MALDI-MSI spatial mapping of phenanthrenes in Bletilla striata leaves uncovers BsLAC1 as the first laccase catalysing...

Bletilla striata, a traditional Chinese medicinal herb, is particularly rich in phenanthrenes and their dimers, which exhibit anti-inflammatory, antibacterial, and antitumor properties. While phenanthrenes are biosynthesized from bibenzyl precursors through free radical oxidative coupling, and that monomers may further dimerize through radical reactions, the enzymatic basis of the dimerisation remains unexplored. In this study, matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was employed to visualize the distribution of metabolites across four tissues-pseudobulbs, stems, petioles, and leaf tips. Bibenzyls, phenanthrenes, and their dimers exhibited distinct spatial patterns, with significantly stronger signals in leaf tips than in petioles. Quantitative analysis revealed that phenanthrene dimers were significantly more abundant in leaf tips, suggesting that enzymes promoting monomer coupling into dimers may be more active there. Given the well-established role of laccases in radical coupling reactions in plants, we hypothesized that these enzymes may catalyse phenanthrene dimerisation. In vitro assays confirmed that commercial laccases from Trametes versicolor and Rhus verniciflua catalysed phenanthrene dimer formation. Transcriptomic comparison between leaf tips and petioles, coupled with AlphaFold3-based structural modelling and homology alignment identified five candidate laccase genes, with Unigene10810 showing the highest similarity to the R. verniciflua laccase. Transient expression of Unigene10810 in Nicotiana benthamiana confirmed its catalytic activity, and it was designated as Bletilla striata laccase1 (BsLAC1). This study provides the first evidence of laccase-mediated radical oxidative coupling in phenanthrene dimer biosynthesis, enhancing our understanding of their biosynthetic pathway in B. striata.