Functional characterization of a putative signalling peptide TAXIMIN in the model plant Arabidopsis thaliana and a medicinal plant Sutherlandia frutescens L. R. Br.

Colling, Janine (2016-03)

Thesis (PhD)--Stellenbosch University, 2016.

Thesis

ENGLISH ABSTRACT: Secondary metabolite production in plants assists with protection against predators and attraction of pollinators. Manipulation of secondary metabolite pathways towards increased production of compounds of interest has become a target. The techniques to assist with understanding regulation of these pathways are therefore important. Several factors influence metabolite synthesis in plants including age, developmental stage, tissue type and environmental factors. In this study we describe a technique, cDNA-AFLP, which can be applied to study changes in whole genome expression to identify genes which are differentially expressed during stress conditions. We also describe the study of the function of a novel signalling peptide TAXIMIN in the model plant Arabidopsis thaliana. This peptide was discovered by cDNA-AFLP analysis of Methyl Jasmonate (MeJA) elicited Taxus baccata cell suspension cultures. TAXIMIN represents a novel signalling peptide which belongs to the cysteine rich peptides and has an N-terminal signal peptide and a C-terminal peptide with six conserved cysteines and three conserved prolines. Two TbTAX homologs (TAX1 and TAX2) were discovered in A. thaliana. Fusion of the full length peptide to the Venus fluorescent protein targeted the peptide to the plasma membrane-cell wall interface and this movement was abolished when the N-terminal signal was removed. Single and double mutants lacked a visible phenotype which can be related to functional redundancy with other genes or lack of environmental factors to induce a phenotypic response. Fusion of the TAX promoters to the GUS reporter gene revealed that TAX2 was expressed in vasculature tissue, whilst TAX1 expression was found in anthers, nectaries, roots and the base of the organs of the paraclade junctions indicating the neo-functionalization of the two peptides. Constitutive expression of TAX1 resulted in a fusion phenotype in the paraclade junctions and a fruit phenotype. Fruits were shorter and wider at the tip which co-insided with a wider replum as well as seed-stacking in this region. The fusion phenotype was similar to the phenotype observed for a mutant of the MYB transcription factor LATERAL ORGAN FUSION (LOF1) which plays a role in boundary formation. However, TAX1 overexpression (TAX1 OE) did not result in reduced LOF expression in paraclade junctions and TAX1 expression was similar to wild type plants in the lof1lof2 mutant paraclade junctions. Dexamethasone induction of TAX1 overexpression also did not result in changes in LOF1 expression in seedling or in the shoot apical meristem. No changes in LOF2 driven GUS expression level or pattern was observed when crossing to the TAX1 overexpression background. TAX1 therefore appears to regulate boundary formation independently from LOF1. However, these pathways may converge later in development. Metabolite analysis of the primary metabolite profiles of the leaf and roots of TAX1 OE lines indicated increases in phosphate (leaves) and serine (root and leaves) levels which were observed in all lines. TAX1 OE lines also appeared to be sensitive to the length of the photoperiod and this may be related to a reduced abundance of sinapoyl malate in leaves. cDNA-AFLP was applied to study changes in genome expression of nitrogen, salinity or MeJA-stressed Sutherlandia frutescens shoots cultivated in vitro. Results indicated that pathways involved in polyamine biosynthesis or regulated by plant hormones such as ethylene or abscisic acid are differentially expressed. Salinity stress caused a reduction in nitrogen uptake, but did not affect photosynthesis or the carbon: nitrogen ratio. On the metabolite level an increase in arginine and proline content was observed. This might be related to the reduction of ammonium toxicity effects or the osmotic response to reduce the damage due to accumulating ions. Plants were generally tolerant to low levels of salinity and no significant changes in sutherlandioside B abundance were observed. Constitutive expression of TAXIMIN genes from T. baccata and Medicago truncatula did not alter the abundance of sutherlandins, sutherlandiosides or soyasaponins in S. frutescens hairy roots. This suggests that these peptides do not directly affect the biosynthesis pathways of these compounds in S. frutescens. Application of MeJA enhanced soyasaponin production confirming previous reports on induction of these pathways by MeJA elicitation. This study describes the establishment of a platform which can be used to study changes in the transcriptome in response to the application of stress in non-model plants and includes the use of tools to study the function of uncharacterized genes in the model plant A. thaliana. This study also describes the transformation of a non-model medicinal plant (S. frutescens) which could be used to study the effects of ‘novel’ heterologous genes on the metabolism of these plants.

AFRIKAANSE OPSOMMING: Plante produseer sekondêre metaboliete vir beskerming teen herbivore en om insekte vir bestuiwing te lok. Die manipulasie van sekondêre metaboliet produksie, ten einde verhoogde produksie van die metaboliete van belang mee te bring, het ‘n teiken geword. Tegnieke wat tot ons kennis van die regulasie van die produksie paaie kan lei is van belang. Verskeie faktore soos bv. ouderdom, ontwikkelingsfase, tipe weefsel en omgewingsfaktore beinvloed die produksie van metaboliete in plante. In hierdie studie beskryf ons ‘n tegniek, cDNA-AFLP, wat toegepas kan word om die verandering in genoom wye geen uitdrukking te bestudeer en dus veranderinge in geen uitdrukking tydens verskillend stress kondisies te identifiseer. In hierdie studie, is die funksie van ‘n nuwe sein-peptied, TAXIMIN ondersoek in die model plant Arabidopsis thaliana ondersoek. Hierdie peptide is tydens die analise van Metiel Jasmonaat behandelde Taxus baccata sel suspensie kultuur m.b.v cDNA-AFLP ontdek. TAXIMIN verteenwoordig ‘n nuwe sein peptide wat aan die cysteine ryke peptied groep behoort. Dit besit ‘n N-terminale sein-peptied en ‘n C-terminale peptied wat gekonserveerde cysteine en proliene bevat. Twee TbTAX homoloë (TAX1 en TAX2) is in die model plant A. thaliana ontdek. Fusie van die vol-lengte peptied aan ‘n Venus fluoreserende protein het die protein na die die plasmambraan-selwand skeiding geteiken. Die beweging is vernietig indien die N-terminal sein peptied verwyder is. Plante met ‘n enkele of dubbel TAX mutasie het geen sigbare fenotipe getoon. Die afwesigheid van ‘n fenotipe mag weens die teenwoordigheid van funksionele ressesiewe gene wees of weens die vereiste van die teenwoordigheid van spesifieke omgewingsfaktore wat ‘n fenotipe kan induseer. Die fusie van die TAX promoters aan die GUS verklikker geen het aangedui dat TAX2 hoofsaaklik in die vaskulêre weefsel uitgedruk word. Terwyl TAX1 geen uitdrukking in die helmknoppie, nektarkliere, wortels en die basis van die organe van die nodes uitgedruk word. Die resultaat kan dui op die ontwikkeling van afsonderlike regulering van geen uitdrukkingspatrone of selfs spesialisering van die funksie van die twee peptiede. Aaneenhoudende uitdrukking van TAX1 het tot ‘n fusie fenotipe in die nodes asook ‘n vrug fenotipe gelei. In vergelyking met die wilde tipe vrugte, het die vrugte van TAX1 ooruitdrukkingslyne (OU) korter en wyer by die punt vertoon. Dit het terselfdertyd met ‘n breër replum asook met saad-stapeling in hierdie area gepaard gegaan het. Die fusie fenotipe was soortgelyk aan die fenotipe wat in plante met ‘n mutasie in die MYB transkripsie faktor LATERAL ORGAN FUSION (LOF1) waargeneem is. LOF1 speel ‘n rol in die skeiding van organe. Analises het getoon dat die TAX1 OU nie tot ‘n vermindering in LOF1 geen uitdrukking in die nodes van die plante gelei nie. Die vlak van geen uitdrukking van die TAX1 geen in die lof1lof2 mutante plante was ook soortelyk aan die geen uitdrukking in wilde tipe plante. Die toediening van dexamethasoon het tot die induksie van ooruitdrukking van TAX1 geen gelei. Dit het egter nie LOF1 geen uitdrukking in saailinge of in die stingel apikale meristem verander nie. ‘n Kruising tussen ‘n lyn wat TAX1 ooruitdruk en ‘n lyn waar die uitdrukking van GUS deur die promoter van LOF2 gedrewe word, het geen verandering in die patroon of vlak van LOF2 geen uitdrukking getoon nie. Dit wil dus voorkom asof TAX1 die skeiding tussen organe op ‘n onafhanklike wyse van LOF1 reguleer. Dit is wel moontlik dat die twee gene ontwikkeling in plante op ‘n latere wyse tesame beheer. Analise van die primêre metaboliet samestelling van die blare en wortels van al die TAX1 OU plante het ‘n toename in fosfaat (blare) en serien (wortels en blare) vlakke getoon. Die TAX1 OU plante was ook sensitief vir die periode van ligblootstelling en hierdie effek mag verwant wees aan die vermindering in sinapoyl malaat produksie in blare. Die cDNA-AFLP tegniek is toegepas om die verandering in genoom-wye geen uitdrukking van in vitro Sutherlandia frutescens plante wat met stikstof, sout of Metiel Jasmonaat behandel is te bestudeer. Die resultate het aangedui dat reaksies wat tot poli-amien produksie of die produksie van plant hormone soos etileen of absisiensuur lei, verandering toon. Sout stress het ‘n verlaging in stikstof opname veroorsaak, maar het geen effek op fotosintese of die koolstof tot stikstof verhouding veroorsaak nie. ‘n Toename in die arginien en prolien vlakke is waargeneem en die verandering mag verwantskap hou met vermindering van ammonium toksisiteit. ‘n Alternatiewe opsie is dat die produksie van die metaboliete tydens die osmotiese reaksie toeneem om die akkumulasie van ione wat skade aanbring, te verminder. Die plante was in die algemeen verdraagsaam vir lae vlakke van sout en geen aansienlike verandering in die sutherlandioside B vlakke is waargeneem nie. Aaneenhoudende uitdrukking van die TAXIMIN gene van T. baccata en Medicago truncatula het nie die vlakke van die sutherlandins, sutherlandiosides of saponiene in die S. frutescens harige wortels geaffekteer nie. Hierdie resultaat dui daarop dat die gene nie direk die produksie van die metaboliete in S. frutescens reguleer nie. Toediening van Metiel Jasmonaat het die produksie van sojasaponiene verhoog en dit bevestig vorige bevindings dat Metiel Jasmonaat behandeling hierdie metaboliese paaie kan induseer. Hierdie studie beskryf die ontwikkeling van ‘n platform wat gebruik kan word om die veranderinge in die transkriptoom weens die toediening van stress op nie-model plante te ondersoek. Dit platform sluit die beskrywing van tegnieke om die funksie van gene wat nie voorheen gekarakteriseer is nie in die model plant A. thaliana te ondersoek. Die studie beskryf ook die transformasie van ‘n nie-model medisinale plant (S. futescens) wat gebruik kan word om die effek van ‘n nuwe heteroloë geen op metabolisme van die plante te bestudeer.

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