RESUMO
Cytokinesis during pollen mitosis I is critical for cell division and differentiation in the male gametophyte development, but the vesicle trafficking mechanisms in this process are largely unknown. Exocyst is an octameric tethering complex which plays multiple important roles in plant cell vesicle trafficking. Here we report the characterization of exocyst subunit SEC6 in the cytokinesis during pollen mitosis I. We found that significantly amount of pollen from two sec6/+ mutant alleles arrested at the transition from unicelluar stage microspore to bicellular stage. Further analysis showed that sec6 mutation impaired cell plate formation and led to vesicles accumulation in cytoplasm. The localization of KNOLLE on the cell plate was compromised. Consistently, SEC6 gene was expressed start from early pollen development stage and SEC6-GFP localized to the cell plate. These results indicated that SEC6 participated in the cell plate formation during pollen mitosis I, where it might help to tether the vesicles before fusion.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Pólen/citologia , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Fluorescência Verde/genética , Mutação , Células Vegetais , Plantas Geneticamente Modificadas , Pólen/fisiologia , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismoRESUMO
In flowering plants, hydration of desiccated pollen grains on stigma is a prerequisite for pollen germination, during which pollen increase markedly in volume through water uptake, requiring them to survive hypoosmotic shock to maintain cellular integrity. However, the mechanisms behind the adaptation of pollen to this hypoosmotic challenge are largely unknown. Here, we identify the Qc-SNARE protein SYP72, which is specifically expressed in male gametophytes, as a critical regulator of pollen survival upon hypoosmotic shock during hydration. SYP72 interacts with the MSCS-LIKE 8 (MSL8) and is required for its localization to the plasma membrane. Intraspecies and interspecies genetic complementation experiments reveal that SYP72 paralogs and orthologs from green algae to angiosperms display conserved molecular functions and rescue the defects of Arabidopsis syp72 mutant pollen facing hypoosmotic shock following hydration. Our findings demonstrate a critical role for SYP72 in pollen resistance to hypoosmotic shock through the MSL8 cascade during pollen hydration.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Canais Iônicos/metabolismo , Pressão Osmótica , Proteínas Qa-SNARE/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Membrana Celular/metabolismo , Fenômenos Químicos , Fertilidade , Canais Iônicos/genética , Desenvolvimento Vegetal , Plantas Geneticamente Modificadas , Pólen/genética , Polinização , Proteínas Qa-SNARE/genética , Água/metabolismoRESUMO
The fecundity of female mammals is resolved by the limited size of the primordial follicle (PF) pool formed perinatally. The establishment of PF pool is accompanied by a significant programmed oocyte death. Long non-coding RNAs (lncRNA) are central modulators in regulating cell apoptosis or autophagy in multiple diseases, however, the significance of lncRNAs governing perinatal oocyte loss remains unknown. Here we find that Yin-Yang 1 (YY1) directly binds to the lncRNA X-inactive-specific transcript (Xist) promoter and facilitates Xist expression in the perinatal mouse ovaries. Xist is highly expressed in fetal ovaries and sharply downregulated along with the establishment of PF pool after birth. Gain or loss of function analysis reveals that Xist accelerates oocyte autophagy, mainly through binding to pre-miR-23b or pre-miR-29a in the nucleus and preventing the export of pre-miR-23b/pre-miR-29a to the cytoplasm, thus resulting in decreased mature of miR-23b-3p/miR-29a-3p expression and upregulation miR-23b-3p/miR-29a-3p co-target, STX17, which is essential for timely control of the degree of oocyte death in prenatal mouse ovaries. Overall, these findings identify Xist as a key non-protein factor that can control the biogenesis of miR-23b-3p/miR-29a-3p, and this YY1-Xist-miR-23b-3p/miR-29a-3p-STX17 regulatory axis is responsible for perinatal oocyte loss through autophagy.
Assuntos
Oócitos/fisiologia , Processamento Pós-Transcricional do RNA/genética , RNA Longo não Codificante/fisiologia , Animais , Animais Recém-Nascidos , Autofagia/genética , Células Cultivadas , Regulação para Baixo/genética , Feminino , Feto/metabolismo , Células HEK293 , Humanos , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Células NIH 3T3 , Ovário/crescimento & desenvolvimento , Ovário/metabolismo , Gravidez , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Transporte de RNA/genética , Regulação para Cima/genética , Fator de Transcrição YY1/fisiologiaRESUMO
Pollen development is a key process for the sexual reproduction of angiosperms. The Golgi plays a critical role in pollen development via the synthesis and transport of cell wall materials. However, little is known about the molecular mechanisms underlying the maintenance of Golgi integrity in plants. In Arabidopsis thaliana, syntaxin of plants (SYP) 3 family proteins SYP31 and SYP32 are the only two Golgi-localized Qa-soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) with unknown endogenous functions. Here, we demonstrate the roles of SYP31 and SYP32 in modulating Golgi morphology and pollen development. Two independent lines of syp31/+ syp32/+ double mutants were male gametophytic lethal; the zero transmission rate of syp31 syp32 mutations was restored to largely normal levels by pSYP32:SYP32 but not pSYP32:SYP31 transgenes, indicating their functional differences in pollen development. The initial arrest of syp31 syp32 pollen occurred during the transition from the microspore to the bicellular stage, where cell plate formation in pollen mitosis I (PMI) and deposition of intine were abnormal. In syp31 syp32 pollen, the number and length of Golgi cisterna were significantly reduced, accompanied by many surrounding vesicles, which could be largely attributed to defects in anterograde and retrograde trafficking routes. SYP31 and SYP32 directly interacted with COG3, a subunit of the conserved oligomeric Golgi (COG) complex and were responsible for its Golgi localization, providing an underlying mechanism for SYP31/32 function in intra-Golgi trafficking. We propose that SYP31 and SYP32 play partially redundant roles in pollen development by modulating protein trafficking and Golgi structure.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Complexo de Golgi , Pólen , Proteínas Qa-SNARE , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Complexo de Golgi/metabolismo , Pólen/genética , Pólen/crescimento & desenvolvimento , Transporte Proteico , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Proteínas SNARE/genética , Proteínas SNARE/metabolismoRESUMO
Endomembrane transport system begins at the endoplasmic reticulum (ER), continues to the Golgi apparatus and subsequent compartment called trans-Golgi network (TGN). We found that SUT2, a tobacco sucrose-transporter ortholog and was localized in the TGN, decreased significantly under a sucrose-starvation condition. The tobacco SNARE protein SYP41, localized in the TGN and secretory vesicle cluster (SVC), also decreased under the starvation. Similarly, the SCAMP2-RFP fusion protein, which is localized in TGN, SVC, and plasma membrane (PM), was distributed solely in the PM under the starvation. Under the same starvation condition, protein secretion was not arrested but pectin deposition to cell wall was suppressed. These data indicated that the protein composition in TGN and existence of the SVC are regulated by sugar availability. Furthermore, our findings as well as the involvement of SVC in pectin secretion suggested that synthesis and transport of pectin are regulated by the level of extracellular sugars. ABBREVIATIONS: ER: endoplasmic reticulum; GI-TGN: Golgi-released independent TGN; GFP: green fluorescent protein; mRFP: monomeric red fluorescent protein; P4H1.1: prolyl 4-hydroxylase 1.1; PM: plasma membrane; SCAMP2: secretory carrier membrane protein 2; SUT2: sucrose transporter 2; SVC: secretory vesicle cluster; SYP41: syntaxin of plant 41; TGN: trans-Golgi network; YFP: yellow fluorescent protein.
Assuntos
Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Membrana Transportadoras/genética , Nicotiana/metabolismo , Pectinas/metabolismo , Sacarose/metabolismo , Rede trans-Golgi/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Células Cultivadas , Meios de Cultura/química , Retículo Endoplasmático/efeitos dos fármacos , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Células Vegetais/efeitos dos fármacos , Células Vegetais/metabolismo , Transporte Proteico , Proteólise/efeitos dos fármacos , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Vesículas Secretórias/metabolismo , Sacarose/farmacologia , Nicotiana/citologia , Nicotiana/efeitos dos fármacos , Nicotiana/genética , Rede trans-Golgi/efeitos dos fármacos , Proteína Vermelha FluorescenteRESUMO
SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) complex formation is necessary for intracellular membrane fusion and thus has a key role in processes such as secretion. However, little is known about the regulatory factors that bind to Qa-SNAREs, which are also known as syntaxins (SYPs) in plants. Here, we characterized Arabidopsis (Arabidopsis thaliana) Tomosyn protein (AtTMS) and demonstrated that it is a conserved regulator of SYPs in plants. AtTMS binds strongly via its R-SNARE motif-containing C terminus to the Qa domain of PM-resident, pollen-expressed SYP1s (SYP111, SYP124, SYP125, SYP131, and SYP132), which were narrowed down from 12 SYPs. AtTMS is highly expressed in pollen from the bicellular stage onwards, and overexpression of AtTMS under the control of the UBIQUITIN10, MSP1, or LAT52 promoter all resulted in defective pollen after the microspore stage in which secretion was inhibited, leading to the failure of intine deposition and cell plate formation during pollen mitosis I. In tobacco (Nicotiana benthamiana) leaf epidermal cells, overexpression of AtTMS inhibited the secretion of secreted GFP. The defects were rescued by mCherry-tagged SYP124, SYP125, SYP131, or SYP132. In vivo, SYP132 partially rescued the pMSP1:AtTMS phenotype. In addition, AtTMS, lacking a transmembrane domain, was recruited to the plasma membrane by SYP124, SYP125, SYP131, and SYP132 and competed with Vesicle-Associated Membrane Protein721/722 for binding to, for example, SYP132. Together, our results demonstrated that AtTMS might serve as a negative regulator of secretion, whereby active secretion might be fine-tuned during pollen development.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas SNARE/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Transporte Biológico , Membrana Celular/metabolismo , Expressão Gênica , Fusão de Membrana , Pólen/genética , Pólen/crescimento & desenvolvimento , Pólen/fisiologia , Ligação Proteica , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Proteínas SNARE/genética , Vesículas Secretórias/metabolismo , Nicotiana/genética , Nicotiana/fisiologiaRESUMO
Folate deficiency during early embryonic development constitutes a risk factor for neural tube defects and potentially for childhood leukemia via unknown mechanisms. We tested whether folate consumption during the 12 months prior to conception induced DNA methylation modifications at birth in healthy neonates with a genome-wide and agnostic approach. We hypothesized that DNA methylation in genes involved in neural tube development and/or cancer susceptibility would be affected by folate exposure. We retrospectively assessed folate exposure at the time of conception by food-frequency questionnaires administered to the mothers of 343 healthy newborns. We measured genome-wide DNA methylation from neonatal blood spots. We implemented a method based on bootstrap resampling to decrease false-positive findings. Folate was inversely associated with DNA methylation throughout the genome. Among the top folate-associated genes that were replicated in an independent Gambian study were TFAP2A, a gene critical for neural crest development, STX11, a gene implicated in acute myeloid leukemia, and CYS1, a candidate gene for cystic kidney disease. Reduced periconceptional folate intake was associated with increased methylation and, in turn, decreased gene expression at these 3 loci. The top folate-sensitive genes defined by their associated CpG sites were enriched for numerous transcription factors by Gene Set Enrichment Analysis, including those implicated in cancer development (e.g., MYC-associated zinc finger protein). The influence of estimated periconceptional folate intake on neonatal DNA methylation levels provides potential mechanistic insights into the role of this vitamin in the development of neural tube defects and childhood cancers.
Assuntos
Metilação de DNA , Deficiência de Ácido Fólico/genética , Ácido Fólico/farmacologia , Regulação da Expressão Gênica no Desenvolvimento , Genes Neoplásicos , Crista Neural/embriologia , Suplementos Nutricionais , Epigenômica , Feminino , Fertilização , Humanos , Recém-Nascido , Proteínas de Membrana/genética , Crista Neural/metabolismo , Defeitos do Tubo Neural/genética , Gravidez , Efeitos Tardios da Exposição Pré-Natal , Proteínas Qa-SNARE/genética , Estudos Retrospectivos , Fatores de Tempo , Fator de Transcrição AP-2/genéticaRESUMO
Fibroblast-like synoviocytes are important mediators of inflammatory joint damage in arthritis through the release of cytokines, but it is unknown whether their exocytosis from these particular cells is SNARE-dependent. Here, the complement of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in human synovial sarcoma cells (SW982) was examined with respect to the secretion of interleukin-6 (IL-6) and tumour necrosis factor α (TNFα), before and after knockdown of a synaptosome-associated protein of molecular mass 23 kDa (SNAP-23) or the vesicle-associated membrane protein 3 (VAMP-3). Wild-type SW982 cells expressed SNAP-23, VAMP-3, syntaxin isoforms 2-4 and synaptic vesicle protein 2C (SV2C). These cells showed Ca²âº-dependent secretion of IL-6 and TNFα when stimulated by interleukin-1ß (IL-1ß) or in combination with K⺠depolarization. Specific knockdown of SNAP-23 or VAMP-3 decreased the exocytosis of IL-6 and TNFα; the reduced expression of SNAP-23 caused accumulation of SV2 in the peri-nuclear area. A monoclonal antibody specific for VAMP-3 precipitated SNAP-23 and syntaxin-2 (and syntaxin-3 to a lesser extent). The formation of SDS-resistant complexes by SNAP-23 and VAMP-3 was reduced upon knockdown of SNAP-23. Although the syntaxin isoforms 2, 3 and 4 are expressed in SW982 cells, knockdown of each did not affect the release of cytokines. Collectively, these results show that SNAP-23 and VAMP-3 participate in IL-1ß-induced Ca²âº-dependent release of IL-6 and TNFα from SW982 cells.
Assuntos
Exocitose , Interleucina-6/metabolismo , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Membrana Sinovial/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteína 3 Associada à Membrana da Vesícula/metabolismo , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Artrite/tratamento farmacológico , Artrite/imunologia , Artrite/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular Tumoral , Exocitose/efeitos dos fármacos , Humanos , Interleucina-1beta/metabolismo , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Terapia de Alvo Molecular , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico/efeitos dos fármacos , Proteínas Qa-SNARE/antagonistas & inibidores , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Proteínas Qb-SNARE/antagonistas & inibidores , Proteínas Qb-SNARE/genética , Proteínas Qc-SNARE/antagonistas & inibidores , Proteínas Qc-SNARE/genética , Interferência de RNA , RNA Interferente Pequeno , Receptores de Interleucina-1/metabolismo , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/imunologia , Proteína 3 Associada à Membrana da Vesícula/antagonistas & inibidores , Proteína 3 Associada à Membrana da Vesícula/genéticaRESUMO
Given that the spinal cord is capable of learning sensorimotor tasks and that dietary interventions can influence learning involving supraspinal centers, we asked whether the presence of omega-3 fatty acid docosahexaenoic acid (DHA) and the curry spice curcumin (Cur) by themselves or in combination with voluntary exercise could affect spinal cord learning in adult spinal mice. Using an instrumental learning paradigm to assess spinal learning we observed that mice fed a diet containing DHA/Cur performed better in the spinal learning paradigm than mice fed a diet deficient in DHA/Cur. The enhanced performance was accompanied by increases in the mRNA levels of molecular markers of learning, i.e., BDNF, CREB, CaMKII, and syntaxin 3. Concurrent exposure to exercise was complementary to the dietary treatment effects on spinal learning. The diet containing DHA/Cur resulted in higher levels of DHA and lower levels of omega-6 fatty acid arachidonic acid (AA) in the spinal cord than the diet deficient in DHA/Cur. The level of spinal learning was inversely related to the ratio of AA:DHA. These results emphasize the capacity of select dietary factors and exercise to foster spinal cord learning. Given the non-invasiveness and safety of the modulation of diet and exercise, these interventions should be considered in light of their potential to enhance relearning of sensorimotor tasks during rehabilitative training paradigms after a spinal cord injury.
Assuntos
Dieta , Aprendizagem , Condicionamento Físico Animal , Desempenho Psicomotor , Traumatismos da Medula Espinal/reabilitação , Animais , Ácido Araquidônico/administração & dosagem , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Curcumina/administração & dosagem , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Ácidos Docosa-Hexaenoicos/administração & dosagem , Ácidos Graxos/metabolismo , Masculino , Camundongos , Desempenho Psicomotor/efeitos dos fármacos , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Traumatismos da Medula Espinal/dietoterapia , Traumatismos da Medula Espinal/metabolismoRESUMO
Vesicle fusion processes in plants are important for both development and stress responses. Transgenic potato plants with reduced expression of SYNTAXIN-RELATED1 (StSYR1), a gene encoding the potato homolog of Arabidopsis PENETRATION1 (AtPEN1), display spontaneous necrosis and chlorosis at later stages of development. In accordance with this developmental defect, tuber number, weight and overall yield are significantly reduced in StSYR1-RNAi lines. Enhanced resistance of StSYR1-RNAi plants to Phytophthora infestans, the causal agent of late blight disease of potato, correlates with enhanced levels of salicylic acid, whereas levels of 12-oxophytodienoic acid and jasmonic acid are unaltered. Cultured cells of StSYR1-RNAi lines secrete at least two compounds which are not detectable in the supernatant of control cells, suggesting an involvement of StSYR1 in secretion processes to the apoplast.
Assuntos
Genes de Plantas , Doenças das Plantas/genética , Imunidade Vegetal/genética , Proteínas de Plantas/genética , Tubérculos/crescimento & desenvolvimento , Proteínas Qa-SNARE/genética , Solanum tuberosum/fisiologia , Ciclopentanos/metabolismo , Ácidos Graxos Insaturados/metabolismo , Expressão Gênica , Oxilipinas/metabolismo , Phytophthora infestans , Desenvolvimento Vegetal/genética , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Tubérculos/fisiologia , Plantas Geneticamente Modificadas , Proteínas Qa-SNARE/metabolismo , Interferência de RNA , Ácido Salicílico/metabolismoRESUMO
The oomycete Phytophthora infestans is the causal agent of late blight, the most devastating disease of potato. The importance of vesicle fusion processes and callose deposition for defense of potato against Phytophthora infestans was analyzed. Transgenic plants were generated, which express RNA interference constructs targeted against plasma membrane-localized SYNTAXIN-RELATED 1 (StSYR1) and SOLUBLE N-ETHYLMALEIMIDE-SENSITIVE FACTOR ADAPTOR PROTEIN 33 (StSNAP33), the potato homologs of Arabidopsis AtSYP121 and AtSNAP33, respectively. Phenotypically, transgenic plants grew normally, but showed spontaneous necrosis and chlorosis formation at later stages. In response to infection with Phytophthora infestans, increased resistance of StSYR1-RNAi plants, but not StSNAP33-RNAi plants, was observed. This increased resistance correlated with the constitutive accumulation of salicylic acid and PR1 transcripts. Aberrant callose deposition in Phytophthora infestans-infected StSYR1-RNAi plants coincided with decreased papilla formation at penetration sites. Resistance against the necrotrophic fungus Botrytis cinerea was not significantly altered. Infiltration experiments with bacterial solutions of Agrobacterium tumefaciens and Escherichia coli revealed a hypersensitive phenotype of both types of RNAi lines. The enhanced defense status and the reduced growth of Phytophthora infestans on StSYR1-RNAi plants suggest an involvement of syntaxins in secretory defense responses of potato and, in particular, in the formation of callose-containing papillae.