The landscape of transcription factor promoter activity during vegetative development in Marchantia.

Transcription factors (TFs) are essential for the regulation of gene expression and cell fate determination. Characterizing the transcriptional activity of TF genes in space and time is a critical step toward understanding complex biological systems. The vegetative gametophyte meristems of bryophytes share some characteristics with the shoot apical meristems of flowering plants. However, the identity and expression profiles of TFs associated with gametophyte organization are largely unknown. With only ∼450 putative TF genes, Marchantia (Marchantia polymorpha) is an outstanding model system for plant systems biology. We have generated a near-complete collection of promoter elements derived from Marchantia TF genes. We experimentally tested reporter fusions for all the TF promoters in the collection and systematically analyzed expression patterns in Marchantia gemmae. This allowed us to build a map of expression domains in early vegetative development and identify a set of TF-derived promoters that are active in the stem-cell zone. The cell markers provide additional tools and insight into the dynamic regulation of the gametophytic meristem and its evolution. In addition, we provide an online database of expression patterns for all promoters in the collection. We expect that these promoter elements will be useful for cell-type-specific expression, synthetic biology applications, and functional genomics.

Optimizing Promoters and Subcellular Localization for Constitutive Transgene Expression in Marchantia polymorpha.

Marchantia polymorpha has become an important model system for comparative studies and synthetic biology. The systematic characterization of genetic elements would make heterologous gene expression more predictable in this test bed for gene circuit assembly and bioproduction. Yet, the toolbox of genetic parts for Marchantia includes only a few constitutive promoters that need benchmarking to assess their utility. We compared the expression patterns of previously characterized and new constitutive promoters. We found that driving expression with the double enhancer version of the cauliflower mosaic virus 35S promoter (pro35S × 2) provided the highest yield of proteins, although it also inhibits the growth of transformants. In contrast, promoters derived from the Marchantia genes for ETHYLENE RESPONSE FACTOR 1 and the CLASS II HOMEODOMAIN-LEUCINE ZIPPER protein drove expression to higher levels across all tissues without a growth penalty and can provide intermediate levels of gene expression. In addition, we showed that the cytosol is the best subcellular compartment to target heterologous proteins for higher levels of expression without a significant growth burden. To demonstrate the potential of these promoters in Marchantia, we expressed RUBY, a polycistronic betalain synthesis cassette linked by P2A sequences, to demonstrate coordinated expression of metabolic enzymes. A heat-shock-inducible promoter was used to further mitigate growth burdens associated with high amounts of betalain accumulation. We have expanded the existing tool kit for gene expression in Marchantia and provided new resources for the Marchantia research community.