FWF - MAT - Monolingnol oxidoreductases in Arabidopsis thaliana

The berberine bridge enzyme (BBE) catalyzes the formation of the so-called “berberine bridge”, which constitutes a unique type of ring-closure leading to the biosynthesis of benzylisoquinoline alkaloids. Higher plants contain a large number of genes coding for BBE-like proteins although alkaloid production was not reported for most of these plants. In the case of Arabidopsis thaliana, 27 genes were identified encoding BBE-like proteins, which can be grouped into seven distinct families. Recently, we have shown that two members of a subfamily, comprising a total of five enzymes, oxidize monolignols to their corresponding aldehydes in vitro, i.e. these enzymes have monolignol oxidoreductase (MOX) activity. Monolignols and their corresponding aldehydes are essential building blocks for the plant polymer lignin and, therefore, we hypothesize that BBE-like MOXs play a role in controlling the extracellular ratio of monolignols to aldehydes. By specifically altering the composition of the extracellular pool of lignin monomers, the five enzymes of the MOX subfamily could be involved in modifying the aldehyde content of lignin in a highly localized manner, for example at certain developmental stages, as well as during a spatio-temporally specific response to environmental factors. To test our hypotheses, we will employ analytical, biochemical, and genetic methods. The expression of the five genes in various plant tissues will be analyzed by qRT-PCR and promoter-GUS reporter lines. The subcellular localization of the proteins will be achieved by generating fusion constructs with a fluorescent protein tag and subsequent analysis by confocal laser scanning microscopy. To obtain further information on the physiological role of the encoded proteins, loss-of-function mutants will be generated by using T-DNA insertion lines in combination with CRISPR/Cas9 editing. We will generate single and multiple knock-out lines to gain information of the potential redundancy in the family of MOX. In addition, the biochemical composition of selected plant tissues will be analyzed by a mass-spectrometry based metabolomics approach (Prof. Boerjan) and solution-state NMR-spectroscopy (Prof. Ralph). The proposed novel role of BBE-like MOXs will provide further insights into mechanisms that alter the composition of lignin in developmental contexts and as a response to abiotic and biotic factors. In this sense, our research will not only promote our understanding of plant development and defense strategies, but may also be valuable for efforts to manipulate the composition of lignin for improved applicability in biotechnology.