flg22诱导的拟南芥转录组分析及芥子油苷代谢途径的变化

flg22是细菌鞭毛蛋白N端的一段保守性极高的区域,能够诱导植物天然的免疫反应,为全面了解植物在受到细菌性病原菌侵害后的系统响应,利用Illumina Hiseq2000对flg22处理和未处理的拟南芥幼苗进行转录组测序。对两组数据进行差异表达分析,共获得1 200个差异表达基因,包括290个下调基因和910个上调基因。对差异表达基因进行GO富集分析和KEGG pathway富集分析,结果显示,flg22处理后,拟南芥在能量代谢、氨基酸代谢及次生代谢产物的生物合成等方面产生了巨大变化。芥子油苷是一类在植物防御病原菌的天然免疫反应中起重要作用的次生代谢产物,因此对芥子油苷代谢途径的变化进行了深入分析。根据测序结果,Flg22处理后吲哚族芥子油苷合成途径的基因表达水平显著提高,而脂肪族芥子油苷代谢途径几乎没有变化,进一步对吲哚族芥子油苷合成途径的关键酶基因进行Real Time RT-PCR的分析,验证了测序结果的正确性,证明了吲哚族芥子油苷在植物抗病防御反应中的重要作用。这为深入理解病原菌诱导的植物防御性反应及吲哚族芥子油苷的抗病机制提供了大量参考数据。     

Approaching Cellular and Molecular Resolution of Auxin Biosynthesis and Metabolism

There is abundant evidence of multiple biosynthesis pathways for the major naturally occurring auxin in plants, indole-3-acetic acid (IAA), and examples of differential use of two general routes of IAA synthesis, namely Trp-dependent and Trp-independent. Although none of these pathways has been completely defined, we now have examples of specific IAA biosynthetic pathways playing a role in developmental processes by way of localized IAA synthesis, causing us to rethink the interactions between IAA synthesis, transport, and signaling. Recent work also points to some IAA biosynthesis pathways being specific to families within the plant kingdom, whereas others appear to be more ubiquitous. An important advance within the past 5 years is our ability to monitor IAA biosynthesis and metabolism at increasingly higher resolution.The topic of auxin biosynthesis and metabolism in plants was comprehensively reviewed in 2005 (Woodward and Bartel 2005). Since then, more genes involved in IAA biosynthesis and metabolism have been identified. A combination of numerous valuable mutants, the manipulation of IAA synthesis in specific cell types, and direct measurement of IAA levels at tissue and cellular resolution now point to localized IAA biosynthesis and metabolism as playing key roles in specific developmental events (reviewed in Cheng and Zhao 2007; Lau et al. 2008; Zhao 2008; Chandler 2009). With apologies to any authors who were not included because of space constraints, this review summarizes those recent findings that require us to rethink yet again, the role of IAA biosynthesis and metabolism in auxin biology.     

Focus on Metabolism: Something Old,Something New: Conserved Enzymes and the Evolution of Novelty in Plant Specialized Metabolism

Plants produce hundreds of thousands of small molecules known as specialized metabolites, many of which are of economic and ecological importance. This remarkable variety is a consequence of the diversity and rapid evolution of specialized metabolic pathways. These novel biosynthetic pathways originate via gene duplication or by functional divergence of existing genes, and they subsequently evolve through selection and/or drift. Studies over the past two decades revealed that diverse specialized metabolic pathways have resulted from the incorporation of primary metabolic enzymes. We discuss examples of enzyme recruitment from primary metabolism and the variety of paths taken by duplicated primary metabolic enzymes toward integration into specialized metabolism. These examples provide insight into processes by which plant specialized metabolic pathways evolve and suggest approaches to discover enzymes of previously uncharacterized metabolic networks.The plant kingdom collectively produces hundreds of thousands of low molecular weight organic molecules traditionally known as secondary metabolites, some of which have been shown to play roles in abiotic and biotic stress responses (e.g. herbivory defense), beneficial insect interactions (e.g. pollinator attraction), and communication with other plant and nonplant species (e.g. allelopathy and legume-rhizobia interactions; Saito and Matsuda, 2010; Pichersky and Lewinsohn, 2011; Wink, 2011). These metabolites have been widely used throughout the course of human history as medicines, spices, perfumes, cosmetics, and pest-control agents as well as in religious and cultural rituals. For the past 150 years, there has been a strong focus on documenting the chemical diversity of secondary metabolites in the plant kingdom, leading to the discovery of diverse classes of compounds such as terpenes, flavonoids, alkaloids, phenylpropanoids, glucosinolates, and polyketides. These secondary compounds were historically differentiated from products of primary metabolism, such as sugars, amino acids, nucleic acids, and fatty acids, as being nonessential for plant survival (Sachs, 1874; Kossel, 1891; Hartmann, 2008). However, by the 1980s, important functional roles began to be elucidated for metabolites previously classified as secondary, such as the phenolics (e.g. plant-microbe interactions and UV-B light protection; Bolton et al., 1986; Peters et al., 1986; Li et al., 1993; Landry et al., 1995), alkaloids (defense against herbivory; Steppuhn et al., 2004), and terpenes (defense against herbivory, antimicrobial activities, and volatile pollinator attractants; Papadopoulou et al., 1999; Schiestl and Ayasse, 2001; Erbilgin et al., 2006; Nieuwenhuizen et al., 2009). This change in our understanding of their roles has led to the coining of a new term, specialized metabolites, for these compounds, both to acknowledge their importance and to reflect the fact that many of them are phylogenetically restricted (Pichersky et al., 2006; Pichersky and Lewinsohn, 2011).Although the structural diversity of specialized metabolites far exceeds that of primary metabolites, all specialized metabolite classes are ultimately derived from primary metabolic precursors (Wink, 2011). For example, phenylpropanoids are derived from the amino acid Phe (Vogt, 2010), while the biosynthetic blocks of terpenes, isopentenyl diphosphate and dimethylallyl diphosphate, originate from mevalonate, a sterol precursor, and alternatively from methylerythritol phosphate, which is derived from glycolytic pathway precursors (Kirby and Keasling, 2009). Nitrogen-containing alkaloids are derived from a variety of primary metabolites, including amino acids and purine nucleosides (Facchini, 2001). Over the past 20 years, an increasing number of specialized metabolic enzymes have also been found to have their origins in primary metabolic pathways (Weng, 2014). Such shifts in enzyme function are made possible primarily by the process of gene duplication, which is very common in plants.     

Focus on Metabolism: Alteration of Plant Primary Metabolism in Response to Insect Herbivory

Plants in nature, which are continuously challenged by diverse insect herbivores, produce constitutive and inducible defenses to reduce insect damage and preserve their own fitness. In addition to inducing pathways that are directly responsible for the production of toxic and deterrent compounds, insect herbivory causes numerous changes in plant primary metabolism. Whereas the functions of defensive metabolites such as alkaloids, terpenes, and glucosinolates have been studied extensively, the fitness benefits of changes in photosynthesis, carbon transport, and nitrogen allocation remain less well understood. Adding to the complexity of the observed responses, the feeding habits of different insect herbivores can significantly influence the induced changes in plant primary metabolism. In this review, we summarize experimental data addressing the significance of insect feeding habits, as related to herbivore-induced changes in plant primary metabolism. Where possible, we link these physiological changes with current understanding of their underlying molecular mechanisms. Finally, we discuss the potential fitness benefits that host plants receive from altering their primary metabolism in response to insect herbivory.Plants in nature are subject to attack by a wide variety of phytophagous insects. Nevertheless, the world is green, and most plants are resistant to most individual species of insect herbivores. To a large extent, this resistance is due to an array of toxic and deterrent small molecules and proteins that can prevent nonadapted insects from feeding. Although many plant defenses are produced constitutively, others are inducible (i.e. defense-related metabolites and proteins that are normally present at low levels become more abundant in response to insect feeding). Inducible defense systems, which allow more energy to be directed toward growth and reproduction in the absence of insect herbivory, represent a form of resource conservation. Well-studied examples of inducible plant defenses include the production of nicotine in tobacco (Nicotiana tabacum; Baldwin et al., 1998), protease inhibitors in tomato (Solanum lycopersicum; Ryan, 2000), benzoxazinoids in maize (Zea mays; Oikawa et al., 2004), and glucosinolates in Arabidopsis (Arabidopsis thaliana; Mewis et al., 2005). Additionally, herbivore-induced plant responses can include the production of physical defenses such as trichomes or thickened cell walls that can make insect feeding more difficult. Some plant defensive metabolites are highly abundant, suggesting that their biosynthesis can have a significant effect on overall plant metabolism. For instance, benzoxazinoids can constitute 1% to 2% of the total dry matter of some Poaceae (Zúñiga et al., 1983), and up to 6% of the nitrogen in herbivore-induced Nicotiana attenuata can be devoted to nicotine production (Baldwin et al., 1998).In addition to the herbivore-induced production of physical and chemical defenses, numerous changes in plant primary metabolism occur in response to insect herbivory. Among other observed effects, these can include either elevated or suppressed photosynthetic efficiency, remobilization of carbon and nitrogen resources, and altered plant growth rate. However, although the defensive value of induced toxins such as nicotine, terpenes, benzoxazinoids, and glucosinolates is clear, it is sometimes more difficult to elucidate the function of herbivore-induced changes in plant primary metabolism. Insects may also manipulate plant primary metabolism for their own benefit, making it challenging to determine whether the observed changes are actually a plant defensive response.Here, we describe commonly observed changes in plant primary metabolism, focusing on carbohydrates and nitrogen, and discuss their possible functions in plant defense against insect herbivory. There are large differences among published studies involving different plant-herbivore combinations, and no universal patterns in the herbivory-induced changes in plant primary metabolism. Therefore, we also discuss how the potential benefits can depend on the tissue that is being attacked, the extent of the tissue damage, and the type of insect herbivore that is involved in the interaction.     

Gene Duplication in the Diversification of Secondary Metabolism: Tandem 2-Oxoglutarate–Dependent Dioxygenases Control Glucosinolate Biosynthesis in Arabidopsis

Secondary metabolites are a diverse set of plant compounds believed to have numerous functions in plant–environment interactions. The large chemical diversity of secondary metabolites undoubtedly arises from an equally diverse set of enzymes responsible for their biosynthesis. However, little is known about the evolution of enzymes involved in secondary metabolism. We are studying the biosynthesis of glucosinolates, a large group of secondary metabolites, in Arabidopsis to investigate the evolution of enzymes involved in secondary metabolism. Arabidopsis contains natural variations in the presence of methylsulfinylalkyl, alkenyl, and hydroxyalkyl glucosinolates. In this article, we report the identification of genes encoding two 2-oxoglutarate–dependent dioxygenases that are responsible for this variation. These genes, AOP2 and AOP3, which map to the same position on chromosome IV, result from an apparent gene duplication and control the conversion of methylsulfinylalkyl glucosinolate to either the alkenyl or the hydroxyalkyl form. By heterologous expression in Escherichia and the correlation of gene expression patterns to the glucosinolate phenotype, we show that AOP2 catalyzes the conversion of methylsulfinylalkyl glucosinolates to alkenyl glucosinolates. Conversely, AOP3 directs the formation of hydroxyalkyl glucosinolates from methylsulfinylalkyl glucosinolates. No ecotype coexpressed both genes. Furthermore, the absence of functional AOP2 and AOP3 leads to the accumulation of the precursor methylsulfinylalkyl glucosinolates. A third member of this gene family, AOP1, is present in at least two forms and found in all ecotypes examined. However, its catalytic role is still uncertain.     

Molecular Characterization of Quinate and Shikimate Metabolism in Populus trichocarpa

The shikimate pathway leads to the biosynthesis of aromatic amino acids essential for protein biosynthesis and the production of a wide array of plant secondary metabolites. Among them, quinate is an astringent feeding deterrent that can be formed in a single step reaction from 3-dehydroquinate catalyzed by quinate dehydrogenase (QDH). 3-Dehydroquinate is also the substrate for shikimate biosynthesis through the sequential actions of dehydroquinate dehydratase (DQD) and shikimate dehydrogenase (SDH) contained in a single protein in plants. The reaction mechanism of QDH resembles that of SDH. The poplar genome encodes five DQD/SDH-like genes (Poptr1 to Poptr5), which have diverged into two distinct groups based on sequence analysis and protein structure prediction. In vitro biochemical assays proved that Poptr1 and -5 are true DQD/SDHs, whereas Poptr2 and -3 instead have QDH activity with only residual DQD/SDH activity. Poplar DQD/SDHs have distinct expression profiles suggesting separate roles in protein and lignin biosynthesis. Also, the QDH genes are differentially expressed. In summary, quinate (secondary metabolism) and shikimate (primary metabolism) metabolic activities are encoded by distinct members of the same gene family, each having different physiological functions.     

植物次生代谢基因工程

植物次生代谢基因工程,是利用基因工程技术对植物次生代谢途径的遗传特性进行改造,进而改变植物次生代谢产物。植物次生代谢基因工程的出现是人类对次生代谢途径的深入了解和分子生物学向纵深发展的结果,同时它又促进了次生代谢分子生物学的发展。调控因子的应用和多基因的协同转化为植物次生代谢基因工程拓宽了思路。从次生代谢图谱、植物基因工程策略和植物转基因方法等方面对植物次生代谢的基因工程研究进展做一简要概述。     

MetaCyc and AraCyc. Metabolic pathway databases for plant research

MetaCyc (http://metacyc.org) contains experimentally determined biochemical pathways to be used as a reference database for metabolism. In conjunction with the Pathway Tools software, MetaCyc can be used to computationally predict the metabolic pathway complement of an annotated genome. To increase the breadth of pathways and enzymes, more than 60 plant-specific pathways have been added or updated in MetaCyc recently. In contrast to MetaCyc, which contains metabolic data for a wide range of organisms, AraCyc is a species-specific database containing only enzymes and pathways found in the model plant Arabidopsis (Arabidopsis thaliana). AraCyc (http://arabidopsis.org/tools/aracyc/) was the first computationally predicted plant metabolism database derived from MetaCyc. Since its initial computational build, AraCyc has been under continued curation to enhance data quality and to increase breadth of pathway coverage. Twenty-eight pathways have been manually curated from the literature recently. Pathway predictions in AraCyc have also been recently updated with the latest functional annotations of Arabidopsis genes that use controlled vocabulary and literature evidence. AraCyc currently features 1,418 unique genes mapped onto 204 pathways with 1,156 literature citations. The Omics Viewer, a user data visualization and analysis tool, allows a list of genes, enzymes, or metabolites with experimental values to be painted on a diagram of the full pathway map of AraCyc. Other recent enhancements to both MetaCyc and AraCyc include implementation of an evidence ontology, which has been used to provide information on data quality, expansion of the secondary metabolism node of the pathway ontology to accommodate curation of secondary metabolic pathways, and enhancement of the cellular component ontology for storing and displaying enzyme and pathway locations within subcellular compartments.     

Changing trends in biotechnology of secondary metabolism in medicinal and aromatic plants

Main conclusion

Medicinal and aromatic plants are known to produce secondary metabolites that find uses as flavoring agents, fragrances, insecticides, dyes and drugs. Biotechnology offers several choices through which secondary metabolism in medicinal plants can be altered in innovative ways, to overproduce phytochemicals of interest, to reduce the content of toxic compounds or even to produce novel chemicals. Detailed investigation of chromatin organization and microRNAs affecting biosynthesis of secondary metabolites as well as exploring cryptic biosynthetic clusters and synthetic biology options, may provide additional ways to harness this resource. Plant secondary metabolites are a fascinating class of phytochemicals exhibiting immense chemical diversity. Considerable enigma regarding their natural biological functions and the vast array of pharmacological activities, amongst other uses, make secondary metabolites interesting and important candidates for research. Here, we present an update on changing trends in the biotechnological approaches that are used to understand and exploit the secondary metabolism in medicinal and aromatic plants. Bioprocessing in the form of suspension culture, organ culture or transformed hairy roots has been successful in scaling up secondary metabolite production in many cases. Pathway elucidation and metabolic engineering have been useful to get enhanced yield of the metabolite of interest; or, for producing novel metabolites. Heterologous expression of putative plant secondary metabolite biosynthesis genes in a microbe is useful to validate their functions, and in some cases, also, to produce plant metabolites in microbes. Endophytes, the microbes that normally colonize plant tissues, may also produce the phytochemicals produced by the host plant. The review also provides perspectives on future research in the field.

     

Identification of novel gene clusters for secondary metabolism in <Emphasis Type="Italic">Trichoderma</Emphasis> genomes

Trichoderma species are widely used in agriculture as biofungicides. These fungi are rich source of secondary metabolites and the mycoparasitic species are enriched in genes for biosynthesis of secondary metabolites. Most often, genes for secondary metabolism are clustered in fungal genomes. Previously, no systematic study was undertaken to identify the secondary-metabolism related gene clusters in Trichoderma genomes. In the present study, a survey of the three Trichoderma genomes viz. T. reesei, T. atroviride and T. virens, was made to identify the putative gene clusters associated with secondary metabolism. In T. reesei genome, we identified one new NRPS and 6 new PKS clusters, which is much less than that found in T. atroviride (4 and 8) and T. virens (8 and 7). This work would pave the way for discovery of novel secondary metabolites and pathways in Trichoderma.