we have established a repertoire of virtually all the wheat (Triticum aestivum) GT sequences, using the large publicly available banks of expressed sequences. Based on sequence similarity with Arabidopsis and rice GTs compiled in the carbohydrate active enzyme database (CAZY), we have identified and classified these wheat sequences. The results were used to feed a searchable database available on the web () that can be used for initiating an exhaustive candidate gene survey in wheat applied to a particular biological process. This is illustrated through the identification of GT families which are expressed during cell wall formation in wheat grain maturation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. This work was funded by a grant of the French ministry of research.     

Cell-specific expression of tryptophan decarboxylase and 10-hydroxygeraniol oxidoreductase,key genes involved in camptothecin biosynthesis in <Emphasis Type="Italic">Camptotheca acuminata</Emphasis> Decne (Nyssaceae)

Background  

Camptotheca acuminata is a major natural source of the terpenoid indole alkaloid camptothecin (CPT). At present, little is known about the cellular distribution of the biosynthesis of CPT, which would be useful knowledge for developing new strategies and technologies for improving alkaloid production.     

The trichomes of pericarp in Juglans (Juglandaceae): scanning microscopy, fluorescent microscopy amd histochemistry

Four types of glandular and non-glandular trichomes of pericarp in four Juglans species (J. ailanthifolia, J. cordiformis, J. mandshurica and J. regia) from Juglandaceae were studied by scanning electron microscopy, fluorescent light microscopy and histochemistry. The capitate trichomes on short stalk, the capitate trichomes on long stalk and the peltate trichomes belong to glandular trichomes; the simple hairs concern to non-glandular trichomes. The investigated species differ one from another in dimensions and distribution oftrichomes as well as the chemical content and the mechanism of secretion. The fluorescent markers and histochemical tests show the presence of flavonoids, tannins and polyphenols in trichomes on short and long stalk. In peltate trichomes the flavonoids and tannins were found in lesser quantity and the polyphenols are absent. In simple hairs the phenolic substances have not been recognized. It has been come out with the suggestion about a functional role of each type of trichomes.     

The plant glycosyltransferase clone collection for functional genomics

The glycosyltransferases (GTs) are an important and functionally diverse family of enzymes involved in glycan and glycoside biosynthesis. Plants have evolved large families of GTs which undertake the array of glycosylation reactions that occur during plant development and growth. Based on the Carbohydrate‐Active enZymes (CAZy) database, the genome of the reference plant Arabidopsis thaliana codes for over 450 GTs, while the rice genome (Oryza sativa) contains over 600 members. Collectively, GTs from these reference plants can be classified into over 40 distinct GT families. Although these enzymes are involved in many important plant specific processes such as cell‐wall and secondary metabolite biosynthesis, few have been functionally characterized. We have sought to develop a plant GTs clone resource that will enable functional genomic approaches to be undertaken by the plant research community. In total, 403 (88%) of CAZy defined Arabidopsis GTs have been cloned, while 96 (15%) of the GTs coded by rice have been cloned. The collection resulted in the update of a number of Arabidopsis GT gene models. The clones represent full‐length coding sequences without termination codons and are Gateway^® compatible. To demonstrate the utility of this JBEI GT Collection, a set of efficient particle bombardment plasmids (pBullet) was also constructed with markers for the endomembrane. The utility of the pBullet collection was demonstrated by localizing all members of the Arabidopsis GT14 family to the Golgi apparatus or the endoplasmic reticulum (ER). Updates to these resources are available at the JBEI GT Collection website http://www.addgene.org/ .     

龙葵腺毛发育的细胞学研究

利用光学显微镜、扫描电镜和透射电镜技术,观察了龙葵“四叶一心”期时叶片及茎表皮的腺毛的种类、分布,探究了不同类型腺毛的起源、生长、成熟、分泌、衰老等发育过程的细胞学特征;通过组织化学染色和荧光显微技术,观察了龙葵腺毛成分、分布,为龙葵的进一步开发利用提供参考。结果表明：（1）龙葵腺毛分为单细胞头腺毛和多细胞头腺毛两类,前者主要分布于茎表面和叶上下表皮,后者主要分布于茎表面的单细胞头腺毛之间、叶脉及叶边缘;（2）龙葵腺毛发育起始于表皮细胞突起,单细胞头腺毛行顶端生长,具1-4个柄细胞,四种类型;多细胞头腺毛可再分为一层、两层与三层多细胞头腺毛,另具三种特殊类型;（3）龙葵成熟腺毛具分泌能力,通过皮下空间的物质积累导致腺毛头细胞表面形成突起、包块、破口,最终释放分泌物;而头细胞与柄细胞随即皱缩、衰老。（4）超微结构显示,腺毛头细胞中内质网与高尔基体极为丰富,合成代谢及分泌活动活跃,产生大量包裹嗜锇物质的囊泡,囊泡与细胞壁融合,进而将嗜锇物质转移至细胞壁并积累,随后储存在角质层下的皮下空间直至分泌释放;（5）组织化学染色结果表明,腺毛含有萜类、生物碱、脂类、蛋白质、酚类和多糖。头细胞中主要含有萜类、生物碱、脂类、蛋白质、酚类和中性多糖;柄细胞中主要含有萜类、生物碱、脂类。     

Nutlet micro‐morphology of the genus Microula (Boraginaceae) from the Qinghai–Tibetan Plateau,and its systematic implications

The micro‐morphology of nutlets in 30 taxa (28 species and 2 varieties) of Microula Benth. (Boraginaceae) was surveyed by light and scanning electron microscopy. A principal coordinate‐based ordination analysis (PCO) (8 morphological and 14 micro‐morphological characters) was applied to explore whether taxa clustered according to the current sectional taxonomy. The nutlet epidermis showed three major features: trichomes, papillae and lamellae. Two basic trichome types could be identified: branched and unbranched. Stellate trichomes (STT) and stipitate stellate trichomes (SST) can be considered as two subtypes of branched trichomes based on shape and branching location. Three kinds of unbranched trichomes were recognized by their shapes: slender trichomes (SLT), spear‐like trichomes (SPT) and conical trichomes (CT). Four types of papillae were revealed in this genus: central apiculate papillae (CAP), debris‐covered convex papillae (DCP), rugous papillae (RP) and tabular papillae (TP). The lamellae was classified in five types: glossy lamellae (GL), thread‐like lamellae (TL), rugous lamellae (RL), interrupted lamellae (IL) and debris‐covered lamellae (DCL). The variation in the nature of surface sculpturing, nutlet shape and size, dorsal aperture shape, and attachment scar positions proved useful diagnostic characters. Taxa belonging to the same section generally clustered together in the PCO analysis. A key based on the morphology and micro‐morphology of nutlets is provided to distinguish the sections of Microula. The presence of SLT, STT, DCP and DCL is consistent with the macro‐morphological classification and provides additional evidence for the subgeneric delimitations. The SST, SLT, CAP, DCP, RP, DCL, GL and RL are useful in discriminating species in Microula. Additionally, the distribution of some trichomes, papillae and lamellae types is correlated with the altitudinal distribution of the species.     

Camptothecin and 10-hydroxycamptothecin from<Emphasis Type="Italic"> Camptotheca acuminata</Emphasis> hairy roots

Camptothecin (CPT) is an anticancer and antiviral alkaloid produced by the Chinese tree Camptotheca acuminata (Nyssaceae) and some other species belonging to the families Apocynaceae, Olacaceae, and Rubiaceae. Bark and seeds are currently used as sources for the drug. Several attempts have been made to produce CPT from cell suspensions; however, the low yields obtained limit this approach. Cultures of differentiated cell types may be an alternative source of alkaloid production. Hairy root cultures of C. acuminata were established from tissue transformed with Agrobacterium rhizogenes strains ATCC 15834 and R-1000. Integration of the genes responsible for the hairy-root phenotype (rol genes) into the plant genome was verified by DNA gel blot analysis. The hairy roots produce and secrete CPT as well as the more potent and less toxic natural derivative, 10-hydroxycamptothecin (HCPT), into the medium. Remarkably, the cultures were able to synthesize the alkaloids at levels equal to, and sometimes greater than, the roots in planta, i.e., 1.0 and 0.15 mg/g dry weight for CPT and the HCPT, respectively.Abbreviations CPT Camptothecin - DR Dry weight - FW Fresh weight - HCPT 10-Hydroxycamptothecin - HPLC High-performance liquid chromatography - kb Kilobases - NAA 1-Napththalenacetic acid - TLC Thin-layer chromatographyCommunicated by K.K. Kamo     

Catnip,Nepeta cataria, a morphological comparison of mutant and wild type specimens to gain an ethnobotanical perspective

Nepeta cataria L, the catnip plant, is important in the pet industry for cats and as an herbal medicinal treatment for the fevers, diarrhea, insomnia, and lacking menstruation of humans. A natural mutation of N. cataria produced a novel morphology that warranted investigation to determine how the mutation affected the microscopic features, including catnip’s ethnobotanical storehouse of glandular hairs. The morphology, anatomy, and physiology of this mutant are compared to that of the wild type of catnip to document the major differences. The secondary plant metabolites which facilitate catnip’s ethnobotanical uses are stored in microscopic glandular hairs (trichomes). The trichomes on the mutant and wild type catnip leaves were not shown to differ (scanning and transmission electron microscopy). The feliobotany (use by cats) of N. cataria is discussed in relation to catnip trichomes.     

A collection of glycosyltransferases from rice (Oryza sativa) exposed to atrazine

The rice (Oryza sativa) GTs belong to a super family possibly with hundreds of members. However, which GTs are involved in plant response to toxic chemicals is unknown. Here, we demonstrated 59 novel GT genes screened from our recent genome-wide sequencing datasets of rice crops exposed to atrazine (a herbicide persistent in ecosystems). Analysis of GT genes showed that most of the GTs contain functional domains typically found in proteins transferring glycosyl moieties to their target compounds. A phylogenetic analysis revealed that many GT genes from different families have diverse cis-elements necessary for response to biotic and environmental stresses. Experimental validation for the GTs was undertaken through a microarray, and 36 GT genes were significantly detected with an expression pattern similar to that from deep-sequencing datasets. Furthermore, 12 GT genes were randomly selected and confirmed by quantitative real-time RT-PCR. Finally, the special activity of total GTs was determined in rice roots and shoots, with an increased activity under the atrazine exposure. This response was closely associated with atrazine absorption in the rice tissues. These results indicate that exposure to atrazine can trigger specific GT genes and enzyme activities in rice.     

Response of <Emphasis Type="Italic">Camptotheca acuminata</Emphasis> calli stimulated by mechanical vibration

Plants in their natural environment are usually affected by mechanical stress because of their sessile growth habit. This work was aimed at determining the effects of mechanical vibration on the growth rate, physiological indexes, and secondary metabolite biosynthesis of Camptotheca acuminata calli. In this study, mechanical vibrations of 1–4 Hz in frequency were applied to stimulate the C. acuminata calli; we found that a mechanical vibration of moderate frequency (2 Hz) can clearly promote the growth rate and increase the soluble protein content and superoxide dismutase (SOD) activity. Under a mechanical vibration of a 2 Hz frequency, camptothecin (CPT), the main secondary metabolite produced by C. acuminata, was increased by about fourfold compared to the control group, In contrast, the increased accumulation disappears at higher frequencies. The optimal vibration time for obtaining the highest levels of biomass and CPT was 60 min. This study showed that there are neutral frequencies and optimal periods of mechanical vibration on C. acuminata calli.     

Dependence of UV-B-induced camptothecin production on nitrate reductase-mediated nitric oxide signaling in Camptotheca acuminata suspension cell cultures

UV-B irradiation induced production of secondary metabolites in plant cells. However, the mechanisms of UV-B-induced secondary metabolite production remained largely unknown. Here we report that UV-B treatment stimulated nitric oxide (NO) generation and camptothecin (CPT) production in Camptotheca acuminata cells. To investigate the origin of the UV-B-triggered NO and the role of NO in UV-B-induced CPT production, we assayed the responses of nitrate reductase (NR) and NO synthase (NOS) activities of the cells to UV-B exposure and examined the effects of NR and NOS inhibitors on CPT production in UV-B-treated cells. The data showed that UV-B irradiation enhanced NR activities in the cells. Pretreatment with NR inhibitors tungstate and okadaic acid not only suppressed the UV-B-triggered NR activities but also inhibited the UV-B-induced NO generation and CPT production in the cells. In contrast, UV-B irradiation had no effects on NOS activity of the cells and treatment of NOS inhibitor did not suppress UV-B-induced CAT production. Together, the results demonstrated that NR activity was essential for UV-B-triggered NO generation and that NR-mediated NO signaling was involved in UV-B-induced CPT production in C. acuminata cells.     

荧光物质(MFA、MFB)的分离、结构鉴定及金华地区高产菌株的筛选

采用硅胶柱层析及制备型液相色谱仪对红曲米中两种荧光物质进行分离纯化,使用高效液相色谱法(HPLC)检测荧光物质纯度,然后使用高分辨质谱(ESI-HRMS)对两种荧光物质进行分析,得到两种荧光物质的分子量分别为356和384,ESI-MS/MS二级质谱把两者鉴定为monasfluore A (MFA)和monasfluore B (MFB);从金华地区红曲米中分离得到10株红曲菌株,经固态发酵采用HPLC法分析,筛选获得1株高产MFA、MFB的菌株WZWZ,该菌株发酵制得红曲米中MFA含量为3.63 g/kg,MFB含量为7.29 g/kg,对WZWZ菌株进行外观形态学及显微观察、ITS基因序列测定与分析,最终将菌株WZWZ鉴定为紫色红曲霉(Monascus purpureus)。     

Micromorphological and chemical characterisation of Stachys recta L. subsp. serpentini (Fiori) Arrigoni in comparison to Stachys recta L. subsp. recta (Lamiaceae)

Stachys recta L. is a very polymorphous species in which numerous subspecies were recognised. S. recta L. subsp. serpentini (Fiori) Arrigoni is a typical endemism growing on serpentine soils in northern Apennines and particularly in Tuscany (Italy). In order to contribute to a better knowledge of this plant and to its differentiation with respect to S. recta L. subsp. recta, the micromorphological characters (non-glandular and glandular trichomes) and the essential oil composition of the two subspecies were investigated. Micromorphological characters were studied using scanning and transmission electron microscopy, while light microscopy was used for histochemical observations. Essential oil analysis was carried out by gas chromatography and mass spectrometry.In the two examined taxa, the morphology and distribution of glandular and non-glandular trichomes, and the different essential oil composition, may be considered distinctive characters at subspecies level. This is consistent with the taxonomic classification considering S. recta subsp. serpentini a subordinate taxon of S. recta.     

Okadaic acid accumulation in macrofilter feeders subjected to natural blooms of Dinophysis acuminata

Thermaikos Gulf is a eutrophic area located in the Northwestern part of the Aegean Sea in the Eastern Mediterranean. Interspecific differences among various filter feeders in their ability to accumulate okadaic acid, were observed during natural blooms of Dinophysis acuminata in the gulf. Okadaic acid analyses by high performance liquid chromatography (HPLC) were performed on benthic specimens and D. acuminata cell densities and cell toxin content were estimated in water samples. Seven filter feeding species were collected in the gulf during two DSP outbreaks in May 2003 and March 2004. The various species showed a different potential to accumulate okadaic acid in their tissues. The highest concentrations were found in the mussel populations (Mytilus galloprovincialis and Modiolus barbatus), while among the non-bivalve filter feeders, ascidians were the main accumulators of okadaic acid. The rest of shellfish populations (Flexopecten proteus, Chlamys varia and Venus verrucosa) were found to contain toxins only during 2004, when D. acuminata densities were found above 10000 cells l⁻¹. M. galloprovincialis was proved to be the most appropriate indicator for a safe warning of okadaic acid contamination in Thermaikos Gulf.