SACM - Netherlands
Permanent URI for this collectionhttps://hdl.handle.net/20.500.14154/68623
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Item Restricted evelopment of a Reporter Toolkit to Study Transcriptional Dynamics in Marchantia polymorpha(Saudi Digital Library, 2025) Alhammad, Ahmed; Weijers, Dolf; Borst, Jan Willem; García, Jorge HernándezTranscriptional 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.8 0