Browsing by Author "Pholo, Motlalepula"

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    Molecular analysis of plant growth promoted with low molecular weight compounds in relation to genetically altered photosynthetic carbohydrate partitioning in higher plants
    (Stellenbosch : Stellenbosch University, 2017-03) Pholo, Motlalepula; Hills, Paul N.; Kossmann, Jens; Lloyd, James Richard; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. Institute for Plant Biotechnology.
    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.