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.

Transformation of Riccia fluitans, an Amphibious Liverwort Dynamically Responding to Environmental Changes.

The colonization of land by streptophyte algae, ancestors of embryophyte plants, was a fundamental event in the history of life on earth. Bryophytes are early diversifying land plants that mark the transition from freshwater to terrestrial ecosystems. The amphibious liverwort Riccia fluitans can thrive in aquatic and terrestrial environments and thus represents an ideal organism to investigate this major transition. Therefore, we aimed to establish a transformation protocol for R. fluitans to make it amenable for genetic analyses. An Agrobacterium transformation procedure using R. fluitans callus tissue allows to generate stably transformed plants within 10 weeks. Furthermore, for comprehensive studies spanning all life stages, we demonstrate that the switch from vegetative to reproductive development can be induced by both flooding and poor nutrient availability. Interestingly, a single R. fluitans plant can consecutively adapt to different growth environments and forms distinctive and reversible features of the thallus, photosynthetically active tissue that is thus functionally similar to leaves of vascular plants. The morphological plasticity affecting vegetative growth, air pore formation, and rhizoid development realized by one genotype in response to two different environments makes R. fluitans ideal to study the adaptive molecular mechanisms enabling the colonialization of land by aquatic plants.

Identification of miRNAs and Their Targets in the Liverwort Marchantia polymorpha by Integrating RNA-Seq and Degradome Analyses.

Bryophytes (liverworts, hornworts and mosses) comprise the three earliest diverging lineages of land plants (embryophytes). Marchantia polymorpha, a complex thalloid Marchantiopsida liverwort that has been developed into a model genetic system, occupies a key phylogenetic position. Therefore, M. polymorpha is useful in studies aiming to elucidate the evolution of gene regulation mechanisms in plants. In this study, we used computational, transcriptomic, small RNA and degradome analyses to characterize microRNA (miRNA)-mediated pathways of gene regulation in M. polymorpha. The data have been integrated into the open access ContigViews-miRNA platform for further reference. In addition to core components of the miRNA pathway, 129 unique miRNA sequences, 11 of which could be classified into seven miRNA families that are conserved in embryophytes (miR166a, miR390, miR529c, miR171-3p, miR408a, miR160 and miR319a), were identified. A combination of computational and degradome analyses allowed us to identify and experimentally validate 249 targets. In some cases, the target genes are orthologous to those of other embryophytes, but in other cases, the conserved miRNAs target either paralogs or members of different gene families. In addition, the newly discovered Mpo-miR11707.1 and Mpo-miR11707.2 are generated from a common precursor and target MpARGONAUTE1 (LW1759). Two other newly discovered miRNAs, Mpo-miR11687.1 and Mpo-miR11681.1, target the MADS-box transcription factors MpMADS1 and MpMADS2, respectively. Interestingly, one of the pentatricopeptide repeat (PPR) gene family members, MpPPR_66 (LW9825), the protein products of which are generally involved in various steps of RNA metabolism, has a long stem-loop transcript that can generate Mpo-miR11692.1 to autoregulate MpPPR_66 (LW9825) mRNA. This study provides a foundation for further investigations of the RNA-mediated silencing mechanism in M. polymorpha as well as of the evolution of this gene silencing pathway in embryophytes.

Comparison of the MpEF1α and CaMV35 promoters for application in Marchantia polymorpha overexpression studies.

Constitutive promoters are essential tools for analyses of gene functions by transgenic approaches. For overexpression and silencing studies of genes, a ubiquitous and strong expression of genes under investigation as well as selection markers is preferred. For future applications in the emerging basal plant model system Marchantia polymorpha, a liverwort, activities of the viral 35S cauliflower mosaic virus promoter and the endogenous elongation factor 1α (MpEF1α) promoter were analyzed. Expression of the reporter gene ß-glucuronidase (GUS), driven by the CaMV35 and MpEF1α promoters, was compared throughout plant development. Significant differences were observed between the two promoter activities. The CaMV35 promoter yields a weak reporter gene expression in the meristematic zones but drives a strong expression in the thallus. The MpEF1α promoter causes a strong meristematic GUS expression and is more active in female sexual tissues. Overall, the MpEF1α promoter seems to be the better option for obtaining a strong and ubiquitous transgene expression. Furthermore, a whole mount in situ hybridization protocol for Marchantia was established. Analysis of MpEF1α mRNA transcript in intact, whole tissues showed an expression pattern that is overall similar to the pattern of the GUS reporter gene expression driven by the MpEF1α promoter, including strong expression in meristematic zones. The whole mount technique reported here can be used to determine the mRNA expression in intact gemmae and archegonia, and has the potential to be applied for screening large numbers of transgenic plants, for instance to identify knock-down mutants.

Whole-Genome Sequence of Aneurinibacillus sp. Ricciae_BoGa-3, Isolated from Riccia fluitans.

Here, we present the Nanopore-only genome sequence of Aneurinibacillus sp. Ricciae_BoGa-3. It was isolated from Riccia fluitans ecotype BoGa-3 and its source was Botanical Garden Osnabrück (Germany). The complete circular genome is 4,981,254 bp with a GC content of 44.8%.

Time-delayed summation as a means of improving resolution on fast rotating computed tomography systems.

PURPOSE: Modern computed tomography (CT) systems are supporting increasingly fast rotation speeds, which are a prerequisite for fast dynamic acquisition, e.g. in perfusion imaging, and for new modalities such as dedicated breast CT, where breathhold scanning is indicated. However, not all detector technologies are supporting the high frame rates that are necessary to retain high resolution for objects far away from the isocenter. Even on systems that would support a sufficiently high frame rate, the necessary bandwidth of the data transfer from the rotating gantry stills remains challenging. The authors evaluated a pixel shifting technique termed time-delayed summation (TDS) as a method of increasing resolution on fast rotating CT systems without the need to increase the frame rate. METHODS: In TDS mode, detector pixel values are shifted along rows during image acquisition to compensate for detector motion. In order to fully exploit TDS, focal spot position control (FSC) was used in combination with TDS. FSC applies a counter movement to the x-ray focal spot during image acquisition such that it is kept fixed in space. As a proof of concept, measurements were performed on a prototype photon counting detector capable of TDS. The detector was mounted on a movable table and a gold wire phantom was imaged with different TDS settings and detector velocities. Additionally, simulations of a broad range of TDS and FSC settings on two different modalities, a clinical CT scanner and a breast CT scanner, and two different detector geometries, flat and cylindrical, were performed to assess the gain in resolution and contrast in cylindrical water phantoms containing a small wire at distances from the phantom center varied from 5% to 90% of the phantom radius. As figures of merit, the modulation transfer function (MTF) at 10% and the maximum contrast were used and compared against the respective values when using step-and-shoot acquisition, which means stopping the rotation when a projection image is acquired. RESULTS: Measurements showed that detector movement and the resulting blurring of the wire projections were compensated to the expected degree when using the appropriate number of TDS shifts per frame (TDS factor). Using simulations it was found that when using the optimal TDS factor, over 90% of the resolution achieved in step-and-shot mode was reached for all investigated wire positions. TDS showed better performance on a cylindrical detector that on the same system with a flat detector. TDS factors that were deviating from the optimum by more than 1 shift led to a performance below that of standard continuous acquisition. CONCLUSIONS: The findings of this study encourage the combined usage of TDS and FSC in systems that require fast rotation. The integration of TDS in state-of-the-art x-ray detectors is feasible.

Developmental Plasticity of the Amphibious Liverwort Riccia fluitans.

The colonization of land by ancestors of embryophyte plants was one of the most significant evolutionary events in the history of life on earth. The lack of a buffering aquatic environment necessitated adaptations for coping with novel abiotic challenges, particularly high light intensities and desiccation as well as the formation of novel anchoring structures. Bryophytes mark the transition from freshwater to terrestrial habitats and form adaptive features such as rhizoids for soil contact and water uptake, devices for gas exchange along with protective and repellent surface layers. The amphibious liverwort Riccia fluitans can grow as a land form (LF) or water form (WF) and was employed to analyze these critical traits in two different habitats. A combination of light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) studies was conducted to characterize and compare WF and LF morphologies. A complete phenotypic adaptation of a WF plant to a terrestrial habitat is accomplished within 15 days after the transition. Stable transgenic R. fluitans lines expressing GFP-TUBULIN and mCherry proteins were generated to study cell division and differentiation processes and revealed a higher cell division activity in enlarged meristematic regions at LF apical notches. Morphological studies demonstrated that the R. fluitans WF initiates air pore formation. However, these pores are arrested at an early four cell stage and do not develop further into open pores that could mediate gas exchange. Similarly, also arrested rhizoid initial cells are formed in the WF, which exhibit a distinctive morphology compared to other ventral epidermal cells. Furthermore, we detected that the LF thallus has a reduced surface permeability compared to the WF, likely mediated by formation of thicker LF cell walls and a distinct cuticle compared to the WF. Our R. fluitans developmental plasticity studies can serve as a basis to further investigate in a single genotype the molecular mechanisms of adaptations essential for plants during the conquest of land.

Gamete expression of TALE class HD genes activates the diploid sporophyte program in Marchantia polymorpha.

Eukaryotic life cycles alternate between haploid and diploid phases and in phylogenetically diverse unicellular eukaryotes, expression of paralogous homeodomain genes in gametes primes the haploid-to-diploid transition. In the unicellular chlorophyte alga Chlamydomonas, KNOX and BELL TALE-homeodomain genes mediate this transition. We demonstrate that in the liverwort Marchantia polymorpha, paternal (sperm) expression of three of five phylogenetically diverse BELL genes, MpBELL234, and maternal (egg) expression of both MpKNOX1 and MpBELL34 mediate the haploid-to-diploid transition. Loss-of-function alleles of MpKNOX1 result in zygotic arrest, whereas a loss of either maternal or paternal MpBELL234 results in variable zygotic and early embryonic arrest. Expression of MpKNOX1 and MpBELL34 during diploid sporophyte development is consistent with a later role for these genes in patterning the sporophyte. These results indicate that the ancestral mechanism to activate diploid gene expression was retained in early diverging land plants and subsequently co-opted during evolution of the diploid sporophyte body.

TALEN-mediated genome-editing approaches in the liverwort Marchantia polymorpha yield high efficiencies for targeted mutagenesis.

BACKGROUND: The liverwort Marchantia polymorpha occupies a crucial position in land plant evolution and provides the opportunity to investigate adaptations to a terrestrial plant life style. Marchantia reverse genetic analyses have thus far been conducted by employing a homologous recombination approach, which yields an efficiency of around 3%. Availability of the characterized and suitable endogenous MpEF1α promoter prompted us to establish the TALEN gene targeting technique for Marchantia. RESULTS: Here, two different TALEN techniques, using custom and self-assembled TALEN constructs, were applied and compared. The MpNOP1 gene was selected as a candidate gene, as the respective knockout mutant has been shown to lack air chamber formation, representing an easily traceable phenotype. We demonstrate that both TALEN approaches are successful in Marchantia yielding high gene targeting efficiencies of over 20%. Investigation of selected G1 up to G4 generations proved the stability of the knockout mutants. In 392 analyzed T1 plants, no additional phenotypes were observed and only one chimeric knockout plant was detected after an extended cultivation period. Interestingly, two out of the 24 sequenced mutants harbored indels causing in-frame mutations and revealed novel Mpnop1-related phenotypes. This demonstrates the potential to detect crucial amino acids and motives of targeted proteins, which is of special interest for essential genes where full knockouts are lethal. The FastTALE™ TALEN assembly kit enables the rapid assembly and ligation of the TALEN arms within half a day. For transformations, custom and assembled constructs were subcloned into Marchantia binary vectors possessing the MpEF1α promoter. CONCLUSION: Considering time, costs and practicability, the assembly TALEN approach represents a rapid and highly efficient gene targeting system to generate Marchantia knockout mutants, which can be further adapted for future advanced genome-editing applications.

Optical measurement of dimensional parameters of the breast with subjects in prone position.

Various applications require information on breast parameters, such as breast length and volume. An optical system was designed and tested for measuring these parameters with subjects in a prone position. The study results were used for optimizing patient positioning and handling for a future breast computed tomography (BCT) system. Measurements were conducted using an optical measurement system. To test the functionality and accuracy of the system, measurements were performed using reference phantoms. Additionally, 20 women and 5 men were examined to calculate breast parameters in alternative positions and breathing states. The results of the optical measurements were compared with magnetic resonance imaging (MRI) measurements. Volume and length of the reference phantoms were determined with errors below 2%. The patient study demonstrated a mean breast volume of 530.7 ml for women during normal breathing. During an exhalation state, breast volume increased significantly by 17.7 ml in comparison with normal breathing. Differences with MRI measurements were found to be 3% for breast length and 9% for breast volume on average. The proposed optical measurement system was found to be suitable for measuring the dimensional parameters of the breast in a prone position and provides a tool for evaluating breast coverage for BCT.