Mahadevan, C. | Search bioRxiv

bioRxiv2025

Polyphenol oxidase silencing avoids protein crosslinking and enzymatic browning in Nicotiana benthamiana leaf extracts

Oxidation in extracts from agroinfiltrated leaves leads to browning and protein precipitation. Here we show that silencing of polyphenol oxidase (PPO) in Nicotiana benthamiana suppresses enzymatic browning and avoids crosslinking of endogenous proteins such as RuBisCo. PPO silencing does not impact plant growth or development and may also enhance transient gene expression. These results show that depletion of PPO activity could be highly beneficial for plant science applications and molecular pharming.

plant biology↗

bioRxiv2024

Discovery of active mouse, plant and fungal cytochrome P450s in endogenous proteomes and upon expression in planta.

Eukaryotes produce a large number of cytochrome P450s that mediate the synthesis and degradation of diverse endogenous and exogenous metabolites. Yet, most of these P450s are uncharacterized and global tools to study these challenging, membrane-resident enzymes remain to be exploited. Here, we applied activity profiling of plant, mouse and fungal P450s with chemical probes that become reactive when oxidized by P450 enzymes. Identification by mass spectrometry revealed labeling of a wide range of active P450s, including six plant P450s, 40 mouse P450s and 13 P450s of the fungal wheat pathogen Zymoseptoria tritici. We next used transient expression of GFP-tagged P450s by agroinfiltration to show ER-targeting and NADPH-dependent, activity-based labeling of plant, mouse and fungal P450s. Both global profiling and transient expression can be used to detect a broad range of active P450s to study e.g. their regulation and discover selective inhibitors.

plant biology↗