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Plant oxylipins are a class of lipid-derived signaling molecules being involved in the regulation of various biotic and abiotic stress responses. A major class of oxylipins are the circular derivatives to which 12-oxo-phytodienoic acid (OPDA) and its metabolite jasmonic acid (JA) belong. While OPDA and its shorter chain homologue dinor-OPDA (dnOPDA) seem to be ubiquitously found in land plants ranging from bryophytes to angiosperms, the occurrence of JA and its derivatives is still under discussion. The bryophyte Physcomitrium patens has received increased scientific interest as a nonvascular plant model organism over the last decade. Therefore, we followed the metabolism upon wounding by metabolite fingerprinting with the aim to identify jasmonates as well as novel oxylipins in P. patens. A non-targeted metabolomics approach was used to reconstruct the metabolic pathways for the synthesis of oxylipins, derived from roughanic, linoleic, a-linolenic, and arachidonic acid in wild type, the oxylipin-deficient mutants of Ppaos1 and Ppaos2, the mutants of Ppdes being deficient in all fatty acids harboring a D6- double bond and the C20-fatty acid-deficient mutants of Ppelo. Beside of OPDA, iso-OPDA, dnOPDA, and iso-dnOPDA, three additional C18- compounds and a metabolite being isobaric to JA were identified to accumulate after wounding. These findings can now serve as foundation for future research in determining, which compound(s) will serve as native ligand(s) for the oxylipin-receptor COI1 in P. patens. [ABSTRACT FROM AUTHOR]

Plant oxylipins are a class of lipid-derived signaling molecules being involved in the regulation of various biotic and abiotic stress responses. A major class of oxylipins are the circular derivatives to which 12-oxo-phytodienoic acid (OPDA) and its metabolite jasmonic acid (JA) belong. While OPDA and its shorter chain homologue dinor-OPDA (dnOPDA) seem to be ubiquitously found in land plants ranging from bryophytes to angiosperms, the occurrence of JA and its derivatives is still under discussion. The bryophyte Physcomitrium patens has received increased scientific interest as a non-vascular plant model organism over the last decade. Therefore, we followed the metabolism upon wounding by metabolite fingerprinting with the aim to identify jasmonates as well as novel oxylipins in P. patens. A non-targeted metabolomics approach was used to reconstruct the metabolic pathways for the synthesis of oxylipins, derived from roughanic, linoleic, α-linolenic, and arachidonic acid in wild type, the oxylipin-deficient mutants of Ppaos1 and Ppaos2, the mutants of Ppdes being deficient in all fatty acids harboring a Δ6-double bond and the C20-fatty acid-deficient mutants of Ppelo. Beside of OPDA, iso-OPDA, dnOPDA, and iso-dnOPDA, three additional C18-compounds and a metabolite being isobaric to JA were identified to accumulate after wounding. These findings can now serve as foundation for future research in determining, which compound(s) will serve as native ligand(s) for the oxylipin-receptor COI1 in P. patens.

Plant phospholipase D (PLD) exerts important roles in various biological processes, such as intracellular signaling and morphological development. Our knowledge about early land plant PLDs is still underdeveloped. In this study, we identified 84 PLD genes in six mosses, i.e., Physcomitrella patens, Ceratodon purpureus, Fontinalis antipyretica, Pleurozium schreberi, Sphagnum magellanicum, and Sphagnum fallax. These PLDs were classified into four clades (I-IV). We showed that PLD underwent rapid expansion in mosses. A total of six conserved domains and two core HKD motifs were detected. Structure analysis uncovered that the moss PLDs from within a clade generally exhibited similar exon-intron organization. Cis-elements prediction and expression analyses indicated that P. patens PLDs had key roles in stress responsiveness and plant development. Particularly, about half of the P. patens PLDs (e.g., PpPLD1, PpPLD2, and PpPLD5) were differentially expressed under biotic and abiotic stresses. We also determined the expression pattern of P. patens PLD genes in various tissues and at different stages of development. Although the moss, clubmoss, liverwort, and fern PLDs evolved largely under functional constraints, we found episodic positive selection in the moss PLDs, e.g., C. purpureus PLD2 and P. patens PLD11. We infer that the evolutionary force acting on the PLDs may have facilitated moss colonization of land. Our work provides valuable insights into the diversification of moss PLD genes, and can be used for future studies of their functions. [ABSTRACT FROM AUTHOR]

Targeted gene knockout is particularly useful for analyzing gene functions in plant growth, signaling, and development. By transforming knockout cassettes consisting of homologous sequences of the target gene into protoplasts, the classical gene targeting method aims to obtain targeted gene replacement, allowing for the characterization of gene functions in vivo. The moss Physcomitrella patens is a known model organism for a high frequency of homologous recombination and thus harbors a remarkable rate of gene targeting. Other moss features, including easy to culture, dominant haploidy phase, and sequenced genome, make gene targeting prevalent in Physcomitrella patens. However, even gene targeting was powerful to generate knockouts, researchers using this method still experienced technical challenges. For example, obtaining a good number of targeted knockouts after protoplast transformation and regeneration disturbed the users. Off-target mutations such as illegitimate random integration mediated by nonhomologous end joining and targeted insertion wherein one junction on-target but the other end off-target is commonly present in the knockouts. Protoplast fusion during transformation and regeneration was also a problem. This review will discuss the advantages and technical challenges of gene targeting. Recently, CRISPR-Cas9 is a revolutionary technology and becoming a hot topic in plant gene editing. In the second part of this review, CRISPR-Cas9 technology will be focused on and compared to gene targeting regarding the practical use in Physcomitrella patens. This review presents an updated perspective of the gene targeting and CRISPR-Cas9 techniques to plant biologists who may consider studying gene functions in the model organism Physcomitrella patens.

Specialized photosynthetic organs have appeared several times independently during the evolution of land plants. Phyllids, the leaf-like organs of bryophytes such as mosses or leafy liverworts, display a simple morphology, with a small number of cells and cell types and lack typical vascular tissue which contrasts greatly with flowering plants. Despite this, the leaf structures of these two plant types share many morphological characteristics. In this review, we summarize the current understanding of leaf morphogenesis in the model moss Physcomitrium patens , focusing on the underlying cellular patterns and molecular regulatory mechanisms. We discuss this knowledge in an evolutionary context and identify parallels between moss and flowering plant leaf development. Finally, we propose potential research directions that may help to answer fundamental questions in plant development using moss leaves as a model system. [ABSTRACT FROM AUTHOR]

Specialized photosynthetic organs have appeared several times independently during the evolution of land plants. Phyllids, the leaf-like organs of bryophytes such as mosses or leafy liverworts, display a simple morphology, with a small number of cells and cell types and lack typical vascular tissue which contrasts greatly with flowering plants. Despite this, the leaf structures of these two plant types share many morphological characteristics. In this review, we summarize the current understanding of leaf morphogenesis in the model moss Physcomitrium patens, focusing on the underlying cellular patterns and molecular regulatory mechanisms. We discuss this knowledge in an evolutionary context and identify parallels between moss and flowering plant leaf development. Finally, we propose potential research directions that may help to answer fundamental questions in plant development using moss leaves as a model system.

C-terminal peptidase (Ctp) cleaves the C-terminal extension of the D1 precursor (pD1) to form mature D1. Among the three homologs CtpA, CtpB, and CtpC in photosynthetic organisms only the first is capable of processing pD1 while the roles of CtpB and CtpC remain elusive. Phylogenetic analysis of Ctps from photosynthetic organisms revealed that CtpA has diverged early from CtpB and CtpC during evolution implying distinct roles for the Ctps. Analysis of Arabidopsis Ctp-deficient mutants revealed that pD1 processing was not affected in atctpb , atctpc , or atctpbatctpc mutants, demonstrating that AtCtpA, not AtCtpB or AtCtpC, is responsible for cleaving the pD1 C-terminal extension. Ectopic expression of CtpAs from Synechococcus elongatus , Chlamydomonas reinhardtii , and Physcomitrella patens in atctpa rescued the lethal phenotype of the mutant indicating that SeCtpA, CrCtpA, and PpCtpA could process pD1 in Arabidopsis. Enzyme activity assays showed that PpCtpA and CrCtpA could convert pD1 into mature D1 in vitro. In contrast, expressing CtpB or CtpC from Arabidopsis, C. reinhardtii , or P. patens in atctpa did not rescue its D1 maturation deficiency, and enzyme activity assays also showed that neither CtpB nor CtpC could process pD1 in vitro. Taken together, we conclude that the function of pD1 processing by CtpA is conserved in photosynthetic organisms. It is possible that among other factors CtpA developed this function to initiate the formation of the oxygenic D1/D2 type PSII complex during evolution whereas CtpB or CtpC have other roles that are still unclear. [ABSTRACT FROM AUTHOR]

Plant glycoproteins display a characteristic type of O -glycosylation where short arabinans or larger arabinogalactans are linked to hydroxyproline. The conversion of proline to 4-hydroxyproline is accomplished by prolyl-hydroxylases (P4Hs). Eleven putative Nicotiana benthamiana P4Hs, which fall in four homology groups, have been identified by homology searches using known Arabidopsis thaliana P4H sequences. One member of each of these groups has been expressed in insect cells using the baculovirus expression system and applied to synthetic peptides representing the O -glycosylated region of erythropoietin (EPO), IgA1, Art v 1 and the Arabidopsis thaliana glycoprotein STRUBBELIG. Unlike the situation in the moss Physcomitrella patens , where one particular P4H was mainly responsible for the oxidation of erythropoietin, the tobacco P4Hs exhibited rather similar activities, albeit with biased substrate preferences and preferred sites of oxidation. From a biotechnological viewpoint, this result means that silencing/knockout of a single P4H in N. benthamiana cannot be expected to result in the abolishment of the plant-specific oxidation of prolyl residues in a recombinant protein. [ABSTRACT FROM AUTHOR]

Introduction:Physcomitrium patens (Hedw.) Mitten (previously known as Physcomitrella patens) was collected by H.L.K. Whitehouse in Gransden Wood (Huntingdonshire, United Kingdom) in 1962 and distributed across the globe starting in 1974. Hence, the Gransden accession has been cultured in vitro in laboratories for half a century. Today, there are more than 13 different pedigrees derived from the original accession. Additionally, accessions from other sites worldwide were collected during the last decades.Methods and Results: In this study, 250 high throughput RNA sequencing (RNA-seq) samples and 25 gDNA samples were used to detect single nucleotide polymorphisms (SNPs). Analyses were performed using five different P. patens accessions and 13 different Gransden pedigrees. SNPs were overlaid with metadata and known phenotypic variations. Unique SNPs defining Gransden pedigrees and accessions were identified and experimentally confirmed. They can be successfully employed for PCR-based identification.Conclusion: We show independent mutations in different Gransden laboratory pedigrees, demonstrating that somatic mutations occur and accumulate during in vitro culture. The frequency of such mutations is similar to those observed in naturally occurring populations. We present evidence that vegetative propagation leads to accumulation of deleterious mutations, and that sexual reproduction purges those. Unique SNP sets for five different P. patens accessions were isolated and can be used to determine individual accessions as well as Gransden pedigrees. Based on that, laboratory methods to easily determine P. patens accessions and Gransden pedigrees are presented.

To colonize different terrestrial habitats, early land plants had to overcome the challenge of coping with harsh new environments. Alternative splicing – an RNA processing mechanism through which splice sites are differentially recognized, originating multiple transcripts and potentially different proteins from the same gene – can be key for plant stress tolerance. Serine/arginine-rich (SR) proteins constitute an evolutionarily conserved family of major alternative splicing regulators that in plants subdivides into six subfamilies. Despite being well studied in animals and a few plant species, such as the model angiosperm Arabidopsis thaliana and the crop Oryza sativa, little is known of these splicing factors in early land plants. Establishing the whole complement of SR proteins in different species is essential to understand the functional and evolutionary significance of alternative splicing. An in silico search for SR proteins in the extant moss Physcomitrella patens revealed inconsistencies both in the published data and available databases, likely arising from automatic annotation lacking adequate manual curation. These misannotations interfere with the description not only of the number and subfamily classification of Physcomitrella SR proteins but also of their domain architecture, potentially hindering the elucidation of their molecular functions. We therefore advise caution when looking into P. patens genomic resources. Our systematic survey nonetheless confidently identified 16 P. patens SR proteins that fall into the six described subfamilies and represent counterparts of well-established members in Arabidopsis and rice. Intensified research efforts should disclose whether SR proteins were already determining alternative splicing modulation and stress tolerance in early land plants.

The function of trehalose metabolism in plants during growth and development has been extensively studied, mostly in the eudicot Arabidopsis thaliana. So far, however, not much is known about trehalose metabolism in the moss Physcomitrella patens. Here, we show that in P. patens, two active trehalose-6-phosphate synthase enzymes exist, PpTPS1 and PpTPS2. Expression of both enzymes in Saccharomyces cerevisiae can complement the glucose-growth defect of the yeast tps1∆ mutant. Truncation of N-terminal extension in PpTPS1 and PpTPS2 resulted in higher TPS activity and high trehalose levels, upon expression in yeast. Physcomitrella knockout plants were generated and analyzed in various conditions to functionally characterize these proteins. tps1∆ and tps2∆ knockouts displayed a lower amount of caulonema filaments and were significantly reduced in size of gametophores as compared to the wild type. These phenotypes were more pronounced in the tps1∆ tps2∆ mutant. Caulonema formation is induced by factors such as high energy and auxins. Only high amounts of supplied energy were able to induce caulonema filaments in the tps1∆ tps2∆ mutant. Furthermore, this mutant was less sensitive to auxins as NAA-induced caulonema development was arrested in the tps1∆ tps2∆ mutant. In contrast, formation of caulonema filaments is repressed by cytokinins. This effect was more severe in the tps1∆ and tps1∆ tps2∆ mutants. Our results demonstrate that PpTPS1 and PpTPS2 are essential for sensing and signaling sugars and plant hormones to monitor the balance between caulonema and chloronema development.

DNMT2 is a DNA/tRNA cytosine methyltransferase that is highly conserved in structure and function in eukaryotes. In plants however, limited information is available on the function of this methyltransferase. We have previously reported that in the moss Physcomitrella patens , DNMT2 plays a crucial role in stress recovery and tRNAAsp transcription/stability under salt stress. To further investigate the role of PpDNMT2 at genome level, in this study we have performed RNA sequencing of ppdnmt2. Transcriptome analysis reveals a number of genes and pathways to function differentially and suggests a close link between PpDNMT2 function and osmotic and ionic stress tolerance. We propose PpDNMT2 to play a pivotal role in regulating salt tolerance by affecting molecular networks involved in stress perception and signal transduction that underlie maintenance of ion homeostasis in cells. We also examined interactome of PpDNMT2 using affinity purification (AP) coupled to mass spectrometry (AP-MS). Quantitative proteomic analysis reveals several chloroplast proteins involved in light reactions and carbon assimilation and proteins involved in stress response and some not implicated in stress to co-immunoprecipitate with PpDNMT2. Comparison between transcriptome and interactome datasets has revealed novel association between PpDNMT2 activity and the antioxidant enzyme Superoxide dismutase (SOD), protein turnover mediated by the Ubiquitin-proteasome system and epigenetic gene regulation. PpDNMT2 possibly exists in complex with CuZn-SODs in vivo and the two proteins also directly interact in the yeast nucleus as observed by yeast two-hybrid assay. Taken together, the work presented in this study sheds light on diverse roles of PpDNMT2 in maintaining molecular and physiological homeostasis in P. patens. This is a first report describing transcriptome and interactome of DNMT2 in any land plant. [ABSTRACT FROM AUTHOR]

Introduction: Physcomitrium patens (Hedw.) Mitten (previously known as Physcomitrella patens) was collected by H.L.K. Whitehouse in Gransden Wood (Huntingdonshire, United Kingdom) in 1962 and distributed across the globe starting in 1974. Hence, the Gransden accession has been cultured in vitro in laboratories for half a century. Today, there are more than 13 different pedigrees derived from the original accession. Additionally, accessions from other sites worldwide were collected during the last decades. Methods and Results: In this study, 250 high throughput RNA sequencing (RNA-seq) samples and 25 gDNA samples were used to detect single nucleotide polymorphisms (SNPs). Analyses were performed using five different P. patens accessions and 13 different Gransden pedigrees. SNPs were overlaid with metadata and known phenotypic variations. Unique SNPs defining Gransden pedigrees and accessions were identified and experimentally confirmed. They can be successfully employed for PCR-based identification. Conclusion: We show independent mutations in different Gransden laboratory pedigrees, demonstrating that somatic mutations occur and accumulate during in vitro culture. The frequency of such mutations is similar to those observed in naturally occurring populations. We present evidence that vegetative propagation leads to accumulation of deleterious mutations, and that sexual reproduction purges those. Unique SNP sets for five different P. patens accessions were isolated and can be used to determine individual accessions as well as Gransden pedigrees. Based on that, laboratory methods to easily determine P. patens accessions and Gransden pedigrees are presented. [ABSTRACT FROM AUTHOR]

COR413 genes belong to a poorly characterized group of plant-specific cold-regulated genes initially identified as part of the transcriptional activation machinery of plants during cold acclimation. They encode multispanning transmembrane proteins predicted to target the plasma membrane or the chloroplast inner membrane. Despite being ubiquitous throughout the plant kingdom, little is known about their biological function. In this study, we used reverse genetics to investigate the relevance of a predicted chloroplast localized COR413 protein (PpCOR413im) from the moss Physcomitrella patens in developmental and abiotic stress responses. Expression of PpCOR413im was strongly induced by abscisic acid (ABA) and by various environmental stimuli, including low temperature, hyperosmosis, salinity and high light. In vivo subcellular localization of PpCOR413im-GFP fusion protein revealed that this protein is localized in chloroplasts, confirming the in silico predictions. Loss-of-function mutants of PpCOR413im exhibited growth and developmental alterations such as growth retardation, reduced caulonema formation and hypersensitivity to ABA. Mutants also displayed altered photochemistry under various abiotic stresses, including dehydration and low temperature, and exhibited a dramatic growth inhibition upon exposure to high light. Disruption of PpCOR413im also caused altered chloroplast ultrastructure, increased ROS accumulation, and enhanced starch and sucrose levels under high light or after ABA treatment. In addition, loss of PpCOR413im affected both nuclear and chloroplast gene expression in response to ABA and high light, suggesting a role for this gene downstream of ABA in the regulation of growth and environmental stress responses. Developmental alterations exhibited by PpCOR413im knockout mutants had remarkable similarities to those exhibited by hxk1 , a mutant lacking a major chloroplastic hexokinase, an enzyme involved in energy homeostasis. Based on these findings, we propose that PpCOR413im is involved in coordinating energy metabolism with ABA-mediated growth and developmental responses. [ABSTRACT FROM AUTHOR]

Mosses are an ancient land plant lineage and are therefore important in studying the evolution of plant developmental processes. Here, we describe stomatal development in the model moss species Physcomitrium patens (previously known as Physcomitrella patens) over the duration of sporophyte development. We dissect the molecular mechanisms guiding cell division and fate and highlight how stomatal function might vary under different environmental conditions. In contrast to the asymmetric entry divisions described in Arabidopsis thaliana , moss protodermal cells can enter the stomatal lineage directly by expanding into an oval shaped guard mother cell (GMC). We observed that when two early stage P. patens GMCs form adjacently, a spacing division can occur, leading to separation of the GMCs by an intervening epidermal spacer cell. We investigated whether orthologs of Arabidopsis stomatal development regulators are required for this spacing division. Our results indicated that bHLH transcription factors PpSMF1 and PpSCRM1 are required for GMC formation. Moreover, the ligand and receptor components PpEPF1 and PpTMM are also required for orientating cell divisions and preventing single or clustered early GMCs from developing adjacent to one another. The identification of GMC spacing divisions in P. patens raises the possibility that the ability to space stomatal lineage cells could have evolved before mosses diverged from the ancestral lineage. This would have enabled plants to integrate stomatal development with sporophyte growth and could underpin the adoption of multiple bHLH transcription factors and EPF ligands to more precisely control stomatal patterning in later diverging plant lineages. We also observed that when P. patens sporophyte capsules mature in wet conditions, stomata are typically plugged whereas under drier conditions this is not the case; instead, mucilage drying leads to hollow sub-stomatal cavities. This appears to aid capsule drying and provides further evidence for early land plant stomata contributing to capsule rupture and spore release. [ABSTRACT FROM AUTHOR]

To colonize different terrestrial habitats, early land plants had to overcome the challenge of coping with harsh new environments. Alternative splicing – an RNA processing mechanism through which splice sites are differentially recognized, originating multiple transcripts and potentially different proteins from the same gene – can be key for plant stress tolerance. Serine/arginine-rich (SR) proteins constitute an evolutionarily conserved family of major alternative splicing regulators that in plants subdivides into six subfamilies. Despite being well studied in animals and a few plant species, such as the model angiosperm Arabidopsis thaliana and the crop Oryza sativa, little is known of these splicing factors in early land plants. Establishing the whole complement of SR proteins in different species is essential to understand the functional and evolutionary significance of alternative splicing. An in silico search for SR proteins in the extant moss Physcomitrella patens revealed inconsistencies both in the published data and available databases, likely arising from automatic annotation lacking adequate manual curation. These misannotations interfere with the description not only of the number and subfamily classification of Physcomitrella SR proteins but also of their domain architecture, potentially hindering the elucidation of their molecular functions. We therefore advise caution when looking into P. patens genomic resources. Our systematic survey nonetheless confidently identified 16 P. patens SR proteins that fall into the six described subfamilies and represent counterparts of well-established members in Arabidopsis and rice. Intensified research efforts should disclose whether SR proteins were already determining alternative splicing modulation and stress tolerance in early land plants. [ABSTRACT FROM AUTHOR]

The function of trehalose metabolism in plants during growth and development has been extensively studied, mostly in the eudicot Arabidopsis thaliana. So far, however, not much is known about trehalose metabolism in the moss Physcomitrella patens. Here, we show that in P. patens , two active trehalose-6-phosphate synthase enzymes exist, PpTPS1 and PpTPS2. Expression of both enzymes in Saccharomyces cerevisiae can complement the glucose-growth defect of the yeast tps1∆ mutant. Truncation of N-terminal extension in PpTPS1 and PpTPS2 resulted in higher TPS activity and high trehalose levels, upon expression in yeast. Physcomitrella knockout plants were generated and analyzed in various conditions to functionally characterize these proteins. tps1∆ and tps2∆ knockouts displayed a lower amount of caulonema filaments and were significantly reduced in size of gametophores as compared to the wild type. These phenotypes were more pronounced in the tps1∆ tps2∆ mutant. Caulonema formation is induced by factors such as high energy and auxins. Only high amounts of supplied energy were able to induce caulonema filaments in the tps1∆ tps2∆ mutant. Furthermore, this mutant was less sensitive to auxins as NAA-induced caulonema development was arrested in the tps1∆ tps2∆ mutant. In contrast, formation of caulonema filaments is repressed by cytokinins. This effect was more severe in the tps1∆ and tps1∆ tps2∆ mutants. Our results demonstrate that PpTPS1 and PpTPS2 are essential for sensing and signaling sugars and plant hormones to monitor the balance between caulonema and chloronema development. [ABSTRACT FROM AUTHOR]

The XPF-ERCC1 complex, a highly conserved structure-specific endonuclease, functions in multiple DNA repair pathways that are pivotal for maintaining genome stability, including nucleotide excision repair, interstrand crosslink repair, and homologous recombination. XPF-ERCC1 incises double-stranded DNA at double-strand/single-strand junctions, making it an ideal enzyme for processing DNA structures that contain partially unwound strands. Here, we have examined the role of the XPF-ERCC1 complex in the model bryophyte Physcomitrella patens which exhibits uniquely high gene targeting frequencies. We undertook targeted knockout of the Physcomitrella ERCC1 and XPF genes. Mutant analysis shows that the endonuclease complex is essential for resistance to UV-B and to the alkylating agent MMS, and contributes to the maintenance of genome integrity but is also involved in gene targeting in this model plant. Using different constructs we determine whether the function of the XPF-ERCC1 endonuclease complex in gene targeting was removal of 3′ non-homologous termini, similar to SSA, or processing of looped-out heteroduplex intermediates. Interestingly, our data suggest a role of the endonuclease in both pathways and have implications for the mechanism of targeted gene replacement in plants and its specificities compared to yeast and mammalian cells.

Tip growth of pollen tubes, root hairs, and apical cells of moss protonemata is controlled by ROP (Rho of plants) GTPases, which were shown to accumulate at the apical plasma membrane of these cells. However, most ROP localization patterns reported in the literature are based on fluorescent protein tagging and need to be interpreted with caution, as ROP fusion proteins were generally overexpressed at undefined levels, in many cases without assessing effects on tip growth. ROP-GEFs, important regulators of ROP activity, were also described to accumulate at the apical plasma membrane during tip growth. However, to date only the localization of fluorescent ROP-GEF fusion proteins strongly overexpressed using highly active promoters have been investigated. Here, the intracellular distributions of fluorescent PpROP1 and PpROP-GEF4 fusion proteins expressed at essentially endogenous levels in apical cells of Physcomitrella patens “knock-in” protonemata were analyzed. Whereas PpROP-GEF4 was found to associate with a small apical plasma membrane domain, PpROP1 expression was below the detection limit. Estradiol-titratable expression of a fluorescent PpROP1 fusion protein at the lowest detectable level, at which plant development was only marginally affected, was therefore employed to show that PpROP1 also accumulates at the apical plasma membrane, although within a substantially larger domain. Interestingly, RNA-Seq data indicated that the majority of all genes active in protonemata are expressed at lower levels than PpROP1, suggesting that estradiol-titratable expression may represent an important alternative to “knock-in” based analysis of the intracellular distribution of fluorescent fusion proteins in protonemal cells.

The human complement system is an important part of the immune system responsible for lysis and elimination of invading microorganisms and apoptotic body cells. Improper activation of the system due to deficiency, mutations, or autoantibodies of complement regulators, mainly factor H (FH) and FH-related proteins (FHRs), causes severe kidney and eye diseases. However, there is no recombinant FH therapeutic available on the market. The first successful recombinant production of FH was accomplished with the moss bioreactor, Physcomitrella patens. Recently, a synthetic regulator, MFHR1, was designed to generate a multitarget complement inhibitor that combines the activities of FH and the FH-related protein 1 (FHR1). The potential of MFHR1 was demonstrated in a proof-of-concept study with transiently transfected insect cells. Here, we present the stable production of recombinant glyco-engineered MFHR1 in the moss bioreactor. The key features of this system are precise genome engineering via homologous recombination, Good Manufacturing Practice-compliant production in photobioreactors, high batch-to-batch reproducibility, and product stability. Several potential biopharmaceuticals are being produced in this system. In some cases, these are even biobetters, i.e., the recombinant proteins produced in moss have a superior quality compared to their counterparts from mammalian systems as for example moss-made aGal, which successfully passed phase I clinical trials. Via mass spectrometry-based analysis of moss-produced MFHR1, we now prove the correct synthesis and modification of this glycoprotein with predominantly complex-type N-glycan attachment. Moss-produced MFHR1 exhibits cofactor and decay acceleration activities comparable to FH, and its mechanism of action on multiple levels within the alternative pathway of complement activation led to a strong inhibitory activity on the whole alternative pathway, which was higher than with the physiological regulator FH.

Arbuscular mycorrhiza is one of the most common plant symbiotic interactions observed today. Due to their nearly ubiquitous occurrence and their beneficial impact on both partners it was suggested that this mutualistic interaction was crucial for plants to colonize the terrestrial habitat approximately 500 Ma ago. On the plant side the association is established via the common symbiotic pathway (CSP). This pathway allows the recognition of the fungal symbiotic partner, subsequent signaling to the nucleus, and initiation of the symbiotic program with respect to specific gene expression and cellular re-organization. The downstream part of the CSP is a regulatory network that coordinates the transcription of genes necessary to establish the symbiosis, comprising multiple GRAS transcription factors (TFs). These regulate their own expression as an intricate transcriptional network. Deduced from non-host genome data the loss of genes encoding CSP components coincides with the loss of the interaction itself. Here, we analyzed bryophyte species with special emphasis on the moss Physcomitrella patens, supposed to be a non-host, for the composition of the GRAS regulatory network components. We show lineage specific losses and expansions of several of these factors in bryophytes, potentially coinciding with the proposed host/non-host status of the lineages. We evaluate losses and expansions and infer clade-specific evolution of GRAS TFs.

Autophagy is a catabolic process for bulk and selective degradation of cytoplasmic components in the vacuole/lysosome. In Saccharomyces cerevisiae, ATG genes were identified as essential genes for autophagy, and most ATG genes are highly conserved among eukaryotes, including plants. Although reverse genetic analyses have revealed that autophagy is involved in responses to abiotic and biotic stresses in land plants, our knowledge of its molecular mechanism remains limited. This limitation is partly because of the multiplication of some ATG genes, including ATG8 , in widely used model plants such as Arabidopsis thaliana , which adds complexity to functional studies. Furthermore, due to limited information on the composition and functions of the ATG genes in basal land plants and charophytes, it remains unclear whether multiplication of ATG genes is associated with neofunctionalization of these genes. To gain insight into the diversification of ATG genes during plant evolution, we compared the composition of ATG genes in plants with a special focus on a liverwort and two charophytes, which have not previously been analyzed. Our results showed that the liverwort Marchantia polymorpha and the charophytes Klebsormidium nitens and Chara braunii harbor fundamental sets of ATG genes with low redundancy compared with those of A. thaliana and the moss Physcomitrella patens , suggesting that multiplication of ATG genes occurred during land plant evolution. We also attempted to establish an experimental system for analyzing autophagy in M. polymorpha. We generated transgenic plants expressing fluorescently tagged MpATG8 to observe its dynamics in M. polymorpha and produced autophagy-defective mutants by genome editing using the CRISPR/Cas9 system. These tools allowed us to demonstrate that MpATG8 is transported into the vacuole in an MpATG2-, MpATG5-, and MpATG7-dependent manner, suggesting that fluorescently tagged MpATG8 can be used as an autophagosome marker in M. polymorpha. M. polymorpha can provide a powerful system for studying the mechanisms and evolution of autophagy in plants. [ABSTRACT FROM AUTHOR]

The XPF-ERCC1 complex, a highly conserved structure-specific endonuclease, functions in multiple DNA repair pathways that are pivotal for maintaining genome stability, including nucleotide excision repair, interstrand crosslink repair, and homologous recombination. XPF-ERCC1 incises double-stranded DNA at double-strand/single-strand junctions, making it an ideal enzyme for processing DNA structures that contain partially unwound strands. Here, we have examined the role of the XPF-ERCC1 complex in the model bryophyte Physcomitrella patens which exhibits uniquely high gene targeting frequencies. We undertook targeted knockout of the Physcomitrella ERCC1 and XPF genes. Mutant analysis shows that the endonuclease complex is essential for resistance to UV-B and to the alkylating agent MMS, and contributes to the maintenance of genome integrity but is also involved in gene targeting in this model plant. Using different constructs we determine whether the function of the XPF-ERCC1 endonuclease complex in gene targeting was removal of 3′ non-homologous termini, similar to SSA, or processing of looped-out heteroduplex intermediates. Interestingly, our data suggest a role of the endonuclease in both pathways and have implications for the mechanism of targeted gene replacement in plants and its specificities compared to yeast and mammalian cells. [ABSTRACT FROM AUTHOR]

The human complement system is an important part of the immune system responsible for lysis and elimination of invading microorganisms and apoptotic body cells. Improper activation of the system due to deficiency, mutations, or autoantibodies of complement regulators, mainly factor H (FH) and FH-related proteins (FHRs), causes severe kidney and eye diseases. However, there is no recombinant FH therapeutic available on the market. The first successful recombinant production of FH was accomplished with the moss bioreactor, Physcomitrella patens. Recently, a synthetic regulator, MFHR1, was designed to generate a multitarget complement inhibitor that combines the activities of FH and the FH-related protein 1 (FHR1). The potential of MFHR1 was demonstrated in a proof-of-concept study with transiently transfected insect cells. Here, we present the stable production of recombinant glyco-engineered MFHR1 in the moss bioreactor. The key features of this system are precise genome engineering via homologous recombination, Good Manufacturing Practice-compliant production in photobioreactors, high batch-to-batch reproducibility, and product stability. Several potential biopharmaceuticals are being produced in this system. In some cases, these are even biobetters, i.e., the recombinant proteins produced in moss have a superior quality compared to their counterparts from mammalian systems as for example moss-made aGal, which successfully passed phase I clinical trials. Via mass spectrometry-based analysis of moss-produced MFHR1, we now prove the correct synthesis and modification of this glycoprotein with predominantly complex-type N-glycan attachment. Moss-produced MFHR1 exhibits cofactor and decay acceleration activities comparable to FH, and its mechanism of action on multiple levels within the alternative pathway of complement activation led to a strong inhibitory activity on the whole alternative pathway, which was higher than with the physiological regulator FH. [ABSTRACT FROM AUTHOR]

Arbuscular mycorrhiza is one of the most common plant symbiotic interactions observed today. Due to their nearly ubiquitous occurrence and their beneficial impact on both partners it was suggested that this mutualistic interaction was crucial for plants to colonize the terrestrial habitat approximately 500 Ma ago. On the plant side the association is established via the common symbiotic pathway (CSP). This pathway allows the recognition of the fungal symbiotic partner, subsequent signaling to the nucleus, and initiation of the symbiotic program with respect to specific gene expression and cellular re-organization. The downstream part of the CSP is a regulatory network that coordinates the transcription of genes necessary to establish the symbiosis, comprising multiple GRAS transcription factors (TFs). These regulate their own expression as an intricate transcriptional network. Deduced from non-host genome data the loss of genes encoding CSP components coincides with the loss of the interaction itself. Here, we analyzed bryophyte species with special emphasis on the moss Physcomitrella patens , supposed to be a non-host, for the composition of the GRAS regulatory network components. We show lineage specific losses and expansions of several of these factors in bryophytes, potentially coinciding with the proposed host/non-host status of the lineages. We evaluate losses and expansions and infer clade-specific evolution of GRAS TFs. [ABSTRACT FROM AUTHOR]

Pseudomonas syringae pv. phaseolicola is the causative agent of halo blight in common bean (Phaseolus vulgaris). Similar to other pathogenic gram-negative bacteria, it secrets a set of type III effectors into host cells to subvert defense mechanisms. HopQ1 (for Hrp outer protein Q) is one of these type III effectors contributing to virulence of bacteria. Upon delivery into a plant cell, HopQ1 undergoes phosphorylation, binds host 14-3-3 proteins and suppresses defense-related signaling. Some plants however, evolved systems to recognize HopQ1 and respond to its presence and thus to prevent infection. HopQ1 shows homology to Nucleoside Hydrolases (NHs), but it contains a modified calcium binding motif not found in the canonical enzymes. CLuster ANalysis of Sequences (CLANS) revealed that HopQ1 and alike proteins make a distinct group of putative NHs located distantly from the classical enzymes. The HopQ1 – like protein (HLP) group comprises sequences from plant pathogenic bacteria, fungi, and lower plants. Our data suggest that the evolution of HopQ1 homologs in bacteria, fungi, and algae was independent. The location of moss HopQ1 homologs inside the fungal clade indicates a possibility of horizontal gene transfer (HGT) between those taxa. We identified a HLP in the moss Physcomitrella patens. Our experiments show that this protein (referred to as PpHLP) extended by a TTSS signal of HopQ1 promoted P. syringae growth in bean and was recognized by Nicotiana benthamiana immune system. Thus, despite the low sequence similarity to HopQ1 the engineered PpHLP acted as a bacterial virulence factor and displayed similar to HopQ1 virulence properties.

Physcomitrella patens has emerged as a model moss system to investigate the evolution of various plant characters in early land plant lineages. Yet, there is merely a disparate body of ultrastructural and physiological evidence from other mosses to draw inferences about the modes of photosynthate transport in the alternating generations of Physcomitrella. We performed a series of ultrastructural, fluorescent tracing, physiological, and immunohistochemical experiments to elucidate a coherent model of photosynthate transport in this moss. Our ultrastructural observations revealed that Physcomitrella is an endohydric moss with water-conducting and putative food-conducting cells in the gametophytic stem and leaves. Movement of fluorescent tracer 5(6)-carboxyfluorescein diacetate revealed that the mode of transport in the gametophytic generation is symplasmic and is mediated by plasmodesmata, while there is a diffusion barrier composed of transfer cells that separates the photoautotrophic gametophyte from the nutritionally dependent heterotrophic sporophyte. We posited that, analogous to what is found in apoplasmically phloem loading higher plants, the primary photosynthate sucrose, is actively imported into the transfer cells by sucrose/H+ symporters (SUTs) that are, in turn, powered by P-type ATPases, and that the transfer cells harbor an ATP-conserving Sucrose Synthase (SUS) pathway. Supporting our hypothesis was the finding that a protonophore (2,4-dinitrophenol) and a SUT-specific inhibitor (diethyl pyrocarbonate) reduced the uptake of radiolabeled sucrose into the sporangia. In situ immunolocalization of P-type ATPase, Sucrose Synthase, and Proton Pyrophosphatase – all key components of the SUS pathway – showed that these proteins were prominently localized in the transfer cells, providing further evidence consistent with our argument.

The moss Physcomitrella patens is unique among plants in that homologous recombination can be used to knock out genes, just like in yeast. Furthermore, transformed plasmids can be rescued from Physcomitrella back into Escherichia coli, similar to yeast. In the present study, we have tested if a third important tool from yeast molecular genetics, auxotrophic selection markers, can be used in Physcomitrella. Two auxotrophic moss strains were made by knocking out the PpHIS3 gene encoding imidazoleglycerol-phosphate dehydratase, and the PpTRP1 gene encoding phosphoribosylanthranilate isomerase, disrupting the biosynthesis of histidine and tryptophan, respectively. The resulting PpHIS3Δ and PpTRP1Δ knockout strains were unable to grow on medium lacking histidine or tryptophan. The PpHIS3Δ strain was used to test selection of transformants by complementation of an auxotrophic marker. We found that the PpHIS3Δ strain could be complemented by transformation with a plasmid expressing the PpHIS3 gene from the CaMV 35S promoter, allowing the strain to grow on medium lacking histidine. Both linearized plasmids and circular supercoiled plasmids could complement the auxotrophic marker, and plasmids from both types of transformants could be rescued back into E. coli. Plasmids rescued from circular transformants were identical to the original plasmid, whereas plasmids rescued from linearized transformants had deletions generated by recombination between micro-homologies in the plasmids. Our results show that cloning by complementation of an auxotrophic marker works in Physcomitrella, which opens the door for using auxotrophic selection markers in moss molecular genetics. This will facilitate the adaptation of shuttle plasmid dependent methods from yeast molecular genetics for use in Physcomitrella.

The moss Physcomitrella patens is used both as an evo-devo model and biotechnological production system for metabolites and pharmaceuticals. Strong in vivo expression of genes of interest is important for production of recombinant proteins, e.g., selectable markers, fluorescent proteins, or enzymes. In this regard, the choice of the promoter sequence as well as codon usage optimization are two important inside factors to consider in order to obtain optimum protein accumulation level. To reliably quantify fluorescence, we transfected protoplasts with promoter:GFP fusion constructs and measured fluorescence intensity of living protoplasts in a plate reader system. We used the red fluorescent protein mCherry under 2x 35S promoter control as second reporter to normalize for different transfection efficiencies. We derived a novel endogenous promoter and compared deletion variants with exogenous promoters. We used different codon-adapted green fluorescent protein (GFP) genes to evaluate the influence of promoter choice and codon optimization on protein accumulation in P. patens, and show that the promoter of the gene of P. patens chlorophyll a/b binding protein lhcsr1 drives expression of GFP in protoplasts significantly (more than twofold) better than the commonly used 2x 35S promoter or the rice actin1 promoter. We identified a shortened 677 bp version of the lhcsr1 promoter that retains full activity in protoplasts. The codon optimized GFP yields significantly (more than twofold) stronger fluorescence signals and thus demonstrates that adjusting codon usage in P. patens can increase expression strength. In combination, new promotor and codon optimized GFP conveyed sixfold increased fluorescence signal.

Pseudomonas syringae pv. phaseolicola is the causative agent of halo blight in common bean (Phaseolus vulgaris). Similar to other pathogenic gram-negative bacteria, it secrets a set of type III effectors into host cells to subvert defense mechanisms. HopQ1 (for Hrp outer protein Q) is one of these type III effectors contributing to virulence of bacteria. Upon delivery into a plant cell, HopQ1 undergoes phosphorylation, binds host 14-3-3 proteins and suppresses defense-related signaling. Some plants however, evolved systems to recognize HopQ1 and respond to its presence and thus to prevent infection. HopQ1 shows homology to Nucleoside Hydrolases (NHs), but it contains a modified calcium binding motif not found in the canonical enzymes. CLuster ANalysis of Sequences (CLANS) revealed that HopQ1 and alike proteins make a distinct group of putative NHs located distantly from the classical enzymes. The HopQ1 – like protein (HLP) group comprises sequences from plant pathogenic bacteria, fungi, and lower plants. Our data suggest that the evolution of HopQ1 homologs in bacteria, fungi, and algae was independent. The location of moss HopQ1 homologs inside the fungal clade indicates a possibility of horizontal gene transfer (HGT) between those taxa. We identified a HLP in the moss Physcomitrella patens. Our experiments show that this protein (referred to as PpHLP) extended by a TTSS signal of HopQ1 promoted P. syringae growth in bean and was recognized by Nicotiana benthamiana immune system. Thus, despite the low sequence similarity to HopQ1 the engineered PpHLP acted as a bacterial virulence factor and displayed similar to HopQ1 virulence properties. [ABSTRACT FROM AUTHOR]

Salicylic acid (SA) is a key signaling molecule in plant immunity. Two types of SA receptors, NPR1 and NPR3/NPR4, were reported to be involved in the perception of SA in Arabidopsis. SA is also synthesized in the non-vascular moss Physcomitrella patens following pathogen infection. Sequence analysis revealed that there is only one NPR1/NPR3/NPR4-like protein in P. patens. This agrees with the phylogenetic study that showed the divergence of NPR1 and NPR3/NPR4 from the same ancestor during the evolution of higher plants. Intriguingly, expression of the P. patens NPR1/NPR3/NPR4- like gene in Arabidopsis does not complement the constitutive defense phenotype of the npr3 npr4 double mutant, but can partially rescue the mutant phenotypes of npr1-1, suggesting that it functions as an NPR1-like positive regulator of SA-mediated immunity and P. patens does not have an SA receptor functioning similarly as NPR3/NPR4. Future characterization of the P. patens NPR1-like protein and analysis of its functions through knockout and biochemical approaches will help us better understand how SA is perceived and what its functions are in P. patens. [ABSTRACT FROM AUTHOR]

Physcomitrella patens has emerged as a model moss system to investigate the evolution of various plant characters in early land plant lineages. Yet, there is merely a disparate body of ultrastructural and physiological evidence from other mosses to draw inferences about the modes of photosynthate transport in the alternating generations of Physcomitrella. We performed a series of ultrastructural, fluorescent tracing, physiological, and immunohistochemical experiments to elucidate a coherent model of photosynthate transport in this moss. Our ultrastructural observations revealed that Physcomitrella is an endohydric moss with water-conducting and putative food-conducting cells in the gametophytic stem and leaves. Movement of fluorescent tracer 5(6)-carboxyfluorescein diacetate revealed that the mode of transport in the gametophytic generation is symplasmic and is mediated by plasmodesmata, while there is a diffusion barrier composed of transfer cells that separates the photoautotrophic gametophyte from the nutritionally dependent heterotrophic sporophyte. We posited that, analogous to what is found in apoplasmically phloem loading higher plants, the primary photosynthate sucrose, is actively imported into the transfer cells by sucrose/H+ symporters (SUTs) that are, in turn, powered by P-type ATPases, and that the transfer cells harbor an ATP-conserving Sucrose Synthase (SUS) pathway. Supporting our hypothesis was the finding that a protonophore (2,4-dinitrophenol) and a SUT-specific inhibitor (diethyl pyrocarbonate) reduced the uptake of radiolabeled sucrose into the sporangia. In situ immunolocalization of P-type ATPase, Sucrose Synthase, and Proton Pyrophosphatase -- all key components of the SUS pathway -- showed that these proteins were prominently localized in the transfer cells, providing further evidence consistent with our argument. [ABSTRACT FROM AUTHOR]

The moss Physcomitrella patens is unique among plants in that homologous recombination can be used to knock out genes, just like in yeast. Furthermore, transformed plasmids can be rescued from Physcomitrella back into Escherichia coli, similar to yeast. In the present study, we have tested if a third important tool from yeast molecular genetics, auxotrophic selection markers, can be used in Physcomitrella. Two auxotrophic moss strains were made by knocking out the PpHIS3 gene encoding imidazoleglycerol-phosphate dehydratase, and the PpTRP1 gene encoding phosphoribosylanthranilate isomerase, disrupting the biosynthesis of histidine and tryptophan, respectively. The resulting PpHIS3Δ and PpTRP1Δ knockout strains were unable to grow on medium lacking histidine or tryptophan. The PpHIS3Δ strain was used to test selection of transformants by complementation of an auxotrophic marker. We found that the PpHIS3Δ strain could be complemented by transformation with a plasmid expressing the PpHIS3 gene from the CaMV 35S promoter, allowing the strain to grow on medium lacking histidine. Both linearized plasmids and circular supercoiled plasmids could complement the auxotrophic marker, and plasmids from both types of transformants could be rescued back into E. coli. Plasmids rescued from circular transformants were identical to the original plasmid, whereas plasmids rescued from linearized transformants had deletions generated by recombination between micro-homologies in the plasmids. Our results show that cloning by complementation of an auxotrophic marker works in Physcomitrella, which opens the door for using auxotrophic selection markers in moss molecular genetics. This will facilitate the adaptation of shuttle plasmid dependent methods from yeast molecular genetics for use in Physcomitrella. [ABSTRACT FROM AUTHOR]

The moss Physcomitrella patens is used both as an evo-devo model and biotechnological production system for metabolites and pharmaceuticals. Strong in vivo expression of genes of interest is important for production of recombinant proteins, e.g., selectable markers, fluorescent proteins, or enzymes. In this regard, the choice of the promoter sequence as well as codon usage optimization are two important inside factors to consider in order to obtain optimum protein accumulation level. To reliably quantify fluorescence, we transfected protoplasts with promoter:GFP fusion constructs and measured fluorescence intensity of living protoplasts in a plate reader system. We used the red fluorescent protein mCherry under 2x 35S promoter control as second reporter to normalize for different transfection efficiencies. We derived a novel endogenous promoter and compared deletion variants with exogenous promoters. We used different codon-adapted green fluorescent protein (GFP) genes to evaluate the influence of promoter choice and codon optimization on protein accumulation in P. patens, and show that the promoter of the gene of P. patens chlorophyll a/b binding protein lhcsr1 drives expression of GFP in protoplasts significantly (more than twofold) better than the commonly used 2x 35S promoter or the rice actin1 promoter. We identified a shortened 677 bp version of the lhcsr1 promoter that retains full activity in protoplasts. The codon optimized GFP yields significantly (more than twofold) stronger fluorescence signals and thus demonstrates that adjusting codon usage in P. patens can increase expression strength. In combination, new promotor and codon optimized GFP conveyed sixfold increased fluorescence signal. [ABSTRACT FROM AUTHOR]

SynopsisThis work demonstrates that PpABI3 contributes to freezing tolerance regulation in Physcomitrella patens.Transcription factor ABSCISIC ACID INSENSITIVE3 (ABI3) is known to play a major role in regulating seed dormancy, germination, seedling development as well as stress responses. ABI3 is conserved among land plants; however, its roles in non-seed plants under stress conditions have not been well characterized. In this study, we report that ABI3 is involved in freezing tolerance regulation during cold acclimation at least in part through ABA signaling pathway in moss Physcomitrella patens (P. patens). Deletion of PpABI3 (Δabi3-1) compromises the induction of genes related to cold response and antioxidative protection, resulting in reduced accumulation of cryoprotectants and antioxidants. In addition, photosystem II (PSII) activity is repressed in Δabi3-1 during cold acclimation partially due to alternations of photosynthetic protein complexes compositions. The gametophyte of Δabi3-1 displays severe growth inhibition and developmental deficiency under low temperature condition, while two independent complementary lines display phenotypes similar to that of wild-type P. patens (WT). Furthermore, the freezing tolerance of Δabi3-1 was significantly affected by deletion of PpABI3. These data revealed that PpABI3 plays an important role in low temperature response and freezing tolerance in P. patens.

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.

Dehydrins (DHNs) as a member of late-embryogenesis-abundant (LEA) proteins are involved in plant abiotic stress tolerance. Two dehydrins PpDHNA and PpDHNC were previously characterized from the moss Physcomitrella patens, which has been suggested to be an ideal model plant to study stress tolerance due to its adaptability to extreme environment. In this study, functions of these two genes were analyzed by heterologous expressions in Arabidopsis. Phenotype analysis revealed that overexpressing PpDHN dehydrin lines had stronger stress resistance than wild type and empty-vector control lines. These stress tolerance mainly due to the up-regulation of stress-related genes expression and mitigation to oxidative damage. The transgenic plants showed strong scavenging ability of reactive oxygen species(ROS), which was attributed to the enhancing of the content of antioxidant enzymes like superoxide dismutase (SOD) and catalase (CAT). Further analysis showed that the contents of chlorophyll and proline tended to be the appropriate level (close to non-stress environment) and the malondialdehyde (MDA) were repressed in these transgenic plants after exposure to stress. All these results suggest the PpDHNA and PpDHNC played a crucial role in response to drought and salt stress.

Plant protoplasts are widely used for genetic manipulation and functional studies in transient expression systems. However, little is known about the molecular pathways involved in a cell response to the combined stress factors resulted from protoplast generation. Plants often face more than one type of stress at a time, and how plants respond to combined stress factors is therefore of great interest. Here, we used protoplasts of the moss Physcomitrella patens as a model to study the effects of short-term stress on the chloroplast proteome. Using label-free comparative quantitative proteomic analysis (SWATH-MS), we quantified 479 chloroplast proteins, 219 of which showed a more than 1.4-fold change in abundance in protoplasts. We additionally quantified 1451 chloroplast proteins using emPAI. We observed degradation of a significant portion of the chloroplast proteome following the first hour of stress imposed by the protoplast isolation process. Electron-transport chain (ETC) components underwent the heaviest degradation, resulting in the decline of photosynthetic activity. We also compared the proteome changes to those in the transcriptional level of nuclear-encoded chloroplast genes. Globally, the levels of the quantified proteins and their corresponding mRNAs showed limited correlation. Genes involved in the biosynthesis of chlorophyll and components of the outer chloroplast membrane showed decreases in both transcript and protein abundance. However, proteins like dehydroascorbate reductase 1 and 2-cys peroxiredoxin B responsible for ROS detoxification increased in abundance. Further, genes such as thylakoid ascorbate peroxidase were induced at the transcriptional level but down-regulated at the proteomic level. Together, our results demonstrate that the initial chloroplast reaction to stress is due changes at the proteomic level. [ABSTRACT FROM AUTHOR]

Phytochromes are the principle photoreceptors in light-regulated plant development, primarily acting via translocation of the light-activated photoreceptor into the nucleus and subsequent gene regulation. However, several independent lines of evidence indicate unambiguously that an additional cytoplasmic signaling mechanism must exist. Directional responses in filament tip cells of the moss Physcomitrella patens are steered by phy4 which has been shown to interact physically with the blue light receptor phototropin at the plasma membrane. This complex might perceive and transduce vectorial information leading to cytoskeleton reorganization and finally a directional growth response. We developed yeast two-hybrid procedures using photochemically-functional, full-length phy4 as bait in Physcomitrella cDNA library screens and growth assays under different light conditions, revealing Pfr-dependent interactions possibly associated with phytochrome cytoplasmic signaling. Candidate proteins were then expressed in planta with fluorescent protein tags to determine their intracellular localization in darkness and red light. Of 14 candidates, 12 were confirmed to interact with phy4 in planta using bimolecular fluorescence complementation. We discuss the roles these putative holophytochrome-interacting proteins (HIP's) might have in signaling.

Plants respond to pathogen infection by activating signaling pathways leading to the accumulation of proteins with diverse roles in defense. Here, we addressed the functional role of PpPR-10, a pathogenesis-related (PR)-10 gene, of the moss Physcomitrella patens, in response to biotic stress. PpPR-10 belongs to a multigene family and encodes a protein twice the usual size of PR-10 proteins due to the presence of two Bet v1 domains. Moss PR-10 genes are differentially regulated during development and inoculation with the fungal pathogen Botrytis cinerea. Specifically, PpPR-10 transcript levels increase significantly by treatments with elicitors of Pectobacterium carotovorum subsp. carotovorum, spores of B. cinerea, and the defense hormone salicylic acid. To characterize the role of PpPR-10 in plant defense against pathogens, we conducted overexpression analysis in P. patens and in Arabidopsis thaliana. We demonstrate that constitutive expression of PpPR-10 in moss tissues increased resistance against the oomycete Pythium irregulare. PpPR-10 overexpressing moss plants developed less symptoms and decreased mycelium growth than wild type plants. In addition, PpPR-10 overexpressing plants constitutively produced cell wall depositions in protonemal tissue. Ectopic expression of PpPR-10 in Arabidopsis resulted in increased resistance against P. irregulare as well, evidenced by smaller lesions and less cellular damage compared to wild type plants. These results indicate that PpPR-10 is functionally active in the defense against the pathogen P. irregulare, in both P. patens and Arabidopsis, two evolutionary distant plants. Thus, P. patens can serve as an interesting source of genes to improve resistance against pathogen infection in flowering plants.

Plants have developed complex defense mechanisms to cope with microbial pathogens. Pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) are perceived by pattern recognition receptors (PRRs), leading to the activation of defense. While substantial progress has been made in understanding the activation of plant defense by PAMPs and DAMPs recognition in tracheophytes, far less information exists on related processes in early divergent plants like mosses. The aim of this study was to identify genes that were induced in P. patens in response to elicitors of Pectobacterium carotovorum subsp. carotovorum, using a cDNA suppression subtractive hybridization (SSH) method. A total of 239 unigenes were identified, including genes involved in defense responses related to the shikimate, phenylpropanoid and oxylipin pathways. The expression levels of selected genes related to these pathways were analyzed using quantitative RT-PCR, confirming their rapid induction by P.c. carotovorum derived elicitors. In addition, P. patens induced cell wall reinforcement after elicitor treatment by incorporation of phenolic compounds, callose deposition, and elevated expression of Dirigent-like encoding genes. Small molecule defense markers and phytohormones such as cinnamic acid, 12-oxo-phytodienoic acid and auxin levels all increased in elicitor-treated moss tissues. In contrast, salicylic acid levels decreased while abscisic acid levels remained unchanged. P. patens reporter lines harboring an auxin-inducible promoter fused to β-glucuronidase revealed GUS activity in protonemal and gametophores tissues treated with elicitors of P.c. carotovorum, consistent with a localized activation of auxin signaling. These results indicate that P. patens activates the shikimate, phenylpropanoid, oxylipins and auxin pathways upon treatment with P.c. carotovorum derived elicitors.

In contrast to homeohydric vascular plants, mosses employ a poikilohydric strategy for surviving in the dry aerial environment. A detailed understanding of the structure, composition, and development of moss cell walls can contribute to our understanding of not only the evolution of overall cell wall complexity, but also the differences that have evolved in response to selection for different survival strategies. The model moss species Physcomitrella patens has a predominantly haploid lifecycle consisting of protonemal filaments that regenerate from protoplasts and enlarge by tip growth, and leafy gametophores composed of cells that enlarge by diffuse growth and differentiate into several different types. Advantages for genetic studies include methods for efficient targeted gene modification and extensive genomic resources. Immuno and affinity cytochemical labeling were used to examine the distribution of polysaccharides and proteins in regenerated protoplasts, protonemal filaments, rhizoids, and sectioned gametophores of P. patens. The cell wall composition of regenerated protoplasts was also characterized by flow cytometry. Crystalline cellulose was abundant in the cell walls of regenerating protoplasts and protonemal cells that developed on media of high osmolarity, whereas homogalacturonan was detected in the walls of protonemal cells that developed on low osmolarity media and not in regenerating protoplasts. Mannan was the major hemicellulose detected in all tissues tested. Arabinogalactan proteins were detected in different cell types by different probes, consistent with structural heterogeneity. The results reveal developmental and cell type specific differences in cell wall composition and provide a basis for analyzing cell wall phenotypes in knockout mutants.

Whereas contact sites between mitochondria and the ER have been in the focus of animal and fungal research for several years, the importance of this organellar interface and the molecular effectors are largely unknown for plants. This work gives an introduction into known evolutionary differences of molecular effectors of mitochondrial dynamics and interactions between animals, fungi and plants. Using the model plant Physcomitrella patens, we provide microscopic evidence for the existence of mitochondria-ER interactions in plants and their correlation with mitochondrial constriction and fission. We further investigate a previously identified protein of unknown function (MELL1), and show that it modulates the amount of mitochondrial association to the ER, as well as mitochondrial shape and number.

Comet assay in plants. From the first description of the comet assay with isolated nuclei rather than whole cells it became evident that assay is well suited to be applied in plants (Koppen & Verschaeve, 1996). Disintegration of tissue by quick chopping with a razor blade, direct collection of released nuclei by patting and pipetting enables to process samples in time shorter than 2 minutes, the time prerequisite to study quick repair (Kozak et al, 2009). Plants are due to their sessile nature permanently exposed to environmental stresses (drought, salinity), ionizing (IR) and UV radiation and genotoxins, which directly or indirectly via generation of reactive oxidative species (ROS) damage their DNA. Radiomimetic Bleomycin functions as a catalyst to produce ROS leading to clusters of oxidized DNA lesions, single (SSB) and double (DSBs) strand breaks similarly as IR (Steighner & Povirk, 1990). Incurred SSBs and DSBs are easily distinguished and measured by comet assay when varying conditions of mainly used protocol with electrophoresis in 0.3 M NaOH, pH>13 solution (A/A assay). DSBs are detected under “neutral” conditions by N/N assay in regular electrophoretic buffer (Kozak et al, 2009; Olive & Banath, 2006), whilst SSBs are revealed by A/N assay, with alkali-unwinding step in 0.3 M NaOH prior electrophoresis (Angelis et al, 1999; Menke et al, 2001). Better resolution in DSBs and SSBs assays is observed when “neutral” conditions are set between pH 9-10, still well bellow DNA denaturing pH

Arginine-rich tandem zinc-finger proteins (RR-TZF) participate in a wide range of plant developmental processes and adaptive responses to abiotic stress, such as cold, salt and drought. This study investigates the conservation of the genes AtTZF1-5 at the level of their sequences and expression across plant species. The genomic sequences of the two RR-TZF genes TdTZF1-A and TdTZF1-B were isolated in durum wheat and assigned to chromosomes 3A and 3B, respectively. Sequence comparisons revealed that they encode proteins that are highly homologous to AtTZF1, AtTZF2 and AtTZF3. The expression profiles of these RR-TZF durum wheat and Arabidopsis proteins support a common function in the regulation of seed germination and responses to abiotic stress. In particular, analysis of plants with attenuated and overexpressed AtTZF3 indicate that AtTZF3 is a negative regulator of seed germination under conditions of salt stress. Finally, comparative sequence analyses establish that the RR-TZF genes are encoded by lower plants, including the bryophyte Physcomitrella patens and the alga Chlamydomonas reinhardtii. The regulation of the Physcomitrella AtTZF1-2-3-like genes by salt stress strongly suggests that a subgroup of the RR-TZF proteins has a function that has been conserved throughout evolution.

Plants biosynthesize a great diversity of biologically active small molecules of interest for fragrances, flavours, and pharmaceuticals. Among specialized metabolites, terpenoids represent the greatest molecular diversity. Many terpenoids are very complex, and total chemical synthesis often requires many steps and difficult chemical reactions, resulting in a low final yield or incorrect stereochemistry. Several drug candidates with terpene skeletons are difficult to obtain by chemical synthesis due to their large number of chiral centres. Thus, biological production remains the preferred method for industrial production for many of these compounds. However because these chemicals are often found in low abundance in the native plant, or are produced in plants which are difficult to cultivate, there is great interest in engineering increased production or expression of the biosynthetic pathways in heterologous hosts. Although there are many examples of successful engineering of microbes such as yeast or bacteria to produce these compounds, this often requires extensive changes to the host organism's metabolism. Optimization of plant gene expression, post-translational protein modifications, subcellular localization, and other factors often present challenges. To address the future demand for natural products used as drugs, new platforms are being established that are better suited for heterologous production of plant metabolites. Specifically, direct metabolic engineering of plants can provide effective heterologous expression for production of valuable plant-derived natural products. In this review, our primary focus is on small terpenoids and we discuss the benefits of plant expression platforms and provide several successful examples of stable production of small terpenoids in plants.

Phytochromes are the principle photoreceptors in light-regulated plant development, primarily acting via translocation of the light-activated photoreceptor into the nucleus and subsequent gene regulation. However, several independent lines of evidence indicate unambiguously that an additional cytoplasmic signaling mechanism must exist. Directional responses in filament tip cells of the moss Physcomitrella patens are steered by phy4 which has been shown to interact physically with the blue light receptor phototropin at the plasma membrane. This complex might perceive and transduce vectorial information leading to cytoskeleton reorganization and finally a directional growth response. We developed yeast two-hybrid procedures using photochemically functional, full-length phy4 as bait in Physcomitrella cDNA library screens and growth assays under different light conditions, revealing Pfr-dependent interactions possibly associated with phytochrome cytoplasmic signaling. Candidate proteins were then expressed in planta with fluorescent protein tags to determine their intracellular localization in darkness and red light. Of 14 candidates, 12 were confirmed to interact with phy4 in planta using bimolecular fluorescence complementation. We also used database information to study their expression patterns relative to those of phy4. We discuss the likely functional characteristics of these holophytochrome-interacting proteins (HIP's) and their possible roles in signaling. [ABSTRACT FROM AUTHOR]

Plants respond to pathogen infection by activating signaling pathways leading to the accumulation of proteins with diverse roles in defense. Here, we addressed the functional role of PpPR-10, a pathogenesis-related (PR)-10 gene, of the moss Physcomitrella patens, in response to biotic stress. PpPR-10 belongs to a multigene family and encodes a protein twice the usual size of PR-10 proteins due to the presence of two Bet v1 domains. Moss PR-10 genes are differentially regulated during development and inoculation with the fungal pathogen Botrytis cinerea. Specifically, PpPR-10 transcript levels increase significantly by treatments with elicitors of Pectobacterium carotovorum subsp. carotovorum, spores of B. cinerea, and the defense hormone salicylic acid. To characterize the role of PpPR-10 in plant defense against pathogens, we conducted overexpression analysis in P. patens and in Arabidopsis thaliana. We demonstrate that constitutive expression of PpPR-10 in moss tissues increased resistance against the oomycete Pythium irregulare. PpPR- 10 overexpressing moss plants developed less symptoms and decreased mycelium growth than wild type plants. In addition, PpPR-10 overexpressing plants constitutively produced cell wall depositions in protonemal tissue. Ectopic expression of PpPR-10 in Arabidopsis resulted in increased resistance against P. irregulare as well, evidenced by smaller lesions and less cellular damage compared to wild type plants. These results indicate that PpPR-10 is functionally active in the defense against the pathogen P. irregulare, in both P. patens and Arabidopsis, two evolutionary distant plants. Thus, P. patens can serve as an interesting source of genes to improve resistance against pathogen infection in flowering plants. [ABSTRACT FROM AUTHOR]

Plants have developed complex defense mechanisms to cope with microbial pathogens. Pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) are perceived by pattern recognition receptors (PRRs), leading to the activation of defense. While substantial progress has been made in understanding the activation of plant defense by PAMPs and DAMPs recognition in tracheophytes, far less information exists on related processes in early divergent plants like mosses. The aim of this study was to identify genes that were induced in P. patens in response to elicitors of Pectobacterium carotovorum subsp. carotovorum, using a cDNA suppression subtractive hybridization (SSH) method. A total of 239 unigenes were identified, including genes involved in defense responses related to the shikimate, phenylpropanoid, and oxylipin pathways. The expression levels of selected genes related to these pathways were analyzed using quantitative RT-PCR, confirming their rapid induction by P.c. carotovorum derived elicitors. In addition, P. patens induced cell wall reinforcement after elicitor treatment by incorporation of phenolic compounds, callose deposition, and elevated expression of Dirigent-like encoding genes. Small molecule defense markers and phytohormones such as cinnamic acid, 12-oxo-phytodienoic acid, and auxin levels all increased in elicitor-treated moss tissues. In contrast, salicylic acid levels decreased while abscisic acid levels remained unchanged. P. patens reporter lines harboring an auxin-inducible promoter fused to β-glucuronidase revealed GUS activity in protonemal and gametophores tissues treated withelicitors of P.c. carotovorum, consistent with a localized activation of auxin signaling. These results indicate that P. patens activates the shikimate, phenylpropanoid, oxylipins, and auxin pathways upon treatment with P.c. carotovorum derived elicitors. [ABSTRACT FROM AUTHOR]