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1.
Nat Plants ; 7(8): 1143-1159, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34253868

RESUMO

The appearance of plant organs mediated the explosive radiation of land plants, which shaped the biosphere and allowed the establishment of terrestrial animal life. The evolution of organs and immobile gametes required the coordinated acquisition of novel gene functions, the co-option of existing genes and the development of novel regulatory programmes. However, no large-scale analyses of genomic and transcriptomic data have been performed for land plants. To remedy this, we generated gene expression atlases for various organs and gametes of ten plant species comprising bryophytes, vascular plants, gymnosperms and flowering plants. A comparative analysis of the atlases identified hundreds of organ- and gamete-specific orthogroups and revealed that most of the specific transcriptomes are significantly conserved. Interestingly, our results suggest that co-option of existing genes is the main mechanism for evolving new organs. In contrast to female gametes, male gametes showed a high number and conservation of specific genes, which indicates that male reproduction is highly specialized. The expression atlas capturing pollen development revealed numerous transcription factors and kinases essential for pollen biogenesis and function.


Assuntos
Embriófitas/crescimento & desenvolvimento , Embriófitas/genética , Perfilação da Expressão Gênica , Magnoliopsida/crescimento & desenvolvimento , Magnoliopsida/genética , Organogênese Vegetal/genética , Reprodução/genética , Regulação da Expressão Gênica de Plantas , Variação Genética , Genótipo , Organogênese Vegetal/fisiologia , Fenótipo , Proteínas de Plantas/metabolismo , Reprodução/fisiologia , Análise de Sequência de RNA , Fatores de Transcrição/metabolismo
3.
Plant Reprod ; 34(1): 47-60, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33258014

RESUMO

KEY MESSAGE: Analyses of secretomes of in vitro grown pollen tubes from Amborella, maize and tobacco identified many components of processes associated with the cell wall, signaling and metabolism as well as novel small secreted peptides. Flowering plants (angiosperms) generate pollen grains that germinate on the stigma and produce tubes to transport their sperm cells cargo deep into the maternal reproductive tissues toward the ovules for a double fertilization process. During their journey, pollen tubes secrete many proteins (secreted proteome or secretome) required, for example, for communication with the maternal reproductive tissues, to build a solid own cell wall that withstands their high turgor pressure while softening simultaneously maternal cell wall tissue. The composition and species specificity or family specificity of the pollen tube secretome is poorly understood. Here, we provide a suitable method to obtain the pollen tube secretome from in vitro grown pollen tubes of the basal angiosperm Amborella trichopoda (Amborella) and the Poaceae model maize. The previously published secretome of tobacco pollen tubes was used as an example of eudicotyledonous plants in this comparative study. The secretome of the three species is each strongly different compared to the respective protein composition of pollen grains and tubes. In Amborella and maize, about 40% proteins are secreted by the conventional "classic" pathway and 30% by unconventional pathways. The latter pathway is expanded in tobacco. Proteins enriched in the secretome are especially involved in functions associated with the cell wall, cell surface, energy and lipid metabolism, proteolysis and redox processes. Expansins, pectin methylesterase inhibitors and RALFs are enriched in maize, while tobacco secretes many proteins involved, for example, in proteolysis and signaling. While the majority of proteins detected in the secretome occur also in pollen grains and pollen tubes, and correlate in the number of mapped peptides with relative gene expression levels, some novel secreted small proteins were identified. Moreover, the identification of secreted proteins containing pro-peptides indicates that these are processed in the apoplast. In conclusion, we provide a proteome resource from three distinct angiosperm clades that can be utilized among others to study the localization, abundance and processing of known secreted proteins and help to identify novel pollen tube secreted proteins for functional studies.


Assuntos
Magnoliopsida , Tubo Polínico , Óvulo Vegetal , Peptídeos , Tabaco , Zea mays
4.
Methods Mol Biol ; 2200: 371-390, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33175388

RESUMO

Mutations in numerous genes affect reproduction in Arabidopsis leading to sterility and abortion of seed development, respectively. These include mutations in regulators of reproductive development and fertilization, but also in house-keeping genes lacking mutant phenotypes during vegetative development. However, during the haploid phase of germline development or during seed development, lethality or failures become visible when gene activity is needed. Plant reproduction is complex and includes many processes from flowering and flower organ development toward the formation of seeds after a double fertilization process. For those who are less familiar with the various reproductive processes in Arabidopsis and who aim to study the cause of reproductive defects during germline development and function, fertilization, or embryogenesis in a given mutant, we provide here a step-by-step guideline and basic protocols to elucidate the reproductive process affected.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Mutação/genética , Sementes/genética
5.
Nat Plants ; 6(10): 1275-1288, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33020609

RESUMO

Polar growth requires the precise tuning of Rho GTPase signalling at distinct plasma membrane domains. The activity of Rho of plant (ROP) GTPases is regulated by the opposing action of guanine nucleotide-exchange factors (GEFs) and GTPase-activating proteins (GAPs). Whereas plant-specific ROPGEFs have been shown to be embedded in higher-level regulatory mechanisms involving membrane-bound receptor-like kinases, the regulation of GAPs has remained enigmatic. Here, we show that three Arabidopsis ARMADILLO REPEAT ONLY (ARO) proteins are essential for the stabilization of growth sites in root hair cells and trichomes. AROs interact with ROP1 enhancer GAPs (RENGAPs) and bind to the plasma membrane via a conserved polybasic region at the ARO amino terminus. The ectopic spreading of ROP2 in aro2/3/4 mutant root hair cells and the preferential interaction of AROs with active ROPs and anionic phospholipids suggests that AROs recruit RENGAPs into complexes with ROPs to confine ROP signalling to distinct membrane regions.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas do Domínio Armadillo/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Transdução de Sinais , Proteínas rho de Ligação ao GTP/metabolismo , Arabidopsis/citologia , Arabidopsis/enzimologia , Arabidopsis/crescimento & desenvolvimento , Polaridade Celular , Proteínas de Ligação a DNA/metabolismo , Evolução Molecular , Raízes de Plantas/citologia , Raízes de Plantas/metabolismo , Tricomas/citologia , Tricomas/metabolismo
6.
Plant Physiol ; 184(4): 1640-1657, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32989009

RESUMO

Flowering plants (angiosperms) are characterized by pollen tubes (PTs; male gametophytes) carrying two immobile sperm cells that grow over long distances through the carpel toward the ovules, where double fertilization is executed. It is not understood how these reproductive structures evolved, which genes occur de novo in male gametophytes of angiosperms, and to which extent PT functions are conserved among angiosperms. To contribute to a deeper understanding of the evolution of gametophyte functions, we generated RNA sequencing data from seven reproductive and two vegetative control tissues of the basal angiosperm Amborella trichopoda and complemented these with proteomic data of pollen grains (PGs) and PTs. The eudicot model plant Arabidopsis (Arabidopsis thaliana) served as a reference organism for data analysis, as more than 200 genes have been associated with male gametophyte functions in this species. We describe methods to collect bicellular A. trichopoda PGs, to induce their germination in vitro, and to monitor PT growth and germ cell division. Transcriptomic and proteomic analyses indicate that A. trichopoda PGs are prepared for germination requiring lipids, energy, but likely also reactive oxygen species, while PTs are especially characterized by catabolic/biosynthetic and transport processes including cell wall biosynthesis and gene regulation. Notably, a number of pollen-specific genes were lacking in Arabidopsis, and the number of genes involved in pollen signaling is significantly reduced in A. trichopoda In conclusion, we provide insight into male gametophyte functions of the most basal angiosperm and establish a valuable resource for future studies on the evolution of flowering plants.


Assuntos
Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Germinação/genética , Magnoliopsida/crescimento & desenvolvimento , Magnoliopsida/genética , Pólen/crescimento & desenvolvimento , Pólen/genética , Evolução Biológica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Germinação/fisiologia , Tubo Polínico/genética , Tubo Polínico/crescimento & desenvolvimento , Proteômica , Transcriptoma
7.
Nature ; 579(7799): 409-414, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32188942

RESUMO

Plants are essential for life and are extremely diverse organisms with unique molecular capabilities1. Here we present a quantitative atlas of the transcriptomes, proteomes and phosphoproteomes of 30 tissues of the model plant Arabidopsis thaliana. Our analysis provides initial answers to how many genes exist as proteins (more than 18,000), where they are expressed, in which approximate quantities (a dynamic range of more than six orders of magnitude) and to what extent they are phosphorylated (over 43,000 sites). We present examples of how the data may be used, such as to discover proteins that are translated from short open-reading frames, to uncover sequence motifs that are involved in the regulation of protein production, and to identify tissue-specific protein complexes or phosphorylation-mediated signalling events. Interactive access to this resource for the plant community is provided by the ProteomicsDB and ATHENA databases, which include powerful bioinformatics tools to explore and characterize Arabidopsis proteins, their modifications and interactions.


Assuntos
Proteínas de Arabidopsis/análise , Proteínas de Arabidopsis/química , Arabidopsis/química , Espectrometria de Massas , Proteoma/análise , Proteoma/química , Proteômica , Motivos de Aminoácidos , Arabidopsis/anatomia & histologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/biossíntese , Proteínas de Arabidopsis/genética , Bases de Dados de Proteínas , Conjuntos de Dados como Assunto , Regulação da Expressão Gênica de Plantas , Anotação de Sequência Molecular , Fases de Leitura Aberta , Especificidade de Órgãos , Fosfoproteínas/análise , Fosfoproteínas/química , Fosfoproteínas/genética , Fosforilação , Proteoma/biossíntese , Proteoma/genética , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Transcriptoma
8.
Curr Opin Plant Biol ; 53: 106-116, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31841779

RESUMO

During sexual reproduction two gametes of opposite sex unite to produce a zygote. Gamete fusion is a highly controlled process and it has become evident that, across species, common concepts apply to this ancient and fundamental event. Sexual reproduction in flowering plants is even more complex in that two sperm cells fertilize two female reproductive cells (egg and central cell) in a process called double fertilization. Due to the coordinated developmental progression and mutual dependency of the two fertilization products (embryo and endosperm), the success and timing of the two fusion events substantially affects seed set. So far, four proteins are known to act on the surfaces of Arabidopsis gametes to accomplish double fertilization. The molecular and evolutionary characteristics of these players prove that flowering plants integrate plant-specific and widely conserved mechanisms to accomplish the timely fusion of each sperm cell with one female reproductive cell.


Assuntos
Arabidopsis , Magnoliopsida , Fertilização , Sementes , Interações Espermatozoide-Óvulo
9.
Front Plant Sci ; 10: 1469, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31824527

RESUMO

MATH-BTB proteins are known to act as substrate-specific adaptors of CUL3-based E3 ligases in the ubiquitin proteasome pathway. Their BTB domain binds to CUL3 scaffold proteins and the less conserved MATH domain targets a highly diverse collection of substrate proteins to promote their ubiquitination and subsequent degradation. In plants, a significant expansion of the MATH-BTB family occurred in the grasses. Here, we report analysis of TaMAB2, a MATH-BTB protein transiently expressed at the onset of embryogenesis in wheat. Due to difficulties in studying its role in zygotes and early embryos, we have overexpressed TaMAB2 in Arabidopsis to generate gain-of-function mutants and to elucidate interaction partners and substrates. Overexpression plants showed severe growth defects as well as disorganization of microtubule bundles indicating that TaMAB2 interacts with substrates in Arabidopsis. In tobacco BY-2 cells, TaMAB2 showed a microtubule and ubiquitin-associated cytoplasmic localization pattern in form of foci. Its direct interaction with CUL3 suggests functions in targeting specific substrates for ubiquitin-dependent degradation. Although direct interactions with tubulin could not be confimed, tandem affinity purification of TaMAB2 interactors point towards cytoskeletal proteins including tubulin and actin as well as the translation initiation machinery. The idenification of various subunits of eucaryotic translation initiation factors eIF3 and eIF4 as TaMAB2 interactors indicate regulation of translation initiation as a major function during onset of embryogenesis in plants.

10.
Nat Plants ; 5(3): 253-257, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30850817

RESUMO

Successful double fertilization in flowering plants relies on two coordinated gamete fusion events, but the underlying molecular processes are not well understood. We show that two sperm-specific DOMAIN OF UNKNOWN FUNCTION 679 membrane proteins (DMP8 and DMP9) facilitate gamete fusion, with a greater effect on sperm-egg fusion than on sperm-central cell fusion. We also show that sperm adhesion and sperm cell separation depend on egg cell-secreted EGG CELL 1 proteins.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Fusão Celular , Proteínas de Membrana/metabolismo , Óvulo Vegetal/fisiologia , Pólen/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Transporte/genética , Fertilização , Regulação da Expressão Gênica de Plantas , Proteínas de Fluorescência Verde/genética , Proteínas de Membrana/genética , Óvulo Vegetal/genética , Plantas Geneticamente Modificadas , Pólen/genética
11.
Plant Reprod ; 32(2): 229, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30805714

RESUMO

The article Transcriptomics of manually isolated Amborella trichopoda egg apparatus cells, written by María Flores-Tornero, Sebastian Proost, Marek Mutwil, Charles P. Scutt, Thomas Dresselhaus, Stefanie Sprunck, was originally published electronically on the publisher's internet portal (currently SpringerLink) on 1 February 2019 without open access.

12.
Plant Reprod ; 32(1): 15-27, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30707279

RESUMO

KEY MESSAGE: A protocol for the isolation of egg apparatus cells from the basal angiosperm Amborella trichopoda to generate RNA-seq data for evolutionary studies of fertilization-associated genes. Sexual reproduction is particularly complex in flowering plants (angiosperms). Studies in eudicot and monocot model species have significantly contributed to our knowledge on cell fate specification of gametophytic cells and on the numerous cellular communication events necessary to deliver the two sperm cells into the embryo sac and to accomplish double fertilization. However, for a deeper understanding of the evolution of these processes, morphological, genomic and gene expression studies in extant basal angiosperms are inevitable. The basal angiosperm Amborella trichopoda is of special importance for evolutionary studies, as it is likely sister to all other living angiosperms. Here, we report about a method to isolate Amborella egg apparatus cells and on genome-wide gene expression profiles in these cells. Our transcriptomics data revealed Amborella-specific genes and genes conserved in eudicots and monocots. Gene products include secreted proteins, such as small cysteine-rich proteins previously reported to act as extracellular signaling molecules with important roles during double fertilization. The detection of transcripts encoding EGG CELL 1 (EC1) and related prolamin-like family proteins in Amborella egg cells demonstrates the potential of the generated data set to study conserved molecular mechanisms and the evolution of fertilization-related genes and their encoded proteins.


Assuntos
Separação Celular/métodos , Genoma de Planta , Magnoliopsida/citologia , Magnoliopsida/genética , Óvulo Vegetal/genética , Óvulo Vegetal/citologia , RNA de Plantas , Transcriptoma
13.
Plant Physiol ; 179(4): 1525-1536, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30700540

RESUMO

In eukaryotes, the regulated transport of mRNAs from the nucleus to the cytosol through nuclear pore complexes represents an important step in the expression of protein-coding genes. In plants, the mechanism of nucleocytosolic mRNA transport and the factors involved are poorly understood. The Arabidopsis (Arabidopsis thaliana) genome encodes two likely orthologs of UAP56-interacting factor, which acts as mRNA export factor in mammalian cells. In yeast and plant cells, both proteins interact directly with the mRNA export-related RNA helicase UAP56 and the interaction was mediated by an N-terminal UAP56-binding motif. Accordingly, the two proteins were termed UAP56-INTERACTING EXPORT FACTOR1 and 2 (UIEF1/2). Despite lacking a known RNA-binding motif, recombinant UIEF1 interacted with RNA, and the C-terminal part of UIEF1 mainly contributed to the RNA interaction. Mutation of UIEF1, UIEF2, or both in the double-mutant 2xuief caused modest growth defects. A cross between the 2xuief and 4xaly (defective in the four ALY1-4 mRNA export factors) mutants produced the sextuple mutant 4xaly 2xuief, which displayed more severe growth impairment than the 4xaly plants. Developmental defects including delayed bolting and reduced seed set were observed in the 4xaly but not the 2xuief plants. Analysis of the cellular distribution of polyadenylated mRNAs revealed more pronounced nuclear mRNA accumulation in 4xaly 2xuief than in 2xuief and 4xaly cells. In conclusion, the results indicate that UIEF1 and UIEF2 act as mRNA export factors in plants and that they cooperate with ALY1-ALY4 to mediate efficient nucleocytosolic mRNA transport.


Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , RNA Helicases DEAD-box/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/fisiologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Genoma de Planta , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
14.
Sci Rep ; 8(1): 10626, 2018 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-30006526

RESUMO

Genomic imprinting confers parent-of-origin-specific gene expression, thus non-equivalent and complementary function of parental genomes. As a consequence, genomic imprinting poses an epigenetic barrier to parthenogenesis in sexual organisms. We report aberrant imprinting in Boechera, a genus in which apomicts evolved from sexuals multiple times. Maternal activation of a MADS-box gene, a homolog of which is imprinted and paternally expressed in the sexual relative Arabidopsis, is accompanied by locus-specific DNA methylation changes in apomicts where parental imprinting seems to be relaxed.


Assuntos
Brassicaceae/genética , Impressão Genômica , Proteínas de Domínio MADS/genética , Partenogênese , Proteínas de Plantas/genética , Evolução Biológica , Metilação de DNA , Epigenômica , Regulação da Expressão Gênica de Plantas
15.
Plant Physiol ; 177(1): 226-240, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29540591

RESUMO

The regulated transport of mRNAs from the cell nucleus to the cytosol is a critical step linking transcript synthesis and processing with translation. However, in plants, only a few of the factors that act in the mRNA export pathway have been functionally characterized. Flowering plant genomes encode several members of the ALY protein family, which function as mRNA export factors in other organisms. Arabidopsis (Arabidopsis thaliana) ALY1 to ALY4 are commonly detected in root and leaf cells, but they are differentially expressed in reproductive tissue. Moreover, the subnuclear distribution of ALY1/2 differs from that of ALY3/4. ALY1 binds with higher affinity to single-stranded RNA than double-stranded RNA and single-stranded DNA and interacts preferentially with 5-methylcytosine-modified single-stranded RNA. Compared with the full-length protein, the individual RNA recognition motif of ALY1 binds RNA only weakly. ALY proteins interact with the RNA helicase UAP56, indicating a link to the mRNA export machinery. Consistently, ALY1 complements the lethal phenotype of yeast cells lacking the ALY1 ortholog Yra1. Whereas individual aly mutants have a wild-type appearance, disruption of ALY1 to ALY4 in 4xaly plants causes vegetative and reproductive defects, including strongly reduced growth, altered flower morphology, as well as abnormal ovules and female gametophytes, causing reduced seed production. Moreover, polyadenylated mRNAs accumulate in the nuclei of 4xaly cells. Our results highlight the requirement of efficient mRNA nucleocytosolic transport for proper plant growth and development and indicate that ALY1 to ALY4 act partly redundantly in this process; however, differences in expression and subnuclear localization suggest distinct functions.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , RNA Mensageiro/metabolismo , Transporte Ativo do Núcleo Celular , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Regulação da Expressão Gênica de Plantas , Plantas Geneticamente Modificadas , Transporte de RNA , RNA de Plantas/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
16.
Methods Mol Biol ; 1669: 221-234, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28936662

RESUMO

The few-celled female gametophyte, or embryo sac, of flowering plants is not easily accessible as it is buried within the sporophytic tissues of the ovule. Nevertheless, it has become an attractive model system to study the molecular mechanisms underlying patterning and cell type specification, as well as fertilization of the two female gametes, the egg and the central cell. While female gametes, zygotes, and early embryos can be manually isolated from the embryo sacs in maize, wheat, tobacco, and rice by micromanipulation, this approach had been considered impossible for the much smaller embryo sac of the model plant Arabidopsis thaliana. Here, we describe a method to isolate living cells from the Arabidopsis female gametophyte by micromanipulation. The manual isolation of egg cells, central cells, and synergid cells is a technique that enables a number of important studies such as cell-type-specific transcriptional profiling or the analysis of DNA methylation profiles. It also offers the possibility to use isolated female gametes for in vitro fertilization studies.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Oryza/genética , Oryza/metabolismo , Tabaco/genética , Tabaco/metabolismo , Triticum/genética , Triticum/metabolismo , Zea mays/genética , Zea mays/metabolismo
17.
Plant Cell ; 29(9): 2106-2125, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28814645

RESUMO

The formation of a zygote via the fusion of an egg and sperm cell and its subsequent asymmetric division herald the start of the plant's life cycle. Zygotic genome activation (ZGA) is thought to occur gradually, with the initial steps of zygote and embryo development being primarily maternally controlled, and subsequent steps being governed by the zygotic genome. Here, using maize (Zea mays) as a model plant system, we determined the timing of zygote development and generated RNA-seq transcriptome profiles of gametes, zygotes, and apical and basal daughter cells. ZGA occurs shortly after fertilization and involves ∼10% of the genome being activated in a highly dynamic pattern. In particular, genes encoding transcriptional regulators of various families are activated shortly after fertilization. Further analyses suggested that chromatin assembly is strongly modified after fertilization, that the egg cell is primed to activate the translational machinery, and that hormones likely play a minor role in the initial steps of early embryo development in maize. Our findings provide important insights into gamete and zygote activity in plants, and our RNA-seq transcriptome profiles represent a comprehensive, unique RNA-seq data set that can be used by the research community.


Assuntos
Fertilização/genética , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Zea mays/genética , Zigoto/metabolismo , Padronização Corporal/genética , Ciclo Celular/genética , Separação Celular , Cromatina/metabolismo , Genes de Plantas , Células Germinativas Vegetais/metabolismo , Histonas/metabolismo , Ácidos Indolacéticos/metabolismo , Oryza/genética , Reprodutibilidade dos Testes , Sementes/citologia , Sementes/genética , Análise de Sequência de RNA , Transdução de Sinais/genética , Fatores de Tempo , Fatores de Transcrição/metabolismo , Transcriptoma/genética
18.
Plant Physiol ; 173(1): 155-166, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27920160

RESUMO

The EGG CELL1 (EC1) gene family of Arabidopsis (Arabidopsis thaliana) comprises five members that are specifically expressed in the egg cell and redundantly control gamete fusion during double fertilization. We investigated the activity of all five EC1 promoters in promoter-deletion studies and identified SUF4 (SUPPRESSOR OF FRIGIDA4), a C2H2 transcription factor, as a direct regulator of the EC1 gene expression. In particular, we demonstrated that SUF4 binds to all five Arabidopsis EC1 promoters, thus regulating their expression. The down-regulation of SUF4 in homozygous suf4-1 ovules results in reduced EC1 expression and delayed sperm fusion, which can be rescued by expressing SUF4-ß-glucuronidase under the control of the SUF4 promoter. To identify more gene products able to regulate EC1 expression together with SUF4, we performed coexpression studies that led to the identification of MOM1 (MORPHEUS' MOLECULE1), a component of a silencing mechanism that is independent of DNA methylation marks. In mom1-3 ovules, both SUF4 and EC1 genes are down-regulated, and EC1 genes show higher levels of histone 3 lysine-9 acetylation, suggesting that MOM1 contributes to the regulation of SUF4 and EC1 gene expression.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Fertilização/genética , Regulação da Expressão Gênica de Plantas , Células Germinativas Vegetais/citologia , Células Germinativas Vegetais/metabolismo , Transativadores/metabolismo , Proteínas de Arabidopsis/metabolismo , Sequência Conservada/genética , Genes de Plantas , Genes Reporter , Proteínas de Fluorescência Verde/metabolismo , Motivos de Nucleotídeos/genética , Óvulo/citologia , Óvulo/metabolismo , Fenótipo , Regiões Promotoras Genéticas , Transcrição Genética
19.
Curr Biol ; 26(3): R125-39, 2016 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-26859271

RESUMO

Compared with the animal kingdom, fertilization is particularly complex in flowering plants (angiosperms). Sperm cells of angiosperms have lost their motility and require transportation as a passive cargo by the pollen tube cell to the egg apparatus (egg cell and accessory synergid cells). Sperm cell release from the pollen tube occurs after intensive communication between the pollen tube cell and the receptive synergid, culminating in the lysis of both interaction partners. Following release of the two sperm cells, they interact and fuse with two dimorphic female gametes (the egg and the central cell) forming the major seed components embryo and endosperm, respectively. This process is known as double fertilization. Here, we review the current understanding of the processes of sperm cell reception, gamete interaction, their pre-fertilization activation and fusion, as well as the mechanisms plants use to prevent the fusion of egg cells with multiple sperm cells. The role of Ca(2+) is highlighted in these various processes and comparisons are drawn between fertilization mechanisms in flowering plants and other eukaryotes, including mammals.


Assuntos
Fertilização , Magnoliopsida/fisiologia , Interações Espermatozoide-Óvulo
20.
Proc Natl Acad Sci U S A ; 112(38): 11841-5, 2015 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-26351695

RESUMO

During microRNA (miRNA)-guided gene silencing, Argonaute (Ago) proteins interact with a member of the TNRC6/GW protein family. Here we used a short GW protein-derived peptide fused to GST and demonstrate that it binds to Ago proteins with high affinity. This allows for the simultaneous isolation of all Ago protein complexes expressed in diverse species to identify associated proteins, small RNAs, or target mRNAs. We refer to our method as "Ago protein Affinity Purification by Peptides" (Ago-APP). Furthermore, expression of this peptide competes for endogenous TNRC6 proteins, leading to global inhibition of miRNA function in mammalian cells.


Assuntos
Proteínas Argonauta/isolamento & purificação , Cromatografia de Afinidade/métodos , Complexos Multiproteicos/isolamento & purificação , Peptídeos/isolamento & purificação , Sequência de Aminoácidos , Animais , Extratos Celulares , Precipitação Química , Drosophila melanogaster , Inativação Gênica , Células HEK293 , Células HeLa , Humanos , MicroRNAs/metabolismo , Dados de Sequência Molecular , Peptídeos/química
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