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1.
Nat Commun ; 11(1): 3143, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561719

RESUMO

Topoisomerase 3ß (Top3ß) is the only dual-activity topoisomerase in animals that can change topology for both DNA and RNA, and facilitate transcription on DNA and translation on mRNAs. Top3ß mutations have been linked to schizophrenia, autism, epilepsy, and cognitive impairment. Here we show that Top3ß knockout mice exhibit behavioural phenotypes related to psychiatric disorders and cognitive impairment. The mice also display impairments in hippocampal neurogenesis and synaptic plasticity. Notably, the brains of the mutant mice exhibit impaired global neuronal activity-dependent transcription in response to fear conditioning stress, and the affected genes include many with known neuronal functions. Our data suggest that Top3ß is essential for normal brain function, and that defective neuronal activity-dependent transcription may be a mechanism by which Top3ß deletion causes cognitive impairment and psychiatric disorders.


Assuntos
Disfunção Cognitiva/genética , DNA Topoisomerases Tipo I/genética , Transtornos Mentais/genética , Neurogênese/genética , Plasticidade Neuronal/genética , Animais , Técnicas de Observação do Comportamento , Comportamento Animal , Disfunção Cognitiva/diagnóstico , Disfunção Cognitiva/patologia , Modelos Animais de Doenças , Feminino , Hipocampo/citologia , Hipocampo/diagnóstico por imagem , Hipocampo/crescimento & desenvolvimento , Hipocampo/patologia , Humanos , Imageamento por Ressonância Magnética , Masculino , Transtornos Mentais/diagnóstico , Transtornos Mentais/patologia , Camundongos , Camundongos Knockout , Neurônios/patologia , Técnicas Estereotáxicas , Potenciais Sinápticos/genética , Transcrição Genética/fisiologia
2.
Neurobiol Dis ; 129: 79-92, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31102766

RESUMO

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease due to motor neuron (MN) loss. The mechanisms causing selective MN death are largely unknown, thus prejudicing successful pharmacological treatments. Major causes of MN damage are effects downstream of the abnormal glutamate (Glu) neurotransmission. Group I metabotropic Glu receptors (mGluR1, mGluR5) actively contribute to the excitotoxicity in ALS and represent druggable molecular targets. We previously demonstrated that halving mGluR1 or mGluR5 expression in the widely studied SOD1G93A mouse model of ALS had a positive impact on disease onset, clinical progression and survival, as well as on cellular and biochemical parameters altered in ALS. Whereas these effects were similar in female and male mGluR1 heterozygous SOD1G93Amice, only male mGluR5 heterozygous SOD1G93A mice showed improved motor skills during disease progression. To further validate the role of Group I mGluRs in ALS, we generated in this study mGluR1 or mGluR5 null mice expressing the SOD1G93A mutation (SOD1G93AGrm1crv4/crv4 or SOD1G93AGrm5-/-, respectively). SOD1G93AGrm1crv4/crv4 mice showed early and progressive motor impairments and died even before SOD1G93A mice, while SOD1G93AGrm5-/- mice exhibited delayed disease onset, longer survival, and ameliorated motor skills than SOD1G93A mice. No difference between female and male SOD1G93AGrm5-/- mice were observed. These effects were associated with enhanced MN preservation and decreased astrocytic and microglial activation. Our results strongly support the assumption that constitutively lowering of mGluR5 expression has a positive impact in mice with ALS by counteracting the abnormal Glu transmission and this could be a potentially effective pharmacological target in ALS.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Receptor de Glutamato Metabotrópico 5/deficiência , Esclerose Amiotrófica Lateral/genética , Animais , Modelos Animais de Doenças , Progressão da Doença , Feminino , Masculino , Camundongos , Camundongos Knockout , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Medula Espinal/metabolismo , Medula Espinal/patologia , Superóxido Dismutase/genética
3.
Eur J Med Genet ; 62(9): 103555, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30359776

RESUMO

The KCNQ5 gene, widely expressed in the brain, encodes a voltage-gated potassium channel (Kv7.5) important for neuronal function. Here, we report a novel KCNQ5 intragenic duplication at 6q13 spanning about 239 Kb of genomic DNA, identified by array comparative genomic hybridization (array-CGH). The duplication was found in heterozygosity in an adult patient affected by mild intellectual disability with history of absence epilepsy in adolescence, with no EEG nor MRI alterations. By in vitro analyses we demonstrated that this copy number variation (CNV) led to an aberrant transcript with exon 2-11 skipping and a premature stop codon causing, most likely, haploinsufficiency. The Kv7.5 channel plays an important role in the regulation of M-type current and afterhyperpolarization conductances which contribute to neuronal excitability. A recently published paper described KCNQ5 missense mutations in individuals with intellectual disability and treatment-resistant epilepsy that were thought to act through either loss-of-function or gain-of-function mechanisms, associated in both cases with altered neuronal excitability. In the case reported here, we showed that no functional protein can be produced from the allele involved by the intragenic duplication. This evidence strongly supports the hypothesis of KCNQ5 haploinsufficiency, which could lead to altered neuronal excitability, thus contributing to seizure susceptibility and intellectual disability.


Assuntos
Duplicação Gênica , Deficiência Intelectual/genética , Canais de Potássio KCNQ/genética , Mutação com Perda de Função , Códon de Terminação , Feminino , Humanos , Deficiência Intelectual/patologia , Splicing de RNA , Adulto Jovem
4.
Front Mol Neurosci ; 11: 324, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30279647

RESUMO

Mouse cortical GABAergic synaptosomes possess presynaptic inhibitory GABAB autoreceptors. Accordingly, (±)baclofen (3 µM) inhibits in a CGP53423-sensitive manner the 12 mM KCl-evoked release of preloaded [3H]GABA. Differently, the existence of presynaptic release-regulating metabotropic glutamate type 1 (mGlu1) heteroreceptors in these terminals is still matter of discussion, although confocal microscopy unveiled the existence of mGlu1α with GABAB1 or GABAB2 proteins in cortical VGAT-positive synaptosomes. The group I mGlu agonist 3,5-DHPG failed to modify on its own the 12 mM KCl-evoked [3H]GABA exocytosis from cortical nerve endings, but, when added concomitantly to the GABAB agonist, it significantly reduced the 3 µM (±)baclofen-induced inhibition of [3H]GABA exocytosis. Conversely, the mGlu1 antagonist LY367385 (0.03-1 µM), inactive on its own on GABA exocytosis, amplified the 3 µM (±)baclofen-induced inhibition of [3H]GABA overflow. The ( ± )baclofen-induced inhibition of [3H]GABA exocytosis was more pronounced in cortical synaptosomes from Grm1crv4/crv4 mice, which bear a spontaneous mutation of the Grm1 gene leading to the functional inactivation of the mGlu1 receptor. Inasmuch, the expression of GABAB2 receptor protein in cortical synaptosomal lysates from Grm1crv4/crv4 mice was increased when compared to controls. Altogether, these observations seem best interpreted by assuming that mGlu1 coexist with GABAB receptors in GABAergic cortical synaptosomes, where they control GABA receptors in an antagonist-like manner. We then asked whether the mGlu1-mediated control of GABAB receptors is restricted to GABAergic terminals, or if it occurs also in other subpopulations of nerve endings. Release-regulating GABAB receptors also exist in glutamatergic nerve endings. (±)baclofen (1 µM) diminished the 12 mM KCl-evoked [3H]D-aspartate overflow. Also in these terminals, the concomitant presence of 1 µM LY367385, inactive on its own, significantly amplified the inhibitory effect exerted by (±)baclofen on [3H]D-aspartate exocytosis. Confocal microscopy confirmed the colocalization of mGlu1 with GABAB1 and GABAB2 labeling in vesicular glutamate type1 transporter-positive particles. Our results support the conclusion that mGlu1 receptors modulate in an antagonist-like manner presynaptic release-regulating GABAB receptors. This receptor-receptor interaction could be neuroprotective in central disease typified by hyperglutamatergicity.

5.
Int J Mol Sci ; 19(9)2018 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-30217050

RESUMO

Following herbivore attacks, plants modify a blend of volatiles organic compounds (VOCs) released, resulting in the attraction of their antagonists. However, volatiles released constitutively may affect herbivores and natural enemies' fitness too. In tomato there is still a lack of information on the genetic bases responsible for the constitutive release of VOC involved in direct and indirect defenses. Here we studied the constitutive emissions related to the two most abundant sesquiterpene synthase genes expressed in tomato and their functional role in plant defense. Using an RNA interference approach, we silenced the expression of TPS9 and TPS12 genes and assessed the effect of this transformation on herbivores and parasitoids. We found that silenced plants displayed a different constitutive volatiles emission from controls, resulting in reduced attractiveness for the aphid parasitoid Aphidius ervi and in an impaired development of Spodoptera exigua larvae. We discussed these data considering the transcriptional regulation of key-genes involved in the pathway of VOC metabolism. We provide several lines of evidence on the metabolic flux from terpenoids to phenylpropanoids. Our results shed more light on constitutive defenses mediated by plant volatiles and on the molecular mechanisms involved in their metabolic regulation.


Assuntos
Herbivoria/fisiologia , Lycopersicon esculentum/metabolismo , Lycopersicon esculentum/parasitologia , Animais , Afídeos/fisiologia , Interações Hospedeiro-Parasita , Spodoptera/fisiologia , Compostos Orgânicos Voláteis/metabolismo , Vespas/fisiologia
6.
Neurobiol Dis ; 109(Pt A): 44-53, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28982591

RESUMO

Deleterious mutations in the glutamate receptor metabotropic 1 gene (GRM1) cause a recessive form of cerebellar ataxia, SCAR13. GRM1 and GRM5 code for the metabotropic glutamate type 1 (mGlu1) and type 5 (mGlu5) receptors, respectively. Their different expression profiles suggest they could have distinct functional roles. In a previous study, homozygous mice lacking mGlu1 receptors (Grm1crv4/crv4) and exhibiting ataxia presented cerebellar overexpression of mGlu5 receptors, that was proposed to contribute to the mouse phenotype. To test this hypothesis, we here crossed Grm1crv4 and Grm5ko mice to generate double mutants (Grm1crv4/crv4Grm5ko/ko) lacking both mGlu1 and mGlu5 receptors. Double mutants and control mice were analyzed for spontaneous behavior and for motor activity by rotarod and footprint analyses. In the same mice, the release of glutamate from cerebellar nerve endings (synaptosomes) elicited by 12mM KCl or by α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) was also evaluated. Motor coordination resulted improved in double mutants when compared to Grm1crv4/crv4 mice. Furthermore, in in vitro studies, glutamate release elicited by both KCl depolarization and activation of AMPA autoreceptors resulted reduced in Grm1crv4/crv4 mice compared to wild type mice, while it presented normal levels in double mutants. Moreover, we found that Grm1crv4/crv4 mice showed reduced expression of GluA2/3 AMPA receptor subunits in cerebellar synaptosomes, while it resulted restored to wild type level in double mutants. To conclude, blocking of mGlu5 receptor reduced the dysregulation of glutamate transmission and improved motor coordination in the Grm1crv4 mouse model of SCAR13, thus suggesting the possible usefulness of pharmacological therapies based on modulation of mGlu5 receptor activity for the treatment of this type of ataxia.


Assuntos
Ataxia Cerebelar/genética , Ataxia Cerebelar/fisiopatologia , Atividade Motora , Receptor de Glutamato Metabotrópico 5/genética , Receptores de Glutamato Metabotrópico/genética , Animais , Autorreceptores/metabolismo , Cerebelo/metabolismo , Modelos Animais de Doenças , Feminino , Ácido Glutâmico/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Subunidades Proteicas , Receptores de AMPA/metabolismo , Teste de Desempenho do Rota-Rod
7.
Neuropharmacology ; 123: 433-445, 2017 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-28645622

RESUMO

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder due to loss of upper and lower motor neurons (MNs). The mechanisms of neuronal death are largely unknown, thus prejudicing the successful pharmacological treatment. One major cause for MN degeneration in ALS is represented by glutamate(Glu)-mediated excitotoxicity. We have previously reported that activation of Group I metabotropic Glu receptors (mGluR1 and mGluR5) at glutamatergic spinal cord nerve terminals produces abnormal Glu release in the widely studied SOD1G93A mouse model of ALS. We also demonstrated that halving mGluR1 expression in the SOD1G93A mouse had a positive impact on survival, disease onset, disease progression, and on a number of cellular and biochemical readouts of ALS. We generated here SOD1G93A mice with reduced expression of mGluR5 (SOD1G93AGrm5-/+) by crossing the SOD1G93A mutant mouse with the mGluR5 heterozigous Grm5-/+ mouse. SOD1G93AGrm5-/+ mice showed prolonged survival probability and delayed pathology onset. These effects were associated to enhanced number of preserved MNs, decreased astrocyte and microglia activation, reduced cytosolic free Ca2+ concentration, and regularization of abnormal Glu release in the spinal cord of SOD1G93AGrm5-/+ mice. Unexpectedly, only male SOD1G93AGrm5-/+ mice showed improved motor skills during disease progression vs. SOD1G93A mice, while SOD1G93AGrm5-/+ females did not. These results demonstrate that a lower constitutive level of mGluR5 has a significant positive impact in mice with ALS and support the idea that blocking Group I mGluRs may represent a potentially effective pharmacological approach to the disease.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Receptor de Glutamato Metabotrópico 5/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Morte Celular/fisiologia , Sobrevivência Celular/fisiologia , Modelos Animais de Doenças , Progressão da Doença , Feminino , Ácido Glutâmico/metabolismo , Humanos , Masculino , Camundongos Transgênicos , Microglia/metabolismo , Microglia/patologia , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Destreza Motora/fisiologia , Receptor de Glutamato Metabotrópico 5/genética , Receptores de Glutamato Metabotrópico/metabolismo , Fatores Sexuais , Medula Espinal/metabolismo , Medula Espinal/patologia , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo
8.
Bone ; 94: 114-123, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27989650

RESUMO

Recent increasing evidence supports a role for neuronal type signaling in bone. Specifically glutamate receptors have been found in cells responsible for bone remodeling, namely the osteoblasts and the osteoclasts. While most studies have focused on ionotropic glutamate receptors, the relevance of the metabotropic glutamate signaling in bone is poorly understood. Specifically type 1 metabotropic glutamate (mGlu1) receptors are expressed in bone, but the effect of its ablation on skeletal development has never been investigated. Here we report that Grm1crv4/crv4 mice, homozygous for an inactivating mutation of the mGlu1 receptor, and mainly characterized by ataxia and renal dysfunction, exhibit decreased body weight, bone length and bone mineral density compared to wild type (WT) animals. Blood analyses of the affected mice demonstrate the absence of changes in circulating factors, such as vitamin D and PTH, suggesting renal damage is not the main culprit of the skeletal phenotype. Cultures of osteoblasts lacking functional mGlu1 receptors exhibit less homogeneous collagen deposition than WT cells, and present increased expression of osteocalcin, a marker of osteoblast maturation. These data suggest that the skeletal damage is directly linked to the absence of the receptor, which in turn leads to osteoblasts dysfunction and earlier maturation. Accordingly, skeletal histomorphology suggests that Grm1crv4/crv4 mice exhibit enhanced bone maturation, resulting in premature fusion of the growth plate and shortened long bones, and further slowdown of bone apposition rate compared to the WT animals. In summary, this work reveals novel functions of mGlu1 receptors in the bone and indicates that in osteoblasts mGlu1 receptors are necessary for production of normal bone matrix, longitudinal bone growth, and normal skeletal development.


Assuntos
Calcificação Fisiológica , Receptores de Glutamato Metabotrópico/metabolismo , Animais , Tamanho Corporal , Densidade Óssea , Desenvolvimento Ósseo , Diferenciação Celular , Feminino , Masculino , Camundongos Endogâmicos BALB C , Tamanho do Órgão , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Fenótipo
9.
BMC Med Genet ; 17(1): 89, 2016 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-27884122

RESUMO

BACKGROUND: Poland Syndrome (PS) is a rare congenital disorder presenting with agenesis/hypoplasia of the pectoralis major muscle variably associated with thoracic and/or upper limb anomalies. Most cases are sporadic, but familial recurrence, with different inheritance patterns, has been observed. The genetic etiology of PS remains unknown. Karyotyping and array-comparative genomic hybridization (CGH) analyses can identify genomic imbalances that can clarify the genetic etiology of congenital and neurodevelopmental disorders. We previously reported a chromosome 11 deletion in twin girls with pectoralis muscle hypoplasia and skeletal anomalies, and a chromosome six deletion in a patient presenting a complex phenotype that included pectoralis muscle hypoplasia. However, the contribution of genomic imbalances to PS remains largely unknown. METHODS: To investigate the prevalence of chromosomal imbalances in PS, standard cytogenetic and array-CGH analyses were performed in 120 PS patients. RESULTS: Following the application of stringent filter criteria, 14 rare copy number variations (CNVs) were identified in 14 PS patients in different regions outside known common copy number variations: seven genomic duplications and seven genomic deletions, enclosing the two previously reported PS associated chromosomal deletions. These CNVs ranged from 0.04 to 4.71 Mb in size. Bioinformatic analysis of array-CGH data indicated gene enrichment in pathways involved in cell-cell adhesion, DNA binding and apoptosis processes. The analysis also provided a number of candidate genes possibly causing the developmental defects observed in PS patients, among others REV3L, a gene coding for an error-prone DNA polymerase previously associated with Möbius Syndrome with variable phenotypes including pectoralis muscle agenesis. CONCLUSIONS: A number of rare CNVs were identified in PS patients, and these involve genes that represent candidates for further evaluation. Rare inherited CNVs may contribute to, or represent risk factors of PS in a multifactorial mode of inheritance.


Assuntos
Hibridização Genômica Comparativa/métodos , Variações do Número de Cópias de DNA , Redes Reguladoras de Genes , Cariotipagem/métodos , Síndrome de Poland/genética , Duplicação Cromossômica , Feminino , Predisposição Genética para Doença , Humanos , Masculino , Deleção de Sequência
10.
J Plant Physiol ; 173: 28-32, 2015 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-25462075

RESUMO

In the last decade plant-to-plant communication has received an increasing attention, particularly for the role of Volatile Organic Compounds as possible elicitors of plant defense. The role of ß-ocimene as an interspecific elicitor of plant defense has been recently assessed in multitrophic systems including different plant species (Solanaceae, Poaceae, legumes) and different pest species including chewer insects and phytophagous mites. Both chewer insects and phytophagous mites are known to elicit specific plant defensive pathways which are different (at least in part) from those elicited by sap feeders. The aim of this research was to fill this gap of knowledge and to assess the role of ß-ocimene as an elicitor of plant defense against aphid pests, which are sap feeders. For this purpose we used as transgenic tobacco plant releasing an odour plume enriched in this compound as emitter and a tomato plant as receiver. We selected the aphid Macrosiphum euphorbiae and its natural enemy, the parasitoid Aphidius ervi, as the targets of plant induced defense. Tomato plant defense induced by ß-ocimene was assessed by characterizing the aphid performance in terms of fixing behaviour, development and reproduction (direct plant defense) and the parasitoid performance in terms of attraction towards tomato plants (indirect plant defense). The characterization of tomato response to ß-ocimene was completed by the identification of Volatile Organic Compounds as released by conditioned tomato plants. Tomato plants that were exposed to the volatiles of transgenic tobacco enriched in ß-ocimene resulted in less suitable for the aphids in respect to control ones (direct defense). On tomato plants "elicited" by ß-ocimene we recorded: a significant lower number of aphids settled; a significant lower number newborn nymphs; a significant lower weight of aphids feeding. In addition, tomato plants "elicited" by ß-ocimene resulted became more attractive towards the parasitoid A. ervi than control ones. These results could be explained at least in part by examining the composition of the Volatile Organic Compounds released by tomato plants "elicited" by ß-ocimene. Indeed, we found a significantly higher release of several compounds including methyl salicylate and cis-3-hexen-1-ol. These two compounds have been demonstrated to impair aphid development and reproduction and to be involved in the attraction of the aphid parasitoid A. ervi. By considering the ubiquity of ß-ocimene and its ability to regulate the communication of plants belonging 30 to different species (if not families), we concluded that this compound is an ideal candidate for new 31 strategies of sustainable control of agricultural pests.


Assuntos
Alcenos/metabolismo , Afídeos/fisiologia , Interações Hospedeiro-Parasita , Lycopersicon esculentum/imunologia , Imunidade Vegetal , Tabaco/química , Monoterpenos Acíclicos , Animais , Expressão Gênica , Hexanóis/metabolismo , Lycopersicon esculentum/efeitos dos fármacos , Lycopersicon esculentum/genética , Plantas Geneticamente Modificadas , Salicilatos/metabolismo , Tabaco/genética , Compostos Orgânicos Voláteis/metabolismo
11.
Mol Plant Microbe Interact ; 26(10): 1249-56, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23718124

RESUMO

Below ground and above ground plant-insect-microorganism interactions are complex and regulate most of the developmental responses of important crop plants such as tomato. We investigated the influence of root colonization by a nonmycorrhizal plant-growth-promoting fungus on direct and indirect defenses of tomato plant against aphids. The multitrophic system included the plant Solanum lycopersicum ('San Marzano nano'), the root-associated biocontrol fungus Trichoderma longibrachiatum strain MK1, the aphid Macrosiphum euphorbiae (a tomato pest), the aphid parasitoid Aphidius ervi, and the aphid predator Macrolophus pygmaeus. Laboratory bioassays were performed to assess the effect of T. longibrachiatum MK1, interacting with the tomato plant, on quantity and quality of volatile organic compounds (VOC) released by tomato plant, aphid development and reproduction, parasitoid behavior, and predator behavior and development. When compared with the uncolonized controls, plants whose roots were colonized by T. longibrachiatum MK1 showed quantitative differences in the release of specific VOC, better aphid population growth indices, a higher attractiveness toward the aphid parasitoid and the aphid predator, and a quicker development of aphid predator. These findings support the development of novel strategies of integrated control of aphid pests. The species-specific or strain-specific characteristics of these below ground-above ground interactions remain to be assessed.


Assuntos
Afídeos/fisiologia , Himenópteros/fisiologia , Lycopersicon esculentum/microbiologia , Controle Biológico de Vetores , Doenças das Plantas/microbiologia , Trichoderma/fisiologia , Animais , Interações Hospedeiro-Patógeno , Lycopersicon esculentum/química , Lycopersicon esculentum/parasitologia , Doenças das Plantas/parasitologia , Raízes de Plantas/química , Raízes de Plantas/microbiologia , Raízes de Plantas/parasitologia , Especificidade da Espécie , Compostos Orgânicos Voláteis/metabolismo
12.
PLoS One ; 7(3): e32822, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22448229

RESUMO

BACKGROUND: Ginkgo biloba (Ginkgoaceae) is one of the most ancient living seed plants and is regarded as a living fossil. G. biloba has a broad spectrum of resistance or tolerance to many pathogens and herbivores because of the presence of toxic leaf compounds. Little is known about early and late events occurring in G. biloba upon herbivory. The aim of this study was to assess whether herbivory by the generalist Spodoptera littoralis was able to induce early signaling and direct defense in G. biloba by evaluating early and late responses. METHODOLOGY/PRINCIPAL FINDINGS: Early and late responses in mechanically wounded leaves and in leaves damaged by S. littoralis included plasma transmembrane potential (Vm) variations, time-course changes in both cytosolic calcium concentration ([Ca(2+)](cyt)) and H(2)O(2) production, the regulation of genes correlated to terpenoid and flavonoid biosynthesis, the induction of direct defense compounds, and the release of volatile organic compounds (VOCs). The results show that G. biloba responded to hebivory with a significant Vm depolarization which was associated to significant increases in both [Ca(2+)](cyt) and H(2)O(2). Several defense genes were regulated by herbivory, including those coding for ROS scavenging enzymes and the synthesis of terpenoids and flavonoids. Metabolomic analyses revealed the herbivore-induced production of several flavonoids and VOCs. Surprisingly, no significant induction by herbivory was found for two of the most characteristic G. biloba classes of bioactive compounds; ginkgolides and bilobalides. CONCLUSIONS/SIGNIFICANCE: By studying early and late responses of G. biloba to herbivory, we provided the first evidence that this "living fossil" plant responds to herbivory with the same defense mechanisms adopted by the most recent angiosperms.


Assuntos
Comportamento Alimentar , Genes de Plantas , Ginkgo biloba/metabolismo , Folhas de Planta/metabolismo , Folhas de Planta/parasitologia , Transdução de Sinais , Spodoptera/fisiologia , Animais , Biomarcadores/metabolismo , Cálcio/metabolismo , Citosol/metabolismo , Flavonoides/metabolismo , Perfilação da Expressão Gênica , Peróxido de Hidrogênio/metabolismo , Potenciais da Membrana , Análise de Sequência com Séries de Oligonucleotídeos , Extratos Vegetais/farmacologia , RNA Mensageiro/genética , RNA de Plantas/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Compostos Orgânicos Voláteis/metabolismo
13.
J Plant Physiol ; 168(17): 2041-7, 2011 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-21831477

RESUMO

Plants react to microbial attack with a number of defense mechanisms, including the synthesis of volatile organic compounds (VOCs) and the production of reactive oxygen species (ROS). These responses are triggered by elicitors derived from either the cell surface of pathogens or the incomplete hydrolysis of the plant cell wall. Here we show the response of rice (Oryza sativa L., cv Gigante Vercelli) cell cultures following treatment with cell wall hydrolysates prepared from the rice blast Magnaporthe oryzae. Elicitation prompted the production of several plant VOCs, which were analyzed by stir bar sorptive extraction from both the liquid and head-space phase (SBSE and HSSE, respectively) and gas chromatography coupled to mass spectrometry (GC-MS) analysis. VOCs included alkanes, alkenes and long-chain alcohols as well as cinnamyl alcohol, myristicin, a sesquiterpene alcohol (caryolan-1-ol), 1-butanamide and 2-pentylfuran. The major released compounds, 1-octanol and 1-decanol, were found to induce ROS production in both elicited and non-elicited rice cells and showed fungistatic activity against the pathogen M. oryzae. The possible role of induced VOCs and ROS production in the plant-pathogen interaction is discussed.


Assuntos
Extratos Celulares/farmacologia , Magnaporthe/química , Oryza/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Compostos Orgânicos Voláteis/metabolismo , 1-Octanol/metabolismo , Extratos Celulares/isolamento & purificação , Parede Celular/metabolismo , Álcoois Graxos/metabolismo , Interações Hospedeiro-Patógeno , Magnaporthe/fisiologia , Oryza/química , Oryza/microbiologia , Oryza/fisiologia , Doenças das Plantas/microbiologia , Explosão Respiratória
14.
PLoS One ; 6(3): e17195, 2011 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-21408066

RESUMO

Interactions between herbivorous insects and plants storing terpenoids are poorly understood. This study describes the ability of Chrysolina herbacea to use volatiles emitted by undamaged Mentha aquatica plants as attractants and the plant's response to herbivory, which involves the production of deterrent molecules. Emitted plant volatiles were analyzed by GC-MS. The insect's response to plant volatiles was tested by Y-tube olfactometer bioassays. Total RNA was extracted from control plants, mechanically damaged leaves, and leaves damaged by herbivores. The terpenoid quantitative gene expressions (qPCR) were then assayed. Upon herbivory, M. aquatica synthesizes and emits (+)-menthofuran, which acts as a deterrent to C. herbacea. Herbivory was found to up-regulate the expression of genes involved in terpenoid biosynthesis. The increased emission of (+)-menthofuran was correlated with the upregulation of (+)-menthofuran synthase.


Assuntos
Besouros/fisiologia , Mentha/metabolismo , Mentha/parasitologia , Terpenos/metabolismo , Animais , Vias Biossintéticas/efeitos dos fármacos , Besouros/efeitos dos fármacos , Comportamento Alimentar/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Mentha/efeitos dos fármacos , Mentha/genética , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/parasitologia , Reação em Cadeia da Polimerase , Terpenos/química , Terpenos/farmacologia , Compostos Orgânicos Voláteis/análise , Compostos Orgânicos Voláteis/farmacologia
15.
FEMS Microbiol Ecol ; 76(2): 342-51, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21255049

RESUMO

Fusarium oxysporum MSA 35 [wild-type (WT) strain] is a nonpathogenic Fusarium strain, which exhibits antagonistic activity to plant pathogenic F. oxysporum isolates. The fungus lives in association with a consortium of ectosymbiotic bacteria. The WT strain, when cured of the bacterial symbionts [the cured (CU) form], is pathogenic, causing wilt symptoms similar to those of pathogenic F. oxysporum f. sp. lactucae. Both WT and CU MSA 35 strains produce microbial volatile organic compounds (MVOCs), but with a different spectrum. In vitro dual culture assays were used to assess the effects of the MVOCs produced by WT and CU strains of F. oxysporum MSA 35 on the growth and expansin gene expression of lettuce seedlings. An increase in the root length (95.6%), shoot length (75.0%) and fresh weight (85.8%) was observed only after WT strain MVOCs exposure. Leaf chlorophyll content was significantly enhanced (68%) in WT strain MVOC-treated seedlings as compared with CU strain volatiles and nontreated controls. ß-Caryophyllene was found to be one of the volatiles released by WT MSA 35 responsible for the plant growth promotion effect. Semi-quantitative and quantitative reverse transcription-PCR assays indicated a significant difference in the expansin gene expression level between leaf (6.7-fold) and roots (4.4-fold) exposed to WT strain volatiles when compared with the CU strain volatiles and those that were nonexposed.


Assuntos
Bactérias/crescimento & desenvolvimento , Fusarium/crescimento & desenvolvimento , Alface/crescimento & desenvolvimento , Proteínas de Plantas/metabolismo , Compostos Orgânicos Voláteis/análise , Bactérias/metabolismo , Parede Celular/metabolismo , DNA de Plantas/genética , Fusarium/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Alface/efeitos dos fármacos , Alface/genética , Alface/microbiologia , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Proteínas de Plantas/genética , Raízes de Plantas/metabolismo , Raízes de Plantas/microbiologia , Sesquiterpenos Policíclicos , Sesquiterpenos/análise , Sesquiterpenos/metabolismo , Sesquiterpenos/farmacologia , Simbiose , Compostos Orgânicos Voláteis/metabolismo , Compostos Orgânicos Voláteis/farmacologia
16.
J Photochem Photobiol B ; 100(2): 67-75, 2010 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-20627615

RESUMO

Modulation of secondary metabolites by UV-B involves changes in gene expression, enzyme activity and accumulation of defence metabolites. After exposing peppermint (Mentha x piperita L.) plants grown in field (FP) and in a growth chamber (GCP) to UV-B irradiation, we analysed by qRT-PCR the expression of genes involved in terpenoid biosynthesis and encoding: 1-deoxy-D-xylulose-5-phosphate synthase (Dxs), 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase (Mds), isopentenyl diphosphate isomerase (Ippi), geranyl diphosphate synthase (Gpps), (-)-limonene synthase (Ls), (-)-limonene-3-hydroxylase (L3oh), (+)-pulegone reductase (Pr), (-)-menthone reductase (Mr), (+)-menthofuran synthase (Mfs), farnesyl diphosphate synthase (Fpps) and a putative sesquiterpene synthase (S-TPS). GCP always showed a higher terpenoid content with respect to FP. We found that in both FP and GCP, most of these genes were regulated by the UV-B treatment. The amount of most of the essential oil components, which were analysed by gas chromatography-mass spectrometry (GC-MS), was not correlated to gene expression. The total phenol composition was found to be always increased after UV-B irradiation; however, FP always showed a higher phenol content with respect to GCP. Liquid chromatography-mass spectrometry (LC-ESI-MS/MS) analyses revealed the presence of UV-B absorbing flavonoids such as eriocitrin, hesperidin, and kaempferol 7-O-rutinoside whose content significantly increased in UV-B irradiated FP, when compared to GCP. The results of this work show that UV-B irradiation differentially modulates the expression of genes involved in peppermint essential oil biogenesis and the content of UV-B absorbing flavonoids. Plants grown in field were better adapted to increasing UV-B irradiation than plants cultivated in growth chambers. The interplay between terpenoid and phenylpropanoid metabolism is also discussed.


Assuntos
Flavonoides/análise , Mentha piperita/efeitos da radiação , Proteínas de Plantas/genética , Terpenos/análise , Raios Ultravioleta , Cromatografia Líquida de Alta Pressão , Flavanonas/análise , Flavonoides/biossíntese , Regulação da Expressão Gênica de Plantas , Hesperidina/análise , Mentha piperita/metabolismo , Óleos Voláteis/metabolismo , Fenóis/metabolismo , Proteínas de Plantas/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Terpenos/metabolismo
17.
New Phytol ; 187(4): 1089-1101, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20546139

RESUMO

*An integrated approach has been used to obtain an understanding of the molecular and chemical mechanisms underlying resistance to aphids in cherry-like tomato (Solanum lycopersicum) landraces from the Campania region (southern Italy). The aphid-parasitoid system Macrosiphum euphorbiae-Aphidius ervi was used to describe the levels of resistance against aphids in two tomato accessions (AN5, AN7) exhibiting high yield and quality traits and lacking the tomato Mi gene. *Aphid development and reproduction, flight response by the aphid parasitoid A. ervi, gas chromatography-mass spectrometry headspace analysis of plant volatile organic compounds and transcriptional analysis of aphid responsive genes were performed on selected tomato accessions and on a susceptible commercial variety (M82). *When compared with the cultivated variety, M82, AN5 and AN7 showed a significant reduction of M. euphorbiae fitness, the release of larger amounts of specific volatile organic compounds that are attractive to the aphid parasitoid A. ervi, a constitutively higher level of expression of plant defence genes and differential enhancement of plant indirect resistance induced by aphid feeding. *These results provide new insights on how local selection can offer the possibility of the development of innovative genetic strategies to increase tomato resistance against aphids.


Assuntos
Afídeos , Expressão Gênica , Genes de Plantas , Interações Hospedeiro-Parasita/genética , Lycopersicon esculentum/genética , Doenças das Plantas , Imunidade Vegetal/genética , Animais , Lycopersicon esculentum/química , Lycopersicon esculentum/metabolismo , Compostos Orgânicos Voláteis/metabolismo
18.
BMC Plant Biol ; 10: 97, 2010 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-20504319

RESUMO

BACKGROUND: Plant Ca2+ signals are involved in a wide array of intracellular signaling pathways after pest invasion. Ca2+-binding sensory proteins such as Ca2+-dependent protein kinases (CPKs) have been predicted to mediate the signaling following Ca2+ influx after insect herbivory. However, until now this prediction was not testable. RESULTS: To investigate the roles CPKs play in a herbivore response-signaling pathway, we screened the characteristics of Arabidopsis CPK mutants damaged by a feeding generalist herbivore, Spodoptera littoralis. Following insect attack, the cpk3 and cpk13 mutants showed lower transcript levels of plant defensin gene PDF1.2 compared to wild-type plants. The CPK cascade was not directly linked to the herbivory-induced signaling pathways that were mediated by defense-related phytohormones such as jasmonic acid and ethylene. CPK3 was also suggested to be involved in a negative feedback regulation of the cytosolic Ca2+ levels after herbivory and wounding damage. In vitro kinase assays of CPK3 protein with a suite of substrates demonstrated that the protein phosphorylates transcription factors (including ERF1, HsfB2a and CZF1/ZFAR1) in the presence of Ca2+. CPK13 strongly phosphorylated only HsfB2a, irrespective of the presence of Ca2+. Furthermore, in vivo agroinfiltration assays showed that CPK3-or CPK13-derived phosphorylation of a heat shock factor (HsfB2a) promotes PDF1.2 transcriptional activation in the defense response. CONCLUSIONS: These results reveal the involvement of two Arabidopsis CPKs (CPK3 and CPK13) in the herbivory-induced signaling network via HsfB2a-mediated regulation of the defense-related transcriptional machinery. This cascade is not involved in the phytohormone-related signaling pathways, but rather directly impacts transcription factors for defense responses.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Sinalização do Cálcio , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Spodoptera , Animais , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Cálcio/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Ciclopentanos/metabolismo , Citosol/metabolismo , Etilenos/metabolismo , Regulação da Expressão Gênica de Plantas , Mutação , Oxilipinas/metabolismo , Fosforilação , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , RNA de Plantas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tabaco/genética , Tabaco/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética
19.
Environ Microbiol ; 11(4): 844-54, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19396945

RESUMO

Fusarium oxysporum MSA35 [wild-type (WT) strain] is an antagonistic Fusarium that lives in association with a consortium of bacteria belonging to the genera Serratia, Achromobacter, Bacillus and Stenotrophomonas in an Italian soil suppressive to Fusarium wilt. Typing experiments and virulence tests provided evidence that the F. oxysporum isolate when cured of the bacterial symbionts [the cured (CU) form], is pathogenic, causing wilt symptoms identical to those caused by F. oxysporum f. sp. lactucae. Here, we demonstrate that small volatile organic compounds (VOCs) emitted from the WT strain negatively influence the mycelial growth of different formae speciales of F. oxysporum. Furthermore, these VOCs repress gene expression of two putative virulence genes in F. oxysporum lactucae strain Fuslat10, a fungus against which the WT strain MSA 35 has antagonistic activity. The VOC profile of the WT and CU fungus shows different compositions. Sesquiterpenes, mainly caryophyllene, were present in the headspace only of WT MSA 35. No sesquiterpenes were found in the volatiles of ectosymbiotic Serratia sp. strain DM1 and Achromobacter sp. strain MM1. Bacterial volatiles had no effects on the growth of the different ff. spp. of F. oxysporum examined. Hyphae grownwithVOCfrom WT F. oxysporum f. sp. lactucae strain MSA 35 were hydrophobic whereas those grown without VOCs were not, suggesting a correlation between the presence of volatiles in the atmosphere and the phenotype of the mycelium. This is the first report of VOC production by antagonistic F. oxysporum MSA35 and their effects on pathogenic F. oxysporum. The results obtained in this work led us to propose a new potential direct long-distance mechanism for antagonism by F. oxysporum MSA 35 mediated by VOCs. Antagonism could be the consequence of both reduction of pathogen mycelial growth and inhibition of pathogen virulence gene expression.


Assuntos
Antibiose , Fusarium/metabolismo , Fusarium/fisiologia , Microbiologia do Solo , Compostos Orgânicos Voláteis/metabolismo , Fusarium/efeitos dos fármacos , Fusarium/crescimento & desenvolvimento , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Itália , Micélio/efeitos dos fármacos , Micélio/crescimento & desenvolvimento , Fatores de Virulência/biossíntese
20.
Planta ; 227(2): 453-64, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17924138

RESUMO

Plant volatiles emitted by Medicago truncatula in response to feeding larvae of Spodoptera exigua are composed of a complex blend of terpenoids. The cDNAs of three terpene synthases (TPSs), which contribute to the blend of terpenoids, were cloned from M. truncatula. Their functional characterization proved MtTPS1 to be a beta-caryophyllene synthase and MtTPS5 to be a multi-product sesquiterpene synthase. MtTPS3 encodes a bifunctional enzyme producing (E)-nerolidol and geranyllinalool (precursors of C11 and C16 homoterpenes) from different prenyl diphosphates serving as substrates. The addition of jasmonic acid (JA) induced expression of the TPS genes, but terpenoid emission was higher from plants treated with JA and the ethylene precursor 1-amino-cyclopropyl-1-carboxylic acid. Compared to infested wild-type M. truncatula plants, lower amounts of various sesquiterpenes and a C11-homoterpene were released from an ethylene-insensitive mutant skl. This difference coincided with lower transcript levels of MtTPS5 and of 1-deoxy-D: -xylulose-5-phosphate synthase (MtDXS2) in the damaged skl leaves. Moreover, ethephon, an ethylene-releasing compound, modified the extent and mode of the herbivore-stimulated Ca2+ variations in the cytoplasm that is necessary for both JA and terpene biosynthesis. Thus, ethylene contributes to the herbivory-induced terpenoid biosynthesis at least twice: by modulating both early signaling events such as cytoplasmic Ca2+-influx and the downstream JA-dependent biosynthesis of terpenoids.


Assuntos
Sinalização do Cálcio/fisiologia , Ciclopentanos/farmacologia , Etilenos/farmacologia , Medicago truncatula/metabolismo , Mariposas/fisiologia , Oxilipinas/farmacologia , Terpenos/metabolismo , Animais , Comportamento Alimentar/fisiologia , Regulação da Expressão Gênica de Plantas , Larva/fisiologia , Medicago truncatula/efeitos dos fármacos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo
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