The authors adapted OpenPlant kit CRISPR/Cas9 tools to enable multiplex gRNA expression from a single transcript using tRNA sequences in the liverwort Marchantia polymorpha, markedly enhancing editing efficiency and scalability. They coupled this vector system with a simplified, optimized thallus transformation protocol, providing a rapid and versatile platform for generating CRISPR/Cas9 mutants and advancing functional genomics in this model species.

Phylogenetic analysis reveals that non‑seed plants, exemplified by the liverwort Marchantia polymorpha, possess a streamlined repertoire of cyclin and CDK genes, with only three cyclins active in a phase‑specific manner during vegetative development. Single‑cell RNA‑seq and fluorescent reporter assays, combined with functional overexpression studies, demonstrate the distinct, non‑redundant roles of MpCYCD;1, MpCYCA, and MpCYCB;1 in G1 entry, S‑phase progression, and G2/M transition, respectively.

Chromatin accessibility profiling and transcriptomics of Marchantia polymorpha heat‑shock transcription factor (HSF) mutants reveal that HSFA1 governs the placement of cis‑regulatory elements for heat‑induced gene activation, a mechanism conserved across plants, mice, and humans. Integrated gene regulatory network modeling identifies MpWRKY10 and MpABI5B as indirect regulators linking phenylpropanoid and stress pathways, while abscisic acid influences gene expression downstream of HSFA1 without broadly reshaping chromatin. A cross‑species, cross‑condition machine‑learning framework successfully predicts chromatin accessibility and expression, underscoring a conserved regulatory logic in stress responses.