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Natural products from plants have served mankind in a wide range of applications, such as medicines, perfumes, or flavoring agents. For this reason, synthesis, regulation and function of plant-derived chemicals, as well as the evolution of metabolic diversity, has attracted researchers all around the world. In particular, vascular plants have been subject to such analyses due to prevalent characteristics such as appearance, fragrance, and ecological settings. In contrast, bryophytes, constituting the second largest group of plants in terms of species number, have been mostly overlooked in this regard, potentially due to their seemingly tiny, simple and obscure nature. However, the identification of highly interesting chemicals from bryophytes with potential for biotechnological exploitation is changing this perception. Bryophytes offer a high degree of biochemical complexity, as a consequence of their ecological and genetic diversification, which enable them to prosper in various, often very harsh habitats. The number of bioactive compounds isolated from bryophytes is growing rapidly. The rapidly increasing wealth of bryophyte genetics opens doors to functional and comparative genomics approaches, including disentangling of the biosynthesis of potentially interesting chemicals, mining for novel gene families and tracing the evolutionary history of metabolic pathways. Throughout the last decades, the moss Physcomitrella (Physcomitrium patens) has moved from being a model plant together with Marchantia polymorpha in fundamental biology into an attractive host for the production of biotechnologically relevant compounds such as biopharmaceuticals. In the future, bryophytes like the moss P. patens might also be attractive candidates for the production of novel bryophyte-derived chemicals of commercial interest. This review provides a comprehensive overview of natural product research in bryophytes from different perspectives together with biotechnological advances throug

CRISPR-Cas9 has proven to be highly valuable for genome editing in plants, including the model plant Physcomitrium patens. However, the fact that most of the editing events produced using the native Cas9 nuclease correspond to small insertions and deletions is a limitation. CRISPR-Cas9 base editors enable targeted mutation of single nucleotides in eukaryotic genomes and therefore overcome this limitation. Here, we report two programmable base-editing systems to induce precise cytosine or adenine conversions in P. patens. Using cytosine or adenine base editors, site-specific single-base mutations can be achieved with an efficiency up to 55%, without off-target mutations. Using the APT gene as a reporter of editing, we could show that both base editors can be used in simplex or multiplex, allowing for the production of protein variants with multiple amino-acid changes. Finally, we set up a co-editing selection system, named selecting modification of APRT to report gene targeting (SMART), allowing up to 90% efficiency site-specific base editing in P. patens. These two base editors will facilitate gene functional analysis in P. patens, allowing for site-specific editing of a given base through single sgRNA base editing or for in planta evolution of a given gene through the production of randomly mutagenised variants using multiple sgRNA base editing.

Cell-to-cell communication is tightly regulated in response to environmental stimuli in plants. We previously used a photoconvertible fluorescent protein Dendra2 as a model reporter to study this process. This experiment revealed that macromolecular trafficking between protonemal cells in Physcomitrella patens is suppressed in response to abscisic acid (ABA). However, it remains unknown which ABA signaling components contribute to this suppression and how. Here, we show that ABA signaling components SUCROSE NON-FERMENTING 1-RELATED PROTEIN KINASE 2 (PpSnRK2) and ABA INSENSITIVE 3 (PpABI3) play roles as an essential and promotive factor, respectively, in regulating ABA-induced suppression of Dendra2 diffusion between cells (ASD). Our quantitative imaging analysis revealed that disruption of PpSnRK2 resulted in defective ASD onset itself, whereas disruption of PpABI3 caused an 81-min delay in the initiation of ASD. Live-cell imaging of callose deposition using aniline blue staining showed that, despite this onset delay, callose deposition on cross walls remained constant in the PpABI3 disruptant, suggesting that PpABI3 facilitates ASD in a callose-independent manner. Given that ABA is an important phytohormone to cope with abiotic stresses, we further explored cellular physiological responses. We found that the acquisition of salt stress tolerance is promoted by PpABI3 in a quantitative manner similar to ASD. Our results suggest that PpABI3-mediated ABA signaling may effectively coordinate cell-to-cell communication during the acquisition of salt stress tolerance. This study will accelerate the quantitative study for ABA signaling mechanism and function in response to various abiotic stresses.

Cell-to-cell communication is tightly regulated in response to environmental stimuli in plants. We previously used a photoconvertible fluorescent protein Dendra2 as a model reporter to study this process. This experiment revealed that macromolecular trafficking between protonemal cells in Physcomitrella patens is suppressed in response to abscisic acid (ABA). However, it remains unknown which ABA signaling components contribute to this suppression and how. Here, we show that ABA signaling components SUCROSE NON-FERMENTING 1-RELATED PROTEIN KINASE 2 (PpSnRK2) and ABA INSENSITIVE 3 (PpABI3) play roles as an essential and promotive factor, respectively, in regulating ABA-induced suppression of Dendra2 diffusion between cells (ASD). Our quantitative imaging analysis revealed that disruption of PpSnRK2 resulted in defective ASD onset itself, whereas disruption of PpABI3 caused an 81-min delay in the initiation of ASD. Live-cell imaging of callose deposition using aniline blue staining showed that, despite this onset delay, callose deposition on cross walls remained constant in the PpABI3 disruptant, suggesting that PpABI3 facilitates ASD in a callose-independent manner. Given that ABA is an important phytohormone to cope with abiotic stresses, we further explored cellular physiological responses. We found that the acquisition of salt stress tolerance is promoted by PpABI3 in a quantitative manner similar to ASD. Our results suggest that PpABI3-mediated ABA signaling may effectively coordinate cell-to-cell communication during the acquisition of salt stress tolerance. This study will accelerate the quantitative study for ABA signaling mechanism and function in response to various abiotic stresses.

Polarized exocytosis is essential for plant development and defence. The exocyst, an octameric protein complex that tethers exocytotic vesicles to the plasma membrane, targets exocytosis. Upon pathogen attack, secreted materials form papillae to halt pathogen penetration. To determine if the exocyst is directly involved in targeting exocytosis to infection sites, information about its localization is instrumental. Here, we investigated exocyst subunit localization in the moss Physcomitrella patens upon pathogen attack and infection by Phytophthora capsici. Time-gated confocal microscopy was used to eliminate autofluorescence of deposited material around infection sites, allowing the visualization of the subcellular localization of exocyst subunits and of v-SNARE Vamp72A1-labelled exocytotic vesicles during infection. This showed that exocyst subunits Sec3a, Sec5b, Sec5d, and Sec6 accumulated at sites of attempted pathogen penetration. Upon pathogen invasion, the exocyst subunits accumulated on the membrane surrounding papilla-like structures and hyphal encasements. Vamp72A1-labelled vesicles were found to localize in the cytoplasm around infection sites. The re-localization of exocyst subunits to infection sites suggests that the exocyst is directly involved in facilitating polarized exocytosis during pathogenesis.

During land colonization, plants acquired a range of body plan adaptations, of which the innovation of three-dimensional (3D) tissues increased organismal complexity and reproductivity. In the moss, Physcomitrella patens, a 3D leafy gametophore originates from filamentous cells that grow in a two-dimensional (2D) plane through a series of asymmetric cell divisions. Asymmetric cell divisions that coincide with different cell division planes and growth directions enable the developmental switch from 2D to 3D, but insights into the underlying mechanisms coordinating this switch are still incomplete. Using 2D and 3D imaging and image segmentation, we characterized two geometric cues, the width of the initial cell and the angle of the transition division plane, which sufficiently distinguished a gametophore initial cell from a branch initial cell. These identified cues were further confirmed in gametophore formation mutants. The identification of a fluorescent marker allowed us to successfully predict the gametophore initial cell with > 90% accuracy before morphological changes, supporting our hypothesis that, before the transition division, parental cells of the gametophore initials possess different properties from those of the branch initials. Our results suggest that the cell fate decision of the initial cell is determined in the parental cell, before the transition division.

Nuclear matrix constituent proteins (NMCPs), the structural components of the plant lamina, are considered to be the analogues of lamins in plants based on numerous structural and functional similarities. Current phylogenetic know-ledge suggests that, in contrast to lamins, which are widely distributed in eukaryotes, NMCPs are taxonomically re-stricted to Streptophyta. At present, most information about NMCPs comes from angiosperms, and virtually no data are available from more ancestral groups. In angiosperms, the NMCP family comprises two phylogenetic groups, NMCP1 and NMCP2, which evolved from the NMCP1 and NMCP2 progenitor genes. Based on sequence conservation and the presence of NMCP-specific domains, we determined the structure and number of NMCP genes present in different Streptophyta clades. We analysed 91 species of embryophytes and report additional NMCP sequences from mosses, liverworts, clubmosses, horsetail, ferns, gymnosperms, and Charophyta algae. Our results confirm an origin of NMCPs in Charophyta (the earliest diverging group of Streptophyta), resolve the number and structure of NMCPs in the different clades, and propose the emergence of additional NMCP homologues by whole-genome duplication events. Immunofluorescence microscopy demonstrated localization of a basal NMCP from the moss Physcomitrella patens at the nuclear envelope, suggesting a functional conservation for basal and more evolved NMCPs.

During plant cytokinesis a radially expanding membrane-enclosed cell plate is formed from fusing vesicles that compartmentalizes the cell in two. How fusion is spatially restricted to the site of cell plate formation is unknown. Aggregation of cell-plate membrane starts near regions of microtubule overlap within the bipolar phragmoplast apparatus of the moss Physcomitrella patens Since vesicle fusion generally requires coordination of vesicle tethering and subsequent fusion activity, we analyzed the subcellular localization of several subunits of the exocyst, a tethering complex active during plant cytokinesis. We found that the exocyst complex subunit Sec6 but not the Sec3 or Sec5 subunits localized to microtubule overlap regions in advance of cell plate construction in moss. Moreover, Sec6 exhibited a conserved physical interaction with an ortholog of the Sec1/Munc18 protein KEULE, an important regulator for cell-plate membrane vesicle fusion in Arabidopsis Recruitment of the P. patens protein KEULE and vesicles to the early cell plate was delayed upon Sec6 gene silencing. Our findings, thus, suggest that vesicle-vesicle fusion is, in part, enabled by a pool of exocyst subunits at microtubule overlaps, which is recruited independently of vesicle delivery.

Metabolic engineering is an integrated bioengineering approach, which has made considerable progress in producing terpenoids in plants and fermentable hosts. Here, the full biosynthetic pathway of artemisinin, originating from Artemisia annua, was integrated into the moss Physcomitrella patens. Different combinations of the five artemisinin biosynthesis genes were ectopically expressed in P. patens to study biosynthesis pathway activity, but also to ensure survival of successful transformants. Transformation of the first pathway gene, ADS, into P. patens resulted in the accumulation of the expected metabolite, amorpha-4,11-diene, and also accumulation of a second product, arteannuin B. This demonstrates the presence of endogenous promiscuous enzyme activity, possibly cytochrome P450s, in P. patens. Introduction of three pathway genes, ADSCYP71AV1- ADH1 or ADS-DBR2-ALDH1 both led to the accumulation of artemisinin, hinting at the presence of one or more endogenous enzymes in P. patens that can complement the partial pathways to full pathway activity. Transgenic P. patens lines containing the different gene combinations produce artemisinin in varying amounts. The pathway gene expression in the transgenic moss lines correlates well with the chemical profile of pathway products. Moreover, expression of the pathway genes resulted in lipid body formation in all transgenic moss lines, suggesting that these may have a function in sequestration of heterologous metabolites. This work thus provides novel insights into the metabolic response of P. patens and its complementation potential for A. annua artemisinin pathway genes. Identification of the related endogenous P. patens genes could contribute to a further successful metabolic engineering of artemisinin biosynthesis, as well as bioengineering of other high-value terpenoids in P. patens.

Metabolic engineering is an integrated bioengineering approach, which has made considerable progress in producing terpenoids in plants and fermentable hosts. Here, the full biosynthetic pathway of artemisinin, originating from Artemisia annua, was integrated into the moss Physcomitrella patens. Different combinations of the five artemisinin biosynthesis genes were ectopically expressed in P. patens to study biosynthesis pathway activity, but also to ensure survival of successful transformants. Transformation of the first pathway gene, ADS, into P. patens resulted in the accumulation of the expected metabolite, amorpha-4,11-diene, and also accumulation of a second product, arteannuin B. This demonstrates the presence of endogenous promiscuous enzyme activity, possibly cytochrome P450s, in P. patens. Introduction of three pathway genes, ADS-CYP71AV1-ADH1 or ADS-DBR2-ALDH1 both led to the accumulation of artemisinin, hinting at the presence of one or more endogenous enzymes in P. patens that can complement the partial pathways to full pathway activity. Transgenic P. patens lines containing the different gene combinations produce artemisinin in varying amounts. The pathway gene expression in the transgenic moss lines correlates well with the chemical profile of pathway products. Moreover, expression of the pathway genes resulted in lipid body formation in all transgenic moss lines, suggesting that these may have a function in sequestration of heterologous metabolites. This work thus provides novel insights into the metabolic response of P. patens and its complementation potential for A. annua artemisinin pathway genes. Identification of the related endogenous P. patens genes could contribute to a further successful metabolic engineering of artemisinin biosynthesis, as well as bioengineering of other high-value terpenoids in P. patens.

The draft genome of the moss model, Physcomitrella patens, comprised approximately 2000 unordered scaffolds. In order to enable analyses of genome structure and evolution we generated a chromosome-scale genome assembly using genetic linkage as well as (end) sequencing of long DNA fragments. We find that 57% of the genome comprises transposable elements (TEs), some of which may be actively transposing during the life cycle. Unlike in flowering plant genomes, gene-and TE-rich regions show an overall even distribution along the chromosomes. However, the chromosomes are mono-centric with peaks of a class of Copia elements potentially coinciding with centromeres. Gene body methylation is evident in 5.7% of the protein-coding genes, typically coinciding with low GC and low expression. Some giant virus insertions are transcriptionally active and might protect gametes from viral infection via siRNA mediated silencing. Structure-based detection methods show that the genome evolved via two rounds of whole genome duplications (WGDs), apparently common in mosses but not in liverworts and hornworts. Several hundred genes are present in colinear regions conserved since the last common ancestor of plants. These syntenic regions are enriched for functions related to plant-specific cell growth and tissue organization. The P. patens genome lacks the TE-rich pericentromeric and gene-rich distal regions typical for most flowering plant genomes. More non-seed plant genomes are needed to unravel how plant genomes evolve, and to understand whether the P. patens genome structure is typical for mosses or bryophytes., De två första författarna delar förstaförfattarskapet.

High-throughput RNA sequencing (RNA-seq) has recently become the method of choice to define and analyze transcriptomes. For the model moss Physcomitrella patens, although this method has been used to help analyze specific perturbations, no overall reference dataset has yet been established. In the framework of the Gene Atlas project, the Joint Genome Institute selected P. patens as a flagship genome, opening the way to generate the first comprehensive transcriptome dataset for this moss. The first round of sequencing described here is composed of 99 independent libraries spanning 34 different developmental stages and conditions. Upon dataset quality control and processing through read mapping, 28 509 of the 34 361 v3.3 gene models (83%) were detected to be expressed across the samples. Differentially expressed genes (DEGs) were calculated across the dataset to permit perturbation comparisons between conditions. The analysis of the three most distinct and abundant P. patens growth stages - protonema, gametophore and sporophyte - allowed us to define both general transcriptional patterns and stage-specific transcripts. As an example of variation of physico-chemical growth conditions, we detail here the impact of ammonium supplementation under standard growth conditions on the protonemal transcriptome. Finally, the cooperative nature of this project allowed us to analyze inter-laboratory variation, as 13 different laboratories around the world provided samples. We compare differences in the replication of experiments in a single laboratory and between different laboratories.

The draft genome of the moss model, Physcomitrella patens, comprised approximately 2000 unordered scaffolds. In order to enable analyses of genome structure and evolution we generated a chromosome-scale genome assembly using genetic linkage as well as (end) sequencing of long DNA fragments. We find that 57% of the genome comprises transposable elements (TEs), some of which may be actively transposing during the life cycle. Unlike in flowering plant genomes, gene- and TE-rich regions show an overall even distribution along the chromosomes. However, the chromosomes are mono-centric with peaks of a class of Copia elements potentially coinciding with centromeres. Gene body methylation is evident in 5.7% of the protein-coding genes, typically coinciding with low GC and low expression. Some giant virus insertions are transcriptionally active and might protect gametes from viral infection via siRNA mediated silencing. Structure-based detection methods show that the genome evolved via two rounds of whole genome duplications (WGDs), apparently common in mosses but not in liverworts and hornworts. Several hundred genes are present in colinear regions conserved since the last common ancestor of plants. These syntenic regions are enriched for functions related to plant-specific cell growth and tissue organization. The P. patens genome lacks the TE-rich pericentromeric and gene-rich distal regions typical for most flowering plant genomes. More non-seed plant genomes are needed to unravel how plant genomes evolve, and to understand whether the P. patens genome structure is typical for mosses or bryophytes.

Today, recombinant expression of proteins is used for a variety of purposes. One of these is the production of allergens, which are vital components in allergy diagnostics. However, traditional expression systems such as ​Escherichia coli​ and ​Pichia pastoris​ might not have the capacity to express all proteins of interest. Thermo Fisher, which is a leading producer of allergy tests, has requested an evaluation of different microorganisms and their capacity for heterologous protein expression in order to expand their existing toolbox of expression systems. This summary was made through a literature study, where twelve organisms were evaluated. Six eukaryotic and six prokaryotic expression systems are compared based on their ability to properly glycosylate protein, need for specific culture conditions, safety, protease activity, duration, protein yield and protein solubility. The prokaryotic systems – Corynebacterium glutamicum​ , ​Lactococcus lactis​ , ​Pseudomonas fluorescens​ , Pseudoalteromonas haloplanktis​ , ​Ralstonia eutropha​ and ​Streptomyces lividans​ – are characterized by being easy to cultivate, operating in different temperature ranges and providing relatively high yields of recombinant protein. The eukaryotic systems – ​Aspergillus fungi, the green algae ​Chlamydomonas reinhardtii​ , the yeast ​Hansenula polymorpha​ , the parasite ​Leishmania tarentolae​ , the moss ​Physcomitrella patens​ and suspension-based plant cells – all have very different morphology and properties. In comparison with the prokaryotic systems, it can be concluded that they are generally better at folding and providing the correct glycosylation patterns for mammalian and plant proteins. However, they require more time and effort to establish a competent cell line. Furthermore, the resulting protein yield is usually less than for the prokaryotic systems. The conclusion can be drawn that no expression system is perfect. The solution is a toolbox, containing various expression syst

High-throughput RNA sequencing (RNA-seq) has recently become the method of choice to define and analyze transcriptomes. For the model moss Physcomitrella patens, although this method has been used to help analyze specific perturbations, no overall reference dataset has yet been established. In the framework of the Gene Atlas project, the Joint Genome Institute selected P. patens as a flagship genome, opening the way to generate the first comprehensive transcriptome dataset for this moss. The first round of sequencing described here is composed of 99 independent libraries spanning 34 different developmental stages and conditions. Upon dataset quality control and processing through read mapping, 28 509 of the 34 361 v3.3 gene models (83%) were detected to be expressed across the samples. Differentially expressed genes (DEGs) were calculated across the dataset to permit perturbation comparisons between conditions. The analysis of the three most distinct and abundant P. patens growth stages - protonema, gametophore and sporophyte - allowed us to define both general transcriptional patterns and stage-specific transcripts. As an example of variation of physico-chemical growth conditions, we detail here the impact of ammonium supplementation under standard growth conditions on the protonemal transcriptome. Finally, the cooperative nature of this project allowed us to analyze inter-laboratory variation, as 13 different laboratories around the world provided samples. We compare differences in the replication of experiments in a single laboratory and between different laboratories.

The draft genome of the moss model, Physcomitrella patens, comprised approximately 2000 unordered scaffolds. In order to enable analyses of genome structure and evolution we generated a chromosome-scale genome assembly using genetic linkage as well as (end) sequencing of long DNA fragments. We find that 57% of the genome comprises transposable elements (TEs), some of which may be actively transposing during the life cycle. Unlike in flowering plant genomes, gene- and TE-rich regions show an overall even distribution along the chromosomes. However, the chromosomes are mono-centric with peaks of a class of Copia elements potentially coinciding with centromeres. Gene body methylation is evident in 5.7% of the protein-coding genes, typically coinciding with low GC and low expression. Some giant virus insertions are transcriptionally active and might protect gametes from viral infection via siRNA mediated silencing. Structure-based detection methods show that the genome evolved via two rounds of whole genome duplications (WGDs), apparently common in mosses but not in liverworts and hornworts. Several hundred genes are present in colinear regions conserved since the last common ancestor of plants. These syntenic regions are enriched for functions related to plant-specific cell growth and tissue organization. The P. patens genome lacks the TE-rich pericentromeric and gene-rich distal regions typical for most flowering plant genomes. More non-seed plant genomes are needed to unravel how plant genomes evolve, and to understand whether the P. patens genome structure is typical for mosses or bryophytes.

Today, recombinant expression of proteins is used for a variety of purposes. One of these is the production of allergens, which are vital components in allergy diagnostics. However, traditional expression systems such as ​Escherichia coli​ and ​Pichia pastoris​ might not have the capacity to express all proteins of interest. Thermo Fisher, which is a leading producer of allergy tests, has requested an evaluation of different microorganisms and their capacity for heterologous protein expression in order to expand their existing toolbox of expression systems. This summary was made through a literature study, where twelve organisms were evaluated. Six eukaryotic and six prokaryotic expression systems are compared based on their ability to properly glycosylate protein, need for specific culture conditions, safety, protease activity, duration, protein yield and protein solubility. The prokaryotic systems – Corynebacterium glutamicum​ , ​Lactococcus lactis​ , ​Pseudomonas fluorescens​ , Pseudoalteromonas haloplanktis​ , ​Ralstonia eutropha​ and ​Streptomyces lividans​ – are characterized by being easy to cultivate, operating in different temperature ranges and providing relatively high yields of recombinant protein. The eukaryotic systems – ​Aspergillus fungi, the green algae ​Chlamydomonas reinhardtii​ , the yeast ​Hansenula polymorpha​ , the parasite ​Leishmania tarentolae​ , the moss ​Physcomitrella patens​ and suspension-based plant cells – all have very different morphology and properties. In comparison with the prokaryotic systems, it can be concluded that they are generally better at folding and providing the correct glycosylation patterns for mammalian and plant proteins. However, they require more time and effort to establish a competent cell line. Furthermore, the resulting protein yield is usually less than for the prokaryotic systems. The conclusion can be drawn that no expression system is perfect. The solution is a toolbox, containing various expression syst

Securing a molecular toolbox including diverse promoters is essential for genome engineering. However, native promoters have limitations such as the available number or the length of the promoter. In this work, three short synthetic promoters were characterized by using the yellow fluorescent protein Venus. All of the tested promoters were active and showed higher mRNA expression than housekeeping gene PpAct7, and similar protein expression level to the AtUBQ10 promoter. This study shows that few cis-regulatory elements are enough to establish a strong promoter for continuous expression of genes in plants. Along with this, the study enhance the number of available promotors to be used in P. patens. It also demonstrates the potential to construct multiple non-native promoters on demand, which would aid to resolve one bottleneck in multiple pathway expression in P. patens and other plants.

In seed plants, strigolactones (SLs) regulate architecture and induce mycorrhizal symbiosis in response to environmental cues. SLs are formed by combined activity of the carotenoid cleavage dioxygenases (CCDs) 7 and 8 from 9-cis-β-carotene, leading to carlactone that is converted by cytochromes P450 (clade 711; MAX1 in Arabidopsis) into various SLs. As Physcomitrella patens possesses CCD7 and CCD8 homologs but lacks MAX1, we investigated if PpCCD7 together with PpCCD8 form carlactone and how deletion of these enzymes influences growth and interactions with the environment. We investigated the enzymatic activity of PpCCD7 and PpCCD8 in vitro, identified the formed products by high performance liquid chromatography (HPLC) and LC-MS, and generated and analysed ΔCCD7 and ΔCCD8 mutants. We defined enzymatic activity of PpCCD7 as a stereospecific 9-cis-CCD and PpCCD8 as a carlactone synthase. ΔCCD7 and ΔCCD8 lines showed enhanced caulonema growth, which was revertible by adding the SL analogue GR24 or carlactone. Wild-type (WT) exudates induced seed germination in Orobanche ramosa. This activity was increased upon phosphate starvation and abolished in exudates of both mutants. Furthermore, both mutants showed increased susceptibility to phytopathogenic fungi. Our study reveals the deep evolutionary conservation of SL biosynthesis, SL function, and its regulation by biotic and abiotic cues.

Autophagy, a major catabolic process in eukaryotes, was initially related to cell tolerance to nutrient depletion. In plants autophagy has also been widely related to tolerance to biotic and abiotic stresses (through the induction or repression of programmed cell death, PCD) as well as to promotion of developmentally regulated PCD, starch degradation or caloric restriction important for life span. Much less is known regarding its role in plant cell differentiation. Here we show that macroautophagy, the autophagy pathway driven by engulfment of cytoplasmic components by autophagosomes and its subsequent degradation in vacuoles, is highly active during germ cell differentiation in the early diverging land plant Physcomitrella patens. Our data provide evidence that suppression of ATG5-mediated autophagy results in reduced density of the egg cell-mediated mucilage that surrounds the mature egg, pointing toward a potential role of autophagy in extracellular mucilage formation. In addition, we found that ATG5- and ATG7-mediated autophagy is essential for the differentiation and cytoplasmic reduction of the flagellated motile sperm and hence for sperm fertility. The similarities between the need of macroautophagy for sperm differentiation in moss and mouse are striking, strongly pointing toward an ancestral function of autophagy not only as a protector against nutrient stress, but also in gamete differentiation.

Autophagy, a major catabolic process in eukaryotes, was initially related to cell tolerance to nutrient depletion. In plants autophagy has also been widely related to tolerance to biotic and abiotic stresses (through the induction or repression of programmed cell death, PCD) as well as to promotion of developmentally regulated PCD, starch degradation or caloric restriction important for life span. Much less is known regarding its role in plant cell differentiation. Here we show that macroautophagy, the autophagy pathway driven by engulfment of cytoplasmic components by autophagosomes and its subsequent degradation in vacuoles, is highly active during germ cell differentiation in the early diverging land plant Physcomitrella patens. Our data provide evidence that suppression of ATG5-mediated autophagy results in reduced density of the egg cell-mediated mucilage that surrounds the mature egg, pointing toward a potential role of autophagy in extracellular mucilage formation. In addition, we found that ATG5- and ATG7-mediated autophagy is essential for the differentiation and cytoplasmic reduction of the flagellated motile sperm and hence for sperm fertility. The similarities between the need of macroautophagy for sperm differentiation in moss and mouse are striking, strongly pointing toward an ancestral function of autophagy not only as a protector against nutrient stress, but also in gamete differentiation.

Autophagy, a major catabolic process in eukaryotes, was initially related to cell tolerance to nutrient depletion. In plants autophagy has also been widely related to tolerance to biotic and abiotic stresses (through the induction or repression of programmed cell death, PCD) as well as to promotion of developmentally regulated PCD, starch degradation or caloric restriction important for life span. Much less is known regarding its role in plant cell differentiation. Here we show that macroautophagy, the autophagy pathway driven by engulfment of cytoplasmic components by autophagosomes and its subsequent degradation in vacuoles, is highly active during germ cell differentiation in the early diverging land plant Physcomitrella patens. Our data provide evidence that suppression of ATG5-mediated autophagy results in reduced density of the egg cell-mediated mucilage that surrounds the mature egg, pointing toward a potential role of autophagy in extracellular mucilage formation. In addition, we found that ATG5- and ATG7-mediated autophagy is essential for the differentiation and cytoplasmic reduction of the flagellated motile sperm and hence for sperm fertility. The similarities between the need of macroautophagy for sperm differentiation in moss and mouse are striking, strongly pointing toward an ancestral function of autophagy not only as a protector against nutrient stress, but also in gamete differentiation.

Autophagy, a major catabolic process in eukaryotes, was initially related to cell tolerance to nutrient depletion. In plants autophagy has also been widely related to tolerance to biotic and abiotic stresses (through the induction or repression of programmed cell death, PCD) as well as to promotion of developmentally regulated PCD, starch degradation or caloric restriction important for life span. Much less is known regarding its role in plant cell differentiation. Here we show that macroautophagy, the autophagy pathway driven by engulfment of cytoplasmic components by autophagosomes and its subsequent degradation in vacuoles, is highly active during germ cell differentiation in the early diverging land plant Physcomitrella patens. Our data provide evidence that suppression of ATG5-mediated autophagy results in reduced density of the egg cell-mediated mucilage that surrounds the mature egg, pointing toward a potential role of autophagy in extracellular mucilage formation. In addition, we found that ATG5- and ATG7-mediated autophagy is essential for the differentiation and cytoplasmic reduction of the flagellated motile sperm and hence for sperm fertility. The similarities between the need of macroautophagy for sperm differentiation in moss and mouse are striking, strongly pointing toward an ancestral function of autophagy not only as a protector against nutrient stress, but also in gamete differentiation.

Autophagy, a major catabolic process in eukaryotes, was initially related to cell tolerance to nutrient depletion. In plants autophagy has also been widely related to tolerance to biotic and abiotic stresses (through the induction or repression of programmed cell death, PCD) as well as to promotion of developmentally regulated PCD, starch degradation or caloric restriction important for life span. Much less is known regarding its role in plant cell differentiation. Here we show that macroautophagy, the autophagy pathway driven by engulfment of cytoplasmic components by autophagosomes and its subsequent degradation in vacuoles, is highly active during germ cell differentiation in the early diverging land plant Physcomitrella patens. Our data provide evidence that suppression of ATG5-mediated autophagy results in reduced density of the egg cell-mediated mucilage that surrounds the mature egg, pointing toward a potential role of autophagy in extracellular mucilage formation. In addition, we found that ATG5- and ATG7-mediated autophagy is essential for the differentiation and cytoplasmic reduction of the flagellated motile sperm and hence for sperm fertility. The similarities between the need of macroautophagy for sperm differentiation in moss and mouse are striking, strongly pointing toward an ancestral function of autophagy not only as a protector against nutrient stress, but also in gamete differentiation.

Autophagy, a major catabolic process in eukaryotes, was initially related to cell tolerance to nutrient depletion. In plants autophagy has also been widely related to tolerance to biotic and abiotic stresses (through the induction or repression of programmed cell death, PCD) as well as to promotion of developmentally regulated PCD, starch degradation or caloric restriction important for life span. Much less is known regarding its role in plant cell differentiation. Here we show that macroautophagy, the autophagy pathway driven by engulfment of cytoplasmic components by autophagosomes and its subsequent degradation in vacuoles, is highly active during germ cell differentiation in the early diverging land plant Physcomitrella patens. Our data provide evidence that suppression of ATG5-mediated autophagy results in reduced density of the egg cell-mediated mucilage that surrounds the mature egg, pointing toward a potential role of autophagy in extracellular mucilage formation. In addition, we found that ATG5- and ATG7-mediated autophagy is essential for the differentiation and cytoplasmic reduction of the flagellated motile sperm and hence for sperm fertility. The similarities between the need of macroautophagy for sperm differentiation in moss and mouse are striking, strongly pointing toward an ancestral function of autophagy not only as a protector against nutrient stress, but also in gamete differentiation.

Malaria is a real and constant danger to nearly half of the world’s population of 7.4 billion people. In 2015, 212 million cases were reported along with 429,000 estimated deaths. The World Health Organization recommends artemisinin-based combinatorial therapies, and the artemisinin for this purpose is mainly isolated from the plant Artemisia annua. However, the plant supply of artemisinin is irregular, leading to fluctuation in prices. Here, we report the development of a simple, sustainable, and scalable production platform of artemisinin. The five genes involved in artemisinin biosynthesis were engineered into the moss Physcomitrella patens via direct in vivo assembly of multiple DNA fragments. In vivo biosynthesis of artemisinin was obtained without further modifications. A high initial production of 0.21 mg/g dry weight artemisinin was observed after only 3 days of cultivation. Our study shows that P. patens can be a sustainable and efficient production platform of artemisinin that without further modifications allow for industrial-scale production. A stable supply of artemisinin will lower the price of artemisinin-based treatments, hence become more affordable to the lower income communities most affected by malaria; an important step toward containment of this deadly disease threatening millions every year.

Malaria is a real and constant danger to nearly half of the world’s population of 7.4 billion people. In 2015, 212 million cases were reported along with 429,000 estimated deaths. The World Health Organization recommends Artemisinin-based Combinatorial Therapies (ACTs), and the artemisinin for this purpose is mainly isolated from the plant Artemisia annua. However, the plant supply of artemisinin is irregular, leading to fluctuation in prices. Here we report the development of a simple, sustainable, and scalable production platform of artemisinin. The five genes involved in artemisinin biosynthesis were engineered into the moss Physcomitrella patens via direct in vivo assembly of multiple DNA fragments. In vivo biosynthesis of artemisinin was obtained without further modifications. A high initial production of 0.21 mg/g dry weight artemisinin was observed after only three days of cultivation. Our study shows that P. patens can be a sustainable and efficient production platform of artemisinin that without further modifications allow for industrial scale production. A stable supply of artemisinin will lower the price of artemisinin-based treatments, hence become more affordable to the lower income communities most affected by malaria; an important step towards containment of this deadly disease threatening millions every year.

Physcomitrella patens is a non-vascular plant with a relatively small genome and is amongst the few eukaryotic organisms that have a high rate of homologous recombination. This is valuable in biological research because it allows for targeted genetic modification of the organism. In vascular plants like Arabidopsis thaliana, a model organism, Cellulose Synthase-like D (CSLD) genes have been discovered to be important in tip growth. This type of growth is observed in the pollen tubes and root hairs of these plant types. The CSLD genes in Arabidopsis were found to play a crucial role in the growth of root hairs and the production of cellulose or cellulose-like β-1,4-glucan chains in root hair tips. The CSLD genes have also been recognized to be important in pollen tube growth of vascular plants. Physcomitrella patens also contains genes similar to the vascular plant CSLDs, but their functions are not yet fully understood. Within the P. patens genome there are eight genes that make up the CSLD gene family. Additionally, the life cycle of P. patens includes a stage that consists primarily of tip growing cells. This growth stage can be optimized in order to study the role of CSLD genes in tip growth of P. patens. In an effort to further study the roles of the CSLD genes in tip growth of P. patens, we constructed a plasmid that expresses the CSLD1 protein with a green fluorescent protein (GFP) tag. This allowed us to visualize the expression of CSLD1 in living cells using fluorescence microscopy. We also constructed plasmids that were designed to remove specific CSLD genes from the genome and transformed them into wild type or CLSD1 knockout tissue of P. patens. This created single or double knockout mutants that could then be compared to the wild type for changes in the phenotypic characteristics of the plant. These findings will aid in uncovering the roles of the CSLD gene family in P. patens and may provide insight into the functions of these genes in other plants.

Phytochrome sind Rotlicht-/Dunkelrotlicht (R/FR)-revertierbare Photorezeptoren, die eine zentrale Rolle in der Licht-regulierten pflanzlichen Entwicklung spielen. Der aktivierte Rezeptor transloziert in den Zellkern, interagiert dort mit diversen Signaltransduktions-Komponenten, was zu differenzieller Genregulation führt. Verschiedene Evidenzen belegen allerdings zweifelsfrei, dass es zusätzlich einen Phytochrom-Signalweg im Cytoplasma gibt. Beispielsweise werden Rotlicht-Phototropismus und -polarotropismus im Laubmoos Physcomitrella patens durch Phytochrom (hauptsächlich phy4) vermittelt, was eine Genregulations-basierte Signaltransduktion ausschließt, da die Richtungsinformation dabei zwangsläufig verloren geht. Das Richtungssignal wird an das Aktin-Cytoskelett weitergeleitet, welches während der Reaktion massiv umorganisiert wird und somit als Effektor der polaren Wachstumsantwort gilt. In einem vorherigen Dissertationsprojekt konnte mittels Yeast-Two-Hybrid (Y2H)- und Split-YFP-Methoden eine R-induzierte bzw. in planta Interaktion von phy4 mit dem Blaulichtrezeptor Phototropin an der Plasmamembran (PM) demonstriert werden. Der gestörte R-Photo- und Polarotropismus einer Phototropin-Triple-Mutante belegt die physiologische Relevanz der Photorezeptor-Co-Aktion. In dieser Arbeit wurde ein ex vivo Co-Immunopräzipitations-Assay etabliert und erfolgreich angewendet, um die Interaktion mit einer unabhängigen Methode zu demonstrieren. Der Fokus der Doktorarbeit lag auf der Identifikation und Charakterisierung von phy4-Interaktionspartnern, die am cytoplasmatischen phy4-Signalweg beteiligt sein könnten. Dazu wurde ein Y2H-cDNA-Library Screen mit photoaktivem Volllängen-Holophytochrom als Bait durchgeführt und in R selektiert, um vorwiegend Pfr-spezifische Interaktoren zu erfassen. 70 von 4 x 105 gescreenten Hefeklonen konnten dabei isoliert und nach Sequenzierung in silico bzgl. funktioneller Domänen und Homologen in anderen Pflanzen analysiert werden. Nach Eliminierung

The effects of the universal signal molecule, abscisic acid (ABA), with limited knowledge functions in non-tracheophyte have been studied in three selected bryophyte species: two mosses, Physcomitrella patens and Atrichum undulatum, and one liverwort, Marchantia polymorpha. While vegetative development as well as total chlorophyll and carotenoid contents tended to decrease in the three bryophyte species with increasing exogenous ABA concentration, the effect on total biomass showed less clear patterns in the bryophytes tested. These differences in response to ABA likely reflect different adaptations of these three species to conditions in situ.

Dynamic reorganization of photosystems I and II is suggested to occur in chloroplast thylakoid membranes to maintain the efficiency of photosynthesis under fluctuating light conditions. To directly observe the process in action, live-cell imaging techniques are necessary. Using live-cell imaging, we have shown that the fine thylakoid structures in the moss Physcomitrella patens are flexible in time. However, the spatiotemporal resolution of a conventional confocal microscopy limits more precise visualization of entire thylakoid structures and understanding of the structural dynamics. Here, we discuss the issues related to observing chlorophyll fluorescence at multiple spatiotemporal scales in vivo and in vitro.

Author Posting. © The Author(s), 2015. This is the author's version of the work. It is posted here by permission of Oxford University Press for personal use, not for redistribution. The definitive version was published in Plant and Cell Physiology 56 (2015): 737-749, doi:10.1093/pcp/pcv002., Proper positioning of the nucleus is critical for the functioning of various cells. Actin and myosin have been shown to be crucial for the localisation of the nucleus in plant cells, whereas microtubule (MT)-based mechanisms are commonly utilised in animal and fungal cells. In this study, we combined live cell microscopy with RNA interference (RNAi) screening or drug treatment and showed that MTs and a plant-specific motor protein, armadillo repeat-containing kinesin (kinesin-ARK), are required for nuclear positioning in the moss Physcomitrella patens. In tip-growing protonemal apical cells, the nucleus was translocated to the centre of the cell after cell division in an MT-dependent manner. When kinesin-ARKs were knocked down using RNAi, the initial movement of the nucleus towards the centre took place normally; however, before reaching the centre, the nucleus was moved back to the basal edge of the cell. In intact (control) cells, MT bundles that are associated with kinesin-ARKs were frequently observed around the moving nucleus. In contrast, such MT bundles were not identified after kinesin-ARK downregulation. An in vitro MT-gliding assay showed that kinesin-ARK is a plus-end-directed motor protein. These results indicate that MTs and the MT-based motor drive nuclear migration in the moss cells, thus showing a conservation of the mechanism underlying nuclear localisation among plant, animal, and fungal cells., This work was supported by the Next Generation grant (Japan Society for Promotion of Science; JSPS), James A. and Faith Miller Memorial Fund, and Human Frontier Science Program (to G.G.). T.M. is a recipient of a pre-doctoral fellowship of the JSPS., 2016-01-13

Author Posting. © The Author(s), 2015. This is the author's version of the work. It is posted here by permission of Oxford University Press for personal use, not for redistribution. The definitive version was published in Plant and Cell Physiology 56 (2015): 737-749, doi:10.1093/pcp/pcv002., Proper positioning of the nucleus is critical for the functioning of various cells. Actin and myosin have been shown to be crucial for the localisation of the nucleus in plant cells, whereas microtubule (MT)-based mechanisms are commonly utilised in animal and fungal cells. In this study, we combined live cell microscopy with RNA interference (RNAi) screening or drug treatment and showed that MTs and a plant-specific motor protein, armadillo repeat-containing kinesin (kinesin-ARK), are required for nuclear positioning in the moss Physcomitrella patens. In tip-growing protonemal apical cells, the nucleus was translocated to the centre of the cell after cell division in an MT-dependent manner. When kinesin-ARKs were knocked down using RNAi, the initial movement of the nucleus towards the centre took place normally; however, before reaching the centre, the nucleus was moved back to the basal edge of the cell. In intact (control) cells, MT bundles that are associated with kinesin-ARKs were frequently observed around the moving nucleus. In contrast, such MT bundles were not identified after kinesin-ARK downregulation. An in vitro MT-gliding assay showed that kinesin-ARK is a plus-end-directed motor protein. These results indicate that MTs and the MT-based motor drive nuclear migration in the moss cells, thus showing a conservation of the mechanism underlying nuclear localisation among plant, animal, and fungal cells., This work was supported by the Next Generation grant (Japan Society for Promotion of Science; JSPS), James A. and Faith Miller Memorial Fund, and Human Frontier Science Program (to G.G.). T.M. is a recipient of a pre-doctoral fellowship of the JSPS., 2016-01-13

Dynamic reorganization of photosystems I and II is suggested to occur in chloroplast thylakoid membranes to maintain the efficiency of photosynthesis under fluctuating light conditions. To directly observe the process in action, live-cell imaging techniques are necessary. Using live-cell imaging, we have shown that the fine thylakoid structures in the moss Physcomitrella patens are flexible in time. However, the spatiotemporal resolution of a conventional confocal microscopy limits more precise visualization of entire thylakoid structures and understanding of the structural dynamics. Here, we discuss the issues related to observing chlorophyll fluorescence at multiple spatiotemporal scales in vivo and in vitro.

[Background] Using motif detection programs it is fairly straightforward to identify conserved cis-sequences in promoters of co-regulated genes. In contrast, the identification of the transcription factors (TFs) interacting with these cis-sequences is much more elaborate. To facilitate this, we explore the possibility of using several bioinformatic and experimental approaches for TF identification. This starts with the selection of co-regulated gene sets and leads first to the prediction and then to the experimental validation of TFs interacting with cis-sequences conserved in the promoters of these co-regulated genes., [Results] Using the PathoPlant database, 32 up-regulated gene groups were identified with microarray data for drought-responsive gene expression from Arabidopsis thaliana. Application of the binding site estimation suite of tools (BEST) discovered 179 conserved sequence motifs within the corresponding promoters. Using the STAMP web-server, 49 sequence motifs were classified into 7 motif families for which similarities with known cis-regulatory sequences were identified. All motifs were subjected to a footprintDB analysis to predict interacting DNA binding domains from plant TF families. Predictions were confirmed by using a yeast-one-hybrid approach to select interacting TFs belonging to the predicted TF families. TF-DNA interactions were further experimentally validated in yeast and with a Physcomitrella patens transient expression system, leading to the discovery of several novel TF-DNA interactions., [Conclusions] The present work demonstrates the successful integration of several bioinformatic resources with experimental approaches to predict and validate TFs interacting with conserved sequence motifs in co-regulated genes.

Studies of cell walls have long contributed to understanding plant function. Relevance to the production of biofuels from biomass has recently added urgency to the study of plant cell walls. Arabinogalactan proteins (AGPs) are highly glycosylated glycoproteins at the plant plasma membrane and cell wall. Consisting of a core polypeptide surrounded by glycans, AGPs contain abundant galactosyl and arabinosyl residues and often some glucuronosyl, rhamnosyl, or other residues. Previous study revealed that AGPs in the moss Physcomitrella patens contain unusual 3-O-methyl-rhamnosyl residues (3-O-Me-Rha) in amounts as high as 15 mole percent. The goals of this dissertation were to identify and evaluate Physcomitrella genes that are candidates to encode the rhamnosyl 3-O-methyltransferase that forms 3-O-Me-Rha. A Mycobacteria gene encoding a rhamnosyl 3-O-methyltransferase and an Arabidopsis gene encoding a glucuronosyl 4-O-methyltransferase were used as queries in bioinformatics searches of the Physcomitrella genome. Eleven candidate genes were identified and, through homologous recombination in Physcomitrella, targeted knockouts of these genes were generated. Based on reductions in 3-O-Me-Rha/Rha content ratio in AGPs of the knockouts, two genes,KO1 and KO9, were selected for further study. A third gene, KO11, was selected for further study because it shares a domain with the Arabidopsis glucuronosyl 4-O-methyltransferase. Further study of KO1 led to revision of its gene model. A phylogenic tree suggested that the revised KO1 protein contains a LpxB domain, which is characteristic of bacterial lipid A synthesis. The Physcomitrella ko1 knockout exhibited phenotypic effects including abnormal growth in protonema and rhizoids, reduced 3-O-Me-Rha/Rha in AGPs, and reduced lignin-like content in cell walls. Heterologous expression of KO1 did not produce detectable 3-O-Me-Rha in tobacco AGPs. Further bioinformatics study of KO9 showed it to be an ortholog of caffeoyl-CoA O-methyl

RNA interference (RNAi) is a mechanism that regulates genes by either transcriptional (TGS) or posttranscriptional gene silencing (PTGS), required for genome maintenance and proper development of an organism. Small non-coding RNAs are the key players in RNAi and have been intensively studied in eukaryotes. In plants, several classes of small RNAs with specific sizes and dedicated functions have evolved. The major classes of small RNAs include microRNAs (miRNAs) and small interfering RNAs (siRNAs), which differ in their biogenesis. miRNAs are synthesized from a short hairpin structure while siRNAs are derived from long double-stranded RNAs (dsRNA). Both miRNA and siRNAs control the expression of cognate target RNAs by binding to reverse complementary sequences mediating cleavage or translational inhibition of the target RNA. They also act on the DNA and cause epigenetic changes such as DNA methylation and histone modifications. In the last years, the analysis of plant RNAi pathways was extended to the bryophyte Physcomitrella patens, a non-flowering, non-vascular ancient land plant that diverged from the lineage of seed plants approximately 450 million years ago. Based on a number of characteristic features and its phylogenetic key position in land plant evolution P. patens emerged as a plant model species to address basic as well as applied topics in plant biology. Here we summarize the current knowledge on the role of RNAi in P. patens that shows functional overlap with RNAi pathways from seed plants, and also unique features specific to this species. 2013 by the authors; licensee MDPI, Basel, Switzerland.

Studies of cell walls have long contributed to understanding plant function. Relevance to the production of biofuels from biomass has recently added urgency to the study of plant cell walls. Arabinogalactan proteins (AGPs) are highly glycosylated glycoproteins at the plant plasma membrane and cell wall. Consisting of a core polypeptide surrounded by glycans, AGPs contain abundant galactosyl and arabinosyl residues and often some glucuronosyl, rhamnosyl, or other residues. Previous study revealed that AGPs in the moss Physcomitrella patens contain unusual 3-O-methyl-rhamnosyl residues (3-O-Me-Rha) in amounts as high as 15 mole percent. The goals of this dissertation were to identify and evaluate Physcomitrella genes that are candidates to encode the rhamnosyl 3-O-methyltransferase that forms 3-O-Me-Rha. A Mycobacteria gene encoding a rhamnosyl 3-O-methyltransferase and an Arabidopsis gene encoding a glucuronosyl 4-O-methyltransferase were used as queries in bioinformatics searches of the Physcomitrella genome. Eleven candidate genes were identified and, through homologous recombination in Physcomitrella, targeted knockouts of these genes were generated. Based on reductions in 3-O-Me-Rha/Rha content ratio in AGPs of the knockouts, two genes,KO1 and KO9, were selected for further study. A third gene, KO11, was selected for further study because it shares a domain with the Arabidopsis glucuronosyl 4-O-methyltransferase. Further study of KO1 led to revision of its gene model. A phylogenic tree suggested that the revised KO1 protein contains a LpxB domain, which is characteristic of bacterial lipid A synthesis. The Physcomitrella ko1 knockout exhibited phenotypic effects including abnormal growth in protonema and rhizoids, reduced 3-O-Me-Rha/Rha in AGPs, and reduced lignin-like content in cell walls. Heterologous expression of KO1 did not produce detectable 3-O-Me-Rha in tobacco AGPs. Further bioinformatics study of KO9 showed it to be an ortholog of caffeoyl-CoA O-methyl

Flavanoids are plant secondary metabolites synthesized by polyketide synthase (PKS) in plants, fungi and bacteria. The members of the chalcone synthase (CHS) superfamily, also known as the type III PKSs, function as the key entry enzyme of the flavonoid biosynthesis. Flavanoids are found in liverworts and mosses (bryophyte) that are thought to be the land plants ancestor. Although hundred of CHSs genes from various plant species have been successfully clone, expressed and studied, limited information is available on the CHS from the bryophytes. The model moss, Physcomitrella patens is currently the only bryophyte whose genome has been sequenced and its genome contains at least 17 putative type III PKS genes. Among them, a CHS gene was shown to be basal to all plant type III PKS genes in phylogenetic trees. P. patens CHS exhibited similar kinetic properties and substrate preference profiles as those of higher plants. This suggest that the P. Patens CHS may exhibit similar mechanism as the other land plants CHSs. Due to the unavailability of the P. Patens CHS crystal structure, the main aim of this work is to gain some insight on the structure and mechanism of the P. Patens CHS. Apart form the unavailability of P. patens CHS crystal structure, the major bottleneck of this work is to obtain the P. patens CHS due to the slow plant growth rate and low (less than 1%) extraction yield. Therefore, to speed up the process of obtaining the enzyme, the P. patens CHS has been successfully cloned and expressed in E. coli strain BL21 (DE3) plysS. However, the formation of inclusion bodies has become a major disadvantage of this approach. As alternative, P. patens CHS was secreted into the medium using a bacteriocin release protein expression vector. Secretion of P. patens CHS into the culture media was achieved by co-expression with a psW1 plasmid encoding bacteriocin release protein in E. coli Tuner (DE3) plysS. The optimized conditions were incubation of cells for 20 hours with

Flavanoids are plant secondary metabolites synthesized by polyketide synthase (PKS) in plants, fungi and bacteria. The members of the chalcone synthase (CHS) superfamily, also known as the type III PKSs, function as the key entry enzyme of the flavonoid biosynthesis. Flavanoids are found in liverworts and mosses (bryophyte) that are thought to be the land plants ancestor. Although hundred of CHSs genes from various plant species have been successfully clone, expressed and studied, limited information is available on the CHS from the bryophytes. The model moss, Physcomitrella patens is currently the only bryophyte whose genome has been sequenced and its genome contains at least 17 putative type III PKS genes. Among them, a CHS gene was shown to be basal to all plant type III PKS genes in phylogenetic trees. P. patens CHS exhibited similar kinetic properties and substrate preference profiles as those of higher plants. This suggest that the P. Patens CHS may exhibit similar mechanism as the other land plants CHSs. Due to the unavailability of the P. Patens CHS crystal structure, the main aim of this work is to gain some insight on the structure and mechanism of the P. Patens CHS. Apart form the unavailability of P. patens CHS crystal structure, the major bottleneck of this work is to obtain the P. patens CHS due to the slow plant growth rate and low (less than 1%) extraction yield. Therefore, to speed up the process of obtaining the enzyme, the P. patens CHS has been successfully cloned and expressed in E. coli strain BL21 (DE3) plysS. However, the formation of inclusion bodies has become a major disadvantage of this approach. As alternative, P. patens CHS was secreted into the medium using a bacteriocin release protein expression vector. Secretion of P. patens CHS into the culture media was achieved by co-expression with a psW1 plasmid encoding bacteriocin release protein in E. coli Tuner (DE3) plysS. The optimized conditions were incubation of cells for 20 hours with

Colonization of the land by multicellular green plants was a fundamental step in the evolution of life on earth. Land plants evolved from fresh-water aquatic algae, and the transition to a terrestrial environment required the acquisition of developmental plasticity appropriate to the conditions of water availability, ranging from drought to flood. Here we show that extant bryophytes exhibit submergence-induced developmental plasticity, suggesting that submergence responses evolved relatively early in the evolution of land plants. We also show that a major component of the bryophyte submergence response is controlled by the phytohormone ethylene, using a perception mechanism that has subsequently been conserved throughout the evolution of land plants. Thus a plant environmental response mechanism with major ecological and agricultural importance probably had its origins in the very earliest stages of the colonization of the land.

The knowledge of programmed cell death (PCD) in plants is limited. The gene family of caspases in animals is known to be important for the execution of PCD. Putative homologues to caspases have been found in plants and are referred to as metacaspases. The model moss Physcomitrella patens is known to possess a high frequency of homologous recombination, which can be exploited for the generation of targeted gene knockout mutants. The female reproductive organ in P. patens shows rapid cell degradation during its maturation. This cell degradation is believed to be caused by PCD, and is therefore used as a model to study the process. A new metacaspase 5 knockout construct was successfully produced during this project, which, together with a metacaspase 3 construct received from a collaborator, was transformed into moss to generate two metacaspase single knockout lines and four double knockout lines.

Plant cell walls are composed of a variety of carbohydrates, among them cellulose, pectin and hemicellulose. Cellulose is deposited in the cell wall as microfibrils via cellulose synthesis complexes (CSCs). These complexes contain the cellulose synthase proteins (CESAs) and come in two different morphological forms: rosettes and linear complexes. Rosette shaped cellulose synthesis complexes occur in land plants, whilst linear complexes are commonly found in red algae. However, some land plants, notably bryophytes (mosses) and seedless vascular plants, contain genes that encode both CESAs of the type that form rosette CSCs and also genes similar to those found in red algae. The moss Physcomitrella patens contains one gene of the latter type that has been named GT2. GT2 may represent a gene that mosses and seedless vascular plants inherited from their algal ancestors and that has been lost in seed plants. This suggests an evolutionary divergence between mosses and seedless vascular plants and seed plants. Although the precise function of the gene is unknown, its similarity to red algal CESAs suggests that it plays a part in cellulose microfibril synthesis in the plant cell wall. The goal of this research project is to investigate the particular function of the gene GT2 in Physcomitrella patens using targeted gene replacement techniques. During the course of this project, mutant lines of P. patens were created by removing GT2 from their genome. The removal of GT2 was performed by using targeted gene replacement techniques and it was replaced with a gene encoding antibiotic resistance. Proper integration of the vector encoding antibiotic resistance was tested using PCR and agarose gel electrophoresis. A promoter construct is being created and will be inserted into P. patens lines to see where GT2 is expressed. Phenotypic analysis will be performed on the mutant lines to gain insight into the function of GT2.

PtdIns-4,5-bisphosphate is a lipid messenger of eukaryotic cells that plays a critical role in processes such as cytoskeleton organization, intracellular vesicular trafficking, secretion, cell motility, regulation of ion channels and nuclear signalling pathways. The enzymes responsible for the synthesis of PtdIns(4,5)P(2) are phosphatidylinositol phosphate kinases (PIPKs). The moss Physcomitrella patens contains two PIPKs, PpPIPK1 and PpPIPK2. To study their physiological role, both genes were disrupted by targeted homologous recombination and as a result mutant plants with lower PtdIns(4,5)P(2) levels were obtained. A strong phenotype for pipk1, but not for pipk2 single knockout lines, was obtained. The pipk1 knockout lines were impaired in rhizoid and caulonemal cell elongation, whereas pipk1-2 double knockout lines showed dramatic defects in protonemal and gametophore morphology manifested by the absence of rapidly elongating caulonemal cells in the protonemal tissue, leafy gametophores with very short rhizoids, and loss of sporophyte production. pipk1 complemented by overexpression of PpPIPK1 fully restored the wild-type phenotype whereas overexpression of the inactive PpPIPK1E885A did not. Overexpression of PpPIPK2 in the pipk1-2 double knockout did not restore the wild-type phenotype demonstrating that PpPIPK1 and PpPIPK2 are not functionally redundant. In vivo imaging of the cytoskeleton network revealed that the shortened caulonemal cells in the pipk1 mutants was the result of the absence of the apicobasal gradient of cortical F-actin cables normally observed in wild-type caulonemal cells. Our data indicate that both PpPIPKs play a crucial role in the development of the moss P. patens, and particularly in the regulation of tip growth.

Serine/Arginine-rich proteins (SR-proteins) have been well characterized in metazoans and in the flowering plant Arabidopsis thaliana. But so far no attempts on characterizing SR-proteins in the moss Physcomitrella patens have been done. SR-proteins are a conserved family of splicing regulators essential for constitutive- and alternative splicing. SR-proteins are mediators of alternative splicing (AS) and may be alternatively spliced themselves as a form of gene regulation. Three novel SR-proteins of the SR-subfamily were identified in P. patens. The three genes show conserved intron-exon structure and protein domain distribution, not surprising since the gene family has evidently evolved through gene duplications. The SR-proteins PpSR40 and PpSR36 show differential tissue-specific expression, whereas PpSR39 does not. Tissue-specific expression of SR-proteins has also been seen in A. thaliana. SR-proteins determine splice-site usage in a concentration dependent manner. SR-protein overexpression experiments in A. thaliana and Oryza sativa have shown alteration of splicing patterns of endogenous SR-proteins. Overexpression of PpSR40 did not alter the splicing patterns of PpSR40, PpSR36 and PpSR39. This suggests that they might not be a substrate for PpSR40. These first results of SR-protein characterization in P. patens may provide insights on the SR-protein regulation mechanisms of the common land plant ancestor.