Het ontrafelen van de regulatie van ethyleen productie door de vorming van N-malonyl 1-aminocyclopropaan-1-carbonzuur De ontdekking van ACC-N-malonyl transferase (AMT)

The phytohormone ethylene is crucial for various aspects of plant development throughout the entire plant life cycle, from germination to organ senescence and abscission. Furthermore, it is also important in relation to stress responses. Plants have developed sophisticated mechanisms to regulate ethylene biosynthesis, controlling its production rate in both time and space. One such regulatory mechanism involves the derivatisation of ethylene's direct precursor 1‑aminocyclopropane-1-carboxylic acid (ACC). Recent studies have shown that ACC functions as a signalling molecule independent of ethylene, which highlights the importance of regulating ACC levels through conjugation. This research focuses on the most abundant ACC derivative, malonyl‑ACC (MACC), formed by the enzyme ACC‑N‑malonyl transferase (AMT). Despite its discovery four decades ago, the identity of the AMT enzyme remained unknown up to now, which was a huge gap in our understanding of ethylene biosynthesis regulation through ACC conjugation. The goal of this project was to identify AMT and elucidate its biological function. To achieve this, three strategies were developed: (1) protein purification by column chromatography, (2) comparative proteomics and (3) comparative transcriptomics. At the same time, an in vitro AMT activity assay was optimized, enabling the identification of protein purification fractions with active AMT and the study of AMT activity in various biological samples. Using this AMT activity assay, we have investigated the feedback mechanism of ethylene on AMT activity. Our findings revealed that AMT activity is regulated not only by ethylene but also by ACC. Additionally, feeding experiments with MACC resulted in an ethylene phenotype in Arabidopsis seedlings, suggesting that MACC is not an inactive end-product, and can have a biologically active function. By investigating multiple ethylene-related mutants in our MACC feeding experiments, we hypothesize that MACC can be reconverted into ACC and subsequently into ethylene, although more research is needed. After many optimization steps and perfecting the column purification strategy, we successfully identified two AMT proteins from tomato using LC-MS/MS. Phylogenetic analysis revealed that AMTs of tomato and Arabidopsis belong to two clades of the larger acyl‑transferase protein family. In total, 12 AMTs were identified of which six are present in tomato and six in Arabidopsis. We confirmed the ACC-malonyl transferase activity of three AMT members of both tomato and Arabidopsis by ectopic expression in Escherichia coli (E. coli) and quantifying MACC formed using an indirect GC and a direct LC-MS method. Deploying site-directed mutagenesis of a crucial HIS‑residue, known the be essential for acyl‑transferase activity, resulted in a drastic decrease in AMT activity. Furthermore, we fully purified four AMT proteins (two from tomato and two from Arabidopsis) using HIS‑tag affinity purification and size-exclusion chromatography. These pure AMTs will be further used for biochemical characterization. The identification of AMT in this study paves the way for extensive future research on the role of ACC conjugation in both ethylene- and ACC-specific responses. By developing knock‑out, overexpression, and transcriptional and translational reporter lines for individual AMTs, we can strongly enhance our understanding of the role of MACC formation in plants, across developmental stages and environmental conditions.