MultiPly · FMO1 Multifunctionality for improved Plant health

FMO1 Multifunctionality for improved Plant health

‘MultiPly: FMO1 Multifunctionality for improved Plant health’ The project 'MultiPly' will investigate the extended role and evolution of the critically important FMO family in plants and examine the physiological mechanisms of how this enzyme can improve plant resilience under environmental stress conditions. The long-term ambition of this project is to identify new and fundamental mechanisms of plant resistance for incorporation into crop breeding programs. FMO1 is an essential enzyme for pathogen resistance in plants, and a valuable biotechnological target for crop health improvement. The importance of FMO1 for plant health has been recognised for more than 20 years, but its biochemical function in Arabidopsis was only recently determined: FMO1 catalyzes the N-hydroxylation of Pipecolic acid (Pip) to form N-hydroxy-pipecolic acid (N-Pip). N-Pip is the essential signalling molecule for Systemic Acquired Resistance (SAR) defense priming in plants (Figure 1). Following pathogen infection, a salicylic acid (SA) response cascade initiates N-Pip production at the site of infection, which is thereafter transported to distal tissues, signalling a new SA/SAR defense cascade in the uninfected tissue. FMO1 is therefore responsible for developing long-lasting and broad-spectrum disease resistance in plants and a potential target for metabolic engineering to improve disease resistance. In addition to that, studies on Barley FMO1 (HvFMO1) suggest that they possesses alternative functionality in vitro, catalysing the C-hydroxylation of indole to form indoxyl which then dimerizes to form the pigment indigo (Figure 2), highlighting potential diversification of function beyond pipecolic acid metabolism. These data suggest diversification of the FMO1 family in barley, both in terms of substrate specificity (indole vs pipecolic acid) and reaction mechanism (C-hydroxylation vs N-hydroxylation). It is possible that FMO1 functionality has diversified in other plant clades as well. Phylogenetic analyses further suggest duplication events in some eudicot species, pointing to evolutionary diversification within the FMO1 family (Figure 3). Together, these findings indicate that FMO1 may possess broader substrate specificity and roles in both biotic and abiotic stress responses. Understanding FMO1 multifunctionality is therefore highly relevant, not only to resolve fundamental questions of enzyme evolution, but also to identify new breeding targets for improving plant resilience and food security under climate change. FMO1 is a flavin-containing monooxygenase (FMO) belonging to the N-hydroxylating (N-OX) clade. Plants also contain two other major FMO clades: the S-oxidizing (S-OX) FMOs and the YUCCA FMOs (involved in auxin biosynthesis). FMOs are notoriously difficult to purify, and comprehensive structure–function studies are rare. To date, only a single plant FMO has been crystallized (an S-OX from garlic) and one YUCCA protein (AtYUCCA6) has been biochemically characterized. No structure–function studies have yet been reported for the plant FMO1 family, leaving major gaps in our understanding of their catalytic mechanisms, evolution, and functional diversification. Also to date, comprehensive substrate specificity analysis of FMO1 and other plant FMOs has not been performed. Two key questions raised in MultiPly are: 1. Does FMO1 have additional biochemical functions or alternative substrates? 2. Is the N-Pip/SAR defence system conserved across all plants, or do alternative mechanisms exist? The MultiPly project aims to addresses these questions by integrating biochemistry, structural biology, and evolutionary approaches (Figure 4). Objectives of MultiPly:- ---------------------------- The project’s research objectives were organized in three work packages (WP1-WP3): (1) Evolution and characterization of FMO1 sequence homology across the plant kingdom (2) Characterization of plant FMO1s and their catalytic functionality in vitro and in vivo (3) In planta characterization of SAR in barley (H. vulgare) Thus by combining wet-lab enzymology with computational analyses, MultiPly aims to generate new insights into plant defense mechanisms, strengthen our molecular understanding of FMO1, and explore its potential as a target for engineering disease-resistant crops.

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