Molecular analysis of plant growth promoted with low molecular weight compounds in relation to genetically altered photosynthetic carbohydrate partitioning in higher plants

Pholo, Motlalepula (2017-03)

Thesis (PhDAgric)--Stellenbosch University, 2017.

Thesis

ENGLISH ABSTRACT: Despite the impressive advances that have been made over the years in improving both plant growth and yields, there is little reason to become complacent about these developments, especially with regard to food supply for the world’s increasing population. Other than conventional and marker assisted breeding, the use of crude extracts from plant growthpromoting rhizobacteria (PGPR) bacteria that possess biostimulatory properties has been considered as an alternative practice for enhancing plant development and crop productivity. One such substance is lumichrome (7,8 dimethylalatloxazine), a novel plant growth promoting multitrophic signal molecule produced by the bacterium Sinorhizobium meliloti. Lumichrome has been shown to elicit growth promotion and trigger a compensatory increase in whole-plant net carbon assimilation through enhanced starch accumulation and altered ethylene metabolism. Despite physiological experimental advances in exploring the growth stimulatory mechanisms of lumichrome, a comprehensive molecular analysis in various plant species still remains elusive. This study focused on understanding the genetic, molecular and biochemical regulatory networks as key determinants for crop growth and productivity in relation to lumichrome in Arabidopsis thaliana. Our specific objectives focused on unravelling i) global changes in gene expression, ii) protein expression and iii) key metabolites or biochemical pathways which were seemingly affected by lumichrome treatments in Arabidopsis thaliana. The study further made use of Arabidopsis mutant lines deficient in the primary gene encoding the ADP-GLUCOSE PYROPHOSPHORYLASE small subunit (APS1) to study the regulatory mechanisms of carbon metabolism. Subsequently, the findings were used to infer functional interactions among genes, proteins and metabolites in order to speculate on the possible control mechanism(s) involved. Application of biologically-active levels of lumichrome has been demonstrated to enhance plant development through changes in photosynthetic rates, leaf stomatal conductance and transpiration in several plant species. Enhanced growth is reported to be attributed to xylem transport and in situ accumulation of lumichrome in leaves, which subsequently triggers events that promote cell division and leaf expansion in both monocots and dicots. However, previous studies have also demonstrated mixed physiological responses between plants species to lumichrome, hence molecular processes responsible for growth promotion in other species remain somewhat elusive. Consistent with previous studies, our study demonstrated that the addition of 5 nM lumichrome to Arabidopsis thaliana plants elicited a growth promoting effect to increase overall plant size and biomass. The increased overall plant size was attributed to enhanced photosynthesis and the use of higher levels of photoassimilates for cell division and cell enlargement. This was further supported by Next Generation Sequencing (Transcriptomic profiling-True-Seq) which revealed that the growth stimulatory effect was effected predominantly through genes associated to cell wall modification, cell division and expansion. Our proteomics study results further suggested that lumichrome treatment enhances and stabilizes photosynthesis, providing increased photoassimilates for growth in wild type Arabidopsis. Although starch levels were increased in lumichrometreated wild type plants, levels of APS1 were unexpectedly decreased. To demonstrate that silencing APS1 enhances Arabidopsis growth, we profiled both proteins and metabolites in Arabidopsis T-DNA knockout lines which were deficient in APS1. The reverse genetics approach revealed that enhanced growth of aps1 mutant plants relative to the wild type can be ascribed to enhanced photosynthetic efficiency, which ensured the provision of energy and carbon supply for Arabidopsis growth. The results further indicated that similar levels of enhanced growth and photosynthesis following lumichrome-treatment of wild type plants could be achieved in the knockout plants even in the absence of lumichrome. There was no further effect on the growth of these mutant lines following lumichrome, strongly suggesting that APS1 is responsible for mediating the lumichrome-associated growth response in Arabidopsis. Unlike wild type plants, starch levels in the aps1 lines were extremely low and were not affected by lumichrome treatment. We therefore conclude that lumichrome enhances growth in Arabidopsis plants via enhanced photosynthesis in a process mediated via APS1, and that the enhanced levels of starch seen in lumichrome treated wild type plants are merely an artifact of this enhanced photosynthesis. It will be interesting to investigate further the means by which APS1 regulates this mechanism. In summary, proteomic and metabolomic analyses all suggest that down-regulation of APS in lumichrometreated plants enhanced photosynthesis, leading to increased availability of C for enhanced plant growth.

AFRIKAANSE OPSOMMING: Ten spyte van die indrukwekkende vooruitgang wat gemaak is oor die jare in die verbetering van beide plantgroei en -opbrengste, is daar min rede om selfvoldaan oor hierdie ontwikkelings te raak, veral ten opsigte van voedselvoorsiening vir die wêreld se groeiende bevolking. Anders as konvensionele en merkerbemiddelde teling, word die gebruik van ruuittreksels uit plantgroei-bevorderende-rhizobakterieë (PGPR) wat biostimulerende eienskappe besit, beskou as 'n alternatiewe praktyk vir die verbetering van plantontwikkeling en gewas-produktiwiteit. Een sulke stof is lumichrome (7,8 dimethylalatloxazine), 'n nuwe plantgroei-bevorderende multitrofiese seinmolekule, wat deur die bakterie Sinorhizobium meliloti afgeskei word. Lumichrome is bewys om groei-bevordering te ontlok, gepaard met 'n kompenserende toename in geheel-plant netto-koolstof-assimilasie deur verbeterde styselopeenhoping en veranderde etileen-metabolisme. Ten spyte van vooruitgang in die fisiologiese verkenning van die groei-stimulerende meganismes van lumichrome, is 'n omvattende molekulêre analise in verskillende plantspesies steeds ontwykend. Hierdie studie fokus op die begrip van die genetiese, molekulêre en biochemiese regulatoriese netwerke as sleutelfaktore vir gewasgroei en produktiwiteit met betrekking tot lumichrome in Arabidopsis thaliana. Ons spesifieke doelwitte het gefokus daarop om die volgende te ontrafel: i) globale veranderinge in geenuitdrukking, ii) proteïen uitdrukking en iii) die belangrikste metaboliete of biochemiese-paaie in Arabidopsis thaliana wat skynbaar beïnvloed word deur behandeling met lumichrome. Om die regulatoriese meganismes van koolstofmetabolisme te bestudeer, het hierdie studie verder gebruik gemaak van gemuteerde Arabidopsis-lyne met ‘n tekort in die primêre geen wat die ADP-GLUKOSE PYROPHOSPHORYLASE klein subeenheid (APS1) enkodeer. Daarna is die bevindinge gebruik om funksionele interaksies tussen gene, proteïene en metaboliete af te lei om sodoende te spekuleer oor die moontlike beheermeganisme(s) wat betrokke is. Die aanwending van biologies-aktiewe vlakke van lumichrome is getoon om plantontwikkeling te verbeter in verskeie plantspesies deur veranderinge aan te bring in die tempo van fotosintese, blaarhuidmondjie geleiding en transpirasie. Verbeterde groei word na bewering toegeskryf aan die vervoer van lumichrome deur die xileem en in situ versameling in die blare, waar dit gevolglik gebeure veroorsaak wat seldeling en blaar-uitbreiding bevorder (in beide monokotiele en dikotiele). Tog het vorige studies ook gemengde fisiologiese reaksies tussen plantspesies getoon ten opsigte van lumichrome-behandeling. Daarom blyk molekulêre prosesse wat verantwoordelik is vir groei bevordering in ander spesies ietwat ontwykend. In ooreenstemming met vorige studies, het ons studie getoon dat die toevoeging van 5 nM lumichrome tot Arabidopsis thaliana plante groei-bevordering ontlok wat ‘n algehele verhoging in plantgrootte en biomassa tot gevolg het. Die verhoging in plantgrootte word toegeskryf aan verbeterde fotosintese en die gebruik van hoër vlakke van fotosintese-produkte vir seldeling en selvergroting. Dit word verder ondersteun deur “Next Generation Sequencing” (Transcriptomic profiling-True-Seq), wat aan die lig gebring het dat die groei-stimulerende effek oorwegend bewerkstellig word deur gene wat verband hou met selwandverandering, seldeling en uitbreiding. Die resultate van ons proteomika studie het verder voorgestel dat lumichrome-behandeling fotosintese verhoog en stabiliseer, en sodoende verhoogde fotosintese-produkte verskaf vir groei in wilde-tipe Arabidopsis. Hoewel styselvlakke verhoog was in wilde tipe plante wat met lumichrome behandel is, het die vlakke van APS1 onverwags afgeneem. Om te demonstreer dat die afskakeling van APS1 groei in Arabidopsis verhoog, het ons beide proteïene en metaboliete bestudeer in Arabidopsis T-DNA ‘knockout’ lyne met ‘n tekort aan APS1. Die tru-genetika benadering het getoon dat verbeterde groei van aps1 gemuteerde plante, in vergelyking met die wilde-tipe plante, toegeskryf kan word aan verbeterde fotosintetiese-doeltreffendheid wat die energie- en koolstoftoevoer verskaf vir groei in Arabidopsis. Die resultate het verder aangedui dat soortgelyke vlakke van verbeterde groei en fotosintese as gevolg van lumichrome-behandeling van wilde tipe plante bereik kan word in die ‘knockout’ gemuteerde plante, selfs in die afwesigheid van lumichrome. Die aanwending van lumichrome het geen verdere effek op die groei van hierdie ‘knockout’ plante gehad nie, wat daarop dui dat APS1 verantwoordelik is vir die bemiddeling van die lumichromegeassosieerde groei reaksie in Arabidopsis. In teenstelling met wilde-tipe plante, was die styselvlakke in die aps1 gemuteerde plante bitter laag en ongeaffekteer deur behandeling met lumichrome. Ons kan dus aflei dat lumichrome groei in Arabidopsis plante verhoog deur verbeterde fotosintese in 'n proses bemiddel deur APS1, en dat die verhoogde styselvlakke wat waargeneem is in lumichrome-behandelde wilde-tipe plante slegs ‘n byproduk is van hierdie verbeterde fotosintese. Dit sal interessant wees om die manier waarop APS1 hierdie meganisme reguleer verder te ondersoek. Om op te som, dui proteomiese en metabolomiese analises daarop dat die vermindering van APS in lumichrome-behandelde plante fotosintese verbeter, wat gevolglik lei tot die verhoogde beskikbaarheid van koolstof vir beter plantgroei.

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