Browsing by Author "Colling, Janine"

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    Functional 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.
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    Towards understanding the metabolism of in vitro Sutherlandia frutescens (L.)R.Br. cultures
    (Stellenbosch : University Stellenbosch, 2009-12) Colling, Janine; Makunga, N. P.; Valentine, A. J.; Kossmann, J. M.; University of Stellenbosch. Faculty of Agrisciences. Dept. of Genetics. Institute for Plant Biotechnology.
    ENGLISH ABSTRACT: Sutherlandia frutescens (L.) R. Br., also regarded as Lessertia frutescens, is a leguminous, perennial shrub indigenous to South Africa. Extracts prepared from the leaves have traditionally been used for the treatment of various diseases. Reports have also indicated that S. frutescens provides certain health benefits to cancer and HIV/AIDS patients. Analysis of extracts indicated the presence of several compounds (bitter triterpenoid glycosides, several flavonoids, amino acids, small amounts of saponins (no alkaloids though), asparagine, Larginine, canavanine, gamma-aminobutyric acid (GABA) and pinitol) which contribute to the medicinal properties of this plant. The first part of this study involved testing the effect of six treatments (light, dark, soaking of seeds, physical scarification, chemical scarification and flaming of seeds) on the in vitro germination of Sutherlandia seeds to elucidate the factors which control seed germination. Those treatments which removed the seed coat were most successful for germination with physical scarification being the most efficient method, resulting in 98.6% of the seeds germinating after 21 days. Although the organogenesis of Sutherlandia explants (cotyledons and hypocotyls) in vitro were investigated (results not included in this thesis), omitting plant growth regulators (PGR) in the cultivation medium was best for shoot multiplication. However, this PGR-free system successfully provided a continuous supply of plant material for further studies. It would be possible to successfully adopt it for commercial production of plants to assist with cultivation of Sutherlandia as a field crop. Another advantage of this system is spontaneous rooting with 85% of the in vitro microshoots rooting in PGR-free medium. These rooted plants were acclimated in the glasshouse using vented lids to harden off the shoots and this method resulted in 100% survival of plants. The second part of this study investigated the induction of hairy root cultures of S. frutescens using Agrobacterium-mediated transformation. The efficiency of three Agrobacterium strains (A4T, LBA9402 and C58C1) to transform different S. frutescens explants (cotyledons and hypocotyls) was analyzed. All three strains were equally efficient at inducing hairy roots in both hypocotyls and cotyledons. However, transformation of S. frutescens was dependent on the type of explant used with the hypocotyls being more efficiently transformed than the cotyledons. Overall the transformation of both the hypocotyl (93%) and cotyledon (47%) was highest when the strain A4T was used. Four hairy root clones were selected and their cultivation in a liquid system was optimized by investigating their growth in four different types of media (Gamborg B5 (Gamborg et al., 1968), White’s (White, 1934; White, 1954), MS (Murashige and Skoog, 1962) and half strength MS medium). All the growth of hairy root clones was best in the B5 and MS medium, with White’s medium being the least effective cultivation medium. Molecular analysis of hairy roots was used to prove the transgenic status of these four putative transgenic clones. This was achieved using polymerase chain reaction (PCR) amplification of rol A (320 bp), B (780 bp) and C (600 bp) genes to determine the presence of the TL-DNA in the plant genome. During Southern hybridization a radioactively labeled rol A probe was used to determine the copy number of the rol A gene. The three rol genes were present in all four hairy root clones. The third part of this study focused on the effect of three abiotic stress factors (nitrogen availability, salinity and drought) on the synthesis of four metabolites (gamma-aminobutyric acid (GABA), asparagine, arginine and canavanine). The effect of nitrogen availability on metabolite synthesis and the morphology was determined using in vitro shoot cultures as well as the hairy root clone C58C1-g. Nitrogen availability studies were conducted by cultivating the microshoots or root tips on modified MS medium. The MS medium contained either the normal amount of nitrogen (1.9 g L-1 KNO3 and 1.65 g L-1 NH4NO3) in the MS medium (1x nitrogen), half the normal nitrogen concentration in MS medium (0.5x nitrogen) or twice the normal nitrogen concentration in MS medium (2x nitrogen). The arginine and asparagine levels in the roots and shoots and the canavanine level in the shoots were directly correlated with the amount of nitrogen in the medium (as the nitrogen level increased, the metabolite levels increased). The GABA level in the shoots was inversely correlated with the amount of nitrogen in the medium. Several reasons may explain these metabolic changes including the assimilation of extra nitrogen into asparagine, canavanine and arginine in the shoots. The reduced GABA levels may indicate the preferential flux of the free GABA into other nitrogen assimilatory pathways such as protein synthesis as well as its rapid utilization to replenish the tricarboxilic acid cycle intermediates. The effect of water (induced by including 3% (w/v) PEG in the medium) and salt stress (induced by including either 50 or 100 mM NaCl in the medium) was only investigated in the shoot cultures as the root cultures lacked the synthesis of canavanine. Water stress did not significantly alter the metabolite levels, but resulted in a significant decrease in the growth (fresh weight and total shoot length) and the rooting response of these microshoots. Salt stress only resulted in a significant increase in arginine levels with increasing salinity and also caused a reduction in the rooting and growth response. Lowered plant vigour may be the first visual sign of water stress. Addition of NaCl may lead to ion toxicity and requires osmotic adjustment resulting in changes at the metabolic level concomitant to physiological growth changes. Finally, the anti-bacterial activity and the phytochemistry of transgenic root cultures and untransformed in vitro and ex vitro plant material was examined. Only the extracts prepared from the wild harvested leaf material exhibited moderate anti-bacterial activity (1.25 mg ml-1) against all the bacteria (Escherichia coli, Klebsiella pneumoniae, Bacillus subtilis and Staphylococcus aureus) tested. Changes to the secondary metabolism of hairy roots were investigated using TLC and LC-MS analysis. Several of the compounds in the hairy root extracts were present in higher levels than in the control root extracts. Transformation also increased the complexity of the phytochemical pattern of the hairy roots, either due the synthesis of novel compounds or upregulated synthesis of existing metabolic pathways. The production of hairy roots and the establishment in a liquid system during this study was an important step towards upscaling these cultures to a bioreactor. In future these roots can assist in developing cultures which produce a high yield of the desired metabolites.