Insights into Land Plant Evolution Garnered from the Marchantia polymorpha Genome.

The evolution of land flora transformed the terrestrial environment. Land plants evolved from an ancestral charophycean alga from which they inherited developmental, biochemical, and cell biological attributes. Additional biochemical and physiological adaptations to land, and a life cycle with an alternation between multicellular haploid and diploid generations that facilitated efficient dispersal of desiccation tolerant spores, evolved in the ancestral land plant. We analyzed the genome of the liverwort Marchantia polymorpha, a member of a basal land plant lineage. Relative to charophycean algae, land plant genomes are characterized by genes encoding novel biochemical pathways, new phytohormone signaling pathways (notably auxin), expanded repertoires of signaling pathways, and increased diversity in some transcription factor families. Compared with other sequenced land plants, M. polymorpha exhibits low genetic redundancy in most regulatory pathways, with this portion of its genome resembling that predicted for the ancestral land plant. PAPERCLIP.

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.

An enhancer trap system to track developmental dynamics in Marchantia polymorpha.

A combination of streamlined genetics, experimental tractability and relative morphological simplicity compared to vascular plants makes the liverwort Marchantia polymorpha an ideal model system for studying many aspects of plant biology. Here we describe a transformation vector combining a constitutive fluorescent membrane marker with a nuclear marker that is regulated by nearby enhancer elements and use this to produce a library of enhancer trap lines for Marchantia. Screening gemmae from these lines allowed the identification and characterization of novel marker lines, including markers for rhizoids and oil cells. The library allowed the identification of a margin tissue running around the thallus edge, highlighted during thallus development. The expression of this marker is correlated with auxin levels. We generated multiple markers for the meristematic apical notch region, which have different spatial expression patterns, reappear at different times during meristem regeneration following apical notch excision and have varying responses to auxin supplementation or inhibition. This reveals that there are proximodistal substructures within the apical notch that could not be observed otherwise. We employed our markers to study Marchantia sporeling development, observing meristem emergence as defining the protonema-to-prothallus stage transition, and subsequent production of margin tissue during the prothallus stage. Exogenous auxin treatment stalls meristem emergence at the protonema stage but does not inhibit cell division, resulting in callus-like sporelings with many rhizoids, whereas pharmacologically inhibiting auxin synthesis and transport does not prevent meristem emergence. This enhancer trap system presents a useful resource for the community and will contribute to future Marchantia research.

An optimized transformation protocol for Anthoceros agrestis and three more hornwort species.

Land plants comprise two large monophyletic lineages, the vascular plants and the bryophytes, which diverged from their most recent common ancestor approximately 480 million years ago. Of the three lineages of bryophytes, only the mosses and the liverworts are systematically investigated, while the hornworts are understudied. Despite their importance for understanding fundamental questions of land plant evolution, they only recently became amenable to experimental investigation, with Anthoceros agrestis being developed as a hornwort model system. Availability of a high-quality genome assembly and a recently developed genetic transformation technique makes A. agrestis an attractive model species for hornworts. Here we describe an updated and optimized transformation protocol for A. agrestis, which can be successfully used to genetically modify one more strain of A. agrestis and three more hornwort species, Anthoceros punctatus, Leiosporoceros dussii, and Phaeoceros carolinianus. The new transformation method is less laborious, faster, and results in the generation of greatly increased numbers of transformants compared with the previous method. We have also developed a new selection marker for transformation. Finally, we report the development of a set of different cellular localization signal peptides for hornworts providing new tools to better understand the hornwort cell biology.

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.

A Simple Reversed Iontophoresis-Based Sensor to Enable In Vivo Multiplexed Measurement of Plant Biomarkers Using Screen-Printed Electrodes.

The direct quantification of plant biomarkers in sap is crucial to enhancing crop production. However, current approaches are inaccurate, involving the measurement of non-specific parameters such as colour intensity of leaves, or requiring highly invasive processes for the extraction of sap. In addition, these methods rely on bulky and expensive equipment, and they are time-consuming. The present work reports for the first time a low-cost sensing device that can be used for the simultaneous determination of sap K+ and pH in living plants by means of reverse iontophoresis. A screen-printed electrode was modified by deposition of a K+-selective membrane, achieving a super-Nernstian sensitivity of 70 mV Log[K+]−1 and a limit of detection within the micromolar level. In addition, the cathode material of the reverse iontophoresis device was modified by electrodeposition of RuOx particles. This electrode could be used for the direct extraction of ions from plant leaves and the amperometric determination of pH within the physiological range (pH 3−8), triggered by the selective reaction of RuOx with H+. A portable and low-cost (<£60) microcontroller-based device was additionally designed to enable its use in low-resource settings. The applicability of this system was demonstrated by measuring the changes in concentration of K+ and pH in tomato plants before and after watering with deionised water. These results represent a step forward in the design of affordable and non-invasive devices for the monitoring of key biomarkers in plants, with a plethora of applications in smart farming and precision agriculture among others.

Root system size and root hair length are key phenes for nitrate acquisition and biomass production across natural variation in Arabidopsis.

The role of root phenes in nitrogen (N) acquisition and biomass production was evaluated in 10 contrasting natural accessions of Arabidopsis thaliana L. Seedlings were grown on vertical agar plates with two different nitrate supplies. The low N treatment increased the root to shoot biomass ratio and promoted the proliferation of lateral roots and root hairs. The cost of a larger root system did not impact shoot biomass. Greater biomass production could be achieved through increased root length or through specific root hair characteristics. A greater number of root hairs may provide a low-resistance pathway under elevated N conditions, while root hair length may enhance root zone exploration under low N conditions. The variability of N uptake and the expression levels of genes encoding nitrate transporters were measured. A positive correlation was found between root system size and high-affinity nitrate uptake, emphasizing the benefits of an exploratory root organ in N acquisition. The expression levels of NRT1.2/NPF4.6, NRT2.2, and NRT1.5/NPF7.3 negatively correlated with some root morphological traits. Such basic knowledge in Arabidopsis demonstrates the importance of root phenes to improve N acquisition and paves the way to design eudicot ideotypes.

DNA methylation in Marchantia polymorpha.

Methylation of DNA is an epigenetic mechanism for the control of gene expression. Alterations in the regulatory pathways involved in the establishment, perpetuation and removal of DNA methylation can lead to severe developmental alterations. Our understanding of the mechanistic aspects and relevance of DNA methylation comes from remarkable studies in well-established angiosperm plant models including maize and Arabidopsis. The study of plant models positioned at basal lineages opens exciting opportunities to expand our knowledge on the function and evolution of the components of DNA methylation. In this Tansley Insight, we summarize current progress in our understanding of the molecular basis and relevance of DNA methylation in the liverwort Marchantia polymorpha.

Loop assembly: a simple and open system for recursive fabrication of DNA circuits.

High-efficiency methods for DNA assembly have enabled the routine assembly of synthetic DNAs of increased size and complexity. However, these techniques require customization, elaborate vector sets or serial manipulations for the different stages of assembly. We have developed Loop assembly based on a recursive approach to DNA fabrication. The system makes use of two Type IIS restriction endonucleases and corresponding vector sets for efficient and parallel assembly of large DNA circuits. Standardized level 0 parts can be assembled into circuits containing 1, 4, 16 or more genes by looping between the two vector sets. The vectors also contain modular sites for hybrid assembly using sequence overlap methods. Loop assembly enables efficient and versatile DNA fabrication for plant transformation. We show the construction of plasmids up to 16 genes and 38 kb with high efficiency (> 80%). We have characterized Loop assembly on over 200 different DNA constructs and validated the fidelity of the method by high-throughput Illumina plasmid sequencing. Our method provides a simple generalized solution for DNA construction with standardized parts. The cloning system is provided under an OpenMTA license for unrestricted sharing and open access.

MarpoDB: An Open Registry for Marchantia Polymorpha Genetic Parts.

Marchantia polymorpha is an extant relative of the earliest terrestrial plants and has attracted a substantial interest as a model organism for evolutionary and developmental studies. Given its relatively simple genome, compact gene families, simple morphology, ease of propagation and transformation, M. polymorpha is becoming a promising platform for plant synthetic biology. Modular genetic parts have been essential for development of synthetic biology approaches, so we sought to design an engineering oriented database for M. polymorpha genetic parts where each gene is a stand-alone functional unit. MarpoDB is a database of M. polymorpha genes and genetic parts, which is tailored to become an integral tool for a synthetic biology workflow. Among its features are precompiled cross-database querying to InterPro, Pfam signatures and non-redundant Viridiplantae BLAST annotations; BLAST querying to M. polymorpha genes; sequence export in GenBank format; recoding of sequences to the common syntax for type IIS assembly and exchange of DNA parts; and a minimalistic, intuitive and interactive user interface for gene models and sequence exploration. Furthermore, we have implemented user input to encourage feedback, collaboration and exchange between the MarpoDB community. MarpoDB source-code is released on GitHub to promote development of computational tools for synthetic biology.

Orthogonal intercellular signaling for programmed spatial behavior.

Bidirectional intercellular signaling is an essential feature of multicellular organisms, and the engineering of complex biological systems will require multiple pathways for intercellular signaling with minimal crosstalk. Natural quorum-sensing systems provide components for cell communication, but their use is often constrained by signal crosstalk. We have established new orthogonal systems for cell-cell communication using acyl homoserine lactone signaling systems. Quantitative measurements in contexts of differing receiver protein expression allowed us to separate different types of crosstalk between 3-oxo-C6- and 3-oxo-C12-homoserine lactones, cognate receiver proteins, and DNA promoters. Mutating promoter sequences minimized interactions with heterologous receiver proteins. We used experimental data to parameterize a computational model for signal crosstalk and to estimate the effect of receiver protein levels on signal crosstalk. We used this model to predict optimal expression levels for receiver proteins, to create an effective two-channel cell communication device. Establishment of a novel spatial assay allowed measurement of interactions between geometrically constrained cell populations via these diffusible signals. We built relay devices capable of long-range signal propagation mediated by cycles of signal induction, communication and response by discrete cell populations. This work demonstrates the ability to systematically reduce crosstalk within intercellular signaling systems and to use these systems to engineer complex spatiotemporal patterning in cell populations.

A Cyan Fluorescent Reporter Expressed from the Chloroplast Genome of Marchantia polymorpha.

Recently, the liverwort Marchantia polymorpha has received increasing attention as a basal plant model for multicellular studies. Its ease of handling, well-characterized plastome and proven protocols for biolistic plastid transformation qualify M. polymorpha as an attractive platform to study the evolution of chloroplasts during the transition from water to land. In addition, chloroplasts of M. polymorpha provide a convenient test-bed for the characterization of genetic elements involved in plastid gene expression due to the absence of mechanisms for RNA editing. While reporter genes have proven valuable to the qualitative and quantitative study of gene expression in chloroplasts, expression of green fluorescent protein (GFP) in chloroplasts of M. polymorpha has proven problematic. We report the design of a codon-optimized gfp varian, mturq2cp, which allowed successful expression of a cyan fluorescent protein under control of the tobacco psbA promoter from the chloroplast genome of M. polymorpha. We demonstrate the utility of mturq2cp in (i) early screening for transplastomic events following biolistic transformation of M. polymorpha spores; (ii) visualization of stromules as elements of plastid structure in Marchantia; and (iii) quantitative microscopy for the analysis of promoter activity.

The Naming of Names: Guidelines for Gene Nomenclature in Marchantia.

While Marchantia polymorpha has been utilized as a model system to investigate fundamental biological questions for over almost two centuries, there is renewed interest in M. polymorpha as a model genetic organism in the genomics era. Here we outline community guidelines for M. polymorpha gene and transgene nomenclature, and we anticipate that these guidelines will promote consistency and reduce both redundancy and confusion in the scientific literature.

Integrated genetic and computation methods for in planta cytometry.

We present the coupled use of specifically localized fluorescent gene markers and image processing for automated quantitative analysis of cell growth and genetic activity across living plant tissues. We used fluorescent protein markers to identify cells, create seeds and boundaries for the automatic segmentation of cell geometries and ratiometrically measure gene expression cell by cell in Arabidopsis thaliana.

Synthetic Biology: opportunities for Chilean bioindustry and education.

In an age of pressing challenges for sustainable production of energy and food, the new field of Synthetic Biology has emerged as a promising approach to engineer biological systems. Synthetic Biology is formulating the design principles to engineer affordable, scalable, predictable and robust functions in biological systems. In addition to efficient transfer of evolved traits from one organism to another, Synthetic Biology offers a new and radical approach to bottom-up engineering of sensors, actuators, dynamical controllers and the biological chassis they are embedded in. Because it abstracts much of the mechanistic details underlying biological component behavior, Synthetic Biology methods and resources can be readily used by interdisciplinary teams to tackle complex problems. In addition, the advent of robust new methods for the assembly of large genetic circuits enables teaching Biology and Bioengineering in a learning-by-making fashion for diverse backgrounds at the graduate, undergraduate and high school levels. Synthetic Biology offers unique opportunities to empower interdisciplinary training, research and industrial development in Chile for a technology that promises a significant role in this century's economy.

Coordination of plant cell division and expansion in a simple morphogenetic system.

Morphogenesis in plants arises from the interplay of genetic and physical interactions within a growing network of cells. The physical aspects of cell proliferation and differentiation are genetically regulated, but constrained by mechanical interactions between the cells. Higher plant tissues consist of an elaborate three-dimensional matrix of active cytoplasm and extracellular matrix, where it is difficult to obtain direct measurements of geometry or cell interactions. To properly understand the workings of plant morphogenesis, it is necessary to have biological systems that allow simple and direct observation of these processes. We have adopted a highly simplified plant system to investigate how cell proliferation and expansion is coordinated during morphogenesis. Coleocheate scutata is a microscopic fresh-water green alga with simple anatomical features that allow for accurate quantification of morphogenetic processes. Image analysis techniques were used to extract precise models for cell geometry and physical parameters for growth. This allowed construction of a deformable finite element model for growth of the whole organism, which incorporated cell biophysical properties, viscous expansion of cell walls, and rules for regulation of cell behavior. The study showed that a simple set of autonomous, cell-based rules are sufficient to account for the morphological and dynamic properties of Coleochaete growth. A variety of morphogenetic behavior emerged from the application of these local rules. Cell shape sensing is sufficient to explain the patterns of cell division during growth. This simplifying principle is likely to have application in modeling and design for engineering of higher plant tissues.

Paper-Based Multiplex Sensors for the Optical Detection of Plant Stress.

The rising population and the ongoing climate crisis call for improved means to monitor and optimise agriculture. A promising approach to tackle current challenges in food production is the early diagnosis of plant diseases through non-invasive methods, such as the detection of volatiles. However, current devices for detection of multiple volatiles are based on electronic noses, which are expensive, require complex circuit assembly, may involve metal oxides with heating elements, and cannot easily be adapted for some applications that require miniaturisation or limit front-end use of electronic components. To address these challenges, a low-cost optoelectronic nose using chemo-responsive colorimetric dyes drop-casted onto filter paper has been developed in the current work. The final sensors could be used for the quantitative detection of up to six plant volatiles through changes in colour intensities with a sub-ppm level limit of detection, one of the lowest limits of detection reported so far using colorimetric gas sensors. Sensor colouration could be analysed using a low-cost spectrometer and the results could be processed using a microcontroller. The measured volatiles could be used for the early detection of plant abiotic stress as early as two days after exposure to two different stresses: high salinity and starvation. This approach allowed a lowering of costs to GBP 1 per diagnostic sensing paper. Furthermore, the small size of the paper sensors allows for their use in confined settings, such as Petri dishes. This detection of abiotic stress could be easily achieved by exposing the devices to living plants for 1 h. This technology has the potential to be used for monitoring of plant development in field applications, early recognition of stress, implementation of preventative measures, and mitigation of harvest losses.

High-resolution live imaging of plant growth in near physiological bright conditions using light sheet fluorescence microscopy.

Most plant growth occurs post-embryonically and is characterized by the constant and iterative formation of new organs. Non-invasive time-resolved imaging of intact, fully functional organisms allows studies of the dynamics involved in shaping complex organisms. Conventional and confocal fluorescence microscopy suffer from limitations when whole living organisms are imaged at single-cell resolution. We applied light sheet-based fluorescence microscopy to overcome these limitations and study the dynamics of plant growth. We designed a special imaging chamber in which the plant is maintained vertically under controlled illumination with its leaves in the air and its root in the medium. We show that minimally invasive, multi-color, three-dimensional imaging of live Arabidopsis thaliana samples can be achieved at organ, cellular and subcellular scales over periods of time ranging from seconds to days with minimal damage to the sample. We illustrate the capabilities of the method by recording the growth of primary root tips and lateral root primordia over several hours. This allowed us to quantify the contribution of cell elongation to the early morphogenesis of lateral root primordia and uncover the diurnal growth rhythm of lateral roots. We demonstrate the applicability of our approach at varying spatial and temporal scales by following the division of plant cells as well as the movement of single endosomes in live growing root samples. This multi-dimensional approach will have an important impact on plant developmental and cell biology and paves the way to a truly quantitative description of growth processes at several scales.

A molecular framework for the inhibition of Arabidopsis root growth in response to boron toxicity.

Boron is an essential micronutrient for plants and is taken up in the form of boric acid (BA). Despite this, a high BA concentration is toxic for the plants, inhibiting root growth and is thus a significant problem in semi-arid areas in the world. In this work, we report the molecular basis for the inhibition of root growth caused by boron. We show that application of BA reduces the size of root meristems, correlating with the inhibition of root growth. The decrease in meristem size is caused by a reduction of cell division. Mitotic cell number significantly decreases and the expression level of key core cell cycle regulators is modulated. The modulation of the cell cycle does not appear to act through cytokinin and auxin signalling. A global expression analysis reveals that boron toxicity induces the expression of genes related with abscisic acid (ABA) signalling, ABA response and cell wall modifications, and represses genes that code for water transporters. These results suggest that boron toxicity produces a reduction of water and BA uptake, triggering a hydric stress response that produces root growth inhibition.

Root gravitropism requires lateral root cap and epidermal cells for transport and response to a mobile auxin signal.

Re-orientation of Arabidopsis seedlings induces a rapid, asymmetric release of the growth regulator auxin from gravity-sensing columella cells at the root apex. The resulting lateral auxin gradient is hypothesized to drive differential cell expansion in elongation-zone tissues. We mapped those root tissues that function to transport or respond to auxin during a gravitropic response. Targeted expression of the auxin influx facilitator AUX1 demonstrated that root gravitropism requires auxin to be transported via the lateral root cap to all elongating epidermal cells. A three-dimensional model of the root elongation zone predicted that AUX1 causes the majority of auxin to accumulate in the epidermis. Selectively disrupting the auxin responsiveness of expanding epidermal cells by expressing a mutant form of the AUX/IAA17 protein, axr3-1, abolished root gravitropism. We conclude that gravitropic curvature in Arabidopsis roots is primarily driven by the differential expansion of epidermal cells in response to an influx-carrier-dependent auxin gradient.