Optimising promoters and subcellular localisation for constitutive transgene expression in Marchantia polymorpha.

Marchantia polymorpha has become an important model system for comparative studies and synthetic biology. The systematic characterisation of genetic elements would make heterologous gene expression more predictable in this testbed 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 ETHYLENE RESPONSE FACTOR 1 (MpERF1) and the CLASS II HOMEODOMAIN-LEUCINE ZIPPER (MpC2HDZ) genes drove expression to higher levels across all tissues without 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 the polycistronic RUBY betalain synthesis cassette 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 toolkit for gene expression in Marchantia and provide new resources for the Marchantia research community.

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.

The Polycomb repressive complex 2 deposits H3K27me3 and represses transposable elements in a broad range of eukaryotes.

The mobility of transposable elements (TEs) contributes to evolution of genomes. Their uncontrolled activity causes genomic instability; therefore, expression of TEs is silenced by host genomes. TEs are marked with DNA and H3K9 methylation, which are associated with silencing in flowering plants, animals, and fungi. However, in distantly related groups of eukaryotes, TEs are marked by H3K27me3 deposited by the Polycomb repressive complex 2 (PRC2), an epigenetic mark associated with gene silencing in flowering plants and animals. The direct silencing of TEs by PRC2 has so far only been shown in one species of ciliates. To test if PRC2 silences TEs in a broader range of eukaryotes, we generated mutants with reduced PRC2 activity and analyzed the role of PRC2 in extant species along the lineage of Archaeplastida and in the diatom P. tricornutum. In this diatom and the red alga C. merolae, a greater proportion of TEs than genes were repressed by PRC2, whereas a greater proportion of genes than TEs were repressed by PRC2 in bryophytes. In flowering plants, TEs contained potential cis-elements recognized by transcription factors and associated with neighbor genes as transcriptional units repressed by PRC2. Thus, silencing of TEs by PRC2 is observed not only in Archaeplastida but also in diatoms and ciliates, suggesting that PRC2 deposited H3K27me3 to silence TEs in the last common ancestor of eukaryotes. We hypothesize that during the evolution of Archaeplastida, TE fragments marked with H3K27me3 were selected to shape transcriptional regulation, controlling networks of genes regulated by PRC2.

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MarpolBase Expression: A Web-Based, Comprehensive Platform for Visualization and Analysis of Transcriptomes in the Liverwort Marchantia polymorpha.

The liverwort Marchantia polymorpha is equipped with a wide range of molecular and genetic tools and resources that have led to its wide use to explore the evo-devo aspects of land plants. Although its diverse transcriptome data are rapidly accumulating, there is no extensive yet user-friendly tool to exploit such a compilation of data and to summarize results with the latest annotations. Here, we have developed a web-based suite of tools, MarpolBase Expression (MBEX, https://marchantia.info/mbex/), where users can visualize gene expression profiles, identify differentially expressed genes, perform co-expression and functional enrichment analyses and summarize their comprehensive output in various portable formats. Using oil body biogenesis as an example, we demonstrated that the results generated by MBEX were consistent with the published experimental evidence and also revealed a novel transcriptional network in this process. MBEX should facilitate the exploration and discovery of the genetic and functional networks behind various biological processes in M. polymorpha and promote our understanding of the evolution of land plants.

The renaissance and enlightenment of Marchantia as a model system.

The liverwort Marchantia polymorpha has been utilized as a model for biological studies since the 18th century. In the past few decades, there has been a Renaissance in its utilization in genomic and genetic approaches to investigating physiological, developmental, and evolutionary aspects of land plant biology. The reasons for its adoption are similar to those of other genetic models, e.g. simple cultivation, ready access via its worldwide distribution, ease of crossing, facile genetics, and more recently, efficient transformation, genome editing, and genomic resources. The haploid gametophyte dominant life cycle of M. polymorpha is conducive to forward genetic approaches. The lack of ancient whole-genome duplications within liverworts facilitates reverse genetic approaches, and possibly related to this genomic stability, liverworts possess sex chromosomes that evolved in the ancestral liverwort. As a representative of one of the three bryophyte lineages, its phylogenetic position allows comparative approaches to provide insights into ancestral land plants. Given the karyotype and genome stability within liverworts, the resources developed for M. polymorpha have facilitated the development of related species as models for biological processes lacking in M. polymorpha.

Liverwort oil bodies: diversity, biochemistry, and molecular cell biology of the earliest secretory structure of land plants.

Liverworts are known for their large chemical diversity. Much of this diversity is synthesized and enclosed within oil bodies (OBs), a synapomorphy of the lineage. OBs contain the enzymes to biosynthesize and store large quantities of sesquiterpenoids and other compounds while limiting their cytotoxicity. Recent important biochemical and molecular discoveries related to OB formation, diversity, and biochemistry allow comparison with other secretory structures of land plants from an evo-devo perspective. This review addresses and discusses the most recent advances in OB origin, development, and function towards understanding the importance of these organelles in liverwort physiology and adaptation to changing environments. Our mapping of OB types and chemical compounds to the current liverwort phylogeny suggests that OBs were present in the most recent common ancestor of liverworts, supporting that OBs evolved as the first secretory structures in land plants. Yet, we require better sampling to define the macroevolutionary pattern governing the ancestral type of OB. We conclude that current efforts to find molecular mechanisms responsible for the morphological and chemical diversity of secretory structures will help understand the evolution of each major group of land plants, and open new avenues in biochemical research on bioactive compounds in bryophytes and vascular plants.

ROS-Scavenging Enzymes as an Antioxidant Response to High Concentration of Anthracene in the Liverwort Marchantia polymorpha L.

Marchantia polymorpha L. responds to environmental changes using a myriad set of physiological responses, some unique to the lineage related to the lack of a vascular- and root-system. This study investigates the physiological response of M. polymorpha to high doses of anthracene analysing the antioxidant enzymes and their relationship with the photosynthetic processes, as well as their transcriptomic response. We found an anthracene dose-dependent response reducing plant biomass and associated to an alteration of the ultrastructure of a 23.6% of chloroplasts. Despite a reduction in total thallus-chlorophyll of 31.6% of Chl a and 38.4% of Chl b, this was not accompanied by a significant change in the net photosynthesis rate and maximum quantum efficiency (Fv/Fm). However, we found an increase in the activity of main ROS-detoxifying enzymes of 34.09% of peroxidase and 692% of ascorbate peroxidase, supported at transcriptional level with the upregulation of ROS-related detoxifying responses. Finally, we found that M. polymorpha tolerated anthracene-stress under the lowest concentration used and can suffer physiological alterations under higher concentrations tested related to the accumulation of anthracene within plant tissues. Our results show that M. polymorpha under PAH stress condition activated two complementary physiological responses including the activation of antioxidant mechanisms and the accumulation of the pollutant within plant tissues to mitigate the damage to the photosynthetic apparatus.

Molecular mechanisms involved in functional macroevolution of plant transcription factors.

Transcription factors (TFs) are key components of the transcriptional regulation machinery. In plants, they accompanied the evolution from unicellular aquatic algae to complex flowering plants that dominate the land environment. The adaptations of the body plan and physiological responses required changes in the biological functions of TFs. Some ancestral gene regulatory networks are highly conserved, while others evolved more recently and only exist in particular lineages. The recent emergence of novel model organisms provided the opportunity for comparative studies, producing new insights to infer these evolutionary trajectories. In this review, we comprehensively revisit the recent literature on TFs of nonseed plants and algae, focusing on the molecular mechanisms driving their functional evolution. We discuss the particular contribution of changes in DNA-binding specificity, protein-protein interactions and cis-regulatory elements to gene regulatory networks. Current advances have shown that these evolutionary processes were shaped by changes in TF expression pattern, not through great innovation in TF protein sequences. We propose that the role of TFs associated with environmental and developmental regulation was unevenly conserved during land plant evolution.

The Intrinsically Disordered Protein CARP9 Bridges HYL1 to AGO1 in the Nucleus to Promote MicroRNA Activity.

In plants, small RNAs are loaded into ARGONAUTE (AGO) proteins to fulfill their regulatory functions. MicroRNAs (miRNAs), one of the most abundant classes of endogenous small RNAs, are preferentially loaded into AGO1. Such loading, long believed to happen exclusively in the cytoplasm, was recently proposed to also occur in the nucleus. Here, we identified CONSTITUTIVE ALTERATIONS IN THE SMALL RNAS PATHWAYS9 (CARP9), a nuclear-localized, intrinsically disordered protein, as a factor promoting miRNA activity in Arabidopsis (Arabidopsis thaliana). Mutations in the CARP9-encoding gene led to a mild reduction of miRNAs levels, impaired gene silencing, and characteristic morphological defects, including young leaf serration and altered flowering time. Intriguingly, we found that CARP9 was able to interact with HYPONASTIC LEAVES1 (HYL1), but not with other proteins of the miRNA biogenesis machinery. In the same way, CARP9 appeared to interact with mature miRNA, but not with primary miRNA, positioning it after miRNA processing in the miRNA pathway. CARP9 was also able to interact with AGO1, promoting its interaction with HYL1 to facilitate miRNA loading in AGO1. Plants deficient in CARP9 displayed reduced levels of AGO1-loaded miRNAs, partial retention of miRNA in the nucleus, and reduced levels of AGO1. Collectively, our data suggest that CARP9 might modulate HYL1-AGO1 cross talk, acting as a scaffold for the formation of a nuclear post-primary miRNA-processing complex that includes at least HYL1, AGO1, and HEAT SHOCK PROTEIN 90. In such a complex, CARP9 stabilizes AGO1 and mature miRNAs, allowing the proper loading of miRNAs in the effector complex.

Oil Body Formation in Marchantia polymorpha Is Controlled by MpC1HDZ and Serves as a Defense against Arthropod Herbivores.

The origin of a terrestrial flora in the Ordovician required adaptation to novel biotic and abiotic stressors. Oil bodies, a synapomorphy of liverworts, accumulate secondary metabolites, but their function and development are poorly understood. Oil bodies of Marchantia polymorpha develop within specialized cells as one single large organelle. Here, we show that a class I homeodomain leucine-zipper (C1HDZ) transcription factor controls the differentiation of oil body cells in two different ecotypes of the liverwort M. polymorpha, a model genetic system for early divergent land plants. In flowering plants, these transcription factors primarily modulate responses to abiotic stress, including drought. However, loss-of-function alleles of the single ortholog gene, MpC1HDZ, in M. polymorpha did not exhibit phenotypes associated with abiotic stress. Rather, Mpc1hdz mutant plants were more susceptible to herbivory, and total plant extracts of the mutant exhibited reduced antibacterial activity. Transcriptomic analysis of the mutant revealed a reduction in expression of genes related to secondary metabolism that was accompanied by a specific depletion of oil body terpenoid compounds. Through time-lapse imaging, we observed that MpC1HDZ expression maxima precede oil body formation, indicating that MpC1HDZ mediates differentiation of oil body cells. Our results indicate that M. polymorpha oil bodies, and MpC1HDZ, are critical for defense against herbivory, but not for abiotic stress tolerance. Thus, C1HDZ genes were co-opted to regulate separate responses to biotic and abiotic stressors in two distinct land plant lineages.

Co-expression and Transcriptome Analysis of Marchantia polymorpha Transcription Factors Supports Class C ARFs as Independent Actors of an Ancient Auxin Regulatory Module.

We performed differential gene expression (DGE) and co-expression analyses with genes encoding components of hormonal signaling pathways and the ∼400 annotated transcription factors (TFs) of M. polymorpha across multiple developmental stages of the life cycle. We identify a putative auxin-related co-expression module that has significant overlap with transcripts induced in auxin-treated tissues. Consistent with phylogenetic and functional studies, the class C ARF, MpARF3, is not part of the auxin-related co-expression module and instead is associated with transcripts enriched in gamete-producing gametangiophores. We analyze the Mparf3 and MpmiR160 mutant transcriptomes in the context of coexpression to suggest that MpARF3 may antagonize the reproductive transition via activating the MpMIR11671 and MpMIR529c precursors whose encoded microRNAs target SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) transcripts of MpSPL1 and MpSPL2. Both MpSPL genes are part of the MpARF3 co-expression group corroborating their functional significance. We provide evidence of the independence of MpARF3 from the auxin-signaling module and provide new testable hypotheses on the role of auxin-related genes in patterning meristems and differentiation events in liverworts.

Evolutionary history of HOMEODOMAIN LEUCINE ZIPPER transcription factors during plant transition to land.

Plant transition to land required several regulatory adaptations. The mechanisms behind these changes remain unknown. Since the evolution of transcription factors (TFs) families accompanied this transition, we studied the HOMEODOMAIN LEUCINE ZIPPER (HDZ) TF family known to control key developmental and environmental responses. We performed a phylogenetic and bioinformatics analysis of HDZ genes using transcriptomic and genomic datasets from a wide range of Viridiplantae species. We found evidence for the existence of HDZ genes in chlorophytes and early-divergent charophytes identifying several HDZ members belonging to the four known classes (I-IV). Furthermore, we inferred a progressive incorporation of auxiliary motifs. Interestingly, most of the structural features were already present in ancient lineages. Our phylogenetic analysis inferred that the origin of classes I, III, and IV is monophyletic in land plants in respect to charophytes. However, class IIHDZ genes have two conserved lineages in charophytes and mosses that differ in the CPSCE motif. Our results indicate that the HDZ family was already present in green algae. Later, the HDZ family expanded accompanying critical plant traits. Once on land, the HDZ family experienced multiple duplication events that promoted fundamental neo- and subfunctionalizations for terrestrial life.

Arabidopsis thaliana homeodomain-leucine zipper type I transcription factors contribute to control leaf venation patterning.

Venation patterning is a taxonomic attribute for classification of plants and it also plays a role in the interaction of plants with the environment. Despite its importance, the molecular physiology controlling this aspect of plant development is still poorly understood. Auxin plays a central role modulating the final vein network and patterning. This addendum discusses recent findings on the role of homeodomain-leucine zipper (HD-Zip) transcription factors on the regulation of leaf venation patterning. Moreno-Piovano et al. reported that ectopic expression of a sunflower HD-Zip I gene, HaHB4, increased the asymmetry of leaf venation. Even more, this work showed that auxin transport in the leaf through LAX carriers controls venation patterning. Here, we provide evidence indicating that some Arabidopsis thaliana HD-Zip I genes play a role in the determination of the final leaf venation patterning. We propose that these genes contribute to regulate vein patterning, likely controlling auxin homeostasis.

A matter of quantity: Common features in the drought response of transgenic plants overexpressing HD-Zip I transcription factors.

Plant responses to water deficit involve complex molecular mechanisms in which transcription factors have key roles. Previous reports ectopically overexpressed a few members of the homeodomain-leucine zipper I (HD-Zip I) family of transcription factors from different species, and the obtained transgenic plants exhibited drought tolerance which extent depended on the level of overexpression, triggering diverse molecular and physiological pathways. Here we show that most HD-Zip I genes are regulated by drought in the vegetative and/or reproductive stages. Moreover, uncharacterized members of this family were expressed as transgenes both in Col-0 and rdr6-12 backgrounds and were able to enhance drought tolerance in host plants. The extent of such tolerance depended on the expression level of the transgene and was significantly higher in transgenic rdr6-12 than in Col-0. Comparative transcriptome analyses of Arabidopsis thaliana plants overexpressing HD-Zip I proteins indicated that many members have common targets. Moreover, the water deficit tolerance exhibited by these plants is likely due to the induction and repression of certain of these common HD-Zip I-regulated genes. However, each HD-Zip I member regulates other pathways, which, in some cases, generate differential and potentially undesirable traits in addition to drought tolerance. In conclusion, only a few members of this family could become valuable tools to improve drought-tolerance.