Systems analysis of plant metabolism under UV-B radiation by integrating metabolomics and 13CO2 labeling(PhD)

Abstract

Deciphering the response of plants to biotic and abiotic stress is of immense scientific interest owing to their role in agriculture and environment. Under optimal and varying environmental parameters, how plants metabolise CO2 and regulate the metabolic flow from source to sink will allow identifying and developing efficient crops with desirable traits (Allen et. al., 2016). Among the abiotic parameters, UV-B radiation (290 nm to 315 nm), an indispensable element signals through photoreceptors and regulate their physiological, developmental and metabolic processes via a family of B-box (BBX) proteins (Song et al., 2020). Among these, BBX31, a transcription factor is reported to play a significant positive regulatory role under UV-B radiation (Yadav et. al., 2019). The metabolic phenotypes of plant systems with altered Bbox protein expression under the visible and UV-B radiation are not well-defined. The main goal of this thesis is to decipher the precise metabolic phenotypes of Arabidopsis thaliana Col-0 and BBX31 mutant (bbx31) and over expressor (35S:BBX31) lines under the influence of visible and UV-B radiation by integrating robust metabolic systems biology approaches of metabolite profiling, metabolomics and 13CO2 tracer-based pathway mapping. First, robust methodologies of metabolite profiling and metabolomics using Gas Chromatography-Mass Spectrometry (GC-MS) data analysis were optimized. This allowed comprehensive analysis of sugars, organic acids, amino acids and secondary metabolites. Much focus is laid on developing a robust methodology for the analysis of free, glycoside and ester bound phenolics, whose levels are known to be influenced under UV-B. Second, the Arabidopsis Col-0 seedlings subjected to UV-B radiation showed negative effects on the morpho-physiological parameters with diminished growth. Also, their metabolic analysis provided detailed insights into the central and secondary metabolic pathways under UV-B. Third, the metabolomics and pathway analysis of Arabidopsis Col-0, bbx31 mutant and 35S:BBX31 over expressor provided novel insights into the UV-B mediated metabolism. The morphological and metabolic phenotypes of 35S:BBX31 over expressor lines are distinct owing to their reprogrammed metabolism under visible as well as UV-B. BBX31 markedly influenced the levels of photosynthetic compounds, central and secondary metabolites. Mainly, the metabolic pathways involving GS/GOGAT, fatty acid, shikimate, phenolics and flavonoids are regulated, eventually conferring photoprotection under UV-B. Finally, with the aim to define the precise metabolic phenotypes, 13CO2 labeling of Col-0 and 35S:BBX31 over expressor lines was undertaken in a novel designed and built prototype of controlled feeding chambers. The prototype includes mass flow controllers for achieving optimal composition of gases, step change for controlled 13CO2 feeding at desired ppm levels, parallel growth chambers with controlled environmental parameters. BBX31 overexpression line in comparison to Col-0 showed enhanced rates of incorporation of 13C in the mass isotopomers of sugars, soluble amino acids and proteinogenic amino acids under visible light as well as UV-B respectively. The 13C analysis highlighted distinct metabolic phenotypes of Col-0 and BBX31, eventually shedding light qualitatively on the activities of various metabolic pathways and source to sink CO2 mapping. Overall, the robust methodologies of metabolic analysis facilitated defining the metabolic phenotypes of Arabidopsis genotypes (Col-0, bbx31, 35S:BBX31) under the influence of visible and UV-B. The outcomes provided novel insights into our understanding of the BBX31 mediated metabolic rewiring that eventually confer UV-B tolerance in plants. Broadly, the optimised metabolic analysis workflow including plant phenolics and the prototype of 13CO2 parallel feeding set up can readily be adopted to investigate other plant systems under biotic and abiotic stress of interest for agricultural applications.