Lignan production inDaphne odora cell cultures

Lignan production in callus and cell suspension cultures ofDaphne odora is reported for the first time. The cell suspension culture produced pinoresinol, lariciresinol, secoisolariciresinol, matairesinol, and wikstromol. The production of matairesinol in the cell suspension culture was much higher than that inDaphne odora stem tissues.Part of this report was presented at the 51th annual meeting of the Japan Wood Research Society, Tokyo, April 2001     

Stereochemistry of matairesinol formation by <Emphasis Type="Italic">Daphne</Emphasis> secoisolariciresinol dehydrogenase

Secoisolariciresinol dehydrogenase activity was detected for the first time from Daphne odora and Daphne genkwa (Thymelaeaceae), which are known to produce optically pure (+)-matairesinol. In sharp contrast, (–)-matairesinol was formed selectively over the (+)-antipode by the secoisolariciresinol dehydrogenase preparation from both D. odora callus and D. genkwa shoots.     

A new flavanol from Daphne genkwa

A new flavanol, 5, 7, 4'-trihydroxy-8-ethoxycarbonylflavanol (1), was isolated from the ethanol extract of Daphne genkwa Sieb. et Zucc. Its structure was defined by spectroscopic methods.     

A new flavan from Daphne odora var. atrocaulis

The isolation of a new flavan, 5,7,4'-trihydroxy-8-ethoxycarbonylflavan (1), from the root of Daphne odora var. atrocaulis is reported.     

Survey and enzymatic formation of lignans ofAnthriscus sylvestris

Gas chromatography — mass spectrometry analysis of the -glucosidase-treated MeOH extracts ofAnthriscus sylvestris showed, based on comparison of the mass spectra and retention times with those of authentic samples, the presence of lignans, yatein, secoisolariciresinol, lariciresinol, matairesinol, hinokinin, and pluviatolide. The existence of small amounts of bursehernin was suggested by mass chromatography. In addition, nemerosin and deoxypodophyllotoxin were tentatively identified by comparing the mass spectra with those reported in the literature. Enzyme preparations fromA. sylvestris catalyzed the formation of secoisolariciresinol and lariciresinol from coniferyl alcohol. Furthermore, the enzyme preparation catalyzed the formation of lariciresinol from (±)-pinoresinols and the formation of secoisolariciresinol from (±)-lariciresinols. Thus, pinoresinol/lariciresinol reductase (PLR) activity was detected. Chiral high-performance liquid chromatography analysis showed selective formation of (+)-lariciresinol and (–)-secoisolariciresinol from (±)pinoresinols with theA. sylvestris PLR preparation, indicating that the stereochemical property ofA. sylvestris PLR-catalyzed reduction was similar to those ofForsythia PLR andArctium lappa ripening seed PLR.Part of this report was presented at the 43rd Lignin Symposium, Fuchu, October 1998     

Chemical constituents investigation of Daphne tangutica

A phytochemical study of an ethanol-soluble extract from the root barks of Daphne tangutica Maxim., a traditional Tibetan herb medicine, led to the isolation of 30 compounds, including eight daphnane diterpenes, nine coumarines, six lignans, five phenylpropanoid derivatives, β-sitosterol and p-hydroxy benzonate. Two compounds out of these isolates are new daphne diterpene analogs, and their structures were established as 1,2α-dihydro-5β-hydroxy-6α,7α-epoxy-resiniferonol-14-benzonate, and 1,2β-dihydro-5β-hydroxy-6α,7α-epoxy-resiniferonol-14-benzonate, respectively, on the basis of spectroscopic methods. Additionally, this is the first time that 13 known compounds have been isolated and identified from this traditional Tibetan medicinal plant.     

Phenolic constituents ofTaxus cuspidata I: lignans from the roots

The phenolic constituents of the roots ofTaxus cuspidata (Japanese yew) were investigated. Four lignans, [(+)-taxiresinol (1), (+)-lariciresinol (2), (–)-secoisolariciresinol (3), and (+)-pinoresinol (4)] were isolated and identified. The assignment of proton and carbon atoms for the lignans were finally solved by one- and twodimensional-nuclear magnetic resonance spectra. The enantiomeric excess of these lignans were determined by chiral high-performance liquid Chromatographic analyses. (+)-Lariciresinol and (–)-secoisolariciresinol were optically pure; (+)-taxiresinol was also suggested to be optically pure, although (+)-pinoresinol was not (77% enantiomeric excess).     

Photodiscoloration of western hemlock (Tsuga heterophylla) sapwood III Early stage of photodiscoloration reaction with lignans

The reaction during the early stage of photodiscoloration of constituents in western hemlock [Tsuga heterophylla (Raf. Sarg., Pinaceae] sapwood was investigated with chemical methods. The main photodiscoloring constituents, hydroxymatairesinol, allohydroxymatairesinol, -conidendrin, and oxomatairesinol, were used as substrates for light-irradiation experiments in vitro. The structures of photodiscoloration reaction products were elucidated by isolation and instrumental analyses and/or co-high-performance liquid chromatography analyses with authentic specimens. The experiment was undertaken to distinguish each series of liquid phases using chloroform, water (both including a trace of methanol), and methanol, and the solid phase. The reaction products allohydroxymatairesi (2), oxomatairesinol (3), -conidendrin (4), allo-7-methoxymatairesinol (5), 7-methoxymatairesinol (6), and vanillin (7) were isolated or detected in the reaction mixture of a hydroxymatairesinol system. The reaction products hydroxymatairesinol (1), 3, 4, 5, 6, and 7 were confirmed in the reaction system of allohydroxymatairesinol, which was an epimer of hydroxymatairesinol. Product 3 was confirmed from the -conidendrin system, and reaction product 7 was confirmed from oxomatairesinol. The photodiscoloration reaction of western hemlock sapwood could be initiated by the formation of phenoxy radicals from the respective constituents. The reaction was then presumed to progress via formation of a quinonemethide intermediate in many of them. It was suggested that the reactive species, such as phenoxy radical or quinonemethide intermediate, formed by lightirradiation might be converted to quinone derivatives and colored oligomers. Products 1, 2, 3, 4, and 7, formed from substrates such as hydroxymatairesinol, allohydroxymatairesinol, -conidendrin, and oxomatairesinol, were the same as the original metabolic constituents of western hemlock. Therefore it was concluded that the photodiscoloration of western hemlock depends not on the quantitative level of a few respective metabolites but, rather, on the coexistence of many metabolites.Part of this paper was presented at the 46th Annual Meeting of Japan Wood Research Society at Kumamoto, April 1996     

Biosynthesis of lignans and norlignans

Lignans and norlignans constitute abundant classes of phenylpropanoids. Biosynthesis of these compounds has received widespread interest, mainly because they have various clinically important biological activities. In addition, lignans and norlignans are often biosynthesized and deposited in significant amounts in the heartwood region of trees as a metabolic event of heartwood formation, probably preventing heart rot by heart-rot fungi. Furthermore, biosynthetic reactions of lignans and norlignans involve unique stereochemical properties that are of great interest in terms of bioorganic chemistry and are expected to provide a model for biomimetic chemistry and its application. We outline the recent advances in the study of lignan and norlignan biosynthesis.     

不同酶解条件对金边瑞香幼叶原生质体分离的影响

以室内培养的金边瑞香浅黄绿色、质地幼嫩的叶片为材料,探讨了影响其原生质分离的因素。结果表明:对原生质分离效果影响最大的是纤维素酶浓度和酶解时间;采用纤维素酶质量分数为0.2%、甘露醇质量浓度为0.6 mol.L-1、酶解时间为10 h时原生质分离效果最好,原生质体的产量为2.2×105个.g-1。     

Stereochemistry and biosynthesis of (+)-lyoniresinol, a syringyl tetrahydronaphthalene lignan in Lyonia ovalifolia var. elliptica I: isolation and stereochemistry of syringyl lignans and predicted precursors to (+)-lyoniresinol from wood

Steps leading to the biosynthesis of syringyl lignans and tetrahydronaphthalene and naphthalene lignans, especially the formation of the C2–C7′ linkage, have not been elucidated. Lyoniresinol is a typical syringyl lignan, as well as a tetrahydronaphthalene lignan found in Lyonia ovalifolia var. elliptica. To demonstrate the biosynthetic pathway for (+)-lyoniresinol, three putative biosynthetic intermediates of lyoniresinol, syringaresinol, 5,5′-dimethoxylariciresinol, and 5,5′-dimethoxysecoisolariciresinol, were isolated from wood. The identity of the putative intermediates was confirmed by spectroscopic analyses, as well as by comparison of spectral and chromatographic data with those of authentic samples previously synthesized. The stereochemistry (enantiomeric composition and absolute configuration) of the isolated lignans were determined as (±)-syringaresinol, (8S,8′S)-(−)-5,5′-dimethoxylariciresinol [46% enantiomeric excess (e.e.)], (8S,8′S)-(+)-5,5′-dimethoxysecoisolariciresinol (91% e.e.), and (8R,8′R)-(+)-lyoniresinol (42% e.e.). The absolute configurations of (+)-and (-)-5,5′-dimethoxylariciresinols, and (+)-and (-)-5,5′-dimethoxysecoisolariciresinols were determined by their synthesis (catalytic reduction) from (8R,8′R)-(+)-and (8S,8′S)-(-)-syringaresinols and by subsequent chiral high-performance liquid chromatography analysis. This report was presented at the 55th Annual Meeting of the Japan Wood Research Society, Kyoto, March 2005     

Enantiomeric compositions and biosynthesis ofWikstroemia sikokiana lignans

Thymelaeaceae plants produce dextrorotatory dibenzylbutyrolactone lignans, which are opposite enantiomers to the lignans isolated from other plants (e.g.,Forsythia spp.). In our previous paper, (–)-pinoresinol (74% enantiomer excess), (+)-matairesinol (optically pure), and (+)-wikstromol (optically pure) were isolated fromWikstroemia sikokiana (Thymelaeaceae). In the present investigation, a survey of lignans and the determination of their enantiomeric compositions were continued. Four lignans, (–)-lariciresinol, (–)-secoisolariciresinol, (+)-kusunokinin, and (+)-methyltrachelogenin, were isolated from MeOH extracts ofW. sikokiana stem. To our knowledge, we have isolated (+)-methyltrachelogenin from plants for the first time. Chiral high-performance liquid chromatographic analysis showed that (+)-kusunokinin and (+)-methyltrachelogenin were optically pure, whereas (–)-lariciresinol and (–)-secoisolariciresinol were not (39% and 45% enantiomer excess, respectively). Feeding experiments with deuterium-labeled substrates demonstrated conversion of coniferyl alcohol to the lignans and interconversion of lignans. These reaction sequences are similar to the sequence catalyzed byForsythia enzymes. However, predominant enantiomers of the lignans, except for secoisolariciresinol isolated fromW. sikokiana, have absolute configurations opposite to those of the corresponding lignans isolated fromForsythia spp. Based on the results of the isolation and the feeding experiments, several differences betweenW. sikokiana andForsythia spp. are pointed out regarding stereochemical mechanisms for lignan biosynthesis.Parts of this report were presented at the 46th annual meeting of the Japan Wood Research Society, Kumamoto, April 1996; and the 47th annual meeting of the Japan Wood Research Society, Kochi, April 1997     

Effects of the lignan, pinoresinol on the moulting cycle of the bloodsucking bug Rhodnius prolixus and of the milkweed bug Oncopeltus fasciatus

The furano-lignan pinoresinol was toxic to fourth-instar larvae of the milkweed bug Oncopeltus fasciatus and of the haematophagous insect Rhodnius prolixus, a vector of Chagas disease. At lower doses, it also exerted antifeedant activity and dose-dependent antimoulting activity on both insects.     

金边瑞香离体培养中芽和愈伤组织诱导初步研究

以MS为基本培养基，附加激素KT、NAA、6-BA、IAA、ZT进行试验，发现不同的激素组合、不同的激素水平处理对芽及愈伤组织诱导影响较大，其中以MS附加ZT1．0mg／L的培养基上芽的诱导率最高．高达94％．且诱导时间短，芽生长健壮。     

Isolation and sequence analysis of a Dof protein gene (PtDof1) in Populus

Both cDNA and DNA clones of PtDof1 (GenBank Accession No. FJ402844 and FJ402845) were isolated from plants grown in tissue culture of Populus tomentosa. The DNA sequence is 1597 bp including two exons and one intron. The cDNA is 969 bp in length with a 765 bp open reading frame which is capable of encoding 255 amino acids. The deduced amino acids sequence of the PtDof1 protein shares 65%, 56% and 55% identity with Vitis vinifera (CAO48618), Nicotiana tabacum (CAA08755) and Glycine max (ABI16022) Dof protein by blast analysis in GenBank. Phylogenic analysis suggests PtDof1 gene could belong to the Dof gene family. PtDof1 protein contains an unusual conserved single zinc finger with the pattern of C-X2-C-X21-C-X2-C, which may play a functional role in tissue-specific expression and possibly the auxin response of endogenous plant genes.     

Seasonal dynamics of wood formation: a comparison between pinning,microcoring and dendrometer measurements

Three different methods were evaluated for analysing wood formation of Norway spruce [Picea abies (L.) Karst.] and Scots pine (Pinus sylvestris L.) in Finland. During two growing seasons, wood formation dynamics were determined both by wounding the cambium with a needle followed by localisation of the wound-associated tissue modification after the growing season (pinning), and by extracting small increment cores during the growing season (microcoring). Stem radius was additionally monitored with band dendrometers. For Norway spruce, pinning and microcoring yielded similar dates for the onset of wood formation. The timing of wood production during the growing season was also similar for pinning and microcoring. For Scots pine, the onset of wood formation was recorded from microcores almost 2 weeks later than from pinning samples. In Scots pine, microcore measurements also produced somewhat later cessation dates for tracheid formation than the pinning samples. For both tree species, the total number of tracheids formed during the growing season was, however, about the same for pinning and microcoring. Dendrometer results clearly differed from those of pinning and microcoring. In particular, the dendrometers showed an increase of stem radius considerably earlier in spring, when the other methods did not detect wood formation. Thus, pinning and microcoring currently represent the most reliable techniques for detailed monitoring of wood formation.     

Constituents from the roots of <Emphasis Type="Italic">Taxus cuspidata</Emphasis>

The known propelargonidin, afzelechin-(48)-afzelechin (1), the known lignans 7-hydroxynortrachelogenin (2), epinortrachelogenin (3), nortrachelogenin (4), hydroxymatairesinol (5), allohydroxymatairesinol (6), matairesinol (7), oxomatairesinol (8), and isotaxiresinol (9), and the known taxoids taxinine M (10), taxayuntin (11), and 10-deacetyltaxol (12), and 10-deacetylbaccatin III (13) were isolated from the roots of Taxus cuspidata (Japanese yew, Taxaceae). The propelargonidin was isolated from Taxus spp. for the first time, and was detected in the roots, bark, and twigs.     

苹果属无融合生殖SERK1基因的克隆与生物信息学分析

[目的]为探讨苹果属植物无融合生殖分子机制。[方法]以苹果属平邑甜茶及杂种后代33#为试材,以苹果基因组CDS序列设计引物,通过PCR扩增技术克隆出SERK同源基因的cDNA全长序列,命名为MhSERK1和MhdSERK1(GenBank登录号JQ231273和JQ231272),利用实时定量RTqPCR的方法检测了这两个基因在平邑甜茶和杂种后代各组织和器官中的表达模式。[结果]序列分析显示MhSERK1和MhdSERK1编码区序列全长为1 899 bp和1 881 bp,分别编码632和626个氨基酸,其氨基酸序列与其他植物的SERK1同源基因所编码的氨基酸同源性都在80%以上,特别是与葡萄科龙眼品种同源性最高,高达92.56%,与模式植物拟南芥、烟草等植物的SERK同源基因都具有很高的同源性。实时定量PCR结果表明,在平邑甜茶和杂种后代不同组织、花器官中SERK1基因的表达量存在差异,其中在子房中的表达量最高,在营养生长的组织中表达量很低,在平邑甜茶花蕾期的子房中表达量最高。[结论]推测该基因在平邑甜茶和杂种后代的生殖发育过程中可能发挥重要作用。     

Biosynthesis of (+)-syringaresinol inLiriodendron tulipifera I: feeding experiments withl-[U-14C]phenylalanine and [8-14C]sinapyl alcohol

To clarify the biosynthesis of syringyl lignans and lignan formation by stereoselective coupling of monolignols, formation of (+)-syringaresinol and (+)-pinoresinol inLiriodendron tulipifera were investigated by means of feeding experiments. Following individual administration ofl-[U-¹⁴C]phenylalanine and [8-¹⁴C]sinapyl alcohol to excised shoots ofL. tulipifera and their subsequent metabolism for 3h, free [¹⁴C] lignans and [¹⁴C] lignan glucosides were extracted from both of the stems and leaves with methanol and divided into an ether fraction and an aqueous one, respectively. The glucosides were hydrolyzed by a combination of cellulase and-glucosidase to liberate [¹⁴C]lignans as aglycones.l-[U-¹⁴C]Phenylalanine was incorporated into free (+)-[¹⁴C]syringaresinol and its glucosides; the (+)-[¹⁴C]syringaresinols in the stems and leaves had 52% enantiomeric excess (% e.e.) and 42% e.e., respectively; and the (+)-[¹⁴C]syringaresinol aglycones from the glucosides in the stems and leaves had 20% e.e. and 22% e.e., respectively. Furthermore, [8-¹⁴C]sinapyl alcohol was incorporated into (+)-[¹⁴C]syringaresinol and its glucosides in the stems. These results suggest that the (+)-enantiomer of syringaresinol was enantioselectively formed from two molecules of sinapyl alcohol inL. tulipifera followed by transformation into the (+)-syringaresinol glucosides, accompanying the formation of racemic syringaresinol by nonselective coupling and the subsequent transformation of the racemate into their glucosides.l-[U-¹⁴C]Phenylalanine was incorporated also into free (+)-[¹⁴C]pinoresinol and its glucosides with 12%–42% e.e.Part of this paper was presented at the 47th Annual Meeting of the Japan Wood Research Society, Kochi, April 1997     

木麻黄青枯病菌的分离及强致病菌株的筛选

[目的]筛选出强致病菌株用于木麻黄抗病育种研究工作。[方法]在广东沿海木麻黄青枯病发病区采集病根,开展病原菌两种不同分离方法的比较研究,对分离出的31个病株进行16s rRNA测序鉴定及致病性测定。[结果]采用稀释分离法及根系溢出法在TTC培养基上共分离出了31个病原菌株,根系溢出法操作简便,杂菌含量低,分离率在60%左右,可作为常规稀释分离法的补充。31个菌株进行分子鉴定,只有22个菌株扩增出了特异性条带,经测序比对确定这22个菌株为青枯菌。青枯菌株致病性测定结果显示菌株致病性在无性系间、菌株间及菌株与无性系间的交互作用均具有极显著差异(P0.01),不同接种方法间菌株致病性相关系数值较小,介于0.496 6~0.731 0之间,即室内水培接种与小苗盆栽接种不存在密切的直线相关关系。[结论]综合选择在不同无性系及不同接种方法中均具有较强致病性的GL-2、H、M、TC-1、F、Q菌株作为下一步木麻黄种质资源抗性鉴定及抗病育种研究试验菌株。