Browsing Doctoral Degrees (Genetics) by Author "Colling, Janine"
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- ItemFunctional characterization of a putative signalling peptide TAXIMIN in the model plant Arabidopsis thaliana and a medicinal plant Sutherlandia frutescens L. R. Br.(Stellenbosch : Stellenbosch University, 2016-03) Colling, Janine; Makunga, N. P.; Goossens, A.; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics. Institute for Plant Biotechnology (IPB).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.