The molecular analysis of the effects of lumichrome as a plant growth promoting substance

Gouws, Liezel Michelle ; Kossmann, Jens (Stellenbosch : University Stellenbosch, 2009-12)

Thesis

ENGLISH ABSTRACT: Through powerful signal molecules, rhizobacteria affect fundamental processes in plants. In recent years, a number of novel rhizobial molecules have been identified that positively affect plant growth and development. Previous studies have shown that Sinorhizobium meliloti, which form symbiotic relationships with leguminous plants, increases CO2 availability by enhancing root respiration in alfalfa. The active compound was identified as lumichrome, a previously unrecognized rhizosphere signal molecule that has been shown to promote plant growth in various studies. Lumichrome is a common breakdown product of riboflavin and produced by both chemical and biological factors. Various studies on lumichrome have proven its growth promoting effect in the interaction with plants. The mechanism through which lumichrome increases plant growth remains to be clarified. This study provides new insight into the molecular effects of the plant growth promoter lumichrome on the root metabolism of plants. The main aim of the work presented in this thesis was to investigate the molecular mechanism of the plant growth promoting substance lumichrome in the roots of the model plants Lotus japonicus and Solanum lycopersicon (tomato). To asses the impact of lumichrome on the root metabolism of Lotus japonicus and tomato and identify key genes involved in the growth stimulation, a comprehensive profile of differentially expressed genes, proteins and metabolites was compiled. As the effects of lumichrome as a plant growth promoter have not previously been tested on Lotus japonicus and tomato, basic growth studies were completed to determine if lumichrome indeed elicits plant growth at nanomolar concentrations, as was proven in numerous previous studies. Both Lotus japonicus and tomato showed significant increases in root biomass when treated with 5 nM of lumichrome. The treatment with lumichrome caused complex changes in gene expression. Generally, transcript profiling showed that the categories that were predominantly affected by lumichrome in both Lotus and tomato, were genes associated with RNA regulation of transcription and signaling, protein synthesis/degradation/modification and stress and defence. Proteomic studies revealed that the majority of the differentially expressed proteins were down-regulated. Lumichrome seems to largely influence proteins involved in protein folding and down-regulate proteins involved in glycolysis. Proteomics studies revealed that GS1 (Lotus) and GAPDH (Lotus and tomato) were present in lower abundance in lumichrome treated roots, therefore targeted analysis utilizing northern blots, western blots and the measurement of enzyme activities were completed to determine and verify their specific role in the lumichrome mediated growth promotion. The results indicated that GAPDH and GS1 seem to be under post-translational modification. The influence of lumichrome on the metabolome of Lotus roots was immense, however minute in tomato roots. The knowledge gained in the parallel analyses of both Lotus japonicus and tomato aided us in finding key genes involved in the growth stimulation. Overall, one of the most significant observations was that for the first time to our knowledge, six genes related to defence and pathogen responses were identified that are concurrently expressed in both Lotus and tomato. Through identifying a small number of genes involved in mediating the growth stimulation, these can be used for their functional analysis in the future, using reverse genetics to provide more insight into the molecular mechanisms that are triggered by lumichrome as a plant growth promoter.

AFRIKAANSE OPSOMMING: Deur kragtige sein-molekules, beïnvloed rhizobakterieë basiese prosesse in plante. In die laaste jare is ʼn aantal nuwe molekules, afkomstig van rhizobakterieë, geidentifiseer wat plantgroei en ontwikkeling positief beïnvloed. Voorafgaande studies het bewys dat Sinorhizobium meliloti, wat simbiotiese verhoudings met peulplante aangaan, die beskikbaarheid van CO2 vermeerder deur wortel respirasie in alfalfa te verhoog. Die aktiewe komponent is as lumikroom geidentifiseer, 'n vroeë onerkenbare risosfeer sein-molekule, wat deur vorige studies bewys is dat dit plantgroei stimuleer. Lumikroom is ʼn algemene afbreekproduk van riboflavin en word geproduseer deur chemiese en biologiese faktore. Verskeie studies op lumikroom het bewys dat dit 'n groei stimuleerende effek het op die groei van plante as dit daarmee in wisselwerking tree. Die meganisme waarmee lumikroom plante groei verhoog, is nog nie opgeklaar nie. Hierdie studie verleen nuwe insigte in die molekulêre effekte van die plantgroei stimuleerende molekuul lumikroom op die wortel metabolisme van plante. Die hoofdoel van die werk wat voorgestel word in hierdie tesis, was om die molekulêre meganisme van die plantgroei stimuleerende stof, genaamd lumikroom, in die wortels van die model plante Lotus japonicus en Solanum lycopersicon (tamatie), te ondersoek. Om die uitwerking van lumikroom op die wortel metabolisme van Lotus japonicus en tamatie te bepaal, asook sleutelgene wat betrokke is by die groei stimulasie te identifiseer, is 'n breedvoerige profiel van differensiële uitgedrukte gene, proteïne en metaboliete saamgestel. Die effekte van lumikroom as 'n plantgroei stimuleerende stof is nog nooit op Lotus japonicus en tamatie getoets nie. Om díe rede is eers basiese plantgroei studies gedoen, om vas te stel of lumikroom inderdaad plantgroei teen nanomolare konsentrasies stimuleer, soos in vele voorafgaande studies bevestig is. Beide Lotus japonicus en tamatie het aansienlike verhogings in wortel biomassa getoon as dit met 5 nM lumikroom behandel is. Die behandeling van plante met lumikroom het komplekse veranderinge in geen-uitdrukking veroorsaak. Oor die algemeen het die transkrip-profiele gewys dat die kategorieë wat die meeste geraak is deur lumikroom behandeling, in beide Lotus en tamatie, gene was wat geassosieer word met RNS regulasie van transkripsie en sein-netwerke, proteïen sintese/degradasie/wysiging en stres en verdedigings prosesse in plante. Proteïen studies het gewys dat daar 'n daling in die meerderheid van die proteïen vlakke was wat differensieël uitgedruk was. Dit blyk dat lumikroom in 'n groot mate proteïene beïnvloed wat betrokke is by proteïen-vouing en veroorsaak dat proteïen vlakke van glikolitiese ensieme daal. Proteïen studies het gewys dat GS1 en GAPDH in laer vlakke teenwoordig was in lumikroom behandelde plante en daarom is 'n meer doelgerigte analiese gedoen deur gebruik te maak van "northern blot", "western blot" en deur die ensiem aktiwiteite te meet om hulle spesifieke rol in die lumikroom bemiddelde groei vas te stel. Die resultate wys daarop dat GAPDH en GS1 mag onder die invloed van na-translasionele verandering wees. Die invloed van lumikroom op die metabolietvlakke was groot in Lotus wortels, maar dit het minder van 'n effek gehad op tamatie wortels. Die kennis wat opgedoen is deur die paralelle analiese van beide Lotus japonicus en tamatie plante help ons om sleutel gene wat betrokke is by groeistimulasie te identifiseer. Een van die betekenisvolste waarnemings van hierdie studie was dat vir die eerste keer, sover ons kennis strek, ses gene wat almal betrekking het tot verdediging en patogene-reaksies, geidentifiseer is wat gelyktydig in beide Lotus en tamatie uitgedruk word. Deur 'n klein aantal gene te identifiseer, wat betrokke is by groeistimulasie, kan die gene in die toekoms vir funksionele analieses gebruik word deur van keerkoppeling-genetika gebruik te maak. Daardeur sal meer insig verkry word in die molekulêre meganisme wat deur lumikroom as 'n plantgroei stof veroorsaak word.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/4825
This item appears in the following collections: