Im Rahmen dieser Disseration wurden im Einzelnen folgende Ergebnisse erzielt: A. Isolierung und Strukturaufklärung von Naphthylisochinolin-Alkaloiden aus verschiedenen Ancistrocladus-Spezies: • Die bisher noch nicht phytochemisch untersuchten Rindenextrakte der vietnamesischen Unterart Ancistrocladus tectorius ssp. cochinchinensis wurden im Hinblick auf ihre Sekundärmetabolite analysiert. Dabei identifizierte man vier bereits bekannte und drei neuartige Naphthylisochinoline-Alkaloide. Die Strukturen dieser drei Metabolite wurden nach Isolierung unter Verwendung diverser 2D-NMR-Techniken aufgeklärt. Die entdeckten Substanzen – Ancistrocladinium A (30) und seine beiden O-Demethylderivate 31 und 32 – waren die drei ersten Vertreter des neuartigen N,8'-Naphthyldihydroisochinolin-Kupplungstyps. Diese Naturstoffe verfügen über vielversprechende pharmakologische Wirkungen – vor allem gegen den Erreger der Leishmaniose. • Die botanisch noch nicht vollständig charakterisierte Lianenart ''A. ikela'', die aus der Demokratischen Republik Kongo stammt, wurde im Laufe der Arbeit morphologisch und phytochemisch untersucht und beschrieben. Neben den beiden N,C-verknüpften Naphthylisochinolinen Ancistrocladinium A (30) und Ancistrocladinium B [(M/P)-39] wurde bei der phytochemischen Analyse ein neuartiges C,C-gekuppeltes Alkaloid – 8-O-Methylancistrogriffin C (40) – isoliert. Des weiteren wurde ein Gradient entwickelt, der die vollständige Trennung der beiden Atrop-Diastereomere von 39 und dadurch HPLC-NMR- und HPLC-CD-Analysen der einzelen Epimere ermöglichte, so dass die Rotationsbarriere der bei Raumtemperatur langsam drehenden Biarylachse bestimmt werden konnte. • Aus Blättern der bereits gut untersuchten indischen Ancistrocladus-Art A. heyneanus wurde mit 6-O-Methyl-8,4'-O-didemethylancistrocladin (42) ein weiteres neues Naphthylisochinolin-Alkaloid isoliert. • Eine phytochemische Untersuchung der Familie der Ancistrocladaceae auf das Vorkommen von N,C-verküpften Naphtylisochinolinen ergab, dass diese strukturell außergewöhnlichen Alkaloide in diesen Lianen weit verbreitet sind. B: Die Rolle des Phloems bei der Pathogen-vermittelten Ausbreitung von Signalen: • Im Rahmen des Teilprojektes B8 des SFBs 567 wurden Untersuchungen zur Rolle des Phloems bei der Weiterleitung von Langstreckensignalen nach Infektion von Arabidopsis-thaliana-Pflanzen mit virulenten oder avirulenten Stämmen von Pseudomonas syringae pv. tomato durchgeführt. Zunächst wurde dazu eine im nL-Maßstab anwendbare Analysenmethode für die Hauptmetabolite von A. thaliana – die Glucosinolate – entwickelt. Mit Hilfe dieser empfindlichen Methode wurden in Pflanzenextrakten von A. thaliana viele bekannte und einige neue Glucosinolate (8-Methylsulfonyl-n-octyl-, 2-Hydroxy-4-methylsulfinyl-n-butyl-, 2-Hydroxy-4-methylsulfonyl-n-butyl- und 4-Hydroxy- benzoyloxymethylglucosinolat) identifiziert. Des weiteren wurden MS/MS-Analysen der Glucosinolate durchgeführt, bei denen neben mehreren typischen Fragmenten für die Thiozucker-Einheit auch einige charakteristische Fragmente für die unterschiedlichen Seitenketten (z.B. Methylsulfinyl-n-alkyl- oder Methylthio-n-alkyl-Struktur) detektiert wurden. Leider ergaben vor allem die aromatischen und heteroaromatischen Seitenketten-Typen kein typisches Fragmentierungs-muster. • Bei der Analyse der Phloemexsudate konnte in Phloemsäften von unbehandelten Pflanzen neben Methoxyglucobrassicin (73) ein für Pflanzen neuartiges Phosphat 87 (1-Glycero-1-myo-inositolphosphat) identifiziert werden. In den Phloemsäften der unterschiedlich behandelten Pflanzen (infiltriert mit MgCl2, einem virulentem oder einem avirulentem Pseudomonas-Stamm) kamen sämtliche Hauptmetabolite der Blätter vor. Lediglich ein leichter, nicht signifikanter Konzentrationsanstieg von Methoxyglucobrassicin (73) wurde im Phloemsaft von mit avirulenten Pathogenen infizierten Pflanzen festgestellt. Dieser Anstieg muss aber kritisch betrachtet werden, da er auch ein Artefakt des starken mechanischen Reizes des Infiltrationsprozesses sein könnte. Andere kleine Konzentrationsänderungen könnten außerdem durch das starke ''Grundrauschen'' der Infiltration überlagert werden. C: Strukturaufklärung polyketidischer Sekundärmetabolite aus Mikroorganismen: • Zwei niedermolekulare Naturstoffe aus dem extremophilen Streptomyceten-Stamm KC 1030, die in der Arbeitsgruppe von Prof. H.-P. Fiedler (Universität Tübingen) isoliert worden waren, wurden strukturell aufgeklärt. Bei dem einen handelt es sich um das bereits bekannte Frigocyclinon (89), bei dem anderen um ein neues Angucyclinon 88 mit Fridamycin-E-Grundkörper. Darüber hinaus wurden aus einem weiteren Streptomyces-Stamm (AK 671) zwei neue (97, 98) und drei (96, 99, 100) bekannte biosynthetisch interessante Sekundärmetabolite isoliert. / In detail, the following results were achieved during this doctoral thesis: A: Isolation and structural elucidation of naphthylisoquinoline alkaloids from Ancistrocladus species: • The not yet phytochemically investigated bark extracts of the Vietnamese subspecies Ancistrocladus tectorius ssp. cochinchinensis were analyzed. Four already known and three unknown naphthylisoquinoline alkaloids were identified. The three new metabolites were isolated and structurally elucidated by the use of different 2D NMR techniques. The defined structures – ancistrocladinium A (30) and its two O-demethyl derivatives 31 and 32 – are the first representatives of the new N,8'-naphthyl dihydroisoquinoline coupling type. These natural products exhibit promising biological activities – above all against the pathogen of leishmaniasis. • The botanically not yet fully characterized liana species ''A. ikela'', which was collected in the Congo Basin (Democratic Republic Congo), was morphologically and phytochemically investigated and described botanically. Besides the two N,C-coupled naphthylisoquinolines, ancistrocladinium A (30) and ancistrocladinium B [(M/P)-39], a new C,C-coupled alkaloid – 8-O-methylancistrogriffine C (40) – was isolated. Furthermore, by establishing a gradient system for the baseline separation of both atropodiastereomers of 39 HPLC-NMR and HPLC-CD analyses of the individual two purified epimers were performed to define the rotational barrier of the biaryl axis, which slowly rotates at room temperature. • From the leaves of the already well-investigated Indian Ancistrocladus species A. heyneanus, 6-O-methyl-8,4'-O-didemethylancistrocladine (42), a hitherto unknown naphthylisoquinoline alkaloid, was isolated. • A phytochemical investigation of the plant family of the Ancistrocladaceae with respect to the occurrence of N,C-coupled naphtylisoquinolines proved that these structurally exceptional alkaloids are quite common in this liana family. B: The role of the phloem during the propagation of pathogen-mediated signals: • Within the scope of the project B8 of the SFB 567, investigations on the role of the phloem during the propagation of long-distance signals after infection of plants of Arabidopsis thaliana with virulental or avirulental strains of Pseudomonas syringae pv. tomato were carried out. For this purpose initially an analytical method for the main metabolites – the glucosinolates – of A. thaliana applicable at a nL-scale was developed. Using this sensitive method, many known but also some new glucosinolates (8-methylsulfonyl-n-octyl-, 2-hydroxy-4-methylsulfinyl-n-butyl-, 2-hydroxy-4-methylsulfonyl-n-butyl-, and 4-hydroxy-benzoyloxymethyl-glucosinolate) were identified in plant extracts of A. thaliana. Furthermore, MS/MS analyses of the glucosinolates were carried out to verify their structures. Next to some typical fragments of the thiosugar moiety a few characteristic fragments of the different side chain structures (e.g. methylsulfinyl-n-alkyl- or methylthio-n-alkyl structure) were detected. Unfortunately, not all of the different types of side chaines led to typical fragmentation pattern. • In the phloem sap of entirely untreated plants, only methoxyglucobrassicin (73) and a phosphate 87 (1-glycero-1-myo-inositolphosphate), which was not kown so far to be located in plants, were identified in the course of the analysis of the phloem exudates. In the phloem saps of all the differently treated plants (infiltrated with MgCl2, virulental or avirulental Pseudomonas strain) all of the main leaf metabolites occurred. Solely a slight but not significant increase of the concentration of methoxyglucobrassicin (73) was measured in the phloem exudates of plants infected with avirulental pathogens. One has to be extremely careful with respect to the observed increase since it is possible that the enhanced glucosinolate concentration was the result of the strong mechanical stimulus of the infiltration procedure. Furthermore, slight changes in the concentration of other metabolites could be superimposed by signals caused by the infiltration procedure. C: Structural elucidation of polyketidic secondary metabolites from microorganisms • The structures of two low-molecular natural products isolated in the working group of Prof. H.-P. Fiedler (University of Tübingen) from the extremophilic Streptomyces strain KC 1030 were elucidated. One of them was the already known frigocyclinone (89) and the other one was the new angucyclinone 88 with a fridamycine E core structure. Moreover, from the Streptomyces strain AK 671, two new (97, 98) and three known (96, 99, 100) biosynthetically interesting secondary metabolites were isolated.

No description available.

Na célula eucariota, os processos metabólicos estão compartimentalizados em organelas e proteínas sintetizadas no citosol são endereçadas para elas por meio de sistema celular específico. Devido a sobreposições funcionais entre organelas, uma dada proteína pode ser requerida em mais de um compartimento. É o caso de proteínas com duplo direcionamento, em que um gene nuclear é capaz de gerar produtos protéicos direcionados para mais de uma organela. Cerca de 60 proteínas de plantas já tiveram seu duplo direcionamento demonstrado, a maioria para mitocôndrias e cloroplastos, portanto um fenômeno não tão raro como imaginado. Estudos recentes procuram esclarecer os mecanismos que permitem esse duplo direcionamento, mesmo existindo fatores celulares que garantem a especificidade do transporte para cada organela. O presente trabalho está dividido em três partes. Na primeira, foram investigados aspectos evolutivos do duplo direcionamento. Na análise comparativa de famílias gênicas com membros cujos produtos protéicos apresentam duplo direcionamento, em Arabidopsis thaliana e Oryza sativa, foi demonstrada a conservação do duplo direcionamento de monodeidroascorbato redutase, metionina aminopeptidase e, provavelmente, de THI1 (enzima da biossíntese de tiazol), entre as duas espécies. Os dados sugeriram um mesmo padrão de evolução em famílias incluindo membros com duplo direcionamento. Na segunda parte, o foco foi a seqüência de duplo direcionamento. Documentado o duplo direcionamento, para mitocôndrias e cloroplastos, de proteínas de ligação ao RNA, RBP1a, RBP1b e RPS19, mutações sítiodirigidas foram introduzidas na seqüência de direcionamento de RBP1b. A importância de aminoácidos positivos para o direcionamento de proteínas para mitocôndrias foi confirmada. Demonstrou-se que a mutação da alanina na posição 2, conservada em seqüências de direcionamento ambíguas para mitocôdrias e cloroplastos, não afeta o duplo direcionamento de RBP1b. A informação para o duplo direcionamento foi localizada entre os 17 primeiros aminoácidos da região amino-terminal. Os sinais de direcionamento para cloroplastos se distribuíram ao longo da seqüência. Enquanto a metade amino-terminal da seqüência foi suficiente para determinar o duplo direcionamento, a seqüência compreendendo os 13 aminoácidos seguintes afetaram a eficiência do transporte. Nessas análises, mostrou-se a adequação de método quantitativo na medida dos sinais de fluorescência da GFP, para ser aplicado em estudos quantitativos do direcionamento de proteínas para mitocôndrias e cloroplastos, in vivo. Na terceira parte, o foco foi o mecanismo traducional do duplo direcionamento de THI1, em A. thaliana. Entretanto, não foi possível testar a hipótese da presença de um sítio interno de entrada do ribossomo (IRES) no mRNA de thi1, pois não se encontrou um sistema de expressão transiente capaz de reproduzir dados da literatura que mostraram o duplo direcionamento de THI1. Nos sistemas testados, a proteína foi encontrada somente em cloroplastos, o que inviabilizou o prosseguimento da investigação. / Compartimentalization of the metabolic processes in organelles, each one having a characteristic protein pool and distinct functions, is a property of eukaryote cells. A highly specific cellular system directs proteins, which are synthesized in the cytosol, to the proper organelles. However, due to functional overlaps between organelles, a given protein may be needed in different compartments. This is the case of dual-targeted proteins, which are the product of single nuclear genes, but are somehow directed to different organelles. About 60 plant proteins have had their dual targeting demonstrated, most of them to mitochondria and cloroplasts, the phenomenon being not so rare as previously supposed. Investigations have focused on the mechanisms, which enable protein dual targeting, even in the presence of other cell mechanisms that guarantee the specific protein transport to each organelle. The present work on the subject can be divided in three parts. In the first part, evolutionary aspects of dual targeting were investigated. A comparative analysis of gene families that included members encoding dual-targeted proteins in Arabidopsis thaliana and Oryza sativa demonstrated that the dual targeting of monodehidro-ascorbate reductase, methyonine aminopeptidase and, problably, of the thiazole biosynthetic enzyme THI1 was evolutionary conserved between the two species. In addition, the data suggested the same pattern of evolution for families with members presenting dual targeting. The focus of the second part was the ambiguous sequence for dual targeting. After showing that the RNA-binding proteins RBP1a, RBP1b and RPS19 were dual-targeted to mitochondria and cloroplasts, sitedirected mutations were introduced in the targeting sequence of RBP1b. The importance of positive-charged amino acids for directing the protein to mitochondria was confirmed. Mutation of alanine at position 2, which is conserved in ambiguous sequences, was shown not to affect RBP1b dual targeting. Information for dual targeting was localized among the 17 first amino acids in the amino-terminal region. The signals for directing the protein to cloroplasts appeared distributed along the targeting sequence. While the amino-terminal half of the sequence was sufficient for RBP1b dual targeting, the sequence comprising the next 13 amino acids appeared to affect the efficiency of the transport. In these analyses, a quantitative method to measure the intensity of fluorescent signals of GFP had its efficacy demonstrated to be adopted for in vivo quantitative analysis of dual targeting to mitochondria and cloroplasts. In the third part, the focus was the translational mechanism enabling THI1 dual targeting to mitochondria and cloroplasts, in A. thaliana. It was hypothesized that an internal ribosomal entry site (IRES) was present in thi1 mRNA. However, a transient expression system could not be found that reproduced the literature data demonstrating THI1 dual targeting. In the tested systems the protein was addressed solely to cloroplasts, thus preventing the objective to be pursued.

Cheung Ming-yan. / Thesis submitted in 2000. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 169-185). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Abstract --- p.iii / Acknowledgement --- p.vi / General abbreviations --- p.viii / Abbreviations of chemicals --- p.x / List of figures --- p.xii / List of tables --- p.xv / Table of Contents --- p.xvii / Chapter Chapter 1 --- General Introduction - Using transgenic plants as bioreactor --- p.1 / Chapter 1.1 --- Plant as Bioreactor --- p.1 / Chapter 1.1.1 --- Plant transformation historical milestones --- p.1 / Chapter 1.1.2 --- Applications of transgenic plants --- p.5 / Chapter 1.1.2.1 --- Examples of in situ Application --- p.5 / Chapter 1.1.2.2 --- Examples of ex situ application of transgenic plant --- p.9 / Chapter 1.2 --- Plant Hosts for Transformation: Arabidopsis thaliana and Glycine max --- p.18 / Chapter 1.2.1 --- Essential components for plant transformation --- p.18 / Chapter 1.2.1.1 --- Marker genes --- p.18 / Chapter 1.2.1.2 --- Promoters --- p.18 / Chapter 1.2.2 --- Arabidopsis thaliana --- p.20 / Chapter 1.2.2.1 --- Agrobacterium-mediated transformation --- p.20 / Chapter 1.2.2.2 --- Transformation methods for A. thaliana --- p.21 / Chapter 1.2.3 --- Glycine max (soybean) --- p.22 / Chapter 1.2.3.1 --- Soybean cultivars for transformation --- p.23 / Chapter 1.2.3.2 --- Soybean regeneration systems --- p.24 / Chapter 1.2.3.3 --- Soybean transformation systems --- p.26 / Chapter 1.3 --- Target Pharmaceutical and Agricultural Proteins: Lymphocytic choriomeningitis virus and Goldfish Growth hormones I and II --- p.29 / Chapter 1.3.1 --- Production of pharmaceutical proteins --- p.29 / Chapter 1.3.1.1 --- Lymphocytic choriomeningitis virus --- p.30 / Chapter 1.3.1.2 --- Nucleoprotein of LCMV --- p.33 / Chapter 1.3.2 --- Agricultural protein category --- p.34 / Chapter 1.3.2.1 --- Carassius auratus --- p.34 / Chapter 1.3.2.2 --- Growth hormones I and II --- p.35 / Chapter 1.4 --- Hypothesis and Objectives --- p.37 / Chapter Chapter 2 --- Materials and Methods --- p.38 / Chapter 2.1 --- Materials --- p.38 / Chapter 2.1.1 --- "Plants, bacterial strains and vectors" --- p.38 / Chapter 2.1.2 --- Chemicals and Regents --- p.43 / Chapter 2.1.3 --- Commercial kits --- p.44 / Chapter 2.1.4 --- Primers and Adaptors --- p.45 / Chapter 2.1.5 --- Equipments and Facilities used --- p.47 / Chapter 2.1.6 --- "Buffer, solution and medium" --- p.47 / Chapter 2.2 --- Methods --- p.48 / Chapter 2.2.1 --- Molecular Techniques --- p.48 / Chapter 2.2.1.1 --- Bacterial cultures for recombinant DNA and plant transformation --- p.48 / Chapter 2.2.1.2 --- Recombinant DNA techniques --- p.48 / Chapter 2.2.1.3 --- "Preparation and transformation of DH5a, DE3 and Agrobacterium competent cells" --- p.49 / Chapter 2.2.1.4 --- Gel electrophoresis --- p.52 / Chapter 2.2.1.5 --- "DNA, RNA and protein extractions" --- p.53 / Chapter 2.2.1.6 --- Generation of cRNA probes for Southern and Northern blot analyses --- p.56 / Chapter 2.2.1.7 --- Southern blot analysis --- p.56 / Chapter 2.2.1.8 --- Northern blot analysis --- p.57 / Chapter 2.2.1.9 --- Expression of Lymphocytic choriomeningitis virus nucleoprotein (LCMV NP) in bacterial system --- p.58 / Chapter 2.2.1.10 --- Western blot analysis for LCMV NP --- p.59 / Chapter 2.2.1.11 --- Protein dot blot for detecting the presence of recombinant LCMV-NP generated from transgenic plants --- p.62 / Chapter 2.2.1.12 --- PCR techniques --- p.62 / Chapter 2.2.1.13 --- Sequencing --- p.63 / Chapter 2.2.2 --- Plant tissue culture and transformation --- p.64 / Chapter 2.2.2.1 --- Arabidopsis thaliana --- p.64 / Chapter 2.2.2.2 --- Soybean --- p.65 / Chapter 2.2.3 --- In vitro transcription and translation of target genes in rabbit reticulocyte and wheat germ systems --- p.68 / Chapter 2.2.3.1 --- In vitro transcription of target genes with with Ribomix large scale RNA production systems-T7 and SP6 (Promega) --- p.68 / Chapter 2.2.3.2 --- In vitro translation with rabbit reticulocyte lysate and wheat germ extract --- p.69 / Chapter Chapter 3 --- Results --- p.71 / Chapter 3.1 --- Expression of Lymphocytic choriomeningitis virus nucleoprotein (LCMV NP) and goldfish growth hormones I and II (GHI and GHII) in transgenic Arabidopsis thaliana --- p.71 / Chapter 3.1.1 --- Expression of LCMV-NP in transgenic Arabidopsis thaliana --- p.71 / Chapter 3.1.1.1 --- Cloning of the gene encoding LCMV NP into the binary vector system W104 --- p.71 / Chapter 3.1.1.2 --- Transformation of W104-LCMV-NP into the Agrobacterium GV3101/pMP90 --- p.78 / Chapter 3.1.1.3 --- Transformation of LCMV-NP cDNA into Arabidopsis thaliana --- p.80 / Chapter 3.1.1.4 --- Southern blot and Northern blot analyses of transgenic plant containing the LCMV-NP cDNA --- p.83 / Chapter 3.1.1.5 --- Production of recombinant LCMV-NP protein in DE3 cells --- p.90 / Chapter 3.1.1.6 --- Detection of recombinant LCMV-NP protein in transgenic A.thaliana --- p.98 / Chapter 3.1.2 --- Expression of goldfish growth hormones I and II (GHI and GHII) in transgenic Arabidopsis thaliana --- p.105 / Chapter 3.1.2.1 --- "Screening of homozygous lines of goldfish, Carassius auratus, growth hormones transgenic Arabidopsis thaliana" --- p.105 / Chapter 3.1.2.2 --- Southern blot and Northern blot analyses of transgenic plant containing the LCMV-NP cDNA --- p.109 / Chapter 3.1.2.3 --- Detection of recombinant GHI and GHII from transgenic plant --- p.112 / Chapter 3.2 --- In vitro transcription and translation of target genes in rabbit reticulocyte and wheat germ systems --- p.117 / Chapter 3.2.1 --- Subcloning of target genes in pGEM-3Zf(+) vector --- p.117 / Chapter 3.2.1.1 --- Subcloning of LCMV-NP fragment into pGEM-3Zf(+) vector --- p.117 / Chapter 3.2.1.2 --- Subcloning of goldfish GHI and GHII fragments into pGEM-3Zf(+) vector --- p.120 / Chapter 3.2.2 --- In vitro transcription of target genes with Ribomix large scale RNA production systems-T7 and SP6 --- p.125 / Chapter 3.2.3 --- In vitro translation with rabbit reticulocyte lysate and wheat germ extract systems --- p.128 / Chapter 3.3 --- Establishment of Glycine max regeneration and transformation systems --- p.130 / Chapter 3.3.1 --- The Establishment of soybean regeneration system --- p.130 / Chapter 3.3.2 --- Establishment of soybean transformation system --- p.133 / Chapter 3.3.2.1 --- Definition of transformation efficiency --- p.133 / Chapter 3.3.2.2 --- Effects of plant hosts --- p.136 / Chapter 3.3.2.3 --- Effects of Agrobacterium strains --- p.138 / Chapter 3.3.2.4 --- The application of vacuum infiltration --- p.139 / Chapter 3.3.2.5 --- Effect of kanamycin --- p.140 / Chapter 3.3.2.6 --- Effect of cocultivation duration and light/ dark treatment during germination --- p.141 / Chapter 3.3.2.7 --- Application of the detergent Silwet-77 --- p.142 / Chapter 3.3.3 --- Verification of transformation results by PCR screening --- p.143 / Chapter Chapter 4 --- Discussion --- p.147 / Chapter 4.1 --- "Expression of LCMV-NP, GHI and GHII in A. thaliana" --- p.148 / Chapter 4.2 --- Establishing a soybean transformation system --- p.157 / Chapter 4.2.1 --- Plant hosts and explants --- p.158 / Chapter 4.2.2 --- Regeneration of explants --- p.159 / Chapter 4.2.3 --- Agrobacterium strains --- p.161 / Chapter 4.2.4 --- Bacteria-plant interaction --- p.161 / Chapter 4.2.5 --- Transient versus stable transformation --- p.165 / Chapter 4.3 --- Conclusion and perspective --- p.167 / References --- p.169 / Appendix --- p.186

Chan Yee-kwan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 108-131). / Abstracts in English and Chinese. / Thesis Committee --- p.i / Abstract --- p.ii / Acknowledgments --- p.v / General Abbreviations --- p.vii / Abbreviation for Chemicals --- p.x / Table of Contents --- p.xi / List of Figures --- p.xiv / List of Tables --- p.xv / Chapter 1. --- Literature Review / Chapter 1.1 --- Salinity as a global problem --- p.1 / Chapter 1.2 --- Salinity and agriculture --- p.2 / Chapter 1.3 --- Plant adaptation to salinity --- p.4 / Chapter 1.3.1 --- Salt secretion --- p.6 / Chapter 1.3.2 --- Ion transport --- p.8 / Chapter 1.3.2.1 --- Role of H+-ATPase in salt tolerance --- p.8 / Chapter 1.3.2.2 --- Potassium and sodium uptake --- p.13 / Chapter 1.3.2.3 --- Sodium efflux --- p.15 / Chapter 1.3.3 --- Osmotic adjustment --- p.20 / Chapter 1.3.3.1 --- Accumulation of mannitol --- p.21 / Chapter 1.3.3.2 --- Accumulation of proline --- p.23 / Chapter 1.3.3.3 --- Accumulation of glycinebetaine --- p.23 / Chapter 2. --- Materials and Methods / Chapter 2.1 --- Plant materials and growth conditions --- p.26 / Chapter 2.1.1 --- Surface sterilization of Arabidopsis seeds --- p.26 / Chapter 2.1.2 --- Determination of sub-lethal inhibitory doses of sodium --- p.27 / Chapter 2.1.3 --- Growth conditions of Arabidopsis seeds for total RNA extraction --- p.27 / Chapter 2.1.4 --- NaCl dosage tests --- p.28 / Chapter 2.1.5 --- Expression kinetic tests --- p.28 / Chapter 2.2 --- Isolation of total RNAs --- p.28 / Chapter 2.3 --- Isolation of genes differentially expressed in NaCl concentration by RAP-PCR --- p.30 / Chapter 2.3.1 --- RNA fingerprinting by RAP-PCR --- p.30 / Chapter 2.3.2 --- PCR reamplificatin of RAP products --- p.31 / Chapter 2.3.3 --- Cloning of differentially expressed genes --- p.33 / Chapter 2.3.3.1 --- Ligation of inserts into pCR-Script vector and transformation --- p.33 / Chapter 2.3.3.2 --- Ligation of inserts into pBluescript II KS (+) T-vector and transformation --- p.36 / Chapter 2.3.3.3 --- Screening of recombinant plasmids --- p.37 / Chapter 2.4 --- Sequencing of differentially expressed genes --- p.39 / Chapter 2.4.1 --- DNA cycle sequencing --- p.39 / Chapter 2.5 --- Northern blot hybridization of NaCl inducible genes --- p.40 / Chapter 2.5.1 --- RNA fractionation by formaldehyde gel electrophoresis --- p.40 / Chapter 2.5.2 --- Northern blotting --- p.41 / Chapter 2.5.3 --- Preparation of single-stranded DIG-labeled PCR probes --- p.41 / Chapter 2.5.3.1 --- Isolation of Total RNA --- p.41 / Chapter 2.5.3.2 --- Primer design --- p.42 / Chapter 2.5.3.3 --- PCR amplification of single-stranded DIG PCR probes --- p.43 / Chapter 2.5.4 --- Hybridization --- p.45 / Chapter 2.5.5 --- Stringency washes --- p.46 / Chapter 2.5.6 --- Chemiluminescent detection --- p.46 / Chapter 3. --- Results / Chapter 3.1 --- Determination of sub-lethal inhibitory doses of sodium --- p.48 / Chapter 3.2 --- Isolation of total RNA from A. thaliana treated with sodium chloride --- p.48 / Chapter 3.3 --- Isolation of genes differentially expressed in sodium concentration by RNA arbitrarily primed polymerase chain reaction RAP-PCR --- p.52 / Chapter 3.3.1 --- Differential cDNA fragments identified by RAP-PCR --- p.52 / Chapter 3.3.2 --- PCR reamplification of RAP products --- p.52 / Chapter 3.3.3 --- Cloning of selected RAP-fragments --- p.62 / Chapter 3.4 --- Nucleotide sequence analysis of selected RAP PCR clones --- p.65 / Chapter 3.5 --- Expression pattern analysis of salt inducible genes by northern blot hybridization --- p.75 / Chapter 3.5.1 --- Preparation of single-stranded digoxigenin (DIG)-labeled probes --- p.75 / Chapter 3.5.2 --- Dosage response of NaCl inducible genes --- p.79 / Chapter 3.5.3 --- Expression kinetics of NaCl inducible genes --- p.80 / Chapter 4. --- Discussion / Chapter 4.1 --- Isolation of RAP-PCR targets --- p.93 / Chapter 4.2 --- Expression of NaCl inducible P450 genes --- p.94 / Chapter 4.2.1 --- Cytochrome P450 CYP73A5 --- p.97 / Chapter 4.2.2 --- Cytochrome P450 CYP83A1 --- p.98 / Chapter 4.3 --- NaCl induction gene related to post-transcriptional activities --- p.99 / Chapter 4.3.1 --- Glycine-rich RNA binding protein (BAC F3F19) --- p.100 / Chapter 4.3.2 --- Chloroplast signal recognition particle (54CP) --- p.103 / Chapter 4.4 --- Conclusion --- p.106 / References --- p.108

Chan Hiu-ki. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 97-112). / Abstracts in English and Chinese. / Thesis Committee --- p.i / 摘要 --- p.ii / Abstract --- p.iii / Acknowledgements --- p.v / Abbreviations --- p.vi / Table of Contents --- p.vii / List of figures --- p.xi / List of tables --- p.xiii / Chapter Chapter 1 --- Literature Review --- p.1 / Chapter 1.1 --- Nitrogen assimilation in plants --- p.1 / Chapter 1.2 --- Importance of asparagine in plants --- p.5 / Chapter 1.3 --- Enzymatic reaction of asparagine synthetase (AS) --- p.8 / Chapter 1.4 --- Asparagine synthetase of non-plant organisms --- p.10 / Chapter 1.5 --- Biochemistry background of plant asparagine synthetases --- p.12 / Chapter 1.6 --- Molecular studies of asparagine synthetase genes in plants --- p.15 / Chapter 1.7 --- Arabidopsis thaliana as a model plant --- p.24 / Chapter 1.8 --- ASN studies in Arabidopsis thaliana --- p.24 / Chapter 1.9 --- Hypothesis --- p.27 / Chapter Chapter 2 --- Materials and Methods --- p.29 / Chapter 2.1 --- Chemicals --- p.29 / Chapter 2.2 --- Plant materials and growth conditions --- p.29 / Chapter 2.2.1 --- Surface sterilization of Arabidopsis seeds --- p.29 / Chapter 2.2.2 --- "Growth conditions of Arabidopsis seeds for total RNA extraction, enzyme assay, chlorophyll content measurement and dry weight measurement" --- p.30 / Chapter 2.3 --- Agrobacterium mediated transformation via vacuum infiltration method --- p.30 / Chapter 2.3.1 --- Principles --- p.30 / Chapter 2.3.2 --- Plant materials and bacterial strains of Agrobacterium mediated transformation --- p.31 / Chapter 2.3.2.1 --- Plant materials --- p.31 / Chapter 2.3.2.2 --- Gene constructs --- p.31 / Chapter 2.3.2.2 --- Bacterial strains --- p.32 / Chapter 2.3.3 --- Agrobacterium mediated transformation via vacuum infilitration --- p.32 / Chapter 2.4 --- Screening of transformants --- p.33 / Chapter 2.5 --- DNA and RNA manipulation --- p.34 / Chapter 2.5.1 --- DNA extraction and quantitation --- p.34 / Chapter 2.5.2 --- PCR amplification and detection of transgenes --- p.36 / Chapter 2.5.2.1 --- PCR amplification and detection of transgenes --- p.36 / Chapter 2.5.2.2 --- Primer sequence --- p.37 / Chapter 2.6 --- RNA analysis of transormants --- p.38 / Chapter 2.6.1 --- General introduction --- p.38 / Chapter 2.6.2 --- RNA extraction --- p.39 / Chapter 2.6.3 --- Making single-strand DIG PCR probes --- p.40 / Chapter 2.6.4 --- Quantitation of single-strand DIG-labeled probes --- p.42 / Chapter 2.7 --- Northern blot analysis --- p.42 / Chapter 2.7.1 --- Detection --- p.43 / Chapter 2.7.2 --- Film development --- p.43 / Chapter 2.8 --- "Amino acid, protein, dry weight and total nitrogen analysis" --- p.43 / Chapter 2.8.1 --- Extraction of free amino acids --- p.43 / Chapter 2.8.2 --- Protein assay --- p.44 / Chapter 2.8.3 --- Determination of nitrogen and carbon content in seeds --- p.45 / Chapter 2.8.4 --- Dry weight measurement --- p.45 / Chapter 2.8.5 --- Seed storage protein analyses --- p.45 / Chapter 2.8.6 --- Detection of chlorophyll content --- p.46 / Chapter 2.9 --- Asparagine synthetase activity analysis --- p.46 / Chapter 2.9.1 --- Crude extracts preparation --- p.46 / Chapter 2.9.2 --- AS enzyme assay --- p.47 / Chapter 2.9.3 --- Asparagine content measurement --- p.47 / Chapter 2.10 --- In situ hybridization --- p.48 / Chapter 2.10.1 --- Making cRNA probe --- p.48 / Chapter 2.10.2 --- In situ hybridization --- p.48 / Chapter Chapter 3 --- Results --- p.50 / Chapter 3.1 --- Construction of ASN1 overexpressing lines --- p.50 / Chapter 3.2 --- Changes in nitrogen status during vegetative growth of ASN1 overexpressing lines --- p.53 / Chapter 3.3 --- Changes in nitrogen status during seed development of ASN1 overexpressing lines --- p.55 / Chapter Chapter 4 --- Discussion --- p.76 / Chapter Chapter 5 --- Conclusion --- p.85 / Chapter Chapter 6 --- Perspective --- p.86 / Appendix --- p.87 / References --- p.97

AN ABSTRACT OF THE DISSERTATION OF BELAN M. KHALIL, for the Doctor of Philosophy degree in Plant Biology, presented July 11, 2018, at Southern Illinois University Carbondale. TITLE: ANALYSIS OF THE CIS-REGULATORY ELEMENT LEXICON IN UPSTREAM GENE PROMOTERS OF ARABIDOPSIS THALIANA AND ORYZA SATIVA. MAJOR PROFESSOR: Dr Matt Geisler Gene expression in plants is partly regulated through an interaction of trans-acting factors with the promoter regions of the gene. Trans-acting factor binding sites consist of short nucleotide sequences most often present in the upstream promoter region. These binding sites, the cis-regulatory elements (CREs), vary in structure, complexity and function. In binding to trans-acting factors, CREs connect genes to signalling and regulatory pathways that affect plant growth, development, and response to the environment. As words in a language, CREs and thus promoters can be analyzed by looking for spelling (patterns of nucleotides) associated with meaning (functions). Considering CREs as words in a language, this kind of analysis provides a great opportunity for comprehensive understanding of promoter language. Identification and characterization of CREs are challenging either experimentally or bioinformatically, and has previously been accomplished by discovering degenerate words, with ambiguous nucleotides. This kind of result implicitly makes a hypothesis that binding of a specific trans-acting factor is somewhat promiscuous (or sloppy) and that all words represented by a degenerate pattern are equally good at binding. In this study, we unpack the “degeneracy hypothesis” by systematically considering each combination of letters independently for CRE function. Our results demonstrate that not all degenerate combinations of published CREs have the same effect on gene expression. A systematic search and comparison of all 65,536 possible 8 bp CRE words were searched in the 500 bp and 1000 bp upstream promoters of all genes in Arabidopsis thaliana and Oryza sativa, respectively. The function of each CRE was evaluated by statistically comparing the presence or absence of the element in the promoter with that genes response (induction or suppression) to stimuli in 1691 public availability transcriptomes of differential gene expression data. Arabidopsis, a model dicot plant had a much larger number of such data sets, than rice, however rice was chosen as a comparison as it had the largest number of datasets for a monocot, the most distantly related plant group with sufficient data available. A comprehensive list of 8 bp words associated with differential gene expression, linguistically known as lexicon, was retrieved for both species by establishing that the presence of a CRE significantly increased the likelihood for differential expression by at least one stimulus. The lexicons were composed of 641 and 856 CREs respectively in Arabidopsis and rice, and there were only 78 shared CREs between the two lexicons. The CRE lexicon was then characterized for their strength and breadth of response, occurrence frequency, sequence complexity, and sequence conservation between two species. In Arabidopsis, evening element (EE) showed the strongest response to a cold stress transcriptome (p-value 10-99). In rice, the element AAACCCTA showed strongest response to a tissue specific transcriptome (p-value 10-79). The breadth of response varied between the two species due to number of transcriptomes used in the study. The element AAACCCTA and GCGGCGGA significantly correlated to 197 and 58 transcriptomes in both Arabidopsis and rice, respectively. On the other side of the breadth scale there were also many CREs with very restricted response. There were 291 and 258 CREs in Arabidopsis and rice, respectively, significantly correlated to a single stimulus. Occurrence frequency revealed that the most abundant CREs in Arabidopsis and rice genes were TATA box and TATA box like CREs. The structure of the CREs in the lexicon was also varied. CREs were distributed on seven levels of complexity. Level one comprised CREs having 8 copies of the same nucleotide, level seven comprised CREs having two copies of the same nucleotide. In Arabidopsis, out of 641 CREs, 314 were of level 6 complexity, which means having 3 copies of the same nucleotide. In rice, the majority of the lexicon, 263 CREs were of level 5 complexity, which means having 4 copies of the same nucleotide. Each CRE of the lexicon was correlated to at least one experimental condition in the differential gene expression data, but many were correlated to multiple and often related conditions such as drought, temperature and salinity. Therefore, each CRE was assigned a “meaning”, i.e. the associated stimuli, thus providing a sort of CRE function dictionary in addition to the lexicon itself. Many CREs possessed different meanings (termed homographs in language), and in many cases the meanings of different CREs overlapped like language synonyms. Sharing meanings (synonyms) was often among CREs with strong sequence similarity (homonyms or homophones), however, not in all cases. Analyzed as a linguistic aspect, CRE homonymity and synonymity was applied to explore the hypothesis “all CRE synonyms are also homonyms and all CRE homonyms are also synonyms.” To the end a single CRE was compared to all possible CREs with only one letter mismatch in their sequences are considered as homonyms. The CREs meaning was converted to a matrix of stimuli to generate clusters of synonyms that were analyzed for similarity of spelling (sequence). This analysis showed that not all homonyms are synonyms, however most synonyms are homonyms. Furthermore, despite a search of all one letter mismatches among homonyms, many of the functional homonyms shared smaller 4-5bp core sequence and only varied at the flanks. Synonyms being homonyms in the language of promoters raises a question, how did this evolve? Duplication of transcription factors in the genome generated transcription factor families where each family member shares the same core domain, usually a DNA recognition site. We here propose that CREs also duplicate during gene duplication process building CRE families in parallel. Members of CRE families may show different connectivity and affinity to individual members of transcription factors in a transcription factor family. In environmental sensors and developmental decision panel, this association of two families of interaction factors is called dense overlapping region (or DOR) and is a highly overrepresented network topology in biological systems. This also explains the degeneracy of initially discovered CREs. The fact is only a portion of nucleotide combinations implied by a degenerate CRE is bioactive, it represents an overlap of different members of a CRE family which is part of the process of family expansion and diversification and done as compensatory mutations as the family of transcription factors expanded and diversified. We also extensively studied CREs involved abiotic stress and identifies shared elements among abiotic stresses as well as abiotic stress specific CREs. Furthermore, CREs follow a time-sensitive response rule, which means some CREs participates in gene expression regulation only at a certain period during the course of exposure to the abiotic stress.

P1B-type ATPases transport a number of monovalent and divalent heavy metals (Cu+, Cu2+, Ag+, Zn2+, Cd2+, Pb2+ and Co+2) across biological membranes. These ATPases are found in archea, bacteria and eukaryotes and are one of the key elements required for maintaining metal homeostasis. Plants have an unusually high number of P1B-type ATPases with distinct metal selectivity compared to other eukaryotes that usually have one or two Cu+-ATPases. Higher plants are the only eukaryotes where Zn2+-ATPases have been identified. Towards understanding the physiological roles of plant Zn2+-ATPases, we characterized Arabidopsis thaliana HMA2. We expressed HMA2 in yeast and measured the metal dependent ATPase activity in membranes. We showed that HMA2 is a Zn2+-ATPase that is also activated by Cd2+. Zn2+ transport determinations showed that this enzyme drives the efflux of metal from the cytoplasm. Analysis of HMA2 mRNA levels showed that the enzyme is present in all plant organs. We analyzed the effect of removal of HMA2 full-length transcript in whole plants by gene knock out. Although hma2 mutants did not show a different visible phenotype from the wild type plants, we observed increased levels of Zn2+ or Cd2+. The observed phenotype of hma2 mutants and plasma membrane location of HMA2, mainly in vasculature (Hussain et al., 2004), indicates that this ATPase might have a central role in Zn2+ uploading into the phloem. P1B-type ATPases have cytoplasmic regulatory metal binding domains (MBDs) in addition to transmembrane metal binding sites (TMBDs). Plant Zn2+-ATPases have distinct sequences in both their N- and C-termini that might contribute to novel metal binding sites. These ATPases contain long C-terminal sequences rich in CC dipeptides and His repeats. Removal of the C-terminus (C-MBD) of HMA2 leads to a 50% reduction in the enzyme turnover suggesting a regulatory role for this domain. Atomic Absorption Spectroscopy (AAS) analysis showed that Zn2+ binds to C-MBD with a stoichiometry of three (3 Zn/C-MBD). Chemical modification studies and Zn K-edge X-ray Absorption Spectroscopy (XAS) of Zn-C-MBD showed that Zn2+ is likely coordinated by His in two sites and the third site slightly differs from the others involving a Cys together with three His. All plant Zn2+-ATPases lack the typical CXXC signature sequences observed in Cu+-ATPases and some bacterial Zn2+-ATPases N-terminus metal binding domains (N-MBDs). Instead, these have conserved CCXXE sequences. Truncation of HMA2 N-MBD results in a 50% decrease in enzyme Vmax suggesting that N-MBD is also a regulatory domain. The results indicate that the N-MBD binds Zn2+ with a stoichiometry of one (1 Zn/N-MBD). Metal binding analysis of individual N-MBD mutants Cys17Ala, Cys18Ala and Glu21Ala/Cys prevented Zn+2 binding to HMA2 N-MBD suggesting the involvement of all these residues in metal coordination. ATPase activity measurements with HMA2 carrying the mutations Cys17Ala, Cys18Ala and Glu21Ala/Cys showed a reduction in the enzyme activity similar to that observed the truncated protein indicating that the enzyme activity reduction observed in the N-terminus truncated forms of the enzyme is related to the removal of the metal binding capability. Summaryzing, these studies show the central role of HMA2 in plant Zn2+ homeostasis. They also describe the mechanism and direction of Zn2+ transport. Finally, they establish the presence of novel metal binding domains in the cytoplasmic portion of the enzyme. Metal binding to these domains is required for full enzymatic activity.

Turnip crinkle virus (TCV) has been implicated in the suppression of the hypersensitive response (HR), a type of programmed cell death induced during active resistance in Arabidopsis thaliana. In order to investigate the involvement of individual viral components in mediating suppression, TCV genes were cloned for use in an Agrobacterium tumefaciens mediated transient expression in Nicotiana benthamiana. Agroinfiltration of the HR-inducing avrPto/Pto system in conjunction with individual TCV genes has identified the p38 gene, which encodes the viral coat protein, as the gene responsible for the cell death suppression phenotype. The extent of cell death suppression by coat protein was quantified and found to be equal to the level of suppression by the whole virus and AvrPtoB, another cell death inhibitor from bacteria. Thus, the coat protein alone is sufficient to inhibit the HR in plants. Further, the effect of TCV on HR initiation by an avirulence factor from an unrelated bacterial pathogen was investigated. The presence of TCV does not affect the production, secretion or cellular processing of the bacterial avirulence factor.

Metal homeostasis in plants is regulated by diverse mechanisms that act together to maintain optimal metal ion concentrations inside the cell. P1B-ATPases are heavy metal transport ATPases that are likely to be related to these processes. The sequencing of the genome of Arabidopsis thaliana revealed the presence of eight putative P1B-ATPases, HMA1-8. The main goal in this work is to characterize of the role of P1B-ATPases in plant metal homeostasis. Toward this goal, the P1B-ATPases HMA1 and HMA6 from Arabidopsis thaliana were cloned from leaves and sequenced. Results from RT-PCR experiments show ubiquitous expression in planta of this two ATPases, except for HMA1 that does not express in roots. Upon Cu2+ exposure during growth, expression of HMA6 increases in seedlings. HMA1 expression increases when seedlings are grown in high Cu2+ and Co2+ media, and decreases when grown in high concentrations of Zn2+ and Ni2+. hma1-1 plants have smaller size and less chlorophyll content than WT plants. Growth is affected in hma1-1 seedlings when grown in Zn2+, Mn2+, Fe2+, Co2+ and Cu2+ deficient media, or when these metals are in excess. Moreover, hma1-1 plants show an increase in Zn2+, Mn2+ and Fe2+ content in whole plants compared to WT plants. Mutant plants also show increased levels of HMA3 and HMA4 transcripts (Zn2+/Cd2+/Pb2+ P1B-ATPases), upregulation of metallothioneins 1a and 2b, downregulation of metallothionein 1c, and a decrease in the phytochellatin synthases 1 and 2 transcripts, compared to WT plants. Homozygous for mutation in HMA6 seems to be lethal, given that none was recovered after screening. These results indicate HMA1 and HMA6 as essential components of plant metal homeostasis in Arabidopsis thaliana.