RESUMO
The CLAVATA pathway plays a key role in the regulation of multicellular shoot and root meristems in flowering plants. In Arabidopsis, CLAVATA 3-like signaling peptides (CLEs) act via receptor-like kinases CLAVATA 1 and CRINKLY 4 (CR4). In the moss Physcomitrium patens, PpCLAVATA and PpCR4 were previously studied independently and shown to play conserved roles in the regulation of cell proliferation and differentiation. The plant calpain DEFECTIVE KERNEL 1 (DEK1) has been identified as another key regulator of cell division and cell fate in vascular plants and bryophytes. The functional interaction between CLAVATA, CR4, and DEK1 remains unknown. Here, we show that P. patens crinkly4 and dek1 mutants respond differently to CLE peptide treatments suggesting their distinct roles in the CLAVATA pathway. Reduced CLAVATA-mediated suppression of leafy shoot growth in Δcr4 mutants indicates that PpCR4 is involved in CLV3p perception, most likely as a receptor. The CLV3p strongly suppressed leaf vein development in Δcr4 mutants, suggesting that other receptors are involved in these processes and indicating a potential role of PpCR4 in organ sensitization to CLEs.
Assuntos
Bryopsida , Proteínas de Plantas , Bryopsida/genética , Bryopsida/crescimento & desenvolvimento , Bryopsida/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Regulação da Expressão Gênica de Plantas , Peptídeos/metabolismo , Células Germinativas Vegetais/crescimento & desenvolvimento , Células Germinativas Vegetais/metabolismoRESUMO
Plants rely on solar energy to synthesize ATP and NADPH for photosynthetic carbon fixation and all cellular need. Mitochondrial respiration is essential in plants, but this may be due to heterotrophic bottlenecks during plant development or because it is also necessary in photosynthetically active cells. In this study, we examined in vivo changes of cytosolic ATP concentration in response to light, employing a biosensing strategy in the moss Physcomitrium patens and revealing increased cytosolic ATP concentration caused by photosynthetic activity. Plants depleted of respiratory Complex I showed decreased cytosolic ATP accumulation, highlighting a critical role of mitochondrial respiration in light-dependent ATP supply of the cytosol. Consistently, targeting mitochondrial ATP production directly, through the construction of mutants deficient in mitochondrial ATPase (complex V), led to drastic growth reduction, despite only minor alterations in photosynthetic electron transport activity. Since P. patens is photoautotrophic throughout its development, we conclude that heterotrophic bottlenecks cannot account for the indispensable role of mitochondrial respiration in plants. Instead, our results support that mitochondrial respiration is essential for ATP provision to the cytosol in photosynthesizing cells. Mitochondrial respiration provides metabolic integration, ensuring supply of cytosolic ATP essential for supporting plant growth and development.
Assuntos
Trifosfato de Adenosina , Bryopsida , Respiração Celular , Citosol , Mitocôndrias , Fotossíntese , Trifosfato de Adenosina/metabolismo , Citosol/metabolismo , Mitocôndrias/metabolismo , Bryopsida/metabolismo , Bryopsida/genética , Bryopsida/crescimento & desenvolvimento , LuzRESUMO
Structural maintenance of chromosome (SMC) complexes play roles in cohesion, condensation, replication, transcription, and DNA repair. Their cores are composed of SMC proteins with a unique structure consisting of an ATPase head, long arm, and hinge. SMC complexes form long rod-like structures, which can change to ring-like and elbow-bent conformations upon binding ATP, DNA, and other regulatory factors. These SMC dynamic conformational changes are involved in their loading, translocation, and DNA loop extrusion. Here, we examined the binding and role of the PpNSE5 regulatory factor of Physcomitrium patens PpSMC5/6 complex. We found that the PpNSE5 C-terminal half (aa230-505) is required for binding to its PpNSE6 partner, while the N-terminal half (aa1-230) binds PpSMC subunits. Specifically, the first 71 amino acids of PpNSE5 were required for binding to PpSMC6. Interestingly, the PpNSE5 binding required the PpSMC6 head-proximal joint region and PpSMC5 hinge-proximal arm, suggesting a long distance between binding sites on PpSMC5 and PpSMC6 arms. Therefore, we hypothesize that PpNSE5 either links two antiparallel SMC5/6 complexes or binds one SMC5/6 in elbow-bent conformation, the later model being consistent with the role of NSE5/NSE6 dimer as SMC5/6 loading factor to DNA lesions. In addition, we generated the P. patens Ppnse5KO1 mutant line with an N-terminally truncated version of PpNSE5, which exhibited DNA repair defects while keeping a normal number of rDNA repeats. As the first 71 amino acids of PpNSE5 are required for PpSMC6 binding, our results suggest the role of PpNSE5-PpSMC6 interaction in SMC5/6 loading to DNA lesions.
Assuntos
Bryopsida , Proteínas de Plantas , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Bryopsida/genética , Bryopsida/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Cromossomos de Plantas/genética , Ligação ProteicaRESUMO
The de novo synthesis of genomes has made unprecedented progress and achieved milestones, particularly in bacteria and yeast. However, the process of synthesizing a multicellular plant genome has not progressed at the same pace, due to the complexity of multicellular plant genomes, technical difficulties associated with large genome size and structure, and the intricacies of gene regulation and expression in plants. Here we outline the bottom-up design principles for the de novo synthesis of the Physcomitrium patens (that is, earthmoss) genome. To facilitate international collaboration and accessibility, we have developed and launched a public online design platform called GenoDesigner. This platform offers an intuitive graphical interface enabling users to efficiently manipulate extensive genome sequences, even up to the gigabase level. This tool is poised to greatly expedite the synthesis of the P. patens genome, offering an essential reference and roadmap for the synthesis of plant genomes.
Assuntos
Bryopsida , Genoma de Planta , Bryopsida/genética , Biologia Sintética/métodos , SoftwareRESUMO
Most drugs that target the complement system are designed to inhibit the complement pathway at either the proximal or terminal levels. The use of a natural complement regulator such as factor H (FH) could provide a superior treatment option by restoring the balance of an overactive complement system while preserving its normal physiological functions. Until now, the systemic treatment of complement-associated disorders with FH has been deemed unfeasible, primarily due to high production costs, risks related to FH purified from donors' blood, and the challenging expression of recombinant FH in different host systems. We recently demonstrated that a moss-based expression system can produce high yields of properly folded, fully functional, recombinant FH. However, the half-life of the initial variant (CPV-101) was relatively short. Here we show that the same polypeptide with modified glycosylation (CPV-104) achieves a pharmacokinetic profile comparable to that of native FH derived from human serum. The treatment of FH-deficient mice with CPV-104 significantly improved important efficacy parameters such as the normalization of serum C3 levels and the rapid degradation of C3 deposits in the kidney compared to treatment with CPV-101. Furthermore, CPV-104 showed comparable functionality to serum-derived FH in vitro, as well as similar performance in ex vivo assays involving samples from patients with atypical hemolytic uremic syndrome, C3 glomerulopathy and paroxysomal nocturnal hematuria. CPV-104 - the human FH analog expressed in moss - will therefore allow the treatment of complement-associated human diseases by rebalancing instead of inhibiting the complement cascade.
Assuntos
Fator H do Complemento , Humanos , Fator H do Complemento/metabolismo , Fator H do Complemento/genética , Animais , Camundongos , Meia-Vida , Polissacarídeos/metabolismo , Bryopsida/metabolismo , Bryopsida/genética , Glicosilação , Proteínas Recombinantes , Camundongos Knockout , Camundongos Endogâmicos C57BL , MasculinoRESUMO
The first chromosome-scale reference genome of the rare narrow-endemic African moss Physcomitrellopsis africana (P. africana) is presented here. Assembled from 73 × Oxford Nanopore Technologies (ONT) long reads and 163 × Beijing Genomics Institute (BGI)-seq short reads, the 414â Mb reference comprises 26 chromosomes and 22,925 protein-coding genes [Benchmarking Universal Single-Copy Ortholog (BUSCO) scores: C:94.8% (D:13.9%)]. This genome holds 2 genes that withstood rigorous filtration of microbial contaminants, have no homolog in other land plants, and are thus interpreted as resulting from 2 unique horizontal gene transfers (HGTs) from microbes. Further, P. africana shares 176 of the 273 published HGT candidates identified in Physcomitrium patens (P. patens), but lacks 98 of these, highlighting that perhaps as many as 91 genes were acquired in P. patens in the last 40 million years following its divergence from its common ancestor with P. africana. These observations suggest rather continuous gene gains via HGT followed by potential losses during the diversification of the Funariaceae. Our findings showcase both dynamic flux in plant HGTs over evolutionarily "short" timescales, alongside enduring impacts of successful integrations, like those still functionally maintained in extant P. africana. Furthermore, this study describes the informatic processes employed to distinguish contaminants from candidate HGT events.
Assuntos
Bryopsida , Transferência Genética Horizontal , Genoma de Planta , Filogenia , Bryopsida/genética , Genômica/métodos , Anotação de Sequência MolecularRESUMO
Plant mitochondrial and chloroplast transcripts are subject to numerous events of specific cytidine-to-uridine (C-to-U) RNA editing to correct genetic information. Key protein factors for this process are specific RNA-binding pentatricopeptide repeat (PPR) proteins, which are encoded in the nucleus and post-translationally imported into the two endosymbiotic organelles. Despite hundreds of C-to-U editing sites in the plant organelles, no comparable editing has been found for nucleo-cytosolic mRNAs raising the question why plant RNA editing is restricted to chloroplasts and mitochondria. Here, we addressed this issue in the model moss Physcomitrium patens, where all PPR-type RNA editing factors comprise specific RNA-binding and cytidine deamination functionalities in single proteins. To explore whether organelle-type RNA editing can principally also take place in the plant cytosol, we expressed PPR56, PPR65 and PPR78, three editing factors recently shown to also function in a bacterial setup, together with cytosolic co-transcribed native targets in Physcomitrium. While we obtained unsatisfying results upon their constitutive expression, we found strong cytosolic RNA editing under hormone-inducible expression. Moreover, RNA-Seq analyses revealed varying numbers of up to more than 900 off-targets in other cytosolic transcripts. We conclude that PPR-mediated C-to-U RNA editing is not per se incompatible with the plant cytosol but that its limited target specificity has restricted its occurrence to the much less complex transcriptomes of mitochondria and chloroplast in the course of evolution.
Assuntos
Bryopsida , Cloroplastos , Citosol , Mitocôndrias , Edição de RNA , RNA de Plantas , Cloroplastos/metabolismo , Cloroplastos/genética , Citosol/metabolismo , Bryopsida/genética , Bryopsida/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/genética , RNA de Plantas/genética , RNA de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Citidina/metabolismo , Citidina/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Regulação da Expressão Gênica de Plantas , Uridina/metabolismo , Uridina/genéticaRESUMO
Moss plants appear in the early stages of land colonization and possess varying degrees of dehydration tolerance. In this study, a protein called PpFAS1.3 was identified, which contains a fasciclin 1-like domain and is essential for the moss Physcomitrium patens' response to short-term rapid dehydration. When the FAS1.3 protein was knocked out, leafyshoots showed a significant decrease in tolerance to rapid dehydration, resulting in accelerated water loss and increased membrane leakage. Phylogenetic analysis suggests that PpFAS1.3 and its homologous proteins may have originated from bacteria and are specifically found in non-vascular plants like mosses and liverworts. As a dehydration-related protein, FAS1.3 plays a significant role in regulating lipid metabolism, particularly in the synthesis of free fatty acids (FFA) and the metabolism of two phospholipids, PC and PA. This discovery highlights the close connection between PpFAS1.3 and lipid metabolism, providing new insights into the molecular mechanisms underlying plant adaptation to stresses.
Assuntos
Bryopsida , Metabolismo dos Lipídeos , Filogenia , Proteínas de Plantas , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Bryopsida/metabolismo , Bryopsida/genética , Desidratação , Regulação da Expressão Gênica de Plantas , Sequência de AminoácidosRESUMO
Genome editing via CRISPR/Cas has enabled targeted genetic modifications in various species, including plants. The requirement for specific protospacer-adjacent motifs (PAMs) near the target gene, as seen with Cas nucleases like SpCas9, limits its application. PAMless SpCas9 variants, designed with a relaxed PAM requirement, have widened targeting options. However, these so-call PAMless SpCas9 still show variation of editing efficiency depending on the PAM and their efficiency lags behind the native SpCas9. Here we assess the potential of a PAMless SpCas9 variant for genome editing in the model plant Physcomitrium patens. For this purpose, we developed a SpRYCas9i variant, where expression was optimized, and tested its editing efficiency using the APT as a reporter gene. We show that the near PAMless SpRYCas9i effectively recognizes specific PAMs in P. patens that are not or poorly recognized by the native SpCas9. Pattern of mutations found using the SpRYCas9i are similar to the ones found with the SpCas9 and we could not detect off-target activity for the sgRNAs tested in this study. These findings contribute to advancing versatile genome editing techniques in plants.
Assuntos
Bryopsida , Edição de Genes , Edição de Genes/métodos , Sistemas CRISPR-Cas/genética , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , RNA Guia de Sistemas CRISPR-Cas , Mutação , Bryopsida/genética , Genoma de Planta/genéticaRESUMO
The conquest of land by plants was concomitant with, and possibly enabled by, the evolution of three-dimensional (3D) growth. The moss Physcomitrium patens provides a model system for elucidating molecular mechanisms in the initiation of 3D growth. Here, we investigate whether the phytohormone ethylene, which is believed to have been a signal before land plant emergence, plays a role in 3D growth regulation in P. patens. We report ethylene controls 3D gametophore formation, based on results from exogenously applied ethylene and genetic manipulation of PpEIN2, which is a central component in the ethylene signaling pathway. Overexpression (OE) of PpEIN2 activates ethylene responses and leads to earlier formation of gametophores with fewer gametophores produced thereafter, phenocopying ethylene-treated wild-type. Conversely, Ppein2 knockout mutants, which are ethylene insensitive, show initially delayed gametophore formation with more gametophores produced later. Furthermore, pharmacological and biochemical analyses reveal auxin levels are decreased in the OE lines but increased in the knockout mutants. Our results suggest that evolutionarily, ethylene and auxin molecular networks were recruited to build the plant body plan in ancestral land plants. This might have played a role in enabling ancient plants to acclimate to the continental surfaces of the planet.
Assuntos
Bryopsida , Etilenos , Regulação da Expressão Gênica de Plantas , Ácidos Indolacéticos , Proteínas de Plantas , Etilenos/metabolismo , Ácidos Indolacéticos/metabolismo , Ácidos Indolacéticos/farmacologia , Bryopsida/crescimento & desenvolvimento , Bryopsida/genética , Bryopsida/efeitos dos fármacos , Bryopsida/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Células Germinativas Vegetais/metabolismo , Células Germinativas Vegetais/crescimento & desenvolvimento , Células Germinativas Vegetais/efeitos dos fármacos , Mutação/genéticaRESUMO
Green feather algae (Bryopsidales) undergo a unique life cycle in which a single cell repeatedly executes nuclear division without cytokinesis, resulting in the development of a thallus (>100 mm) with characteristic morphology called coenocyte. Bryopsis is a representative coenocytic alga that has exceptionally high regeneration ability: extruded cytoplasm aggregates rapidly in seawater, leading to the formation of protoplasts. However, the genetic basis of the unique cell biology of Bryopsis remains poorly understood. Here, we present a high-quality assembly and annotation of the nuclear genome of Bryopsis sp. (90.7 Mbp, 27 contigs, N50 = 6.7 Mbp, 14 034 protein-coding genes). Comparative genomic analyses indicate that the genes encoding BPL-1/Bryohealin, the aggregation-promoting lectin, are heavily duplicated in Bryopsis, whereas homologous genes are absent in other ulvophyceans, suggesting the basis of regeneration capability of Bryopsis. Bryopsis sp. possesses >30 kinesins but only a single myosin, which differs from other green algae that have multiple types of myosin genes. Consistent with this biased motor toolkit, we observed that the bidirectional motility of chloroplasts in the cytoplasm was dependent on microtubules but not actin in Bryopsis sp. Most genes required for cytokinesis in plants are present in Bryopsis, including those in the SNARE or kinesin superfamily. Nevertheless, a kinesin crucial for cytokinesis initiation in plants (NACK/Kinesin-7II) is hardly expressed in the coenocytic part of the thallus, possibly underlying the lack of cytokinesis in this portion. The present genome sequence lays the foundation for experimental biology in coenocytic macroalgae.
Assuntos
Genoma de Planta , Genoma de Planta/genética , Filogenia , Clorófitas/genética , Clorófitas/fisiologia , Regeneração/genética , Bryopsida/genética , Bryopsida/fisiologia , Bryopsida/citologia , Cinesinas/genética , Cinesinas/metabolismo , Miosinas/genética , Miosinas/metabolismoRESUMO
Moss-cyanobacteria symbioses were proposed to be based on nutrient exchange, with hosts providing C and S while bacteria provide N, but we still lack understanding of the underlying molecular mechanisms of their interactions. We investigated how contact between the ubiquitous moss Hylocomium splendens and its cyanobiont affects nutrient-related gene expression of both partners. We isolated a cyanobacterium from H. splendens and co-incubated it with washed H. splendens shoots. Cyanobacterium and moss were also incubated separately. After 1 week, we performed acetylene reduction assays to estimate N2 fixation and RNAseq to evaluate metatranscriptomes. Genes related to N2 fixation and the biosynthesis of several amino acids were up-regulated in the cyanobiont when hosted by the moss. However, S-uptake and the biosynthesis of the S-containing amino acids methionine and cysteine were down-regulated in the cyanobiont while the degradation of selenocysteine was up-regulated. In contrast, the number of differentially expressed genes in the moss was much lower, and almost no transcripts related to nutrient metabolism were affected. It is possible that, at least during the early stage of this symbiosis, the cyanobiont receives few if any nutrients from the host in return for N, suggesting that moss-cyanobacteria symbioses encompass relationships that are more plastic than a constant mutualist flow of nutrients.
Assuntos
Briófitas , Bryopsida , Cianobactérias , Simbiose , Fixação de Nitrogênio , Bryopsida/genética , Bryopsida/metabolismo , Bryopsida/microbiologia , Cianobactérias/metabolismo , Aminoácidos/metabolismoRESUMO
The plant cuticle is a hydrophobic barrier, which seals the epidermal surface of most aboveground organs. While the cuticle biosynthesis of angiosperms has been intensively studied, knowledge about its existence and composition in nonvascular plants is scarce. Here, we identified and characterized homologs of Arabidopsis thaliana fatty acyl-CoA reductase (FAR) ECERIFERUM 4 (AtCER4) and bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferase 1 (AtWSD1) in the liverwort Marchantia polymorpha (MpFAR2 and MpWSD1) and the moss Physcomitrium patens (PpFAR2A, PpFAR2B, and PpWSD1). Although bryophyte harbor similar compound classes as described for angiosperm cuticles, their biosynthesis may not be fully conserved between the bryophytes M. polymorpha and P. patens or between these bryophytes and angiosperms. While PpFAR2A and PpFAR2B contribute to the production of primary alcohols in P. patens, loss of MpFAR2 function does not affect the wax profile of M. polymorpha. By contrast, MpWSD1 acts as the major wax ester-producing enzyme in M. polymorpha, whereas mutations of PpWSD1 do not affect the wax ester levels of P. patens. Our results suggest that the biosynthetic enzymes involved in primary alcohol and wax ester formation in land plants have either evolved multiple times independently or undergone pronounced radiation followed by the formation of lineage-specific toolkits.
Assuntos
Ceras , Ceras/metabolismo , Álcoois/metabolismo , Filogenia , Marchantia/genética , Marchantia/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Bryopsida/genética , Bryopsida/metabolismo , Briófitas/genética , Briófitas/metabolismo , Aldeído Oxirredutases/metabolismo , Aldeído Oxirredutases/genética , Vias Biossintéticas/genética , Evolução Molecular , Regulação da Expressão Gênica de Plantas , Aciltransferases/metabolismo , Aciltransferases/genética , Evolução Biológica , Arabidopsis/genética , Arabidopsis/metabolismo , Mutação/genéticaRESUMO
Bryophytes, known as poikilohydric plants, possess vegetative desiccation-tolerant (DT) ability to withstand water deficit stress. Consequently, they offer valuable genetic resources for enhancing resistance to water scarcity stress. In this research, we examined the physiological, phytohormonal, and transcriptomic changes in DT mosses Calohypnum plumiforme from two populations, with and without desiccation treatment. Comparative analysis revealed population differentiation at physiological, gene sequence, and expression levels. Under desiccation stress, the activities of superoxide dismutase (SOD) and peroxidase (POD) showed significant increases, along with elevation of soluble sugars and proteins, consistent with the transcriptome changes. Notable activation of the bypass pathway of JA biosynthesis suggested their roles in compensating for JA accumulation. Furthermore, our analysis revealed significant correlations among phytohormones and DEGs in their respective signaling pathway, indicating potential complex interplays of hormones in C plumiforme. Protein phosphatase 2C (PP2C) in the abscisic acid signaling pathway emerged as the pivotal hub in the phytohormone crosstalk regulation network. Overall, this study was one of the first comprehensive transcriptome analyses of moss C. plumiforme under slow desiccation rates, expanding our knowledge of bryophyte transcriptomes and shedding light on the gene regulatory network involved in response to desiccation, as well as the evolutionary processes of local adaptation across moss populations.
Assuntos
Briófitas , Bryopsida , Transcriptoma/genética , Secas , Perfilação da Expressão Gênica , Reguladores de Crescimento de Plantas/metabolismo , Bryopsida/genética , Briófitas/genética , Estresse Fisiológico/genética , Regulação da Expressão Gênica de PlantasRESUMO
KEY MESSAGE: To establish a sterile culture system and protoplast regeneration system for Bryum argenteum, and to establish and apply CRISPR/Cas9 system in Bryum argenteum. Bryum argenteum is a fascinating, cosmopolitan, and versatile moss species that thrives in various disturbed environments. Because of its comprehensive tolerance to the desiccation, high UV and extreme temperatures, it is emerging as a model moss for studying the molecular mechanisms underlying plant responses to abiotic stresses. However, the lack of basic tools such as gene transformation and targeted genome modification has hindered the understanding of the molecular mechanisms underlying the survival of B. argenteum in different environments. Here, we reported the protonema of B. argenteum can survive up to 95.4% water loss. In addition, the genome size of B. argenteum is approximately 313 Mb by kmer analysis, which is smaller than the previously reported 700 Mb. We also developed a simple method for protonema induction and an efficient protoplast isolation and regeneration protocol for B. argenteum. Furthermore, we established a PEG-mediated protoplast transient transfection and stable transformation system for B. argenteum. Two homologues of ABI3(ABA-INSENSITIVE 3) gene were successfully cloned from B. argenteum. To further investigate the function of the ABI3 gene in B. argenteum, we used the CRISPR/Cas9 genetic editing system to target the BaABI3A and BaABI3B gene in B. argenteum protoplasts. This resulted in mutagenesis at the target in about 2-5% of the regenerated plants. The isolated abi3a and abi3b mutants exhibited increased sensitivity to desiccation, suggesting that BaABI3A and BaABI3B play redundant roles in desiccation stress. Overall, our results provide a rapid and simple approach for molecular genetics in B. argenteum. This study contributes to a better understanding of the molecular mechanisms of plant adaptation to extreme environmental.
Assuntos
Briófitas , Bryopsida , Edição de Genes , Bryopsida/genética , Briófitas/genética , Estresse Fisiológico/genética , Transformação Genética , Sistemas CRISPR-Cas/genética , ProtoplastosRESUMO
Scientists make partially synthetic version of moss chromosome, aiming to harness plant for industry.
Assuntos
Bryopsida , Cromossomos Artificiais , Genoma de Planta , Bryopsida/genética , IndústriasRESUMO
Water scarcity, resulting from climate change, poses a significant threat to ecosystems. Syntrichia ruralis, a dryland desiccation-tolerant moss, provides valuable insights into survival of water-limited conditions. We sequenced the genome of S. ruralis, conducted transcriptomic analyses, and performed comparative genomic and transcriptomic analyses with existing genomes and transcriptomes, including with the close relative S. caninervis. We took a genetic approach to characterize the role of an S. ruralis transcription factor, identified in transcriptomic analyses, in Arabidopsis thaliana. The genome was assembled into 12 chromosomes encompassing 21 169 protein-coding genes. Comparative analysis revealed copy number and transcript abundance differences in known desiccation-associated gene families, and highlighted genome-level variation among species that may reflect adaptation to different habitats. A significant number of abscisic acid (ABA)-responsive genes were found to be negatively regulated by a MYB transcription factor (MYB55) that was upstream of the S. ruralis ortholog of ABA-insensitive 3 (ABI3). We determined that this conserved MYB transcription factor, uncharacterized in Arabidopsis, acts as a negative regulator of an ABA-dependent stress response in Arabidopsis. The new genomic resources from this emerging model moss offer novel insights into how plants regulate their responses to water deprivation.
Assuntos
Arabidopsis , Dessecação , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ácido Abscísico/farmacologia , Ácido Abscísico/metabolismo , Filogenia , Sequência Conservada/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Bryopsida/genética , Bryopsida/fisiologia , Genes de Plantas , Estresse Fisiológico/genética , Modelos Biológicos , Transcriptoma/genéticaRESUMO
The bZIP60, XBP1 and HAC1 mRNAs encode transcription factors that mediate the unfolded protein response (UPR) in plants, animals and yeasts, respectively. Upon UPR, these mRNAs undergo unconventional cytoplasmic splicing on the endoplasmic reticulum (ER) to produce active transcription factors. Although cytoplasmic splicing is conserved, the ER targeting mechanism differs between XBP1 and HAC1. The ER targeting of HAC1 mRNA occurs before translation, whereas that of XBP1 mRNA involves a ribosome-nascent chain complex that is stalled when a hydrophobic peptide emerges from the ribosome; the corresponding mechanism is unknown for bZIP60. Here, we analyzed ribosome stalling on bZIP60 orthologs of plants. Using a cell-free translation system, we detected nascent peptide-mediated ribosome stalling during the translation elongation of the mRNAs of Arabidopsis, rice and Physcomitrium (moss) orthologs, and the termination-step stalling in the Selaginella (lycopod) ortholog, all of which occurred â¼50 amino acids downstream of a hydrophobic region. Transfection experiments showed that ribosome stalling contributes to cytoplasmic splicing in bZIP60u orthologs of Arabidopsis and Selaginella. In contrast, ribosome stalling was undetectable for liverwort, Klebsormidium (basal land plant), and green algae orthologs. This study highlights the evolutionary diversity of ribosome stalling and its contribution to ER targeting in plants.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Fatores de Transcrição de Zíper de Leucina Básica , Filogenia , RNA Mensageiro , Ribossomos , Resposta a Proteínas não Dobradas , Arabidopsis/genética , Arabidopsis/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ribossomos/metabolismo , Ribossomos/genética , Resposta a Proteínas não Dobradas/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Oryza/genética , Oryza/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/genética , Splicing de RNA , Bryopsida/genética , Bryopsida/metabolismo , Biossíntese de ProteínasRESUMO
KEY MESSAGE: Characterization of Physcomitrella 3'UTRs across different promoters yields endogenous single and double terminators for usage in molecular pharming. The production of recombinant proteins for health applications accounts for a large share of the biopharmaceutical market. While many drugs are produced in microbial and mammalian systems, plants gain more attention as expression hosts to produce eukaryotic proteins. In particular, the good manufacturing practice (GMP)-compliant moss Physcomitrella (Physcomitrium patens) has outstanding features, such as excellent genetic amenability, reproducible bioreactor cultivation, and humanized protein glycosylation patterns. In this study, we selected and characterized novel terminators for their effects on heterologous gene expression. The Physcomitrella genome contains 53,346 unique 3'UTRs (untranslated regions) of which 7964 transcripts contain at least one intron. Over 91% of 3'UTRs exhibit more than one polyadenylation site, indicating the prevalence of alternative polyadenylation in Physcomitrella. Out of all 3'UTRs, 14 terminator candidates were selected and characterized via transient Dual-Luciferase assays, yielding a collection of endogenous terminators performing equally high as established heterologous terminators CaMV35S, AtHSP90, and NOS. High performing candidates were selected for testing as double terminators which impact reporter levels, dependent on terminator identity and positioning. Testing of 3'UTRs among the different promoters NOS, CaMV35S, and PpActin5 showed an increase of more than 1000-fold between promoters PpActin5 and NOS, whereas terminators increased reporter levels by less than tenfold, demonstrating the stronger effect promoters play as compared to terminators. Among selected terminator attributes, the number of polyadenylation sites as well as polyadenylation signals were found to influence terminator performance the most. Our results improve the biotechnology platform Physcomitrella and further our understanding of how terminators influence gene expression in plants in general.
Assuntos
Briófitas , Bryopsida , Animais , Bryopsida/genética , Regiões 3' não Traduzidas , Agricultura Molecular , Expressão Gênica , MamíferosRESUMO
Alfalfa (Medicago sativa L.), a perennial forage plant, is a rich source of nutrients such as vitamins, minerals, and proteins. Salt stress, however, impedes its growth. The plant-specific transcription factor abscisic acid insensitive 3 (ABI3) has a critical contribution to the control of abscisic acid (ABA) signaling pathway and abiotic stress response. The gene ScABI3 from Syntrichia caninervis, a moss species tolerant to desiccation, could be considered a potential candidate gene to modify alfalfa's nutritional and growth aspects. However, it remains unclear how ScABI3 affects the salt stress response of transgenic alfalfa. Therefore, we elucidated the role and molecular mechanism of ScABI3 from S. caninervis as an ABA signaling factor in transgenic alfalfa. Our findings demonstrate that ScABI3 overexpression in transgenic alfalfa improves salt tolerance by promoting relative water content, antioxidant enzyme activity, and photosynthetic parameters. Furthermore, the key genes of plant hormone signaling and the classical salt tolerance pathway were activated in ScABI3 transgenic lines under salt stress. Based on these results, ScABI3 could be considered a potentially critical candidate gene to alleviate salt stress in alfalfa. The present study provides valuable insights for developing transgenic crop breeding strategies for saline-alkaline soils.