RESUMO
Regulatory T (Treg) cells require T-cell receptor (TCR) signalling to exert their immunosuppressive activity, but the precise organization of the TCR signalling network compared to conventional T (Tconv) cells remains elusive. By using accurate mass spectrometry and multi-epitope ligand cartography (MELC) we characterized TCR signalling and recruitment of TCR signalling components to the immunological synapse (IS) in Treg cells and Tconv cells. With the exception of Themis which we detected in lower amounts in Treg cells, other major TCR signalling components were found equally abundant, however, their phosphorylation-status notably discriminates Treg cells from Tconv cells. Overall, this study identified 121 Treg cell-specific phosphorylations. Short-term triggering of T cell subsets via CD3 and CD28 widely harmonized these variations with the exception of eleven TCR signalling components that mainly regulate cytoskeleton dynamics and molecular transport. Accordingly, conjugation with B cells indeed caused variant cellular morphology and revealed a Treg cell-specific recruitment of TCR signalling components such as PKCθ, PLCγ1 and ZAP70 as well as B cell-derived CD86 into the IS. Together, results from this study support the existence of a Treg cell-specific IS and suggest Treg cell-specific cytoskeleton dynamics as a novel determinant for the unique functional properties of Treg cells.
Assuntos
Sinapses Imunológicas/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologia , Animais , Células Cultivadas , Feminino , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Fosforilação , Proteoma/imunologia , Proteoma/metabolismo , Proteômica/métodos , Receptores de Antígenos de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/metabolismo , Proteína-Tirosina Quinase ZAP-70/imunologia , Proteína-Tirosina Quinase ZAP-70/metabolismoRESUMO
Adaptive immunity critically contributes to control acute infection with enteropathogenic Yersinia pseudotuberculosis; however, the role of CD4+ T cell subsets in establishing infection and allowing pathogen persistence remains elusive. Here, we assessed the modulatory capacity of Y. pseudotuberculosis on CD4+ T cell differentiation. Using in vivo assays, we report that infection with Y. pseudotuberculosis resulted in enhanced priming of IL-17-producing T cells (Th17 cells), whereas induction of Foxp3+ regulatory T cells (Tregs) was severely disrupted in gut-draining mesenteric lymph nodes (mLNs), in line with altered frequencies of tolerogenic and proinflammatory dendritic cell (DC) subsets within mLNs. Additionally, by using a DC-free in vitro system, we could demonstrate that Y. pseudotuberculosis can directly modulate T cell receptor (TCR) downstream signaling within naïve CD4+ T cells and Tregs via injection of effector molecules through the type III secretion system, thereby affecting their functional properties. Importantly, modulation of naïve CD4+ T cells by Y. pseudotuberculosis resulted in an enhanced Th17 differentiation and decreased induction of Foxp3+ Tregs in vitro. These findings shed light to the adjustment of the Th17-Treg axis in response to acute Y. pseudotuberculosis infection and highlight the direct modulation of CD4+ T cell subsets by altering their TCR downstream signaling.
Assuntos
Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Reguladores/microbiologia , Células Th17/microbiologia , Infecções por Yersinia pseudotuberculosis/imunologia , Yersinia pseudotuberculosis/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/microbiologia , Diferenciação Celular , Feminino , Fatores de Transcrição Forkhead/imunologia , Interações Hospedeiro-Patógeno , Intestinos/imunologia , Intestinos/microbiologia , Camundongos Endogâmicos BALB C , Transdução de Sinais , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Yersinia pseudotuberculosis/fisiologiaRESUMO
BACKGROUND: Theiler's murine encephalomyelitis virus (TMEV) infection represents a commonly used infectious animal model to study various aspects of the pathogenesis of multiple sclerosis (MS). In susceptible SJL mice, dominant activity of Foxp3(+) CD4(+) regulatory T cells (Tregs) in the CNS partly contributes to viral persistence and progressive demyelination. On the other hand, resistant C57BL/6 mice rapidly clear the virus by mounting a strong antiviral immune response. However, very little is known about the role of Tregs in regulating antiviral responses during acute encephalitis in resistant mouse strains. METHODS: In this study, we used DEREG mice that express the diphtheria toxin (DT) receptor under control of the foxp3 locus to selectively deplete Foxp3(+) Tregs by injection of DT prior to infection and studied the effect of Treg depletion on the course of acute Theiler's murine encephalomyelitis (TME). RESULTS: As expected, DEREG mice that are on a C57BL/6 background were resistant to TMEV infection and cleared the virus within days of infection, regardless of the presence or absence of Tregs. Nevertheless, in the absence of Tregs we observed priming of stronger effector T cell responses in the periphery, which subsequently resulted in a transient increase in the frequency of IFNγ-producing T cells in the brain at an early stage of infection. Histological and flow cytometric analysis revealed that this transiently increased frequency of brain-infiltrating IFNγ-producing T cells in Treg-depleted mice neither led to an augmented antiviral response nor enhanced inflammation-mediated tissue damage. Intriguingly, Treg depletion did not change the expression of IL-10 in the infected brain, which might play a role for dampening the inflammatory damage caused by the increased number of effector T cells. CONCLUSION: We therefore propose that unlike susceptible mice strains, interfering with the Treg compartment of resistant mice only has negligible effects on virus-induced pathologies in the CNS. Furthermore, in the absence of Tregs, local anti-inflammatory mechanisms might limit the extent of damage caused by strong anti-viral response in the CNS.
Assuntos
Infecções por Cardiovirus/imunologia , Resistência à Doença/imunologia , Encefalite Viral/imunologia , Linfócitos T Reguladores/fisiologia , Theilovirus/imunologia , Doença Aguda , Animais , Infecções por Cardiovirus/prevenção & controle , Encefalite Viral/prevenção & controle , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
BACKGROUND: Epigenetic changes in DNA methylation have recently been demonstrated to be involved in effector T-cell polarization, resulting in differential secretion of T(H)1 and T(H)2 cytokines. However, the contribution to the development of a chronic inflammatory phenotype remains still unclear. OBJECTIVE: We sought to investigate changes in DNA methylation in marker genes of T-cell subsets during allergen sensitization/challenge and their influence on the development of an allergic airway inflammatory response. METHODS: The relationship between changes in DNA methylation and phenotype development were examined in a well-established model of experimental asthma. DNA methylation was investigated at genomic loci associated with T(H)1 (IFNG promoter) or T(H)2 (conserved noncoding sequence 1 [CNS1]) cytokine production by using bisulfite pyrosequencing. RESULTS: Analysis of CD4(+) T cells revealed a significant increase in DNA methylation at the IFNG promoter after allergen sensitization/challenge, which correlated with decreased IFN-γ cytokine expression, whereas only minor changes were observed at the CNS1 locus. Furthermore, the increase in DNA methylation at the IFNG promoter could be reversed with a DNA methyltransferase (DNMT) inhibitor in vitro and in vivo with beneficial effects on sensitization status and allergic phenotype. The specific importance of the DNA methylation status in CD4(+) T cells could be confirmed by using adoptive transfer experiments. CONCLUSION: We here report the novel finding that epigenetic regulation in T cells contributes to the development of experimental asthma and can be targeted pharmacologically.
Assuntos
Asma/genética , Citocinas/genética , Metilação de DNA , Células Th1/imunologia , Células Th2/imunologia , Animais , Asma/imunologia , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Metilação de DNA/efeitos dos fármacos , Decitabina , Epigênese Genética , Feminino , Interferon gama/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Regiões Promotoras Genéticas , Células Th1/metabolismo , Células Th2/efeitos dos fármacos , Células Th2/metabolismoRESUMO
BACKGROUND: Bronchial asthma is a chronic inflammatory disease resulting from complex gene-environment interactions. Natural microbial exposure has been identified as an important environmental condition that provides asthma protection in a prenatal window of opportunity. Epigenetic regulation is an important mechanism by which environmental factors might interact with genes involved in allergy and asthma development. OBJECTIVE: This study was designed to test whether epigenetic mechanisms might contribute to asthma protection conferred by early microbial exposure. METHODS: Pregnant maternal mice were exposed to the farm-derived gram-negative bacterium Acinetobacter lwoffii F78. Epigenetic modifications in the offspring were analyzed in T(H)1- and T(H)2-relevant genes of CD4(+) T cells. RESULTS: Prenatal administration of A lwoffii F78 prevented the development of an asthmatic phenotype in the progeny, and this effect was IFN-γ dependent. Furthermore, the IFNG promoter of CD4(+) T cells in the offspring revealed a significant protection against loss of histone 4 (H4) acetylation, which was closely associated with IFN-γ expression. Pharmacologic inhibition of H4 acetylation in the offspring abolished the asthma-protective phenotype. Regarding T(H)2-relevant genes only at the IL4 promoter, a decrease could be detected for H4 acetylation but not at the IL5 promoter or the intergenic T(H)2 regulatory region conserved noncoding sequence 1 (CNS1). CONCLUSION: These data support the hygiene concept and indicate that microbes operate by means of epigenetic mechanisms. This provides a new mechanism in the understanding of gene-environment interactions in the context of allergy protection.
Assuntos
Acinetobacter/imunologia , Asma/prevenção & controle , Epigênese Genética , Hipersensibilidade/prevenção & controle , Imunidade Materno-Adquirida/genética , Complicações na Gravidez/imunologia , Acetilação , Animais , Asma/genética , Asma/imunologia , Meio Ambiente , Feminino , Histonas/metabolismo , Hipersensibilidade/genética , Hipersensibilidade/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Gravidez , Complicações na Gravidez/genética , Fatores de Risco , Linfócitos TRESUMO
Theiler's murine encephalomyelitis (TME) of susceptible mouse strains is a commonly used infectious animal model for multiple sclerosis. The study aim was to test the hypothesis whether cytotoxic T cell responses account for the limited impact of regulatory T cells on antiviral immunity in TME virus-induced demyelinating disease (TMEV-IDD) resistant C57BL/6 mice. TME virus-infected C57BL/6 mice were treated with (i) interleukin-2/-anti-interleukin-2-antibody-complexes to expand regulatory T cells ("Treg-expansion"), (ii) anti-CD8-antibodies to deplete cytotoxic T cells ("CD8-depletion") or (iii) with a combination of Treg-expansion and CD8-depletion ("combined treatment") prior to infection. Results showed that "combined treatment", but neither sole "Treg-expansion" nor "CD8-depletion," leads to sustained hippocampal infection and virus spread to the spinal cord in C57BL/6 mice. Prolonged infection reduces myelin basic protein expression in the spinal cord together with increased accumulation of ß-amyloid precursor protein in axons, characteristic of myelin loss and axonal damage, respectively. Chronic spinal cord infection upon "combined treatment" was also associated with increased T and B cell recruitment, accumulation of CD107b+ microglia/macrophages and enhanced mRNA expression of interleukin (IL)-1α, IL-10 and tumor necrosis factor α. In conclusion, data revealed that the suppressive capacity of Treg on viral elimination is efficiently boosted by CD8-depletion, which renders C57BL/6 mice susceptible to develop chronic neuroinfection and TMEV-IDD.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Doenças Desmielinizantes/imunologia , Linfócitos T Reguladores/imunologia , Theilovirus/imunologia , Animais , Encéfalo/imunologia , Encéfalo/virologia , Infecções por Cardiovirus/imunologia , Doenças Desmielinizantes/virologia , Modelos Animais de Doenças , Feminino , Camundongos Endogâmicos C57BL , Medula Espinal/imunologia , Medula Espinal/virologiaRESUMO
Theiler's murine encephalomyelitis virus (TMEV) infection represents an experimental mouse model to study hippocampal damage induced by neurotropic viruses. IL-10 is a pleiotropic cytokine with profound anti-inflammatory properties, which critically controls immune homeostasis. In order to analyze IL-10R signaling following virus-induced polioencephalitis, SJL mice were intracerebrally infected with TMEV. RNA-based next generation sequencing revealed an up-regulation of Il10, Il10rα and further genes involved in IL-10 downstream signaling, including Jak1, Socs3 and Stat3 in the brain upon infection. Subsequent antibody-mediated blockade of IL-10R signaling led to enhanced hippocampal damage with neuronal loss and increased recruitment of CD3+ T cells, CD45R+ B cells and an up-regulation of Il1α mRNA. Increased expression of Tgfß and Foxp3 as well as accumulation of Foxp3+ regulatory T cells and arginase-1+ macrophages/microglia was detected in the hippocampus, representing a potential compensatory mechanism following disturbed IL-10R signaling. Additionally, an increased peripheral Chi3l3 expression was found in spleens of infected mice, which may embody reactive regulatory mechanisms for prevention of excessive immunopathology. The present study highlights the importance of IL-10R signaling for immune regulation and its neuroprotective properties in the context of an acute neurotropic virus infection.
Assuntos
Infecções por Cardiovirus/imunologia , Hipocampo/imunologia , Receptores de Interleucina-10/imunologia , Theilovirus/imunologia , Animais , Infecções por Cardiovirus/genética , Infecções por Cardiovirus/patologia , Infecções por Cardiovirus/virologia , Feminino , Hipocampo/patologia , Hipocampo/virologia , Camundongos Endogâmicos C57BL , Receptores de Interleucina-10/genética , Transdução de Sinais , Regulação para CimaRESUMO
Little is known about the evolution of the intron-exon organization in the more primitive groups of land plants, and the intron distribution among Plantae (glauco-, rhodo-, chloro- and streptophytes) has not been investigated so far. The present study is focused on some key species such as the liverwort Marchantia polymorpha, representing the most ancient lineage of land plants, and the streptophycean green alga Mesostigma viride, branching prior to charophycean green algae and terrestrial plants. The intron distribution of six genes for sugar phosphate metabolism was analyzed including four different glyceraldehyde-3-phosphate dehydrogenases (GAPDH), the sedoheptulose-1,7-bisphosphatase (SBP) and the glucose-6-phosphate isomerase (GPI). We established 15 new sequences including three cDNA and twelve genomic clones with up to 24 introns per gene, which were identified in the GPI of Marchantia. The intron patterns of all six genes are completely conserved among seed plants, lycopods, mosses and even liverworts. This intron stasis without any gain of novel introns seem to last for nearly 500 million years and may be characteristic for land plants in general. Some unique intron positions in Mesostigma document that a uniform distribution is no common trait of all streptophytes, but it may correlate with the transition to terrestrial habitats. However, the respective genes of chlorophycean green algae display largely different patterns, thus indicating at least one phase of massive intron rearrangement in the green lineage. We moreover included rhodophyte and glaucophyte reference sequences in our analyses and, even if the well documented monophyly of Plantae is not reflected by a uniform intron distribution, at least one GPI intron is strictly conserved for 1.5 billion years.
Assuntos
Evolução Molecular , Íntrons , Plantas/genética , Sequência de Bases , Clorófitas/classificação , Clorófitas/genética , Clorófitas/metabolismo , Cyanophora/classificação , Cyanophora/genética , Cyanophora/metabolismo , DNA de Algas/genética , DNA de Plantas/genética , Duplicação Gênica , Genes de Plantas , Marchantia/classificação , Marchantia/genética , Marchantia/metabolismo , Filogenia , Plantas/classificação , Plantas/metabolismo , Fosfatos Açúcares/genética , Fosfatos Açúcares/metabolismo , Fatores de TempoRESUMO
Sedoheptulose-1,7-bisphosphatase (SBPase) and fructose-1,6-bisphosphatase (FBPase) are essential nuclear-encoded enzymes involved in land plant Calvin cycle and gluconeogenesis. In this study, we cloned seven SBP and seven FBP cDNAs/genes and established sequences from all lineages of photosynthetic eukaryotes, in order to investigate their origin and evolution. Our data are best explained by a single recruitment of plastid-targeted SBP in Plantae after primary endosymbiosis and a further distribution to algae with complex plastids. While SBP is universally found in photosynthetic lineages, its presence in apicomplexa, ciliates, trypanosomes, and ascomycetes is surprising given that no metabolic function beyond the one in the plastid Calvin cycle is described so far. Sequences of haptophytes, cryptophytes, diatoms, and peridinin-containing dinoflagellates (complex red lineage) strongly group together in the SBP tree and the same assemblage is recovered for plastid-targeted FBP sequences, although this is less supported. Both SBP and plastid-targeted FBP are most likely of red algal origin. Including phosphoribulokinase, fructose bisphosphate aldolase, and glyceraldehyde-3-phosphate dehydrogenase, a total of five independent plastid-related nuclear-encoded markers support a common origin of all complex rhodoplasts via a single secondary endosymbiosis event. However, plastid phylogenies are incongruent with those of the host cell, as illustrated by the cytosolic FBP isoenzyme. These results are discussed in the context of Cavalier-Smith's far-reaching chromalveolate hypothesis. In our opinion, a more plausible evolutionary scenario would be the establishment of a unique secondary rhodoplast and its subsequent spread via tertiary endosymbioses.
Assuntos
Proteínas de Algas/genética , Eucariotos/enzimologia , Frutose-Bifosfatase/genética , Monoéster Fosfórico Hidrolases/genética , Proteínas de Plantas/genética , Plantas/enzimologia , Animais , Apicomplexa/genética , Ascomicetos/genética , Cilióforos/genética , DNA de Algas/genética , DNA de Plantas/genética , Diatomáceas/genética , Dinoflagellida/genética , Emigração e Imigração , Eucariotos/genética , Eucariotos/fisiologia , Evolução Molecular , Transferência Genética Horizontal , Dados de Sequência Molecular , Filogenia , Fenômenos Fisiológicos Vegetais , Plantas/genética , Plastídeos/genética , Análise de Sequência de DNA , Homologia de Sequência , Simbiose , Trypanosoma/genéticaRESUMO
Theiler´s murine encephalomyelitis virus (TMEV)-infection is a widely used animal model for studying demyelinating disorders, including multiple sclerosis (MS). The immunosuppressive cytokine Interleukin (IL)-10 counteracts hyperactive immune responses and critically controls immune homeostasis in infectious and autoimmune disorders. In order to investigate the effect of signaling via Interleukin-10 receptor (IL-10R) in infectious neurological diseases, TMEV-infected SJL mice were treated with IL-10R blocking antibody (Ab) in the acute and chronic phase of the disease. The findings demonstrate that (i) Ab-mediated IL-10 neutralization leads to progressive colitis with a reduction in Foxp3+ regulatory T cells and increased numbers of CD8+CD44+ memory T cells as well as activated CD4+CD69+ and CD8+CD69+ T cells in uninfected mice. (ii) Concurrent acute TMEV-infection worsened enteric disease-mediated by IL-10R neutralization. Virus-triggered effects were associated with an enhanced activation of CD4+ T helper cells and CD8+ cytotoxic T lymphocytes and augmented cytokine expression. By contrast, (iii) IL-10R neutralization during chronic TMEV-infection was not associated with enhanced peripheral immunopathology but an increased CD3+ T cell influx in the spinal cord. IL-10R neutralization causes a breakdown in peripheral immune tolerance in genetically predisposed mice, which leads to immune-mediated colitis, resembling inflammatory bowel disease. Hyperactive immune state following IL-10R blockade is enhanced by central nervous system-restricted viral infection in a disease phase-dependent manner.
Assuntos
Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/virologia , Colite/metabolismo , Colite/patologia , Colite/virologia , Receptores de Interleucina-10/metabolismo , Animais , Anticorpos/farmacologia , Antígenos CD , Antígenos de Diferenciação de Linfócitos T , Complexo CD3/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Feminino , Fatores de Transcrição Forkhead/metabolismo , Receptores de Hialuronatos/metabolismo , Lectinas Tipo C , Camundongos , Receptores de Interleucina-10/antagonistas & inibidores , Receptores de Interleucina-10/deficiência , Linfócitos T/metabolismo , Theilovirus/fisiologiaRESUMO
Food antigens are harmless environmental components. The physiological response is the development of clinical and immunological tolerance. It is now well appreciated that tolerance development is the result of active immunoregulation and depends on a close interaction between the innate and adaptive immune system resulting in the development of tolerance-mediating T-cell responses. Programming of the immune system, particularly with regard to tolerance development, already starts before birth and stays under close control of the maternal immune system. Therefore, the pre-and postnatal period represents an important 'window of opportunity' for immunoprogramming. Underlying mechanisms include maternal cell transmission, antibody transfer, transfer of mediates/cytokines, and transmission of antigens and allergens. Immunoprogramming is fostered and augmented in the context of microbial components. Recently, several microbes have been identified which possess the capacity of immunoprogramming early in life. Epigenetic regulation represents an important novel mechanism in this regard. This concept opens new avenues for the development of preventive strategies to avoid inappropriate immune responses against food antigens.
Assuntos
Proteínas Alimentares/imunologia , Hipersensibilidade Alimentar , Tolerância Imunológica/fisiologia , Fatores Imunológicos/fisiologia , Troca Materno-Fetal/imunologia , Envelhecimento/imunologia , Alérgenos/fisiologia , Anticorpos/fisiologia , Epigênese Genética , Feminino , Hipersensibilidade Alimentar/embriologia , Hipersensibilidade Alimentar/imunologia , Hipersensibilidade Alimentar/prevenção & controle , Trato Gastrointestinal/citologia , Trato Gastrointestinal/crescimento & desenvolvimento , Trato Gastrointestinal/microbiologia , Humanos , Imunidade , Imunidade Inata , Imunidade Materno-Adquirida , Masculino , Gravidez , Linfócitos T/imunologiaRESUMO
The pre- and postnatal environment may represent a window of opportunity for allergy and asthma prevention, and the hygiene hypothesis implies that microbial agents may play an important role in this regard. Using the cowshed-derived bacterium Acinetobacter lwoffii F78 together with a mouse model of experimental allergic airway inflammation, this study investigated the hygiene hypothesis, maternal (prenatal) microbial exposure, and the involvement of Toll-like receptor (TLR) signaling in prenatal protection from asthma. Maternal intranasal exposure to A. lwoffii F78 protected against the development of experimental asthma in the progeny. Maternally, A. lwoffii F78 exposure resulted in a transient increase in lung and serum proinflammatory cytokine production and up-regulation of lung TLR messenger RNA. Conversely, suppression of TLRs was observed in placental tissue. To investigate further, the functional relevance of maternal TLR signaling was tested in TLR2/3/4/7/9(-/-) knockout mice. The asthma-preventive effect was completely abolished in heterozygous offspring from A. lwoffii F78-treated TLR2/3/4/7/9(-/-) homozygous mother mice. Furthermore, the mild local and systemic inflammatory response was also absent in these A. lwoffii F78-exposed mothers. These data establish a direct relationship between maternal bacterial exposures, functional maternal TLR signaling, and asthma protection in the progeny.
Assuntos
Acinetobacter/imunologia , Asma/prevenção & controle , Feto/imunologia , Transdução de Sinais/fisiologia , Receptores Toll-Like/fisiologia , Líquido Amniótico/química , Animais , Endotoxinas/análise , Feminino , Imunidade Inata , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovalbumina/imunologia , Gravidez , RNA Mensageiro/análise , Receptores Toll-Like/genéticaRESUMO
Independent evidence from morphological, ultrastructural, biochemical, and molecular data have shown that land plants originated from charophycean green algae. However, the branching order within charophytes is still unresolved, and contradictory phylogenies about, for example,the position of the unicellular green alga Mesostigma viride are difficult to reconcile. A comparison of nuclear-encoded Calvin cycle glyceraldehyde-3-phosphate dehydrogenases (GAPDH) indicates that a crucial duplication of the GapA gene occurred early in land plant evolution. The duplicate called GapB acquired a characteristic carboxy-terminal extension (CTE) from the general regulator of the Calvin cycle CP12. This CTE is responsible for thioredoxin-dependent light/dark regulation. In this work, we established GapA, GapB, and CP12 sequences from bryophytes, all orders of charophyte as well as chlorophyte green algae, and the glaucophyte Cyanophora paradoxa. Comprehensive phylogenetic analyses of all available plastid GAPDH sequences suggest that glaucophytes and green plants are sister lineages and support a positioning of Mesostigma basal to all charophycean algae. The exclusive presence of GapB in terrestrial plants, charophytes, and Mesostigma dates the GapA/B gene duplication to the common ancestor of Streptophyta. The conspicuously high degree of GapB sequence conservation suggests an important metabolic role of the newly gained regulatory function. Because the GapB-mediated protein aggregation most likely ensures the complete blockage of the Calvin cycle at night, we propose that this mechanism is also crucial for efficient starch mobilization. This innovation may be one prerequisite for the development of storage tissues in land plants.
Assuntos
Caráceas/genética , Duplicação Gênica , Gliceraldeído-3-Fosfato Desidrogenases/genética , Plantas/genética , Sequência de Aminoácidos , Teorema de Bayes , Briófitas/genética , Caráceas/classificação , Clorófitas/genética , Cianobactérias/genética , Cyanophora/genética , Evolução Molecular , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/classificaçãoRESUMO
Phosphoribulokinase (PRK) is an essential enzyme of photosynthetic eukaryotes which is active in the plastid-located Calvin cycle and regenerates the substrate for ribulose-bisphosphate carboxylase/oxygenase (Rubisco). Rhodophytes and chlorophytes (red and green algae) recruited their nuclear-encoded PRK from the cyanobacterial ancestor of plastids. The plastids of these organisms can be traced back to a single primary endosymbiosis, whereas, for example, haptophytes, dinoflagellates, and euglenophytes obtained their "complex" plastids through secondary endosymbioses, comprising the engulfment of a unicellular red or green alga by a eukaryotic host cell. We have cloned eight new PRK sequences from complex algae as well as a rhodophyte in order to investigate their evolutionary origin. All available PRK sequences were used for phylogenetic analyses and the significance of alternative topologies was estimated by the approximately unbiased test. Our analyses led to several astonishing findings. First, the close relationship of PRK genes of haptophytes, heterokontophytes, cryptophytes, and dinophytes (complex red lineage) supports a monophyletic origin of their sequences and hence their plastids. Second, based on PRK genes the complex red lineage forms a highly supported assemblage together with chlorophytes and land plants, to the exclusion of the rhodophytes. This green affinity is in striking contrast to the expected red algal origin and our analyses suggest that the PRK gene was acquired once via lateral transfer from a green alga. Third, surprisingly the complex green lineages leading to Bigelowiella and Euglena probably also obtained their PRK genes via lateral gene transfers from a red alga and a complex alga with red plastids, respectively.