Molecular cloning and expression profile of a jasmonate biosynthetic pathway gene for allene oxide cyclase from <Emphasis Type="Italic">Hyoscyamus niger</Emphasis>

Hyoscyamus niger L. is a medicinal plant which produces a class of jasmonate-responsive pharmaceutical secondary metabolites named tropane alkaloids. As a family of signaling phytohormones, jasmonates play significant roles in the biosynthesis of many plant secondary metabolites. In the jasmonate biosynthetic pathway of plants, allene oxide cyclase (AOC, EC 5.3.99.6) catalyzes the most important step. Here we cloned a cDNA from H. niger, named HnAOC (GenBank accession no.: AY708383), which was 1044 bp long, with a 747-bp open reading frame (ORF) encoding a polypeptide of 248 amino acid residues. Southern blot analysis indicated that it was a multicopy gene. RT-PCR analysis revealed that the expression of HnAOC was regulated by various stresses and elicitors, with methyl-jasmonate showing the most prominent inducement. The characterization of HnAOC would be helpful for improving the production of valuable secondary metabolites by regulating the biosynthesis of jasmonates. The text was submitted by the authors in English.     

Molecular cloning and mRNA expression profiling of the first specific jasmonate biosynthetic pathway gene allene oxide synthase from <Emphasis Type="Italic">Hyoscyamus niger</Emphasis>

In the endeavor to enhance the production of pharmaceutically valuable tropane alkaloids including hyoscyamine and scopolamine in Hyoscyamus niger, methyl jasmonate (MeJA) showed significant stimulation both in tropane biosynthetic pathway enzymes activities and tropane alkaloids yields. Therefore it was speculated that genetic engineering of jasmonate biosynthetic pathway might enhance the endogenous jasmonates concentration, followed by stimulating the production of tropane alkaloids. Herein a full-length cDNA encoding allene oxide synthase (AOS, EC 4.2.1.92), the first committed step enzyme in jasmonate biosynthetic pathway was reported (named HnAOS, GenBank accession: EF532599). HnAOS was a novel member of the cytochrome P450 (CYP74A) subfamily. Real-time quantitative PCR analysis showed that HnAOS mRNA accumulated mainly in stems, and responded significantly to wounding or methyl jasmonate. The article is published in the original.     

植物内源茉莉酸类物质的生物合成途径及其生物学意义

茉莉酸类物质（JAs）是新确认的一类广泛存在于植物体内的内源激素,在植物的生长发育、应激反应和次生代谢过程中起着重要的调控作用。该文主要概述了植物中茉莉酸类物质的生物合成途径、各关键酶的生理作用及其在植物次生代谢工程等方面的研究进展,并探讨了茉莉酸类物质的潜在应用价值。     

Molecular cloning and expression profile of a jasmonate biosynthetic pathway gene allene oxide cyclase from Hyoscyamus niger

Hyoscyamus niger L. is a medicinal plant which produces a class of jasmonate-responsive pharmaceutical secondary metabolites named as tropane alkaloids. As a family of signaling phytohormones, jasmonates play significant roles in the biosynthesis of many plant secondary metabolites. In jasmonate biosynthetic pathway of plants, allene oxide cyclase (AOC, [...] EC 5.3.99.6 [...]) catalyzes the most important step. Here we cloned a cDNA from H. niger, named HnAOC (GenBank accession: AY708383), which was 1044 bp long, with a 747 bp open reading frame (ORF) encoding a polypeptide of 248 amino acid residues. Southern blot analysis indicated that it was a multi-copy gene. RT-PCR analysis revealed that the expression of HnAOC was regulated by various stresses and elicitors, with methyl-jasmonate showing the most prominent inducement. The characterization of HnAOC would be helpful for improving the production of valuable secondary metabolites by regulating the biosynthesis ofjasmonates.     

植物内源茉莉酸类物质的生物合成途径及其生物学意义

茉莉酸类物质(JAs)是新确认的一类广泛存在于植物体内的内源激素, 在植物的生长发育、应激反应和次生代谢过程中起着重要的调控作用。该文主要概述了植物中茉莉酸类物质的生物合成途径、各关键酶的生理作用及其在植物次生代谢工程等方面的研究进展, 并探讨了茉莉酸类物质的潜在应用价值。     

Molecular cloning and expression profiling of the first specific jasmonate biosynthetic pathway gene allene oxide synthase from <Emphasis Type="Italic">Lonicera japonica</Emphasis>

In jasmonate biosynthetic pathway, allene oxide synthase (AOS, EC 4.2.1.92), which is a cytochrome P450 (CYP74A), catalyzes the first committed step. We herein cloned a novel cDNA from Lonicera japonica Thunb., named LjAOS (GenBank accession: DQ303120), which was homologous to other AOSs. Southern blot analysis revealed that it was a multi-copy gene. Real-time quantitative PCR analysis showed that LjAOS mRNA accumulated most abundantly in alabastrums, in which the content of chlorogenic acid (CA, the major important active ingredient indicator) was previously proven to be the highest.     

Efficient elicitation of ginsenoside biosynthesis in cell cultures ofPanax notoginseng by using self-chemically-synthesized jasmonates

A series of fluorine and hydroxyl containing jasmonate derivatives, which were chemically synthesized in our institute, were investigated for their effects on the biosynthesis and heterogeneity of ginsenosides in suspension cultures ofPanax notoginseng cells. Compared to the control (without addition of elicitors), 100 μM of each of the jasmonate was added on day 4 to the suspension cultures ofP. notoginseng cells. It was observed that, jasmonates greatly enhanced the ginsenoside content and the ratio of Rb group to Rg group (i.e. (Rb₁+Rd)/(Rg₁+Re)) in theP. notoginseng cells. Some of the synthetic jasmonates, such as pentafluoropropyl jasmonate (PFPJA), 2-hydroxyethyl jasmonate (HEJA) and 2-hydroxyethoxyethyl jasmonate (HEEJA), could promote the ginsenoside content to 2.55±0.11, 3.65±0.13 and 2.94±0.06 mg/100 mg DW, respectively, compared to that of 0.64±0.06 mg/100 mg DW for the control and 2.17±0.04 mg/100 mg DW by the commercially available methyl jasmonate (MJA); and they could change the respective Rb: Rg ratio to 1.60±0.04, 1.87±0.01 and 1.56±0.05, compared to that of 0.47±0.01 for the control and 1.42±0.06 by MJA. The results suggest that suitable esterification of MJA with fluorine or hydroxyl group could increase the elicitation activity to induce plant secondary metabolism. The information obtained from this study is useful for hyper-production of heterogeneous products by plant cell cultures.     

卡伍尔氏链霉菌NA4的Ⅱ型聚酮基因簇的靶向激活及其产物鉴定

【目的】以基因组信息为导向,定向激活海洋来源卡伍尔氏链霉菌（Streptomyces cavourensis） NA4中沉默的Ⅱ型聚酮类次级代谢产物生物合成基因簇,鉴定新产生的次级代谢产物的结构和抑菌活性。【方法】通过添加启动子和敲除负调控基因的方法激活实验室培养条件下沉默或低表达的生物合成基因簇,并完成目标化合物的分离与纯化,通过电喷雾质谱（electrospray ionization-mass spectrometry,ESI-MS）和核磁共振（nuclear magnetic resonance,NMR）数据分析鉴定目标化合物结构,对目标化合物进行抑菌活性鉴定,基于生物信息学信息推导化合物的生物合成途径。【结果】根据基因组生物信息学分析,从海洋来源链霉菌Streptomyces cavourensis NA4中选取一个编码PKSⅡ型次级代谢产物的生物合成基因簇开展研究,成功激活目标基因簇,从中分离到1个PKSⅡ型化合物,推导了其生物合成途径并进行了抑菌活性鉴定。【结论】基因组导向下的天然产物挖掘,可以目标明确地分离产物,充分挖掘链霉菌编码次级代谢产物的潜力。     

Promotive effect of jasmonates on the senescence of detached maize leaves

Promotion of senescence of detached maize leaves by jasmonates was investigated. Senescence of detached maize leaves was promoted by linolenic acid, the precursor of biosynthesis of jasmonic acid, and retarded by inhibitors of lipoxygenase, the first enzyme in the biosynthetic pathway of jasmonic acid. Results support a role of endogenous jasmonates in the regulation of senescence of detached maize leaves. Silver thiosulfate, an inhibitor of ethylene action, was found to inhibit methyl jasmonate, linolenic acid- and abscisic acid-promoted senescence of detached maize leaves. It seems that jasmonate-promoted senescence is mediated through an increase in ethylene sensitivity in detached maize leaves.Abbreviations ABA abscisic acid - MJ methyl jasmonate - STS silver thiosulfate     

Jasmonates induce Nod factor production by Bradyrhizobium japonicum

Jasmonates are signaling molecules involved in induced systemic resistance, wounding and stress responses of plants. We have previously demonstrated that jasmonates can induce nod genes of Bradyrhizobium japonicum when measured by beta-galactosidase activity. In order to test whether jasmonates can effectively induce the production and secretion of Nod factors (lipo-chitooligosaccharides, LCOs) from B. japonicum, we induced two B. japonicum strains, 532C and USDA3, with jasmonic acid (JA), methyl jasmonate (MeJA) and genistein (Ge). As genistein is well characterized as an inducer of nod genes it was used a positive control. The high-performance liquid chromatography (HPLC) profile of LCOs isolated following treatment with jasmonates or genistein showed that both JA and MeJA effectively induced nod genes and caused production of LCOs from bacterial cultures. JA and MeJA are more efficacious inducers of LCO production than genistein. Genistein plus JA or MeJA resulted in greater LCO production than either alone. A soybean root hair deformation assay showed that jasmonate induced LCOs were as effective as those induced by genistein. This is the first report that jasmonates induce Nod factor production by B. japonicum. This report establishes the role of jasmonates as a new class of signaling molecules in the Bradyrhizobium-soybean symbiosis.     

Biosynthesis of secondary metabolites in the rice blast fungus Magnaporthe grisea: the role of hybrid PKS-NRPS in pathogenicity

Fungal secondary metabolites are an important source of bioactive compounds for agrochemistry and pharmacology. Over the past decade, many studies have been undertaken to characterize the biosynthetic pathways of fungal secondary metabolites. This effort has led to the discovery of new compounds, gene clusters, and key enzymes, and has been greatly supported by the recent releases of fungal genome sequences. In this review, we present results from a search for genes involved in secondary metabolism and their clusters in the genome of the rice pathogen, Magnaporthe grisea, as well as in other fungal genomes. We have also performed a phylogenetic analysis of recently discovered genes encoding hybrids between a polyketide synthase and a single non-ribosomal peptide synthetase module (PKS–NRPS), as M. grisea seems rich in these enzymes compared with other fungi. Using results from expression and functional studies, we discuss the role of these PKS-NRPS in the avirulence and pathogenicity of M. grisea.     

Polyamines and plant alkaloids

Naturally occurring alkaloids are nitrogenous compounds that constitute the pharmacogenically active basic principles of flowering plants. Alkaloids are classified into several biogenically related groups. Tobacco alkaloids are metabolised from polyamines and diamines putrescine and cadaverine. N-methyl transferase is the first enzyme in alkaloid biosynthetic pathway which drives the flow of nitrogen away from polyamine biosynthesis to alkaloid biosynthesis. Arginine decarboxylase has been suggested to be primarily responsible for providing putrescine for nicotine synthesis. Tryptophan is the precursor of indole alkaloids. However, the biosynthetic pathway of tropane and isoquinoline alkaloids are not clear. Genes for several key biosynthetic enzymes like arginine decarboxylase, ornithine decarboxylase, putrescine N-methyl transferase and spermidine synthase, hyoscyamine 6 beta hydroxylase,tryptophan decarboxylase etc have been cloned from different plant species. These genes are regulated by plant hormones, light, different kinds of stress and elicitors like jasmonates and their strong expression is primarily in the cultured roots. In view of this, the axenic hairy root cultures induced by Agrobacterium rhizogenes have been utilised to synthesise secondary metabolites. The current development in the knowledge of alkaloid biosynthesis, particularly molecular analysis, has been discussed in this review that may help to open up new avenues of investigation for the researchers.     

Jasmonoyl isoleucine accumulation is needed for abscisic acid build‐up in roots of Arabidopsis under water stress conditions

Phytohormones are central players in sensing and signalling numerous environmental conditions like drought. In this work, hormone profiling together with gene expression of key enzymes involved in abscisic acid (ABA) and jasmonate biosynthesis were studied in desiccating Arabidopsis roots. Jasmonic acid (JA) content transiently increased after stress imposition whereas progressive and concomitant ABA and Jasmonoyl Isoleucine (JA‐Ile) accumulations were detected. Molecular data suggest that, at least, part of the hormonal regulation takes place at the biosynthetic level. These observations also point to a possible involvement of jasmonates on ABA biosynthesis under stress. To test this hypothesis, mutants impaired in jasmonate biosynthesis (opr3, lox6 and jar1‐1) and in JA‐dependent signalling (coi1) were employed. Results showed that the early JA accumulation leading to JA‐Ile build up was necessary for an ABA increase in roots under two different water stress conditions. Signal transduction between water stress‐induced JA‐Ile accumulation and COI1 is necessary for a full induction of the ABA biosynthesis pathway and subsequent hormone accumulation in roots of Arabidopsis plants. The present work adds a level of interaction between jasmonates and ABA at the biosynthetic level.     

Expression profiling of the lignin biosynthetic pathway in Norway spruce using EST sequencing and real-time RT-PCR

Lignin biosynthesis is a major carbon sink in gymnosperms and woody angiosperms. Many of the enzymes involved are encoded for by several genes, some of which are also related to the biosynthesis of other phenylpropanoids. In this study, we aimed at the identification of those gene family members that are responsible for developmental lignification in Norway spruce (Picea abies (L.) Karst.). Gene expression across the whole lignin biosynthetic pathway was profiled using EST sequencing and quantitative real-time RT-PCR. Stress-induced lignification during bending stress and Heterobasidion annosum infection was also studied. Altogether 7,189 ESTs were sequenced from a lignin forming tissue culture and developing xylem of spruce, and clustered into 3,831 unigenes. Several paralogous genes were found for both monolignol biosynthetic and polymerisation-related enzymes. Real-time RT-PCR results highlighted the set of monolignol biosynthetic genes that are likely to be responsible for developmental lignification in Norway spruce. Potential genes for monolignol polymerisation were also identified. In compression wood, mostly the same monolignol biosynthetic gene set was expressed, but peroxidase expression differed from the vertically grown control. Pathogen infection in phloem resulted in a general up-regulation of the monolignol biosynthetic pathway, and in an induction of a few new gene family members. Based on the up-regulation under both pathogen attack and in compression wood, PaPAL2, PaPX2 and PaPX3 appeared to have a general stress-induced function. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

藤黄生孢链霉菌NRRL 2401遗传操作系统和基因文库的构建北大核心CSCD

【目的】建立藤黄生孢链霉菌NRRL 2401的遗传操作系统和基因文库,以便筛选次级代谢产物生物合成基因。【方法】利用大肠杆菌和链霉菌的属间接合转移的方法,以整合型载体pPM927、pSET152和复制型载体pJTU1278构建链霉菌遗传操作系统。以pCClFOS^(TM)载体,大肠杆菌EP1300^(TM)-T1~R为宿主菌构建Fosmid文库。随后,设计引物,利用"板池-行池-单克隆"的三级PCR方法对文库进行快速筛选。【结果】pPM927、pSET152和pJTU1278均成功转入藤黄生孢链霉菌NRRL 2401,其中pSET152载体的转化效率最高。构建了稳定高效的藤黄生孢链霉菌NRRL 2401的基因文库,含2880个克隆,平均插入片段大小约为35 kb,空载率小于1%,文库覆盖率为99.99%,覆盖基因组16.5倍。同时,初步筛选出可能含有吲哚霉素生物合成基因簇的9个阳性克隆。【结论】成功构建了稳定高效的藤黄生孢链霉菌NRRL 2401遗传操作系统和高质量的基因文库,为克隆该菌中次级代谢产物生物合成基因簇以及进一步遗传改造奠定了基础。