RESUMO
Plant cells are surrounded by a cell wall and do not migrate, which makes the regulation of cell division orientation crucial for development. Regulatory mechanisms controlling cell division orientation may have contributed to the evolution of body organization in land plants. The GRAS family of transcription factors was transferred horizontally from soil bacteria to an algal common ancestor of land plants. SHORTROOT (SHR) and SCARECROW (SCR) genes in this family regulate formative periclinal cell divisions in the roots of flowering plants, but their roles in nonflowering plants and their evolution have not been studied in relation to body organization. Here, we show that SHR cell autonomously inhibits formative periclinal cell divisions indispensable for leaf vein formation in the moss Physcomitrium patens, and SHR expression is positively and negatively regulated by SCR and the GRAS member LATERAL SUPPRESSOR, respectively. While precursor cells of a leaf vein lacking SHR usually follow the geometry rule of dividing along the division plane with the minimum surface area, SHR overrides this rule and forces cells to divide nonpericlinally. Together, these results imply that these bacterially derived GRAS transcription factors were involved in the establishment of the genetic regulatory networks modulating cell division orientation in the common ancestor of land plants and were later adapted to function in flowering plant and moss lineages for their specific body organizations.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Divisão Celular/genética , Raízes de Plantas/metabolismo , Regulação da Expressão Gênica de PlantasRESUMO
Mosses of the genus Sphagnum are the main components of peatlands, a major carbon-storing ecosystem. Changes in precipitation patterns are predicted to affect water relations in this ecosystem, but the effect of desiccation on the physiological and molecular processes in Sphagnum is still largely unexplored. Here we show that different Sphagnum species have differential physiological and molecular responses to desiccation but, surprisingly, this is not directly correlated with their position in relation to the water table. In addition, the expression of drought responsive genes is increased upon water withdrawal in all species. This increase in gene expression is accompanied by an increase in abscisic acid (ABA), supporting a role for ABA during desiccation responses in Sphagnum. Not only do ABA levels increase upon desiccation, but Sphagnum plants pre-treated with ABA display increased tolerance to desiccation, suggesting that ABA levels play a functional role in the response. In addition, many of the ABA signalling components are present in Sphagnum and we demonstrate, by complementation in Physcomitrium patens, that Sphagnum ABI3 is functionally conserved. The data presented here, therefore, support a conserved role for ABA in desiccation responses in Sphagnum.
Assuntos
Ácido Abscísico , Sphagnopsida , Ácido Abscísico/metabolismo , Dessecação , Ecossistema , Solo , Sphagnopsida/metabolismo , Água/metabolismoRESUMO
Plants have evolved and grown under the selection pressure of gravitational force at 1 g on Earth. In response to this selection pressure, plants have acquired gravitropism to sense gravity and change their growth direction. In addition, plants also adjust their morphogenesis in response to different gravitational forces in a phenomenon known as gravity resistance. However, the gravity resistance phenomenon in plants is poorly understood due to the prevalence of 1 g gravitational force on Earth: not only it is difficult to culture plants at gravity > 1 g(hypergravity) for a long period of time but it is also impossible to create a < 1 genvironment (µg, micro g) on Earth without specialized facilities. Despite these technical challenges, it is important to understand how plants grow in different gravity conditions in order to understand land plant adaptation to the 1 g environment or for outer space exploration. To address this, we have developed a centrifugal device for a prolonged duration of plant culture in hypergravity conditions, and a project to grow plants under the µg environment in the International Space Station is also underway. Our plant material of choice is Physcomitrium (Physcomitrella) patens, one of the pioneer plants on land and a model bryophyte often used in plant biology. In this review, we summarize our latest findings regarding P. patens growth response to hypergravity, with reference to our on-going "Space moss" project. In our ground-based hypergravity experiments, we analyzed the morphological and physiological changes and found unexpected increments of chloroplast size and photosynthesis rate, which might underlie the enhancement of growth and increase in the number of gametophores and rhizoids. We further discussed our approaches at the cellular level and compare the gravity resistance in mosses and that in angiosperms. Finally, we highlight the advantages and perspectives from the space experiments and conclude that research with bryophytes is beneficial to comprehensively and precisely understand gravitational responses in plants.
Assuntos
Bryopsida/crescimento & desenvolvimento , Gravitação , Hipergravidade , Meristema/crescimento & desenvolvimento , Brotos de Planta/crescimento & desenvolvimento , Voo Espacial/métodos , Bryopsida/citologia , Bryopsida/metabolismo , Divisão Celular/fisiologia , Citoesqueleto/metabolismo , Meristema/citologia , Meristema/metabolismo , Modelos Biológicos , Fotossíntese/fisiologia , Brotos de Planta/citologia , Brotos de Planta/metabolismoRESUMO
Many differentiated plant cells can dedifferentiate into stem cells, reflecting the remarkable developmental plasticity of plants. In the moss Physcomitrella patens, cells at the wound margin of detached leaves become reprogrammed into stem cells. Here, we report that two paralogous P. patens WUSCHEL-related homeobox 13-like (PpWOX13L) genes, homologs of stem cell regulators in flowering plants, are transiently upregulated and required for the initiation of cell growth during stem cell formation. Concordantly, Δppwox13l deletion mutants fail to upregulate genes encoding homologs of cell wall loosening factors during this process. During the moss life cycle, most of the Δppwox13l mutant zygotes fail to expand and initiate an apical stem cell to form the embryo. Our data show that PpWOX13L genes are required for the initiation of cell growth specifically during stem cell formation, in analogy to WOX stem cell functions in seed plants, but using a different cellular mechanism.
Assuntos
Bryopsida/citologia , Bryopsida/genética , Genes de Plantas/genética , Folhas de Planta/citologia , Proteínas de Plantas/genética , Protoplastos/citologia , Células-Tronco/citologia , Sequência de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Bryopsida/crescimento & desenvolvimento , Proliferação de Células , Parede Celular/genética , Deleção de Genes , Regulação da Expressão Gênica de Plantas , Meristema/citologia , Meristema/crescimento & desenvolvimento , Dados de Sequência Molecular , Folhas de Planta/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Protoplastos/metabolismo , Regeneração , Células-Tronco/metabolismo , Regulação para Cima/genética , Zigoto/citologia , Zigoto/crescimento & desenvolvimentoRESUMO
Microtubules (MTs) play a crucial role in the anisotropic deposition of cell wall material, thereby affecting the direction of growth. A wide range of tip-growing cells display highly polarized cell growth, and MTs have been implicated in regulating directionality and expansion. However, the molecular machinery underlying MT dynamics in tip-growing plant cells remains unclear. Here, we show that highly dynamic MT bundles form cyclically in the polarized expansion zone of the moss Physcomitrella patens caulonemal cells through the coalescence of growing MT plus ends. Furthermore, the plant-specific kinesins (KINID1) that are is essential for the proper MT organization at cytokinesis also regulate the turnover of the tip MT bundles as well as the directionality and rate of cell growth. The plus ends of MTs grow toward the expansion zone, and KINID1 is necessary for the stability of a single coherent focus of MTs in the center of the zone, whose formation coincides with the accumulation of KINID1. We propose that KINID-dependent MT bundling is essential for the correct directionality of growth as well as for promoting growth per se. Our findings indicate that two localized cell wall deposition processes, tip growth and cytokinesis, previously believed to be functionally and evolutionarily distinct, share common and plant-specific MT regulatory components.
Assuntos
Bryopsida/crescimento & desenvolvimento , Citocinese/fisiologia , Cinesinas/fisiologia , Microtúbulos/fisiologia , Bryopsida/citologia , Cinesinas/genética , Dados de Sequência MolecularRESUMO
Stem cells are formed at particular times and positions during the development of multicellular organisms. Whereas flowering plants form stem cells only in the sporophyte generation, non-seed plants form stem cells in both the sporophyte and gametophyte generations. Although the molecular mechanisms underlying stem cell formation in the sporophyte generation have been extensively studied, only a few transcription factors involved in the regulation of gametophyte stem cell formation have been reported. The moss Physcomitrella patens forms a hypha-like body (protonema) and a shoot-like body (gametophore) from a protonema apical cell and a gametophore apical cell, respectively. These apical cells have stem cell characteristics and are formed as side branches of differentiated protonema cells. Here, we show that four AP2-type transcription factors orthologous to Arabidopsis thaliana AINTEGUMENTA, PLETHORA and BABY BOOM (APB) are indispensable for the formation of gametophore apical cells from protonema cells. Quadruple disruption of all APB genes blocked gametophore formation, even in the presence of cytokinin, which enhances gametophore apical cell formation in the wild type. All APB genes were expressed in emerging gametophore apical cells, but not in protonema apical cells. Heat-shock induction of an APB4 transgene driven by a heat-shock promoter increased the number of gametophores. Expression of all APB genes was induced by auxin but not by cytokinin. Thus, the APB genes function synergistically with cytokinin signaling to determine the identity of the two types of stem cells.
Assuntos
Proteínas de Arabidopsis/metabolismo , Bryopsida/citologia , Bryopsida/genética , Diferenciação Celular/fisiologia , Regulação da Expressão Gênica de Plantas/genética , Células-Tronco/fisiologia , Fatores de Transcrição/metabolismo , Southern Blotting , Bryopsida/crescimento & desenvolvimento , Análise por Conglomerados , Biologia Computacional , Citocininas/metabolismo , Células Germinativas Vegetais/crescimento & desenvolvimento , Histocitoquímica , Ácidos Indolacéticos/metabolismo , Funções Verossimilhança , Microscopia de Fluorescência , Modelos Genéticos , Filogenia , Plasmídeos/genética , Reação em Cadeia da Polimerase em Tempo Real , Especificidade da EspécieRESUMO
Mitosis is a fundamental process of eukaryotic cell proliferation. However, the molecular mechanisms underlying mitosis remain poorly understood in plants partly because of the lack of an appropriate model cell system in which loss-of-function analyses can be easily combined with high-resolution microscopy. Here, we developed an inducible RNA interference (RNAi) system and three-dimensional time-lapse confocal microscopy in the moss Physcomitrella patens that allowed in-depth phenotype characterization of the moss genes essential for cell division. We applied this technique to two microtubule regulators, augmin and γ-tubulin complexes, whose mitotic roles remain obscure in plant cells. Live imaging of caulonemal cells showed that they proceed through mitosis with continual generation and self-organization of acentrosomal microtubules. We demonstrated that augmin plays an important role in γ-tubulin localization and microtubule generation from prometaphase to cytokinesis. Most evidently, microtubule formation in phragmoplasts was severely compromised after RNAi knockdown of an augmin subunit, leading to incomplete expansion of phragmoplasts and cytokinesis failure. Knockdown of the γ-tubulin complex affected microtubule formation throughout mitosis. We conclude that postanaphase microtubule generation is predominantly stimulated by the augmin/γ-tubulin machinery in moss and further propose that this RNAi system serves as a powerful tool to dissect the molecular mechanisms underlying mitosis in land plants.
Assuntos
Bryopsida/genética , Bryopsida/metabolismo , Microtúbulos/metabolismo , Proteínas de Plantas/metabolismo , Interferência de RNA , Fuso Acromático/metabolismo , Anáfase , Bryopsida/citologia , Técnicas de Silenciamento de Genes , Genes de Plantas/genética , Humanos , Fenótipo , Plantas Geneticamente Modificadas , Transporte Proteico , Reprodutibilidade dos Testes , Tubulina (Proteína)/metabolismoRESUMO
During regeneration, differentiated plant cells can be reprogrammed to produce stem cells, a process that requires coordination of cell cycle reactivation with acquisition of other cellular characteristics. However, the factors that coordinate the two functions during reprogramming have not been determined. Here, we report a link between cell cycle reactivation and the acquisition of new cell-type characteristics through the activity of cyclin-dependent kinase A (CDKA) during reprogramming in the moss Physcomitrella patens. Excised gametophore leaf cells of P. patens are readily reprogrammed, initiate tip growth, and form chloronema apical cells with stem cell characteristics at their first cell division. We found that leaf cells facing the cut undergo CDK activation along with induction of a D-type cyclin, tip growth, and transcriptional activation of protonema-specific genes. A DNA synthesis inhibitor, aphidicolin, inhibited cell cycle progression but prevented neither tip growth nor protonemal gene expression, indicating that cell cycle progression is not required for acquisition of protonema cell-type characteristics. By contrast, treatment with a CDK inhibitor or induction of dominant-negative CDKA;1 protein inhibited not only cell cycle progression but also tip growth and protonemal gene expression. These findings indicate that cell cycle progression is coordinated with other cellular changes by the concomitant regulation through CDKA;1.
Assuntos
Bryopsida/fisiologia , Ciclo Celular/fisiologia , Desdiferenciação Celular/fisiologia , Quinases Ciclina-Dependentes/metabolismo , Afidicolina/farmacologia , Sequência de Bases , Bryopsida/citologia , Bryopsida/efeitos dos fármacos , Bryopsida/genética , Ciclo Celular/efeitos dos fármacos , Ciclina D/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/genética , DNA de Plantas/química , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica de Plantas/fisiologia , Dados de Sequência Molecular , Mutação , Folhas de Planta/citologia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Folhas de Planta/fisiologia , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Análise de Sequência de DNA , Células-Tronco/fisiologia , Fatores de Tempo , Ativação Transcricional/fisiologiaRESUMO
The shoot apical meristem (SAM) contains pluripotent stem cells that produce all the aerial parts of the plant. Stem cells undergo asymmetric cell divisions to self-renew and to produce differentiating cells. Our research focused on unraveling the mechanisms governing the specification of these two distinct cell fates following the stem cell division. For this purpose, we used the model organism Physcomitrium patens, which features a singular pluripotent stem cell known as the gametophore apical cell. We show that the activity of cytokinins, critical stem cell regulators, is restricted to the gametophore apical cell due to the specific localization of PpLOG, the enzyme responsible for cytokinin activation. In turn, PpTAW, which promotes differentiating cell identity of the merophyte, is excluded from the gametophore apical cell by the action of cytokinins. We propose a cytokinin-based model for the establishment of asymmetry in the pluripotent stem cell division.
Assuntos
Bryopsida , Citocininas , Proteínas de Plantas , Células-Tronco Pluripotentes , Citocininas/metabolismo , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , Bryopsida/metabolismo , Bryopsida/citologia , Proteínas de Plantas/metabolismo , Meristema/metabolismo , Meristema/citologia , Diferenciação Celular , Regulação da Expressão Gênica de PlantasRESUMO
Protonemata of the moss Physcomitrium patens are ideal structures in which to observe cytoskeletal organization and dynamics. Special care is needed to prepare P. patens cultures for high-resolution microscopy. Here, we describe methods for spinning disk microscopy of dividing P. patens cells expressing sGFP-tubulin and H2B-mCherry, including detailed methods for culturing P. patens.
Assuntos
Bryopsida , Microscopia , Citocinese , Tubulina (Proteína)RESUMO
Chloroplasts require association with the plasma membrane for movement in response to light and for appropriate positioning within the cell to capture photosynthetic light efficiently. In Arabidopsis, CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1), KINESIN-LIKE PROTEIN FOR ACTIN-BASED CHLOROPLAST MOVEMENT 1 (KAC1) and KAC2 are required for both the proper movement of chloroplasts and the association of chloroplasts with the plasma membrane, through the reorganization of short actin filaments located on the periphery of the chloroplasts. Here, we show that KAC and CHUP1 orthologs (AcKAC1, AcCHUP1A and AcCHUP1B, and PpKAC1 and PpKAC2) play important roles in chloroplast positioning in the fern Adiantum capillus-veneris and the moss Physcomitrella patens. The knockdown of AcKAC1 and two AcCHUP1 genes induced the aggregation of chloroplasts around the nucleus. Analyses of A. capillus-veneris mutants containing perinuclear-aggregated chloroplasts confirmed that AcKAC1 is required for chloroplast-plasma membrane association. In addition, P. patens lines in which two KAC genes had been knocked out showed an aggregated chloroplast phenotype similar to that of the fern kac1 mutants. These results indicate that chloroplast positioning and movement are mediated through the activities of KAC and CHUP1 proteins, which are conserved in land plants.
Assuntos
Adiantum/metabolismo , Membrana Celular/metabolismo , Proteínas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Cinesinas/metabolismo , Proteínas de Plantas/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Adiantum/anatomia & histologia , Adiantum/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas de Cloroplastos/genética , Cloroplastos/genética , Clonagem Molecular , Técnicas de Inativação de Genes , Inativação Gênica , Genes de Plantas , Teste de Complementação Genética , Cinesinas/genética , Folhas de Planta/anatomia & histologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismoRESUMO
Land plants have distinct developmental programs in haploid (gametophyte) and diploid (sporophyte) generations. Although usually the two programs strictly alternate at fertilization and meiosis, one program can be induced during the other program. In a process called apogamy, cells of the gametophyte other than the egg cell initiate sporophyte development. Here, we report for the moss Physcomitrella patens that apogamy resulted from deletion of the gene orthologous to the Arabidopsis thaliana CURLY LEAF (PpCLF), which encodes a component of polycomb repressive complex 2 (PRC2). In the deletion lines, a gametophytic vegetative cell frequently gave rise to a sporophyte-like body. This body grew indeterminately from an apical cell with the character of a sporophytic pluripotent stem cell but did not form a sporangium. Furthermore, with continued culture, the sporophyte-like body branched. Sporophyte branching is almost unknown among extant bryophytes. When PpCLF was expressed in the deletion lines once the sporophyte-like bodies had formed, pluripotent stem cell activity was arrested and a sporangium-like organ formed. Supported by the observed pattern of PpCLF expression, these results demonstrate that, in the gametophyte, PpCLF represses initiation of a sporophytic pluripotent stem cell and, in the sporophyte, represses that stem cell activity and induces reproductive organ development. In land plants, branching, along with indeterminate apical growth and delayed initiation of spore-bearing reproductive organs, were conspicuous innovations for the evolution of a dominant sporophyte plant body. Our study provides insights into the role of PRC2 gene regulation for sustaining evolutionary innovation in land plants.
Assuntos
Bryopsida/genética , Evolução Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/fisiologia , Bryopsida/citologia , Bryopsida/fisiologia , Clonagem Molecular , Diploide , Deleção de Genes , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Haploidia , Temperatura Alta , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/classificação , Plantas Geneticamente Modificadas , Reprodução/genética , Reprodução/fisiologiaRESUMO
Light is a critical signal perceived by plants to adapt their growth rate and direction. Although many signaling components have been studied, how plants respond to constantly fluctuating light remains underexplored. Here, we showed that in the moss Physcomitrium (Physcomitrella) patens, the PSTAIRE-type cyclin-dependent kinase PpCDKA is dispensable for growth. Instead, PpCDKA and its homolog in Arabidopsis thaliana control light-induced tropisms and chloroplast movements by probably influencing the cytoskeleton organization independently of the cell cycle. In addition, lower PpCDKA kinase activity was required to elicit light responses relative to cell cycle regulation. Thus, our study suggests that plant CDKAs may have been co-opted to control multiple light responses, and owing to the bistable switch properties of PSTAIRE-type CDKs, the noncanonical functions are widely conserved for eukaryotic environmental adaptation.
RESUMO
This comprehensive overview of the xyloglucan endotransglucosylase/hydrolase (XTH) family of genes and proteins in bryophytes, based on research using genomic resources that are newly available for the moss Physcomitrella patens, provides new insights into plant evolution. In angiosperms, the XTH genes are found in large multi-gene families, probably reflecting the diverse roles of individual XTHs in various cell types. As there are fewer cell types in P. patens than in angiosperms such as Arabidopsis and rice, it is tempting to deduce that there are fewer XTH family genes in bryophytes. However, the present study unexpectedly identified as many as 32 genes that potentially encode XTH family proteins in the genome of P. patens, constituting a fairly large multi-gene family that is comparable in size with those of Arabidopsis and rice. In situ localization of xyloglucan endotransglucosylase activity in this moss indicates that some P. patens XTH proteins exhibit biochemical functions similar to those found in angiosperms, and that their expression profiles are tissue-dependent. However, comparison of structural features of families of XTH genes between P. patens and angiosperms demonstrated the existence of several bryophyte-specific XTH genes with distinct structural and functional features that are not found in angiosperms. These bryophyte-specific XTH genes might have evolved to meet morphological and functional needs specific to the bryophyte. These findings raise interesting questions about the biological implications of the XTH family of proteins in non-seed plants.
Assuntos
Bryopsida/genética , Evolução Molecular , Glicosiltransferases/genética , Família Multigênica , Sequência de Aminoácidos , Bryopsida/enzimologia , Perfilação da Expressão Gênica , Genes de Plantas , Glicosiltransferases/metabolismo , Imuno-Histoquímica , Dados de Sequência Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
Phosphatidylinositol phosphate kinase (PIPK) is an enzyme involved in the regulation of cellular levels of phosphoinositides involved in various physiological processes, such as cytoskeletal organization, ion channel activation, and vesicle trafficking. In animals, research has focused on the modes of activation and function of PIPKs, providing an understanding of the importance of plasma membrane localization. However, it still remains unclear how this issue is regulated in plant PIPKs. Here, we demonstrate that the carboxyl-terminal catalytic domain, which contains the activation loop, is sufficient for plasma membrane localization of PpPIPK1, a type I/II B PIPK from the moss Physcomitrella patens. The importance of the carboxyl-terminal catalytic domain for plasma membrane localization was confirmed with Arabidopsis (Arabidopsis thaliana) AtPIP5K1. Our findings, in which substitution of a conserved dibasic amino acid pair in the activation loop of PpPIPK1 completely prevented plasma membrane targeting and abolished enzymatic activity, demonstrate its critical role in these processes. Placing our results in the context of studies of eukaryotic PIPKs led us to conclude that the function of the dibasic amino acid pair in the activation loop in type I/II PIPKs is plant specific.
Assuntos
Diamino Aminoácidos/química , Bryopsida/enzimologia , Membrana Celular/enzimologia , Sequência Conservada , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Sequência de Aminoácidos , Animais , Arabidopsis/citologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/enzimologia , Bryopsida/efeitos dos fármacos , Domínio Catalítico , Membrana Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Dados de Sequência Molecular , Cebolas/citologia , Cebolas/efeitos dos fármacos , Cebolas/enzimologia , Ácidos Fosfatídicos/farmacologia , Transporte Proteico/efeitos dos fármacos , Protoplastos/efeitos dos fármacos , Protoplastos/enzimologia , Relação Estrutura-Atividade , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/enzimologiaRESUMO
Gibberellins (GAs) are a group of diterpene-type plant hormones biosynthesized from ent-kaurene via ent-kaurenoic acid. GAs are ubiquitously present in seed plants. The GA signal is perceived and transduced by the GID1 GA receptor/DELLA repressor pathway. The lycopod Selaginella moellendorffii biosynthesizes GA and has functional GID1-DELLA signaling components. In contrast, no GAs or functionally orthologous GID1-DELLA components have been found in the moss Physcomitrella patens. However, P. patens produces ent-kaurene, a common precursor for GAs, and possesses a functional ent-kaurene synthase, PpCPS/KS. To assess the biological role of ent-kaurene in P. patens, we generated a PpCPS/KS disruption mutant that does not accumulate ent-kaurene. Phenotypic analysis demonstrates that the mutant has a defect in the protonemal differentiation of the chloronemata to caulonemata. Gas chromatography-mass spectrometry analysis shows that P. patens produces ent-kaurenoic acid, an ent-kaurene metabolite in the GA biosynthesis pathway. The phenotypic defect of the disruptant was recovered by the application of ent-kaurene or ent-kaurenoic acid, suggesting that ent-kaurenoic acid, or a downstream metabolite, is involved in protonemal differentiation. Treatment with uniconazole, an inhibitor of ent-kaurene oxidase in GA biosynthesis, mimics the protonemal phenotypes of the PpCPS/KS mutant, which were also restored by ent-kaurenoic acid treatment. Interestingly, the GA(9) methyl ester, a fern antheridiogen, rescued the protonemal defect of the disruption mutant, while GA(3) and GA(4), both of which are active GAs in angiosperms, did not. Our results suggest that the moss P. patens utilizes a diterpene metabolite from ent-kaurene as an endogenous developmental regulator and provide insights into the evolution of GA functions in land plants.
Assuntos
Bryopsida/citologia , Bryopsida/metabolismo , Diferenciação Celular , Giberelinas/metabolismo , Alquil e Aril Transferases/metabolismo , Vias Biossintéticas/efeitos dos fármacos , Bryopsida/enzimologia , Bryopsida/genética , Diferenciação Celular/efeitos dos fármacos , Ésteres/farmacologia , Técnicas de Inativação de Genes , Genes de Plantas/genética , Germinação/efeitos dos fármacos , Giberelinas/química , Giberelinas/farmacologia , Ácidos Indolacéticos/farmacologia , Modelos Biológicos , Mutação/genética , Fenótipo , Esporos/efeitos dos fármacos , Esporos/metabolismoRESUMO
A wide range of tip-growing cells in plants display polarized cell growth, which is an essential cellular process for the form and function of individual cells. Understanding of the regulatory mechanisms underlying tip growth in terrestrial plants has improved. Cellular processes involved in tip growth have also been investigated in some algae species that form filamentous cells, but their regulatory mechanisms remain unclear. In the macro red alga Neopyropia yezoensis, for which genome information has recently been released, the conchocelis apical cell exhibits tip growth and forms a filamentous structure. Here, we report a live-imaging technique using high-resolution microscopy to analyze the tip growth and cell division of N. yezoensis conchocelis. This imaging analysis addressed tip growth dynamics and cell division in conchocelis apical cells. The directionality and tip growth expansion were disrupted by the application of cytoskeletal drugs, suggesting the involvement of microtubules (MTs) and actin filaments (AFs) in these processes. A growing apical cell mostly contained a single chloroplast that moved toward the expanding part of the apical cell. Drug application also inhibited chloroplast movement, implying that the movement may be dependent on the cytoskeleton. The study determined that live-imaging analysis is a versatile approach for exploring the dynamics of tip growth and cell division in N. yezoensis conchocelis, which provides insights into the regulatory mechanisms underlying cellular growth in multicellular red algae.
RESUMO
Phosphoinositides (PIs) play a major role in eukaryotic cells, despite being a minor component of most membranes. This is the first report on PI metabolism in a bryophyte, the moss Physcomitrella patens. Moss PI composition is similar to that of other land plants growing under normal conditions. In contrast to the large number of PIPK genes present in flowering plants, the P. patens genome encodes only two type I/II PIPK genes, PpPIPK1 and PpPIPK2, which are very similar at both the nucleotide and protein product levels. However, the expression of the two genes is differentially regulated, and in vitro biochemical characterization shows that the resulting enzymes have different substrate specificities. PpPIPK1 uses PtdIns4P and PtdIns3P with similar preference and also metabolizes PtdIns(3,4)P(2) to produce PtdIns(3,4,5)P(3), a PI not yet detected in intact plant cells. PpPIPK2 prefers PtdIns as substrate and is much less active towards PtdIns4P and PtdIns3P. Thus, PpPIPK2 shows properties reminiscent of both PtdInsP-kinase and PtdIns-kinases. Moreover, a substitution of glutamic acid by alanine in the activation loop drastically reduced PpPIPK1 activity and altered the substrate specificity to PtdIns5P being the preferred substrate compared with PtdIns4P and PtdIns3P. These findings demonstrate that the substrate specificity of plant PIPKs is determined in a plant-specific manner, which provides new insights into the regulatory modes of PIPK activity in plants.
Assuntos
Bryopsida/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Bryopsida/genética , Membrana Celular/enzimologia , Clonagem Molecular , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Dados de Sequência Molecular , Fosfatos de Fosfatidilinositol/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Filogenia , RNA de Plantas/genética , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
Eukaryotic cells have developed several essential membrane components. In flowering plants, appropriate structures and distributions of the major membrane components are predominantly regulated by actin microfilaments. In this study, we have focused on the regulatory mechanism of vacuolar structures in the moss, Physcomitrella patens. The high ability of P. patens to undergo homologous recombination enabled us stably to express green fluorescent protein (GFP) or red fluorescent protein (RFP) fusion proteins, and the simple body structure of P. patens enabled us to perform detailed visualization of the intracellular vacuolar and cytoskeletal structures. Three-dimensional analysis and high-speed time-lapse observations revealed surprisingly complex structures and dynamics of the vacuole, with inner sheets and tubular protrusions, and frequent rearrangements by separation and fusion of the membranes. Depolymerization of microtubules dramatically affected these structures and movements. Dual observation of microtubules and vacuolar membranes revealed that microtubules induced tubular protrusions and cytoplasmic strands of the vacuoles, indicative of interactions between microtubules and vacuolar membranes. These results demonstrate a novel function of microtubules in maintaining the distribution of the vacuole and suggest a functional divergence of cytoskeletal functions in land plant evolution.
Assuntos
Bryopsida/citologia , Membranas Intracelulares/ultraestrutura , Microtúbulos/metabolismo , Vacúolos/ultraestrutura , Bryopsida/genética , Bryopsida/metabolismo , Citoesqueleto/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imageamento Tridimensional , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Plantas Geneticamente Modificadas/citologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Vacúolos/metabolismo , Proteína Vermelha FluorescenteRESUMO
Epigenetic modifications, including histone modifications, stabilize cell-specific gene expression programmes to maintain cell identities in both metazoans and land plants1-3. Notwithstanding the existence of these stable cell states, in land plants, stem cells are formed from differentiated cells during post-embryonic development and regeneration4-6, indicating that land plants have an intrinsic ability to regulate epigenetic memory to initiate a new gene regulatory network. However, it is less well understood how epigenetic modifications are locally regulated to influence the specific genes necessary for cellular changes without affecting other genes in a genome. In this study, we found that ectopic induction of the AP2/ERF transcription factor STEMIN1 in leaf cells of the moss Physcomitrella patens decreases a repressive chromatin mark, histone H3 lysine 27 trimethylation (H3K27me3), on its direct target genes before cell division, resulting in the conversion of leaf cells to chloronema apical stem cells. STEMIN1 and its homologues positively regulate the formation of secondary chloronema apical stem cells from chloronema cells during development. Our results suggest that STEMIN1 functions within an intrinsic mechanism underlying local H3K27me3 reprogramming to initiate stem cell formation.