LC-MS Thermal proteome profiling in Arabidopsis thaliana to study ascorbate interaction

Ascorbate is a major plant metabolite that plays crucial roles in various processes, from reactive oxygen scavenging to epigenetic regulation. However, to what extent and how ascorbate modulates metabolism is largely unknown. To address this, we investigated the consequences of chloroplastic and total cellular ascorbate-deficiencies by studying chloroplastic ascorbate transporter pht4;4 mutant lines, and the ascorbate-deficient vtc2-4 mutant of Arabidopsis thaliana. Under regular growth conditions, both ascorbate-deficiencies caused minor alterations in photosynthesis, with no apparent signs of oxidative damage. In contrast, metabolomics analysis revealed a global and largely overlapping metabolome rewiring in both ascorbate deficiencies, suggesting that chloroplastic ascorbate modulates plant metabolism. We observed significant alterations in amino acid metabolism, particularly in arginine metabolism, activation of nucleotide salvage pathways, and changes in secondary metabolism. In addition, proteome wide analysis of thermostability revealed that ascorbate may interact with enzymes involved in arginine metabolism, the Calvin-Benson cycle, and several photosynthetic electron transport components. Overall, our results suggest that, independently of oxidative stress, chloroplastic ascorbate interconnects and coordinates diverse metabolic pathways in vascular plants and thus acts as a regulatory hub.