DOCSLIB.ORG

Rough Draft of Dissertation

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Rough Draft of Dissertation Tropane alkaloid biosynthesis in Erythroxylum coca involves an atypical type III polyketide synthase by Neill Kim, B.A. A Dissertation In Chemistry Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Approved Dr. Michael Latham Chair of Committee Dr. John D’Auria Dr. Joachim Weber Mark Sheridan Dean of the Graduate School May, 2020 Copyright 2020, Neill Kim Texas Tech University, Neill Kim, May 2020 ACKNOWLEDGMENTS I would like to thank Texas Tech University for the resources and support they provided, Dr. John D’Auria for all the guidance and support he has given me, and Dr. Michael Latham. I would also like the thank Dr. Charles Stewart for helping with the crystallography of the enzyme. This research was funded by the National Science Foundation under grant No. NSF-171423326 given to Dr. John D’Auria. ii Texas Tech University, Neill Kim, May 2020 TABLE OF CONTENTS ACKNOWLEDGMENTS ........................................................................................... ii ABSTRACT ................................................................................................................. vi LIST OF TABLES ..................................................................................................... vii LIST OF FIGURES .................................................................................................. viii LIST OF SCHEMES ................................................................................................. xii LIST OF ABBREVIATIONS .................................................................................. xiv INTRODUCTION ........................................................................................................ 1 1.1 TROPANE ALKALOIDS .................................................................................. 1 1.2 GRANATANE ALKALOIDS ............................................................................ 3 1.3 TYPE III POLYKETIDE SYNTHASES .............................................................. 4 TROPANE AND GRANATANE ALKALOID BIOSYNTHESIS: A SYSTEMATIC ANALYSIS ........................................................................................ 9 2.1 ABSTRACT: ................................................................................................. 9 2.2 INTRODUCTION.......................................................................................... 10 2.2.1 SIMILARITIES AND DIFFERENCES IN MEDICINAL PROPERTIES ........... 11 2.2.2 THE SCATTERED DISTRIBUTION OF TROPANES AND GRANATANES AMONGST ANGIOSPERMS .................................................................. 13 2.2.3 BIOSYNTHESIS OF TAS AND GAS ...................................................... 18 2.3 TROPANE ALKALOID BIOSYNTHESIS ......................................................... 18 2.4 GRANATANE ALKALOID BIOSYNTHESIS .................................................... 29 2.5 METABOLIC ENGINEERING ........................................................................ 34 2.6 CONCLUSIONS ........................................................................................... 40 TROPANE ALKALOIDS: PATHWAY, POTENTIAL AND BIOTECHNOLOGICAL APPLICATIONS ........................................................... 41 3.1 ABSTRACT ................................................................................................. 41 3.2 INTRODUCTION.......................................................................................... 41 3.3 TROPANE ALKALOID BIOSYNTHESIS .......................................................... 43 3.4 TROPANE ALKALOID SIDE CHAIN MODIFICATIONS ..................................... 65 3.5 CONCLUSION ............................................................................................. 68 TROPANE ALKALOID BIOSYNTHESIS IN ERYTHROXYLUM COCA INVOLVES AN ATYPICAL TYPE III POLYKETIDE SYNTHASE ................. 70 4.1 ABSTRACT ................................................................................................. 70 4.2 INTRODUCTION.......................................................................................... 71 4.3 RESULTS ................................................................................................... 75 4.3.1 SEQUENCE ANALYSIS OF ECPYKS1, ECPYKS2, AND ECCHS .......... 75 4.3.2 QUANTITATIVE REAL-TIME PCR OF GENE EXPRESSION IN E. COCA .... 78 4.3.3 PRODUCT FORMATION IN THE AERIAL TISSUES OF E. COCA USING CRUDE EXTRACTS .............................................................................. 80 iii Texas Tech University, Neill Kim, May 2020 4.3.4 ECPYKS DOES NOT USE THE N-METHYL-∆1-PYRROLINIUM CATION AS A SUBSTRATE ................................................................................ 81 4.3.5 ECPYKS1 AND ECPYKS2 ARE ATYPICAL TYPE III PKSS THAT CATALYZE THE FORMATION OF 3-OGA ............................................. 82 4.3.6 MUTAGENESIS EXPERIMENTS ............................................................ 88 4.3.7 STRUCTURAL DATA BASED ON CRYSTALLOGRAPHY .......................... 91 4.3.8 TRANSIENT EXPRESSION SYSTEM IN N. BENTHAMIANA ....................... 94 4.4 DISCUSSION .............................................................................................. 95 4.5 MATERIALS AND METHODS ....................................................................... 97 4.5.1 CHEMICAL REAGENTS AND PLANT MATERIAL .................................... 97 4.5.2 CLONING, HETEROLOGOUS EXPRESSION AND PURIFICATION OF WILD-TYPE ECPYKS1, ECPYKS2 AND ECCHS ............................... 98 4.5.3 SITE DIRECT MUTAGENESIS FOR PYKS MUTANTS .............................. 99 4.5.4 CRUDE PROTEIN EXTRACTION FROM E. COCA LEAVES ...................... 101 4.5.5 RNA EXTRACTION AND CDNA SYNTHESIS ...................................... 101 4.5.6 QUANTITATIVE REAL-TIME PCR ...................................................... 101 4.5.7 PREPARATION OF THE 4-(1-METHYL-2-PYRROLIDINYL)-3- OXOBUTANOATE STANDARD ............................................................ 102 4.5.8 KINETIC ANALYSIS OF ENZYMES AND THE DETERMINATION OF PLANT ACTIVITY .............................................................................. 103 4.5.9 THROMBIN CLEAVAGE ..................................................................... 105 4.5.10 CRYSTALLIZATION OF ABPYKS AND ECPYKS2 ............................. 105 4.5.11 TRANSIENT EXPRESSION OF N. BENTHAMIANA ................................... 106 PYKS IN P. GRANATUM ...................................................................................... 107 5.1 INTRODUCTION........................................................................................ 107 5.2 RESULTS ................................................................................................. 108 5.2.1 SEQUENCE ANALYSIS OF PUTATIVE P. GRANATUM GENES ................ 108 5.2.2 CLONING, EXPRESSION AND PURIFICATION OF PUTATIVE PGPYKS CANDIDATES .................................................................................... 109 5.2.3 KINETIC ANALYSIS OF PYPYKS CANDIDATES ................................. 113 5.3 MATERIALS AND METHODS ..................................................................... 115 5.3.1 PLANT MATERIALS ........................................................................... 115 5.3.2 TOTAL RNA EXTRACTION OF PUNICA GRANATUM ROOT BARK ......... 116 5.3.3 CDNA SYNTHESIS ............................................................................ 117 5.3.4 PCR AMPLIFICATION OF CANDIDATE GENES FROM CDNA ............... 117 5.3.5 COLONY PCR OF K. PHAFFII ............................................................ 117 5.3.6 CHEMICAL COMPETENT TRANSFORMATIONS ................................... 118 5.3.7 ELECTROCOMPETENT TRANSFORMATIONS OF K. PHAFFII CELLS ...... 118 5.3.8 E. COLI PROTEIN EXPRESSION AND PURIFICATION ............................ 118 5.3.9 K. PHAFFII PROTEIN EXPRESSION AND PURIFICATION ....................... 120 5.3.10 ENZYME ASSAY CONDITIONS FOR PYKS ASSAY AND KINETIC ANALYSIS ........................................................................................ 121 5.3.11 ENZYME ASSAY CONDITIONS FOR CHS ASSAY ................................. 122 5.4 CONCLUSION ........................................................................................... 123 iv Texas Tech University, Neill Kim, May 2020 BIBLIOGRAPHY .................................................................................................... 125 v Texas Tech University, Neill Kim, May 2020 ABSTRACT Alkaloids represent 20% of all specialized metabolites. These important compounds are produced by plants to protect themselves from biotic or abiotic forces they may encounter. Tropane alkaloids and granatane alkaloids possess a wide array of pharmaceutical properties. Understanding the enzymes involved in the biosynthesis of these compounds will give a better insight on their evolutionary origins. This will aid synthetic biology endeavors to produce high yields of these important specialized metabolites. Although the biosynthesis of tropane alkaloids is well studied in the Solanaceae family, it is still not fully elucidated in the Erythroxylaceae family. Previous studies on these two families hypothesize a polyphyletic origin of enzymes. As more enzymes within the pathway are characterized, endeavors to metabolically engineer these pharmacologically active metabolites will increase. Here, I report a new atypical type III polyketide synthase enzyme from the Erythroxylaceae
Load more

Recommended publications
  • Title ALKALOID BIOSYNTHESIS in CULTURED TISSUES OF
    ALKALOID BIOSYNTHESIS IN CULTURED TISSUES OF Title DUBOISIA( Dissertation_全文 ) Author(s) Endo, Tsuyoshi Citation 京都大学 Issue Date 1989-03-23 URL https://doi.org/10.14989/doctor.k4307 Right Type Thesis or Dissertation Textversion author Kyoto University ALKALOID BIOSYNTHESIS IN C;ULTURED TISSUES OF DUBOISIA . , . ; . ,­ " 1. :'. '. o , " ::,,~./ ~ ~';-~::::> ,/ . , , .~ - '.'~ . / -.-.........."~l . ~·_l:""· .... : .. { ." , :: I i i , (, ' ALKALOID BIOSYNTHESIS IN CULTURED TISSUES OF DUBOISIA TSUYOSHIENDO 1989 CONTENTS INTRODUCTION ----------1 CHAPTER I ALKALOID PRODUCTION IN CULTURED DUBOISIA TISSUES. INTRODUCTION ----------6 SECTION 1 Alkaloid Production and Plant Regeneration from ~ leichhardtii Calluses. ----------8 SECTION 2 Alkaloid Production in Cultured Roots of Three Species of Duboisia. ---------16 SECTION 3 Non-enzymatic Synthesis of Hygrine from Acetoacetic Acid and from Acetonedicar- boxylic Acid. ---------25 CHAPTER II SOMATIC HYBRIDIZATION OF DUBOISIA AND NICOTIANA. INTRODUCTION ---------35 SECTION 1 Establishment of an Intergeneric Hybrid Cell Line of ~ hopwoodii and ~ tabacum. ---------38 SECTION 2 Genetic Diversity Originating from a Single Somatic Hybrid Cell. ---------47 SECTION 3 Alkaloid Biosynthesis in Somatic Hybrids, D. leichhardtii + ~ tabacum ---------59 CONCLUSIONS ---------76 ACKNOWLEDGMENTS ---------79 REFERENCES ---------80 PUBLICATIONS ---------90 ABBREVIATIONS BA 6-benzyladenine OAPI 4',6-diamino-2-phenylindoledihydrochloride EDTA ethylenediaminetetraacetic acid GC-MS gas chromatography - mass spectrometry
    [Show full text]
  • Duboisia Myoporoides R.Br. Family: Solanaceae Brown, R
    Australian Tropical Rainforest Plants - Online edition Duboisia myoporoides R.Br. Family: Solanaceae Brown, R. (1810) Prodromus Florae Novae Hollandiae : 448. Type: New South Wales, Port Jackson, R. Brown, syn: BM, K, MEL, NSW, P. (Fide Purdie et al. 1982.). Common name: Soft Corkwood; Mgmeo; Poison Corkwood; Poisonous Corkwood; Corkwood Tree; Eye-opening Tree; Eye-plant; Duboisia; Yellow Basswood; Elm; Corkwood Stem Seldom exceeds 30 cm dbh. Bark pale brown, thick and corky, blaze usually darkening to greenish- brown on exposure. Leaves Leaf blades about 4-12 x 0.8-2.5 cm, soft and fleshy, indistinctly veined. Midrib raised on the upper surface. Flowers. © G. Sankowsky Flowers Small bell-shaped flowers present during most months of the year. Calyx about 1 mm long, lobes short, less than 0.5 mm long. Corolla induplicate-valvate in the bud. Induplicate sections of the corolla and inner surfaces of the corolla lobes clothed in somewhat matted, stellate hairs. Corolla tube about 4 mm long, lobes about 2 mm long. Fruit Fruits globular, about 6-8 mm diam. Seed and embryo curved like a banana or sausage. Seed +/- reniform, about 3-3.5 x 1 mm. Testa reticulate. Habit, leaves and flowers. © Seedlings CSIRO Cotyledons narrowly elliptic to almost linear, about 5-8 mm long. First pair of true leaves obovate, margins entire. At the tenth leaf stage: leaf blade +/- spathulate, apex rounded, base attenuate; midrib raised in a channel on the upper surface; petiole with a ridge down the middle. Seed germination time 31 to 264 days. Distribution and Ecology Occurs in CYP, NEQ, CEQ and southwards as far as south-eastern New South Wales.
    [Show full text]
  • Appendix Color Plates of Solanales Species
    Appendix Color Plates of Solanales Species The first half of the color plates (Plates 1–8) shows a selection of phytochemically prominent solanaceous species, the second half (Plates 9–16) a selection of convol- vulaceous counterparts. The scientific name of the species in bold (for authorities see text and tables) may be followed (in brackets) by a frequently used though invalid synonym and/or a common name if existent. The next information refers to the habitus, origin/natural distribution, and – if applicable – cultivation. If more than one photograph is shown for a certain species there will be explanations for each of them. Finally, section numbers of the phytochemical Chapters 3–8 are given, where the respective species are discussed. The individually combined occurrence of sec- ondary metabolites from different structural classes characterizes every species. However, it has to be remembered that a small number of citations does not neces- sarily indicate a poorer secondary metabolism in a respective species compared with others; this may just be due to less studies being carried out. Solanaceae Plate 1a Anthocercis littorea (yellow tailflower): erect or rarely sprawling shrub (to 3 m); W- and SW-Australia; Sects. 3.1 / 3.4 Plate 1b, c Atropa belladonna (deadly nightshade): erect herbaceous perennial plant (to 1.5 m); Europe to central Asia (naturalized: N-USA; cultivated as a medicinal plant); b fruiting twig; c flowers, unripe (green) and ripe (black) berries; Sects. 3.1 / 3.3.2 / 3.4 / 3.5 / 6.5.2 / 7.5.1 / 7.7.2 / 7.7.4.3 Plate 1d Brugmansia versicolor (angel’s trumpet): shrub or small tree (to 5 m); tropical parts of Ecuador west of the Andes (cultivated as an ornamental in tropical and subtropical regions); Sect.
    [Show full text]
  • Enzyme DHRS7
    Toward the identification of a function of the “orphan” enzyme DHRS7 Inauguraldissertation zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel von Selene Araya, aus Lugano, Tessin Basel, 2018 Originaldokument gespeichert auf dem Dokumentenserver der Universität Basel edoc.unibas.ch Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von Prof. Dr. Alex Odermatt (Fakultätsverantwortlicher) und Prof. Dr. Michael Arand (Korreferent) Basel, den 26.6.2018 ________________________ Dekan Prof. Dr. Martin Spiess I. List of Abbreviations 3α/βAdiol 3α/β-Androstanediol (5α-Androstane-3α/β,17β-diol) 3α/βHSD 3α/β-hydroxysteroid dehydrogenase 17β-HSD 17β-Hydroxysteroid Dehydrogenase 17αOHProg 17α-Hydroxyprogesterone 20α/βOHProg 20α/β-Hydroxyprogesterone 17α,20α/βdiOHProg 20α/βdihydroxyprogesterone ADT Androgen deprivation therapy ANOVA Analysis of variance AR Androgen Receptor AKR Aldo-Keto Reductase ATCC American Type Culture Collection CAM Cell Adhesion Molecule CYP Cytochrome P450 CBR1 Carbonyl reductase 1 CRPC Castration resistant prostate cancer Ct-value Cycle threshold-value DHRS7 (B/C) Dehydrogenase/Reductase Short Chain Dehydrogenase Family Member 7 (B/C) DHEA Dehydroepiandrosterone DHP Dehydroprogesterone DHT 5α-Dihydrotestosterone DMEM Dulbecco's Modified Eagle's Medium DMSO Dimethyl Sulfoxide DTT Dithiothreitol E1 Estrone E2 Estradiol ECM Extracellular Membrane EDTA Ethylenediaminetetraacetic acid EMT Epithelial-mesenchymal transition ER Endoplasmic Reticulum ERα/β Estrogen Receptor α/β FBS Fetal Bovine Serum 3 FDR False discovery rate FGF Fibroblast growth factor HEPES 4-(2-Hydroxyethyl)-1-Piperazineethanesulfonic Acid HMDB Human Metabolome Database HPLC High Performance Liquid Chromatography HSD Hydroxysteroid Dehydrogenase IC50 Half-Maximal Inhibitory Concentration LNCaP Lymph node carcinoma of the prostate mRNA Messenger Ribonucleic Acid n.d.
    [Show full text]
  • Tropinone Synthesis Via an Atypical Polyketide Synthase and P450-Mediated Cyclization
    ARTICLE DOI: 10.1038/s41467-018-07671-3 OPEN Tropinone synthesis via an atypical polyketide synthase and P450-mediated cyclization Matthew A. Bedewitz 1, A. Daniel Jones 2,3, John C. D’Auria 4 & Cornelius S. Barry 1 Tropinone is the first intermediate in the biosynthesis of the pharmacologically important tropane alkaloids that possesses the 8-azabicyclo[3.2.1]octane core bicyclic structure that defines this alkaloid class. Chemical synthesis of tropinone was achieved in 1901 but the 1234567890():,; mechanism of tropinone biosynthesis has remained elusive. In this study, we identify a root- expressed type III polyketide synthase from Atropa belladonna (AbPYKS) that catalyzes the formation of 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid. This catalysis proceeds through a non-canonical mechanism that directly utilizes an unconjugated N-methyl-Δ1-pyrrolinium cation as the starter substrate for two rounds of malonyl-Coenzyme A mediated decarbox- ylative condensation. Subsequent formation of tropinone from 4-(1-methyl-2-pyrrolidinyl)-3- oxobutanoic acid is achieved through cytochrome P450-mediated catalysis by AbCYP82M3. Silencing of AbPYKS and AbCYP82M3 reduces tropane levels in A. belladonna. This study reveals the mechanism of tropinone biosynthesis, explains the in planta co-occurrence of pyrrolidines and tropanes, and demonstrates the feasibility of tropane engineering in a non- tropane producing plant. 1 Department of Horticulture, Michigan State University, East Lansing, MI 48824, USA. 2 Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA. 3 Department of Chemistry, Michigan State University, East Lansing, MI 48824, USA. 4 Department of Chemistry & Biochemistry, Texas Tech University, Lubbock, TX 79409, USA.
    [Show full text]
  • Download Article (PDF)
    Enzymatic Biosynthesis of Vomilenine, a Key Intermediate of the Ajmaline Pathway, Catalyzed by a Novel Cytochrome P 450-Dependent Enzyme from Plant Cell Cultures of Rauwolfia serpentina Heike Falkenhagen and Joachim Stöckigt Lehrstuhl für Pharmazeutische Biologie der Johannes Gutenberg-Universität Mainz, Institut für Pharmazie, Staudinger Weg 5, D-55099 Mainz, Bundesrepublik Deutschland Z. Naturforsch. 50c, 45-53 (1995); received September 26, 1994 Vomilenine, Vinorine, Vinorine Hydroxylase, Cytochrome P450, Rauwolfia serpentina Microsomal preparations from Rauwolfia serpentina Benth. cell suspension cultures cata­ lyze a key step in the biosynthesis of ajmaline - the enzymatic hydroxylation of the indole alkaloid vinorine at the allylic C-21 resulting in vomilenine. Vomilenine is an important branch-point intermediate, leading not only to ajmaline but also to several side reactions of the biosynthetic pathway to ajmaline. The investigation of the taxonomical distribution of the enzyme indicated that vinorine hydroxylase is exclusively present in ajmaline-producing plant cells. The novel enzyme is strictly dependent on NADPH2 and 0 2 and can be inhibited by typical cytochrome P450 inhibitors such as cytochrome c, ketoconazole and carbon mon­ oxide (the effect of CO is reversible with light of 450 nm). This suggests that vinorine hy­ droxylase is a cytochrome P450-dependent monooxygenase. A pH optimum of 8.3 and a temperature optimum of 40 °C were found. The Km value was 3 for NADPH2 and 26 [i,M for vinorine. The optimum enzyme activity could be determined at day 4 after inoculation of the cell cultures in AP I medium. Vinorine hydroxylase could be stored with 20% sucrose at -28 °C without significant loss of activity.
    [Show full text]
  • Tropane and Granatane Alkaloid Biosynthesis: a Systematic Analysis
    Office of Biotechnology Publications Office of Biotechnology 11-11-2016 Tropane and Granatane Alkaloid Biosynthesis: A Systematic Analysis Neill Kim Texas Tech University Olga Estrada Texas Tech University Benjamin Chavez Texas Tech University Charles Stewart Jr. Iowa State University, [email protected] John C. D’Auria Texas Tech University Follow this and additional works at: https://lib.dr.iastate.edu/biotech_pubs Part of the Biochemical and Biomolecular Engineering Commons, and the Biotechnology Commons Recommended Citation Kim, Neill; Estrada, Olga; Chavez, Benjamin; Stewart, Charles Jr.; and D’Auria, John C., "Tropane and Granatane Alkaloid Biosynthesis: A Systematic Analysis" (2016). Office of Biotechnology Publications. 11. https://lib.dr.iastate.edu/biotech_pubs/11 This Article is brought to you for free and open access by the Office of Biotechnology at Iowa State University Digital Repository. It has been accepted for inclusion in Office of Biotechnology Publicationsy b an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Tropane and Granatane Alkaloid Biosynthesis: A Systematic Analysis Abstract The tropane and granatane alkaloids belong to the larger pyrroline and piperidine classes of plant alkaloids, respectively. Their core structures share common moieties and their scattered distribution among angiosperms suggest that their biosynthesis may share common ancestry in some orders, while they may be independently derived in others. Tropane and granatane alkaloid diversity arises from the myriad modifications occurring ot their core ring structures. Throughout much of human history, humans have cultivated tropane- and granatane-producing plants for their medicinal properties. This manuscript will discuss the diversity of their biological and ecological roles as well as what is known about the structural genes and enzymes responsible for their biosynthesis.
    [Show full text]
  • A Molecular Phylogeny of the Solanaceae
    TAXON 57 (4) • November 2008: 1159–1181 Olmstead & al. • Molecular phylogeny of Solanaceae MOLECULAR PHYLOGENETICS A molecular phylogeny of the Solanaceae Richard G. Olmstead1*, Lynn Bohs2, Hala Abdel Migid1,3, Eugenio Santiago-Valentin1,4, Vicente F. Garcia1,5 & Sarah M. Collier1,6 1 Department of Biology, University of Washington, Seattle, Washington 98195, U.S.A. *olmstead@ u.washington.edu (author for correspondence) 2 Department of Biology, University of Utah, Salt Lake City, Utah 84112, U.S.A. 3 Present address: Botany Department, Faculty of Science, Mansoura University, Mansoura, Egypt 4 Present address: Jardin Botanico de Puerto Rico, Universidad de Puerto Rico, Apartado Postal 364984, San Juan 00936, Puerto Rico 5 Present address: Department of Integrative Biology, 3060 Valley Life Sciences Building, University of California, Berkeley, California 94720, U.S.A. 6 Present address: Department of Plant Breeding and Genetics, Cornell University, Ithaca, New York 14853, U.S.A. A phylogeny of Solanaceae is presented based on the chloroplast DNA regions ndhF and trnLF. With 89 genera and 190 species included, this represents a nearly comprehensive genus-level sampling and provides a framework phylogeny for the entire family that helps integrate many previously-published phylogenetic studies within So- lanaceae. The four genera comprising the family Goetzeaceae and the monotypic families Duckeodendraceae, Nolanaceae, and Sclerophylaceae, often recognized in traditional classifications, are shown to be included in Solanaceae. The current results corroborate previous studies that identify a monophyletic subfamily Solanoideae and the more inclusive “x = 12” clade, which includes Nicotiana and the Australian tribe Anthocercideae. These results also provide greater resolution among lineages within Solanoideae, confirming Jaltomata as sister to Solanum and identifying a clade comprised primarily of tribes Capsiceae (Capsicum and Lycianthes) and Physaleae.
    [Show full text]
  • Evolutionary Routes to Biochemical Innovation Revealed by Integrative
    RESEARCH ARTICLE Evolutionary routes to biochemical innovation revealed by integrative analysis of a plant-defense related specialized metabolic pathway Gaurav D Moghe1†, Bryan J Leong1,2, Steven M Hurney1,3, A Daniel Jones1,3, Robert L Last1,2* 1Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, United States; 2Department of Plant Biology, Michigan State University, East Lansing, United States; 3Department of Chemistry, Michigan State University, East Lansing, United States Abstract The diversity of life on Earth is a result of continual innovations in molecular networks influencing morphology and physiology. Plant specialized metabolism produces hundreds of thousands of compounds, offering striking examples of these innovations. To understand how this novelty is generated, we investigated the evolution of the Solanaceae family-specific, trichome- localized acylsugar biosynthetic pathway using a combination of mass spectrometry, RNA-seq, enzyme assays, RNAi and phylogenomics in different non-model species. Our results reveal hundreds of acylsugars produced across the Solanaceae family and even within a single plant, built on simple sugar cores. The relatively short biosynthetic pathway experienced repeated cycles of *For correspondence: [email protected] innovation over the last 100 million years that include gene duplication and divergence, gene loss, evolution of substrate preference and promiscuity. This study provides mechanistic insights into the † Present address: Section of emergence of plant chemical novelty, and offers a template for investigating the ~300,000 non- Plant Biology, School of model plant species that remain underexplored. Integrative Plant Sciences, DOI: https://doi.org/10.7554/eLife.28468.001 Cornell University, Ithaca, United States Competing interests: The authors declare that no Introduction competing interests exist.
    [Show full text]
  • Corolla Retention After Pollination Facilitates the Development
    www.nature.com/scientificreports OPEN Corolla retention after pollination facilitates the development of fertilized ovules in Fritillaria Received: 9 May 2018 Accepted: 30 November 2018 delavayi (Liliaceae) Published: xx xx xxxx Yongqian Gao1,2, Changming Wang3, Bo Song4 & Fan Du3 Corollas (or perianths), considered to contribute to pollinator attraction during anthesis, persist after anthesis in many plants. However, their post-foral function has been little investigated within a cost-beneft framework. We explored the adaptive signifcance of corolla retention after anthesis for reproduction in Fritillaria delavayi, a perennial herb endemic to the alpine areas of the Hengduan Mountains, southwestern China. We examined whether the persistent corollas enhance reproductive success during seed development. Persistent corollas increased fruit temperature on sunny days, and greatly decreased the intensity of ultraviolet-B/C (UV-B/C) radiation reaching fruits. When corollas were removed immediately after pollination, fecundity and progeny quality were adversely afected. Measurements of fower mass and size showed no further corolla growth during fruiting, and respiration and transpiration tests demonstrated that both respiration rate and transpiration rate of corollas were much lower during fruiting than during fowering, indicating a slight additional resource investment in corolla retention after anthesis. Thus, seed production by F. delavayi may be facilitated by corolla retention during seed development at only a small physiological cost. We conclude that corolla retention may be an adaptive strategy that enhances female reproductive success by having a protective role for ripening seeds in the harsh conditions at high elevation. In order to ensure reproductive success, fowering plants exhibit an astonishing diversity of foral traits; these include diferent colors of petals1,2, variable fower orientation3,4, individual fower movement5,6, and extrafo- ral structures7,8.
    [Show full text]
  • Hops Humulus Lupulus L
    HERB PROFILE Hops Humulus lupulus L. Family: Cannabaceae INTRODUCTION as noted from observations of young women who reportedly often ops is a perennial vine growing vertically to 33 feet experienced early menstrual periods after harvesting the strobiles with dark green, heart-shaped leaves.l,2,3 The male and in hops fields. lB H female flowers grow on separate vines.l.3 Hops are the Traditionally hops were used for nervousness, insomnia, excit­ dried yellowish-green, cone-like female flowers or fruits (tech­ ability, ulcers, indigestion, and restlessness associated with tension nically referred to as strobiles).l.4 Originally native to Europe, headache.l3 Additional folk medicine uses include pain relief, Asia, and North America,5 several varieties of hops are now culti­ improved appetite, and as a tonic.9 A tea made of hops was vated in Germany, the United States, the British Isles, the Czech ingested for inflammation of the bladder.? Native American Republic, South America, and Australia.4,6 Although still wild tribes used hops for insomnia and pain.5,8 Hops are employed in in Europe and North America, commercial hops come exclu­ Ayurvedic (Indian) medicine for restlessness and in traditional sively from cultivated plants.1,7 The leaves, shoots, female flowers C hinese medicine for insomnia, stomach upset and cramping, (hops), and oil are the parts of the plant used commercially. 8 and lack of appetite.5 Clinical studies in China report promise for the treatment of tuberculosis, leprosy, acute bacterial dysen­ HISTORY AND CULTURAL SIGNIFICANCE
    [Show full text]
  • Effects of a Bacterial ACC Deaminase on Plant Growth
    Effects of a bacterial ACC deaminase on plant growth-promotion by Jennifer Claire Czarny A thesis presented to the University of Waterloo in fulfilment of the thesis requirement for the degree of Doctor of Philosophy in Biology Waterloo Ontario, Canada, 2008 c Jennifer Claire Czarny 2008 Author's declaration I hereby declare that I am the sole author of this thesis. This is a true copy of the thesis, including any required final revisions, as accepted by my examiners. I understand that my thesis may be made electronically available to the public. ii Abstract Plants often live in association with growth-promoting bacteria, which provide them with several benefits. One such benefit is the lowering of plant ethylene levels through the action of the bacterial enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase that cleaves the immediate biosynthetic precursor of ethylene, ACC. The plant hormone ethylene is responsible for many aspects of plant growth and development but under stressful conditions ethylene exacerbates stress symptoms. The ACC deaminase-containing bacterium Pseudomonas putida UW4, isolated from the rhizosphere of reeds, is a potent plant growth- promoting strain and as such was used, along with an ACC deaminase minus mutant of this strain, to study the role of ACC deaminase in plant growth-promotion. Also, transgenic plants expressing a bacterial ACC deaminase gene were used to study the role of this enzyme in plant growth and stress tolerance in the presence and absence of nickel. Transcriptional changes occurring within plant tissues were investigated with the use of an Arabidopsis oligonucleotide microarray. The results showed that transcription of genes involved in hormone regulation, secondary metabolism and the stress response changed in all treatments.
    [Show full text]