Tools for strigolactone research : Towards a strigolactone-responsive promoter and a strigolactone-activity inhibitor

Loubser, Johannes (2016-03)

Thesis (MSc)--Stellenbosch University, 2016.

Thesis

ENGLISH ABSTRACT: Strigolactones are a novel group of phytohormones reported to control branching in plants. Strigolactones also plays a pivotal role in the establishment of symbiotic relationships between plants and symbiotic fungi. Furthermore, its presence in the soil is responsible for the germination of the seeds of devastating plant parasitic plants known as broomrapes and witchweeds. They have also been implicated in playing roles in root development and architecture, secondary growth, adventitious root formation and leaf senescence. These phytohormones are derived from the carotenoid synthetic pathway, with β-carotene as the precursor. Several genes and the proteins (enzymes) they code for have been identified by reverse and forward genetics. A few components of the perception and signalling mechanism have been identified, but most of the pathway remains unknown. This highlights the need for more innovative scientific tools in order to fully elucidate the role of strigolactones in higher plants. A specific inhibitor of strigolactone signalling and strigolactone-responsive reporter are two tools that could aid in the further characterization of this poorly defined pathway. In this study, the effects of furanone-derivatives, with the main focus being on trimethylbutenolide (TMB), on the growth of Arabidopsis thaliana were investigated. The aim of this study was to determine if TMB is a competitive inhibitor of strigolactone signalling, because currently there are no known inhibitors of strigolactone signalling available. Having such an inhibitor would enable researchers to study the effects of strigolactones in non-model plant species where no mutants are available. Such an inhibitor would also aid in the further elucidation of the strigolactone signalling pathway. For the second part of this study, an attempt was made to create a strigolactone-response reporter construct in A. thaliana that is activated only in the presence of exogenously applied strigolactone. Having such a reporter-construct in A. thaliana would be valuable, as strigolactones are difficult to detect and quantify in these plants due to them being bioactive at picomolar concentrations in the plant. Such a reporter-construct would also aid in the further elucidation of the strigolactone pathway, the discovery of more functions and any interactions with other phytohormones and biochemical processes in the plant. During this study, it was found that the presence of TMB induced an increase in lateral root formation in wild-type A. thaliana seedlings, suggesting that it may act as a competitive inhibitor of strigolactone signalling, at least in terms of lateral rooting. It was also found that TMB affects the expression of a small group of strigolactone-responsive genes in an opposite way than GR24, a racemic mixture of strigolactone analogues. This effect on gene expression was observed after prolonged treatment of A. thaliana seedlings with TMB. For the second part of the project, we were unable to create a strigolactone-specific reporter construct, although the results suggest that the 990 bp region immediately upstream of AtBRC1 does respond more strongly to the presence of GR24 than the full, native promoter. It was also found that the 1480 bp and 990 bp regions immediately upstream of AtBRC1 is already severely deregulated version of the native AtBRC1 promoter. From these results, it was concluded that DNA binding motif/s for SMAX-like repressor proteins probably lie upstream of the 1480 bp promoter region of AtBRC1, while possible strigolactone-responsive motif/s lie downstream of the 990bp promoter region of AtBRC1.

AFRIKAANSE OPSOMMING: Strigolaktone is ‘n nuwe groep fitohormone waaroor daar berig is dat hul vertakking in plante reguleer en beheer. Strigolaktone speel ook ‘n deurslaggewende rol in die aanknoping van simbiotiese verhoudings tussen plante en arbuskulêre mikorisaie swamme. Verder is die teenwoordigheid van strigolaktone in die grond ook verantwoordellik vir die ontkieming van die saadjies van verwoestende parasitiese plante beter bekend as besemrape en kopseerblomme. Daar word geïmpliseer dat hulle rolle speel in wortelontwikkeling en argitektuur, sekondêre groei, onverwagte wortelformasie en blaarveroudering. Hierdie fitohormone word afgelei van die karotenoïed sintese padweg, met beta-karoteen as die voorloper molekule. Verskeie gene en die proteine (ensieme) waarvoor hulle kodeer is reeds geidentifiseer met behulp van keer en stuur genetika. ‘n Paar komponente van die persepsie en sein meganismes is reeds geïdentifiseer, maar meeste van die padweg bly grootliks onbekend. Dit beklemtoon die behoefte vir meer innoverende wetenskaplike gereedskap om die volledige rol van strigolactone in hoër plante te ontrafel. ‘n Strigolaktoon sein-inhibeerder en strigolaktoon-spesifieke verklikker is twee instrumente wat kan help met die verdere karakterisering van hierdie swak gedefinieërde padweg. Deur die loop van hierdie studie is die effek van furanoon-afgeleides, met trimetielbutenoliet (TMB) as die hoof fokus, op die groei van Arabidopsis thaliana ondersoek. Die doel van hierdie studie was om te bepaal of TMB ‘n kompeterende inhibeerder van strigolaktoon-seine is, want tans is daar geen bekende inhibeerder van strigolaktoon-seine beskikbaar nie. Met so ‘n inhibeerder sal wetenskaplikes in staat wees om die effek van strigolaktone in nie-model plant spesies waar geen mutante beskikbaar is nie te ondersoek. So ‘n inhibeerder sal ook help met die verdure ontrafeling van die strigolaktoon sein padweg. Vir die tweede gedeel van hierdie studie is ‘n poging aangewend om ‘n strigolaktoon-reagerende verklikkerkonstruk te skep in A. thaliana wat slegs geaktiveer word in die teenwoordigheid van eksogene toegevoegde strigolaktone. So ‘n verklikker-konstruk in A. thaliana sal waardevol wees, want strigolaktone is moeilik om te bepaal en te kwantifiseer in hierdie plante, te danke aan die feit dat hul aktief is by piko-molêre konsentrasies in die plant. So ‘n verklikker-konstruk sou ook help met die verdure ontrafeling van volledige strigolaktoon-padweg, die ontdekking van meer funksies en enige interaksies met ander fitohormone and biochemiese prosesse in die plant. Tydens hierdie studie is daar bevind dat die teenwordigheid van TMB ‘n toename in laterale wortelvorming in wilde-tipe A. thaliana saailinge veroorsaak, wat daarop dui dat dit kan dien as ‘n kompeterende inhibeerder van strigolaktoon-seine, ten minste in terme van laterale wortels. Dit is ook bevind dat TMB ‘n invloed het op ‘n klein groupie van strigolaktoon-reagerende gene in ‘n teenoorgestelde manier as GR24, ‘n rasemiese mengsel van strigolaktoon-analoë. Hierdie effek op die uitdrukking van gene was waargeneem na langdurige behandeling van A. thaliana saailinge met TMB. Vir die tweede gedeelte van hierdie projek, was ons nie in staat gewees om ‘m strigolaktoon-spesifieke verklikker-konstruk te skep nie, alhoewel die resultate daarop dui dat die 900 bp streek onmiddellik stroomop van AtBRC1 sterker reageer in die teenwordigheid van GR24 as die volle, inheemse promotor. Daar is ook bevind dat die 1480 bp en 990 bp streke onmiddellik stroomop van AtBRC1 reeds ‘n dereguleerde weergawe van die inheemse AtBRC1 promotor is. Vanaf hierdie resultate is die gevolgtrekking gemaak dat DNS-bindende motief/motiewe vir SMAX-agtige onderdrukker proteïne waarskynlik verder stroomop van die 1480 bp promotor streek van AtBRC1 lê, terwyl moontlike strigolaktoon-reagerende motief/motiewe stroomaf van die 990 bp promoter streek geleë is.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/98736
This item appears in the following collections: