Transcriptomics of the Rooibos (Aspalathus linearis) Species Complex

Rooibos (Aspalathus linearis), widely known as a herbal tea, is endemic to the Cape Floristic Region of South Africa (SA). It produces a wide range of phenolic compounds that have been associated with diverse health-promoting properties of the plant. The species comprises several growth forms that differ in their morphology and biochemical composition, only one of which is cultivated and used commercially. Here, we established methodologies for non-invasive transcriptome research of wild-growing South African plant species, including (1) harvesting and transport of plant material suitable for RNA sequencing; (2) inexpensive, high-throughput biochemical sample screening; (3) extraction of high-quality RNA from recalcitrant, polysaccharide- and polyphenol-rich plant material; and (4) biocomputational analysis of Illumina sequencing data, together with the evaluation of programs for transcriptome assembly (Trinity, IDBA-Trans, SOAPdenovo-Trans, CLC), protein prediction, as well as functional and taxonomic transcript annotation. In the process, we established a biochemically characterized sample pool from 44 distinct rooibos ecotypes (1–5 harvests) and generated four in-depth annotated transcriptomes (each comprising on average ≈86,000 transcripts) from rooibos plants that represent distinct growth forms and differ in their biochemical profiles. These resources will serve future rooibos research and plant breeding endeavors

Unraveling the transcriptome of Aspalathus linearis (Rooibos) towards identification of novel genes involved in polyphenol biosynthesis

Philosophiae Doctor - PhDSouth Africa (SA) is home to one of the six floral kingdoms of the world, and hosts a very diverse flora comprising an astonishing ~30,000 species. Herbal medicines play an important role in many of the diverse cultures of this country. Yet, agricultural production systems for most of these species are missing, and medicinal plants are usually collected in the wild. The endemic medicinal plants of SA produce a wide range of rare medicinally active compounds, which could be developed into drugs. Knowledge on the genes involved in the biosynthesis of these compounds could not only promote establishment of plant production systems, but also their biotechnological exploitation. Transcriptomics has been revolutionized by Next Generation Sequencing technologies, which can cost-efficiently provide a lot of information on plant genes and biosynthetic pathways. This thesis focuses on the establishment of methodologies for high-throughput plant transcriptome research, including: 1) harvesting plant material suitable for high-quality RNA analysis from distant locations, 2) high-throughput, and inexpensive biochemical sample screening, 3) extraction of high-quality RNA from recalcitrant, polysaccharide- and polyphenol rich plant material, and 4) biocomputational analysis of Illumina sequencing data, including quality control and pre-processing of data, de novo assembly of reads, protein prediction and functional transcriptome annotation. Rooibos (Aspalathus linearis) was chosen as the pilot plant, because it is one of the few indigenous SA medicinal plants that has been successfully cultivated as a commercial crop. It produces a wide range of phenolic compounds with health promoting properties (e.g. aspalathin and a phenylpropenoic acid glucoside with scientifically verified antidiabetic and cardioprotective effects). In the course of this study, seven rooibos transcriptomes were produced, assembled and functionally annotated, providing a first extensive dataset for identification of genes associated with economically important traits such as medicinal compound production, rooibos growth type characteristics and stress resistance

Roles of disproportionating enzymes in the moss Physcomitrella patens

Thesis (MSc)--Stellenbosch University, 2015.ENGLISH ABSTRACT: Starch is a polyglucan made up of the two glucose polymers, amylose and amylopectin. Plants use starch to store excess carbohydrates from photosynthesis which get used for growth during the night. Starch metabolism is well undertood in higher plants such as A. thaliana thaliana and Solanum tuberosum with well-established pathways worked out for the enzymes involved in its synthesis and degradation. The bryophyte Physcomitrella patens has emerged as a popular choice for studying gene function in lower plants both because its genome has been sequenced and because of the ease of establishing knockout mutants via homologous recombination. Many metabolic functions have been studied in P. patens but, until now, little has been done in examining starch metabolism in moss. This study focused on two enzymes that have been found to be involved in starch degradation in higher plants, Disproportionating enzyme 1 (DPE1) and Disproportionating enzyme 2 (DPE2). DPE1 isoforms have been found to break down malto-oligosaccharides, which are products of starch degradation, into glucose within the chloroplast. On the other hand DPE2 catabolizes maltose to glucose in the cytosol. Higher plants that were silenced in these two genes were unable to degrade starch effectively, which lead to an increase in starch, malto-oligosaccharides or maltose and reduced growth. Three orthologs were identified for DPE1 in P. patens (PpDPE1A, B and C) and one for DPE2 (PpDPE2). Only PpDPE1B and PpDPE1C were found to be expressed in P. patens at the beginning of the light period but further investigation would be necessary at different time points as these genes were shown to be optimally expressed at the end of the light period. Targeted gene knockouts were made for each in P. patens which showed a reduced growth phenotype for all, indicating that these genes do play a role in starch catabolism that influences growth. There was, however, no significant change in starch content between the mutant lines and wild type (Wt). GFP fusion proteins showed PpDPE2 to be localized in cytosol, in close proximity to the chloroplast membrane. Similar findings have been found for DPE2 in A. thaliana and S. tuberosum. We hypothesize that PpDPE2 may play a role in cold tolerance in moss as an increase in starch breakdown has been witnessed in cold treated moss as well as increased transcript levels of starch metabolism genes and a maltose transporter. This opens a door to the further study of these generated mutant lines under cold stress.AFRIKAANSE OPSOMMING: Stysel is ‘n poliglukaan wat bestaan uit die twee glukose polimere: amilose en amilopektien. Plante gebruik stysel om oortollige koolhidrate van fotosintese wat vir groei gebruik word gedurende die nag te berg. Styselmetabolisme in hoër plante soos A. thaliana thaliana en Solanum tuberosum word goed verstaan, met gevestigde paaie uitgewerk vir die ensieme wat betrokke is by die sintese en afbreek daarvan. Die briofiet Physcomitrella patens is ‘n populêre keuse vir die bestudering van geenfunksie in laer plante, omdat die genoomvolgorde bepaal is en as gevolg van die gemak waarmee ‘uitklop’-mutante via homoloë rekombinasie gevorm kan word. Baie metaboliese funksies is bestudeer in P. patens maar tot nou is min gedoen om die styselmetabolisme in mos te ondersoek. Hierdie studie het gefokus op twee ensieme, DPE1 and DPE2, wat gevind is om betrokke is afbreek van stysel in hoër plante. Dit is voorheen bevind dat DPE1 isoforme malto-oligosakkariedes (wat produkte is van styselafbraak) afbreek na glukose in the chloroplast. Aan die ander kant kataboliseer DPE2 maltose na glukose in die sitosol. Hoër plante waarin hierdie gene stilgemaak is, is nie instaat daartoe om stysel effektief af te breek nie. Dit lei tot ‘n verhoging in stysel, malto-oligosakkariede of maltose en verminderde groei. Drie ortoloë is geïdentifiseer vir DPE1 in P. patens (PpDPE1A, B en C) en een vir DPE2 (PpDPE2). Slegs PpDPE1B en PpDPE1C word uitgedruk in P. patens aan die begin van die ligperiode, maar verder ondersoek sal nodig wees op verskillende tydpunte, omdat dit bewys is dat hierdie gene optimaal uitgedruk word tydens die einde van die ligperiode. Geteikende geen uiklop-mutante is gemaak vir elk in P. patens wat ‘n verminderde-groei fenotipe vertoon het vir almal, wat aandui dat hierdie gene ‘n rol speel in styselkatobolisme wat groei beïnvloed. Daar was egter geen beduidende verskil in styselinhoud van die mutante lyne en die wilde tipe nie. GFP-fusieproteïne het gewys dat PpDPE2 gelokaliseer is in die sitosol, naby aan die chloroplast membraan. Soorgelyke bevindinge is ook gemaak in DPE2 in A. thaliana en S. tuberosum. Dit word gestel dat PpDPE2 moontlik ‘n rol speel in kouetoleransie in moss, omdat ‘n verhoging in styselafbraak opgemerk is in koue-behandelde moss sowel as verhoogde transkripsievlakke van styselmetabolisme gene en ‘n maltose transporter. Dit maak ‘n deur oop vir verdere studie van hierdie gegenereerde mutant-lyne onder kouestres

RNA-seq Analysis of Monoterpene Indole Alkaloid Biosynthetic Pathway Elucidation in Catharanthus roseus

International audienc

Computational biotechnology guides elucidation of the biosynthesis of the plant anticancer drug camptothecin

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Chromosome-scale genomes throw light on plant drug biosynthesis

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Predicting Monoterpene Indole Alkaloid-Related Genes from Expression Data with Artificial Neural Networks

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Improved virus-induced gene silencing allows discovery of a serpentine synthase gene in Catharanthus roseus

International audienceAbstract Specialized metabolites are chemically complex small molecules with a myriad of biological functions. To investigate plant-specialized metabolite biosynthesis more effectively, we developed an improved method for virus-induced gene silencing (VIGS). We designed a plasmid that incorporates fragments of both the target gene and knockdown marker gene (phytoene desaturase, PDS), which identifies tissues that have been successfully silenced in planta. To demonstrate the utility of this method, we used the terpenoid indole alkaloid (TIA) pathway in Madagascar periwinkle (Catharanthus roseus) as a model system. Catharanthus roseus is a medicinal plant well known for producing many bioactive compounds, such as vinblastine and vincristine. Our VIGS method enabled the discovery of a previously unknown biosynthetic enzyme, serpentine synthase (SS). This enzyme is a cytochrome P450 (CYP) that produces the β-carboline alkaloids serpentine and alstonine, compounds with strong blue autofluorescence and potential pharmacological activity. The discovery of this enzyme highlights the complexity of TIA biosynthesis and demonstrates the utility of this improved VIGS method for discovering unidentified metabolic enzymes in plants

Visualization of Aspalathin in Rooibos (Aspalathus linearis) Plant and Herbal Tea Extracts Using Thin-Layer Chromatography

Aspalathin, the main polyphenol of rooibos (Aspalathus linearis), is associated with diverse health promoting properties of the tea. During fermentation, aspalathin is oxidized and concentrations are significantly reduced. Standardized methods for quality control of rooibos products do not investigate aspalathin, since current techniques of aspalathin detection require expensive equipment and expertise. Here, we describe a simple and fast thin-layer chromatography (TLC) method that can reproducibly visualize aspalathin in rooibos herbal tea and plant extracts at a limit of detection (LOD) equal to 178.7 ng and a limit of quantification (LOQ) equal to 541.6 ng. Aspalathin is a rare compound, so far only found in A. linearis and its (rare) sister species A. pendula. Therefore, aspalathin could serve as a marker compound for authentication and quality control of rooibos products, and the described TLC method represents a cost-effective approach for high-throughput screening of plant and herbal tea extracts

Transcriptomics of the Rooibos (Aspalathus linearis) Species Complex

Rooibos (Aspalathus linearis), widely known as a herbal tea, is endemic to the Cape Floristic Region of South Africa (SA). It produces a wide range of phenolic compounds that have been associated with diverse health promoting properties of the plant. The species comprises several growth forms that differ in their morphology and biochemical composition, only one of which is cultivated and used commercially. Here, we established methodologies for non-invasive transcriptome research of wild-growing South African plant species, including (1) harvesting and transport of plant material suitable for RNA sequencing; (2) inexpensive, high-throughput biochemical sample screening; (3) extraction of high-quality RNA from recalcitrant, polysaccharide- and polyphenol rich plant material; and (4) biocomputational analysis of Illumina sequencing data, together with the evaluation of programs for transcriptome assembly (Trinity, IDBA-Trans, SOAPdenovo-Trans, CLC), protein prediction, as well as functional and taxonomic transcript annotation. In the process, we established a biochemically characterized sample pool from 44 distinct rooibos ecotypes (1–5 harvests) and generated four in-depth annotated transcriptomes (each comprising on average ≈86,000 transcripts) from rooibos plants that represent distinct growth forms and differ in their biochemical profiles. These resources will serve future rooibos research and plant breeding endeavours