Overexpression of α-acetolactate decarboxylase and acetoin reductase/2,3-butanediol dehydrogenase in Arabidopsis thaliana

Thesis (MScAgric)--Stellenbosch University, 2015.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.AFRIKAANSE OPSOMMING: Sekere risobakterieë is geïdentifiseer as plantgroeibevorderende risobakterieë, alhoewel die meganismes wat betrokke is by die bevorderende eienskappe nog nie ten volle verstaanbaar is nie. Hierdie bakterieë lewe in symbiose met plante deur die wortels te koloniseer of selfs om die plant selle te infiltreer as endofiete. Sodra ‘n symbiose gevestig is kan voordelige stowwe vir die plant vrygestel word, bv. vlugtige organiese verbindings. Acetoin en 2,3-butaandiol is twee sulke vlugtige organiese verbindings wat voorheen bewys is om plant groei te bevorder en om ‘n geïnduseerde sistemiese weerstand reaksie te inisieër. In hierdie studie is die gene verantwoordelik vir die vervaardiging van acetoin en 2,3-butaandiol, α-acetolactate decarboxylase (ALDC) and acetoin reductase/2,3-butanediol dehydrogenase (BDH1) onderskeidelik geïsoleer vanaf Aspergillus niger ATCC 1015 and Saccharomyces cerevisiae W303. Die acetoin voorloper, acetolactate, is geleë binne die chloroplast, daarom was die volledige volgorde bepaalde gene geklooneer binne-in plant uitdrukkings vektore (pCambia2300 and pCambia1300) bevattend ‘n ferredoxin-NADP+ reductase (FNR) transito peptied volgorde vir chloroplast fokus. Die gene was in Arabidopsis thaliana Col-0 in getransformeer, deur gebruik te maak van ‘n Agrobacterium-bemiddelde blom dompel metode. Getransformeerde plante was getoets vir geen invoeging en uitdrukking. Sekere van die lyne was onderhewig aan transgeen onderdrukking in die T3 generasie. Voor groei bevordering analiese tussen transgeiese plante en die kontroles uitgevoer kon word, was geen invoeging en uitdrukking voor-af getoets op dubbel getransformeerde T2 generasie en enkel getransformeerde T3 generasie plante. Die transgeniese ALDC lyne en een van die dubbel lyne het potensiaal getoon aangesien hulle aansienlik groter as die kontroles gegroei het in terme van blaar area, vars en droë massa. Wisselende resultate was ondervind vir kort en lang dae toe wilde tipe kontrole plante getoets was teen die sintetiese acetoin en 2,3-butaandiol. Die fisisie teenwoordigheid van acetoin en 2,3-butaandiol was voor getoets deur ensiem toetse, gas chromatography-mass spectrometry (GC-MS) en high-performance liquid chromatography (HPLC) analieses in die transgeniese plante. Die ensiem toetse was onvanpas aangesien die gereduseerde nicotinamide adenine dinucleotide (NADH) oksidasie teen soortgelyke wyses vir kontrole en transgeniese plante plaasgevind het. Geen waarneembare vlakke van acetoin kon deur middel van die HPLC of GC-MS metodes geïdentifiseer word nie. As alles saam gevat word, het hierdie studie die begin blokke van die invoeging van ALDC en BDH1 in Arabidopsis neergelê. Voorlopige groei studies wys dat die enkel ALDC en die dubbel ALDC/BDH1 transgeniese lyne belowende resultate bied. ‘n Gepasde deteksie metode moet nog gestig word vir acetoin en 2,3-butaandiol vir toekomstige studies