The Arabidopsis GolS1 promotor as a potential biosensor for heat stress and fungal infection?

Janse van Rensburg, Henry Christopher (2016-12)

Thesis (MSc)--Stellenbosch University, 2016.

Thesis

ENGLISH ABSTRACT: Galactinol (Gol) has classically been considered to serve as a galactose donor during the biosynthesis of raffinose family oligosaccharides (RFOs). These sucrosyl oligosaccharides have been well characterised in their roles in carbon translocation and storage and, abiotic stress protection in plants. However, recent findings have demonstrated Gol to be an efficient free radical scavenger and it has also been suggested to act as signalling molecule during induced systemic resistance (ISR), upon pathogen infection. Collectively, these findings centres to the involvement of only a single galactinol synthase gene (GolS, synthesising Gol) in Arabidopsis (AtGolS1, At2g47180). The AtGolS1 isoform has been shown to be transcriptionally up-regulated during heat stress and Botrytis cinerea infection. Further, it is also responsive to jasmonic acid, a key component of the ISR pathway. Here we targeted the AtGolS1 promotor containing well defined heat shock transcription factor elements and a single putative jasmonate binding element, to develop a dual-functional biosensor with the ability to detect both heat stress and Botrytis cinerea infection. We created transgenic Arabidopsis lines where the reporter genes β-glucuronidase (GUS) and the green florescent protein (GFP) were under the control of the AtGolS1 promotor. Using the native AtGolS1 gene as a point of reference, we confirmed that the reporter genes were transcriptionally responsive to both heat stress and methyl jasmonate treatment in transgenic Arabidopsis. Under the same experimental conditions, both GUS assays and GFP imaging correlated with these transcriptional responses. Finally, we infected the transgenic lines with Botrytis cinerea infections to analyse reporter activity. Transcript analysis of transgenic lines clearly showed an increase in transcript abundance for both the native AtGolS1 and the reporter genes in reponse to B. cinerea infection. Similarly, reporter assays revealed a distinct difference in activity between infected and uninfected plants from 24h to 96h after Botrytis cinerea infection. These results provide sufficient proof-of-concept for the AtGolS1 promotor to be used as a dual functional biosensor for both heat stress and fungal infection.

AFRIKAANS OPSOMMING: Galaktinol (Gol) is aanvanklik beskou as ʼn galaktose skenker tydens die biosintese van raffinose familie van oligosakkariede (RFO). Hierdie sukrosiel oligosakkariede is goed gekenmerk vir hul funksies in koolstof translokasie en storing, sowel as die beskerming teen abiotiese stres in plante. Onlangse bevindinge het Gol geklassifiseer as 'n doeltreffende vry radikaal werwer, en is voorgestel om op te tree as 'n sein molekule tydens geïnduseerde sistemiese weerstand (ISR), tydens patogeen infeksie. Gesamentlik plaas hierdie bevindinge klem op die betrokkenheid van 'n enkele galaktinol sintase geen (GolS, sintetiseer Gol) in Arabidopsis (AtGolS1, At2g47180). Dit is voorheen bewys dat die AtGolS1 isoform transkripsioneel op-gereguleer word tydens hitte-stres en Botrytis cinerea infeksie. Verder is dit ook sensitief vir jasmijnsuur, 'n belangrike komponent van die ISR pad. Gedurende hierdie studie het ons die AtGolS1 promotor geteiken, wat die goed gedefinieërde hitte-skok transkripsie faktor bindings elemente en 'n enkele vermeende jasmijnsuur bindings element bevat, om 'n dubbele-funksionele biosensor te ontwikkel met die vermoë om beide hitte-stres en Botrytis cinerea infeksie op te spoor. Ons het transgeniese Arabidopsis lyne gegenereer waar die rapporteerder gene β--glukuronidase (GUS) en die groen fluoressent proteïen (GFP) onder die beheer van die AtGolS1 promotor is. Deur gebruik te maak van die inheemse AtGolS1 geen as 'n verwysingspunt, het ons bevestig dat die rapporteerder gene op ‘n transkriptionele vlak reageer op beide hitte-stres en metiel jasmijnsuur behandeling in transgeniese Arabidopsis. Onder dieselfde eksperimentele kondisies het beide GUS toetse en GFP fotografie gekorreleer met die transkripsie analise. Ten slotte, het ons die transgeniese lyne aan Botrytis cinerea infeksies blootgetel om die rapporteerder aktiwiteit te ontleed. Transkripsie analise van transgeniese lyne het ʼn duidelik toename in transkripsie vlakke getoon vir beide die plaaslike AtGolS1 geen en die rapporteerder gene in reaksie op B. cinerea infeksie. Eenders, het rapporteerder toetse 'n duidelike toename in aktiwiteit tussen geïnfekteerde en ongeïnflekteerde plante getoon vanaf 24 h tot 96 h na Botrytis cinerea infeksie. Hierdie resultate bied voldoende bewys-van-konsep vir die AtGolS1 promotor om gebruik te word as 'n dubbele funksionele biosensor vir beide hitte-stres en swam infeksie.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/100248
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