Profiling Thimet Oligopeptidase‐Mediated Proteolysis in Arabidopsis thaliana

Abstract

Proteostasis is essential for proper cellular function, including the production of peptides with biological functions via the breakdown of proteins/peptides through controlled proteolysis. Proteostasis contributes to the maintenance of cellular functions and plant‐environment interactions under physiological conditions. Plant stress continues to reduce agricultural yields resulting in significant economic losses; thus, it is crucial to understand how plants perceive stress signals to elicit responses for survival. As previously shown in Arabidopsis thaliana, thimet oligopeptidases (TOPs) are essential components in plant response to oxidative stress triggered by pathogens. However, further characterization of TOP1 (also referred to as organellar oligopeptidase), TOP2 (also referred to as cytosolic oligopeptidase) and their peptide substrates is required to understand their contributions to stress perception and defense signaling. Label‐free peptidomics via liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) was used to differentially quantify 1111 peptides, originating from 369 proteins, between the Arabidopsis Col‐0 wild type and top1top2 knock‐out mutant. This revealed 350 peptides as significantly more abundant in the mutant, representing accumulation of these potential TOP substrates. Ten direct substrates were validated using in vitro enzyme assays and electrospray ionization mass spectrometry (ESI‐MS) with recombinant TOPs and synthetic candidate peptides. The TOP substrates discovered are derived from proteins involved in photosynthesis, biogenesis, glycolysis, protein folding, and antioxidant defense, implicating TOP involvement in processes beyond defense signaling. Identification of these substrates provides a framework for TOP signaling networks, through which the interplay between proteolytic pathways and defense signaling can be further characterized.