evelopment of a Reporter Toolkit to Study Transcriptional Dynamics in Marchantia polymorpha

Abstract

Transcriptional regulation is pivotal for plant development and adaptation, yet mechanistic insights remain limited in liverwort plants. To bridge this, we developed a modular fluorescent reporter toolkit to enable real-time quantitative exploration of chromatin accessibility and polymerase recruitment in Marchantia polymorpha. Using a modular Golden Gate Cloning strategy, we engineered constructs tagging Histone 2B variants and RNA Polymerase II subunits with fluorescent proteins to monitor chromatin organization and polymerase activity. Furthermore, we designed a dCas9-based system coupled with the MpARF1 activation domain developed for targeted gene activation studies. Subsequently, stable transgenic lines expressing H2B.3-mScarlet-I exhibited nuclear-localized fluorescence, validating chromatin-specific labelling. While constructs for several RNAPII subunits were successfully assembled and transformed into Marchantia polymorpha, microscopy validation remains ongoing. Collectively, this toolkit provides a foundational resource for quantifying transcriptional dynamics, chromatin behavior, and RNAPII activity in non-vascular plants, thereby advancing synthetic biology applications and evolutionary studies of gene regulation. This adaptable toolkit provides essential molecular instruments for quantitative live-cell imaging and synthetic biology approaches in Marchantia polymorpha.