Browsing by Author "Dempers, Dehan"

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    Overexpression of α-acetolactate decarboxylase and acetoin reductase/2,3-butanediol dehydrogenase in Arabidopsis thaliana
    (Stellenbosch : Stellenbosch University, 2015-03) Dempers, Dehan; Hills, Paul N.; Kossmann, Jens; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. Institute for Plant Biotechnology.
    ENGLISH ABSTRACT: Certain rhizobacteria have been identified as plant growth promoting rhizobacteria (PGPR), but the mechanisms involved and the exact mechanisms via which they operate still need to be fully elucidated. These bacteria live in symbiosis with the plants, by colonizing the roots or even entering the plant cells as endophytes. Once a symbiosis is established, certain beneficial substances are released to the plant including, but not limited to, volatile organic compounds (VOCs). Two such VOCs, acetoin and 2,3-butanediol have been shown to enhance general plant growth and initiate an induced systemic resistance (ISR) response. In this study the genes responsible for the production of acetoin and 2,3-butanediol, α-acetolactate decarboxylase (ALDC) and acetoin reductase/2,3-butanediol dehydrogenase (BDH1) respectively, were isolated from Aspergillus niger ATCC 1015 and Saccharomyces cerevisiae W303. The acetoin precursor, acetolactate, is located in the chloroplast, thus the fully sequenced genes were cloned into plant expression vectors (pCambia2300 and pCambia1300) containing a ferredoxin-NADP+ reductase (FNR) transit peptide sequence for chloroplastic targeting. The genes were transformed into Arabidopsis thaliana Col-0 using an Agrobacterium-mediated floral dip method. Transformed plants were tested for gene insertion and expression, and some of the lines were found to have undergone transgene silencing in the T3 generation. Before growth promotion analysis between transgenic plants and untransformed control plants could commence, transformed double transgenic T2 generation and single transgenic T3 generation plants were tested for gene insertion and expression. The transgenic ALDC lines and one of the double transgenic lines showed some promise as they were significantly bigger than untransformed control plants in a number of physiological parameters, including leaf area, fresh and dry mass. Varying results were observed when wild type plants were tested against synthetic acetoin and 2,3-butanediol under short and long day lengths. The physical presence of acetoin and 2,3-butanediol in the transgenic lines was tested by means of enzyme assays, gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC) analysis. The enzymes assays could not be utilized in the plant system tested, however, as identical trends in reduced nicotinamide adenine dinucleotide (NADH) oxidation were observed between transgenic and control plants. No detectable levels of acetoin could be identified by GC-MS or HPLC methods. In general this study laid out the ground work for the incorporation of the ALDC and BDH1 genes in Arabidopsis, with some preliminary growth comparison studies showing promise in the single ALDC and double ALDC/BDH1 transgenic lines. A suitable detection method for acetoin and 2,3-butanediol still needs to be established for future studies.