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1.
BMC Genomics ; 20(1): 421, 2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-31138116

RESUMO

BACKGROUND: Cotton is the most essential textile crop worldwide, and phytohormones are critical for cotton fiber development. One example is the role of auxin in fiber initiation, but we know little molecular basis. MicroRNAs (miRNAs) have a significant function in cotton development; nevertheless their role in fiber initiation remains unclear. Here, exogenous IAA was applied to cotton plant before anthesis. Utilizing small RNA sequencing, the mechanism underlying miRNA-mediated regulation of fiber initiation under exogenous IAA treatment was investigated. RESULTS: With exogenous IAA application, the endogenous IAA and GA contents of IAA treated (IT) ovules were higher than control (CK) ovules at the fiber initiation stage, while endogenous ABA content was lower in IT than CK. Using scanning electron microscopy, we found the fiber number and size were significantly promoted in IT at 0 DPA. Fiber quality analysis showed that fiber length, uniformity, strength, elongation, and micronaire of IT were higher than CK, though not statistically significant, while lint percent was significantly higher in IT. We generated six small RNA libraries using - 3, 0, and 3 DPA ovules of IT and CK, and identified 58 known miRNAs and 83 novel miRNAs together with the target genes. The differential expressed miRNAs number between IT and CK at - 3, 0, 3 DPA was 34, 16 and 24, respectively. Gene ontology and KEGG pathway enrichment analyses for the target genes of the miRNAs expressed in a differential manner showed that they were significantly enriched in 30 terms and 8 pathways. QRT-PCR for those identified miRNAs and the target genes related to phytohormones and fiber development was performed, and results suggested a potential role of these miRNAs in fiber initiation. CONCLUSIONS: The exogenous IAA application affected the relative phytohormone contents in ovule and promoted fiber initiation in cotton. Identification and profiling of miRNAs and their targets at the fiber initiation stage provided insights for miRNAs' regulation function of fiber initiation. These findings not only shed light on the regulatory network of fiber growth but also offer clues for cotton fiber amelioration strategies in cotton.


Assuntos
Gossypium/genética , Ácidos Indolacéticos/farmacologia , MicroRNAs/metabolismo , Reguladores de Crescimento de Planta/farmacologia , Perfilação da Expressão Gênica , Genes de Plantas , Gossypium/efeitos dos fármacos , Gossypium/crescimento & desenvolvimento , Gossypium/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Óvulo Vegetal/efeitos dos fármacos , Óvulo Vegetal/genética , Óvulo Vegetal/crescimento & desenvolvimento , Óvulo Vegetal/ultraestrutura , Reguladores de Crescimento de Planta/metabolismo , Análise de Sequência de RNA
2.
Ann Bot ; 120(4): 529-538, 2017 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-28961769

RESUMO

Background and Aims: Cellular morphogenesis in land plants and brown algae is typically a slow process involving growth established by an interplay of turgor pressure and cell wall rigidity. However, a recent study showed that zygotes of the brown alga Dictyota dichotoma undergo a rapid shape change from a sphere to an elongated spheroid in about 90 s, establishing the first body axis. Methods: Using a combination of pharmacology, staining techniques, membrane depolarization and microscopy techniques (brightfield, transmission electron microscopy and confocal laser scanning microscopy), egg activation and the shape change of the egg cell of D. dichotoma was studied. Key Results: It was established that elongation of the zygote does not involve growth, i.e. a positive change in size. The elongation is dependent on F-actin and myosin but independent of microtubules. Secretion was also found to be necessary for elongation after addition of brefeldin A. Moreover, a temporal correlation between extracellular matrix secretion and elongation was observed. Ionomycin and high potassium seawater are capable of triggering the onset of elongation, suggesting a role for membrane depolarization and calcium influx in the signalling mechanism. The elongated cells are shorter in the presence of ionomycin, suggesting a role for calcium in elongation. Conclusions: A model is proposed in which the fast elongation of the fertilized egg in Dictyota is accomplished by a force generated by F-actin and myosin, regulated by cytoplasmic calcium concentrations and by secretion during elongation lowering the antagonistic force. The finding of early extracellular matrix secretion, membrane depolarization and ionophore-triggered egg activation suggest significant differences in the mechanism of egg activation signalling between D. dichotoma and the oogamous brown algal model system Fucus .


Assuntos
Actinas/fisiologia , Miosinas/fisiologia , Óvulo Vegetal/fisiologia , Feófitas/fisiologia , Sementes/fisiologia , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Óvulo Vegetal/anatomia & histologia , Óvulo Vegetal/ultraestrutura , Feófitas/metabolismo , Feófitas/ultraestrutura , Sementes/anatomia & histologia , Sementes/ultraestrutura
3.
Protoplasma ; 254(2): 657-668, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27154759

RESUMO

In apomictic Taraxacum species, the development of both the embryo and the endosperm does not require double fertilisation. However, a structural reduction of ovular transmitting tissue was not observed in apomictic dandelions. The aim of this study was to analyse the chemical composition of the cell walls to describe the presence of arabinogalactan proteins (AGPs), hemicellulose and some pectic epitopes in the micropylar transmitting tissue of apomictic Taraxacum. The results point to (1) the similar distribution of AGPs in different developmental stages, (2) the absence of highly methyl-esterified homogalacturonan (HG) in transmitting tissue of ovule containing a mature embryo sac and the appearance of this pectin domain in the young seed containing the embryo and endosperm, (3) the similar pattern of low methyl-esterified pectin occurrence in both an ovule and a young seed with an embryo and endosperm in apomictic Taraxacum and (4) the presence of hemicelluloses recognised by LM25 and LM21 antibodies in the reproductive structure of Taraxacum.


Assuntos
Apomixia , Epitopos/metabolismo , Mucoproteínas/metabolismo , Óvulo Vegetal/metabolismo , Pectinas/metabolismo , Polissacarídeos/metabolismo , Taraxacum/metabolismo , Taraxacum/fisiologia , Endosperma/citologia , Imuno-Histoquímica , Óvulo Vegetal/citologia , Óvulo Vegetal/crescimento & desenvolvimento , Óvulo Vegetal/ultraestrutura , Proteínas de Plantas/metabolismo , Taraxacum/embriologia , Taraxacum/ultraestrutura
4.
Planta ; 245(4): 717-728, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27999988

RESUMO

MAIN CONCLUSION: AtPLC2 is an essential gene in Arabidopsis, since it is required for female gametogenesis and embryo development. AtPLC2 might play a role in cell division during embryo-sac development and early embryogenesis. Phosphoinositide-specific phospholipase C (PI-PLC) plays an important role in signal transduction during plant development and in the response to various biotic- and abiotic stresses. The Arabidopsis PI-PLC gene family is composed of nine members, named PLC1 to PLC9. Here, we report that PLC2 is involved in female gametophyte development and early embryogenesis. Using two Arabidopsis allelic T-DNA insertion lines with different phenotypic penetrations, we observed both female gametophytic defects and aberrant embryos. For the plc2-1 mutant (Ws background), no homozygous plants could be recovered in the offspring from self-pollinated plants. Nonetheless, plc2-1 hemizygous mutants are affected in female gametogenesis, showing embryo sacs arrested at early developmental stages. Allelic hemizygous plc2-2 mutant plants (Col-0 background) present reduced seed set and embryos arrested at the pre-globular stage with abnormal patterns of cell division. A low proportion (0.8%) of plc2-2 homozygous mutants was found to escape lethality and showed morphological defects and disrupted megagametogenesis. PLC2-promoter activity was observed during early megagametogenesis, and after fertilization in the embryo proper. Immunolocalization studies in early stage embryos revealed that PLC2 is restricted to the plasma membrane. Altogether, these results establish a role for PLC2 in both reproductive- and embryo development, presumably by controlling mitosis and/or the formation of cell-division planes.


Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/crescimento & desenvolvimento , Gametogênese Vegetal/fisiologia , Sementes/crescimento & desenvolvimento , Fosfolipases Tipo C/fisiologia , Arabidopsis/enzimologia , Arabidopsis/ultraestrutura , Western Blotting , Glucuronidase/metabolismo , Microscopia Confocal , Microscopia de Fluorescência , Óvulo Vegetal/enzimologia , Óvulo Vegetal/fisiologia , Óvulo Vegetal/ultraestrutura , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sementes/enzimologia
5.
Am J Bot ; 103(12): 2028-2057, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27919924

RESUMO

PREMISE OF THE STUDY: Revealing the relative roles of gradual and abrupt transformations of morphological characters is an important topic of evolutionary biology. Gynoecia apparently consisting of one carpel have evolved from pluricarpellate syncarpous gynoecia in several angiosperm clades. The process of reduction can involve intermediate stages, with one fertile and one or more sterile carpels (pseudomonomery). The possible origin of monomery directly via an abrupt change of gynoecium merism has been a matter of dispute. We explore the nature of gynoecium reduction in a clade of Araliaceae. METHODS: The anatomy and development of unilocular gynoecia are investigated using light and scanning electron microscopy in two members of Polyscias subg. Arthrophyllum. Gynoecium diversity in the genus is discussed in a phylogenetic framework. KEY RESULTS: Unilocular gynoecia with one fertile ovule have evolved at least four times in Polyscias, including one newly discovered case. The two unilocular taxa investigated are unicarpellate, without any traces of reduced sterile carpels. Carpel orientation is unstable, and the ovary roof and style contain numerous vascular bundles without clearly recognizable dorsals or ventrals. In contrast to pluricarpellate Araliaceae and Apiaceae, the cross zone is apparently oblique in the unicarpellate species. CONCLUSIONS: No support was found for gradual gynoecium reduction via pseudomonomery. The abrupt origin of monomery via direct change of gynoecium merism and the unstable carpel orientation observed are related to the general lability of the flower groundplan in Polyscias. The apparent occurrence of the unusual oblique cross zone in unicarpellate Araliaceae can be explained by developmental constraints.


Assuntos
Araliaceae/ultraestrutura , Flores/ultraestrutura , Araliaceae/genética , Araliaceae/crescimento & desenvolvimento , Evolução Biológica , Flores/genética , Flores/crescimento & desenvolvimento , Microscopia , Microscopia Eletrônica de Varredura , Óvulo Vegetal/genética , Óvulo Vegetal/crescimento & desenvolvimento , Óvulo Vegetal/ultraestrutura , Filogenia , Sementes/genética , Sementes/crescimento & desenvolvimento , Sementes/ultraestrutura , Análise de Sequência de DNA
6.
Plant Physiol ; 172(4): 2403-2415, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27794102

RESUMO

Gibberellins (GAs) are plant hormones that regulate most plant life cycle aspects, including flowering and fruit development. Here, we demonstrate the implication of GAs in ovule development. DELLA proteins, negative GA response regulators, act as positive factors for ovule integument development in a mechanism that involves transcription factor ABERRANT TESTA SHAPE (ATS). The seeds of the della global mutant, a complete loss-of-function of DELLA, and the ats-1 mutant are remarkably similar, with a round shape, a disorganized testa, and viviparism. These defects are the result of an alteration in integuments that fail to fully develop and are shorter than in wild-type plants. ats-1 also shows some GA-related phenotypes, for example, higher germination rates and early flowering. In fact, ats-1 has elevated GA levels due to the activation of GA biosynthesis genes, which indicates that ATS inhibits GA biosynthesis. Moreover, DELLAs and ATS proteins interact, which suggests the formation of a transcriptional complex that regulates the expression of genes involved in integument growth. Therefore, the repression of GA biosynthesis by ATS would result in the stabilization of DELLAs to ensure correct ATS-DELLA complex formation. The requirement of both activities to coordinate proper ovule development strongly argues that the ATS-DELLA complex acts as a key molecular factor. This work provides the first evidence for a role of GAs in ovule and seed development.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/embriologia , Arabidopsis/metabolismo , Giberelinas/metabolismo , Óvulo Vegetal/embriologia , Óvulo Vegetal/metabolismo , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Flores/fisiologia , Regulação da Expressão Gênica de Plantas , Modelos Biológicos , Mutação/genética , Óvulo Vegetal/ultraestrutura , Fenótipo , Ligação Proteica , Sementes/embriologia , Sementes/metabolismo , Sementes/ultraestrutura , Transdução de Sinais , Fatores de Transcrição/genética
7.
Sci Rep ; 6: 26829, 2016 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-27311358

RESUMO

The cotton fibers are seed trichomes that elongate from the ovule epidermis. Polar lipids are required for the quick enlargement of cell membrane and fiber cell growth, however, how lipids are transported from the ovules into the developing fibers remains less known. Here, we reported the functional characterization of GhLTPG1, a GPI-anchored lipid transport protein, during cotton fiber elongation. GhLTPG1 was abundantly expressed in elongating cotton fibers and outer integument of the ovules, and GhLTPG1 protein was located on cell membrane. Biochemical analysis showed that GhLTPG1 specifically bound to phosphatidylinositol mono-phosphates (PtdIns3P, PtdIns4P and PtdIns5P) in vitro and transported PtdInsPs from the synthesis places to the plasma membranes in vivo. Expression of GhLTPG1 in Arabidopsis caused an increased number of trichomes, and fibers in GhLTPG1-knockdown cotton plants exhibited significantly reduced length, decreased polar lipid content, and repression of fiber elongation-related genes expression. These results suggested that GhLTPG1 protein regulates the cotton fiber elongation through mediating the transport of phosphatidylinositol monophosphates.


Assuntos
Antígenos de Plantas/fisiologia , Proteínas de Transporte/fisiologia , Fibra de Algodão , Gossypium/metabolismo , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas de Plantas/fisiologia , Tricomas/crescimento & desenvolvimento , Arabidopsis/crescimento & desenvolvimento , Transporte Biológico , Regulação da Expressão Gênica de Plantas , Genes Reporter , Gossypium/genética , Gossypium/crescimento & desenvolvimento , Lipossomos , Óvulo Vegetal/crescimento & desenvolvimento , Óvulo Vegetal/metabolismo , Óvulo Vegetal/ultraestrutura , Estruturas Vegetais/metabolismo , Ligação Proteica , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA de Plantas/biossíntese , RNA de Plantas/genética , Proteínas Recombinantes de Fusão/metabolismo , Especificidade da Espécie , Tricomas/metabolismo
8.
Methods ; 98: 66-73, 2016 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-26521978

RESUMO

First evidence on gene function and regulation is provided by the cellular expression pattern in complex tissues. However, to understand the activity of a specific gene, it is essential to analyze the regulatory network, which controls the spatio-temporal translation pattern during the entire life span of the transcribed mRNA. To explore mechanisms which control mRNA abundance and localization in space and time, it is necessary to visualize mRNAs quantitatively with a subcellular resolution, without sectioning the tissues. We have adapted and optimized a protocol for colorimetric whole-mount RNA in situ hybridization (WISH) using egg cell-specific digoxigenin (DIG) labeled probes (Hejátko et al., 2006) [1] on ovules and early seeds of Arabidopsis. Furthermore, we established a fluorescent whole-mount RNA in situ hybridization (F-WISH) protocol, which allows mRNA visualization on a subcellular level. The polar localized mRNA of SBT4.13, encoding a subtilase, was identified using this protocol. Both methods are described and discussed in detail. Additionally a (F)-WISH flow-chart is provided along with a troubleshooting table.


Assuntos
Arabidopsis/ultraestrutura , Células Germinativas Vegetais/ultraestrutura , Hibridização in Situ Fluorescente/métodos , Óvulo Vegetal/ultraestrutura , RNA Mensageiro/química , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Digoxigenina/química , Corantes Fluorescentes/química , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Células Germinativas Vegetais/crescimento & desenvolvimento , Células Germinativas Vegetais/metabolismo , Óvulo Vegetal/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Razão Sinal-Ruído , Subtilisinas/química , Fixação de Tecidos/métodos , Transcrição Genética , Tiramina/química
9.
Development ; 143(3): 422-6, 2016 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-26700684

RESUMO

Angiosperm ovules consist of three proximal-distal domains - the nucellus, chalaza and funiculus - demarcated by developmental fate and specific gene expression. Mutation in three paralogous class III homeodomain leucine zipper (HD-ZIPIII) genes leads to aberrations in ovule integument development. Expression of WUSCHEL (WUS) is normally confined to the nucellar domain, but in this triple mutant expression expands into the chalaza. MicroRNA-induced suppression of this expansion partially suppresses the effects of the HD-ZIPIII mutations on ovule development, implicating ectopic WUS expression as a component of the mutant phenotype. bell1 (bel1) mutants produce aberrant structures in place of the integuments and WUS is ectopically expressed in these structures. Combination of bel1 with the HD-ZIPIII triple mutant leads to a striking phenotype in which ectopic ovules emerge from nodes of ectopic WUS expression along the funiculi of the primary ovules. The synergistic phenotype indicates that BEL1 and the HD-ZIPIII genes act in at least partial independence in confining WUS expression to the nucellus and maintaining ovule morphology. The branching ovules of the mutant resemble those of some fossil gymnosperms, implicating BEL1 and HD-ZIPIII genes as players in the evolution of the unbranched ovule form in extant angiosperms.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Óvulo Vegetal/metabolismo , Arabidopsis/genética , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/genética , Padronização Corporal/genética , Citocininas/metabolismo , Proteínas de Homeodomínio/metabolismo , Modelos Biológicos , Mutação/genética , Óvulo Vegetal/genética , Óvulo Vegetal/ultraestrutura , Fenótipo , Fatores de Transcrição/metabolismo
10.
J Exp Bot ; 67(5): 1447-59, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26712826

RESUMO

Meiosis is essential for gametogenesis in sexual reproduction in rice (Oryza sativa L.). We identified a MutS-homolog (MSH) family gene OsMSH4 in a trisomic plant. Cytological analysis showed that developments of both pollen and embryo sacs in an Osmsh4 mutant were blocked due to defective chromosome pairing. Compared with the wild type, the Osmsh4 mutant displayed a significant ~21.9% reduction in chiasma frequency, which followed a Poisson distribution, suggesting that class I crossover formation in the mutant was impaired. Temporal and spatial expression pattern analyses showed that OsMSH4 was preferentially expressed in meiocytes during their meiosis, indicating a critical role in gametogenesis. Subcellular localization showed that OsMSH4-green fluorescent protein was predominantly located in the nucleus. OsMSH4 could interact with another MSH member (OsMSH5) through the N-terminus and C-terminus, respectively. Direct physical interaction between OsMSH5, OsRPA1a, OsRPA2b, OsRPA1c, and OsRPA2c was identified by yeast two-hybrid assays and further validated by pull-down assays. Our results supported the conclusion that the OsMSH4/5 heterodimer plays a key role in regulation of crossover formation during rice meiosis by interaction with the RPA complex.


Assuntos
Gametogênese Vegetal , Meiose , Oryza/citologia , Oryza/metabolismo , Óvulo Vegetal/metabolismo , Proteínas de Plantas/metabolismo , Pólen/metabolismo , Pareamento Cromossômico , Cromossomos de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Teste de Complementação Genética , Mutação/genética , Oryza/embriologia , Oryza/genética , Óvulo Vegetal/ultraestrutura , Proteínas de Plantas/genética , Pólen/ultraestrutura , Ligação Proteica , Multimerização Proteica , Transporte Proteico , Frações Subcelulares/metabolismo
11.
PLoS One ; 10(10): e0140507, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26485030

RESUMO

Xanthoceras sorbifolium, a tree species endemic to northern China, has high oil content in its seeds and is recognized as an important biodiesel crop. The plant is characterized by late-acting self-incompatibility (LSI). LSI was found to occur in many angiosperm species and plays an important role in reducing inbreeding and its harmful effects, as do gametophytic self-incompatibility (GSI) and sporophytic self-incompatibility (SSI). Molecular mechanisms of conventional GSI and SSI have been well characterized in several families, but no effort has been made to identify the genes involved in the LSI process. The present studies indicated that there were no significant differences in structural and histological features between the self- and cross-pollinated ovules during the early stages of ovule development until 5 days after pollination (DAP). This suggests that 5 DAP is likely to be a turning point for the development of the selfed ovules. Comparative de novo transcriptome analysis of the selfed and crossed ovules at 5 DAP identified 274 genes expressed specifically or preferentially in the selfed ovules. These genes contained a significant proportion of genes predicted to function in the biosynthesis of secondary metabolites, consistent with our histological observations in the fertilized ovules. The genes encoding signal transduction-related components, such as protein kinases and protein phosphatases, are overrepresented in the selfed ovules. X. sorbifolium selfed ovules also specifically or preferentially express many unique transcription factor (TF) genes that could potentially be involved in the novel mechanisms of LSI. We also identified 42 genes significantly up-regulated in the crossed ovules compared to the selfed ovules. The expression of all 16 genes selected from the RNA-seq data was validated using PCR in the selfed and crossed ovules. This study represents the first genome-wide identification of genes expressed in the fertilized ovules of an LSI species. The availability of a pool of specifically or preferentially expressed genes from selfed ovules for X. sorbifolium will be a valuable resource for future genetic analyses of candidate genes involved in the LSI response.


Assuntos
Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica de Plantas , Óvulo Vegetal/genética , Polinização/genética , Sapindaceae/genética , Autoincompatibilidade em Angiospermas/genética , Ontologia Genética , Genes de Plantas/genética , Microscopia Eletrônica de Varredura , Óvulo Vegetal/metabolismo , Óvulo Vegetal/ultraestrutura , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sapindaceae/metabolismo , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
12.
Dev Biol ; 405(1): 158-72, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-26123745

RESUMO

Gene duplications result in paralogs that may be maintained due to the gain of novel functions (neo-functionalization) or the partitioning of ancestral function (sub-functionalization). Plant genomes are especially prone to duplication; paralogs are particularly widespread in the floral MADS box transcription factors that control organ identity through the ABC model of flower development. C class genes establish stamen and carpel identity and control floral meristem determinacy, and are largely conserved across the angiosperm phylogeny. Originally, an additional D class had been identified as controlling ovule identity; yet subsequent studies indicated that both C and D lineage genes more commonly control ovule development redundantly. The ranunculid Thalictrum thalictroides has two orthologs of the Arabidopsis thaliana C class gene AGAMOUS (AG), ThtAG1 and ThtAG2 (Thalictrum thalictroides AGAMOUS1/2). We previously showed that ThtAG1 exhibits typical C class function; here we examine the role of its paralog, ThtAG2. Our phylogenetic analysis shows that ThtAG2 falls within the C lineage, together with ThtAG1, and is consistent with previous findings of a Ranunculales-specific duplication in this clade. However, ThtAG2 is not expressed in stamens, but rather solely in carpels and ovules. This female-specific expression pattern is consistent with D lineage genes, and with other C lineage genes known to be involved in ovule identity. Given the divergent expression of ThtAG2, we tested the hypothesis that it has acquired ovule identity function. Molecular evolution analyses showed evidence of positive selection on ThtAG2-a pattern that supports divergence of function by sub-functionalization. Down-regulation of ThtAG2 by virus-induced gene silencing resulted in homeotic conversions of ovules into carpel-like structures. Taken together, our results suggest that, although ThtAG2 falls within the C lineage, it has diverged to acquire "D function" as an ovule identity gene, and does not appear to require a direct interaction with the ThtAG1 protein. We therefore present a functional example of ovule identity being specified by either a single gene or a gene pair within the C lineage, with no D lineage contribution. In conclusion, following a Ranunculales-wide duplication in the AG lineage, functional divergence has led to the evolution of ovule identity-specificity in a T. thalictroides C lineage gene.


Assuntos
Duplicação Gênica , Genes de Plantas , Óvulo Vegetal/crescimento & desenvolvimento , Óvulo Vegetal/genética , Thalictrum/crescimento & desenvolvimento , Thalictrum/genética , Sequência de Aminoácidos , Aminoácidos/metabolismo , Regulação para Baixo/genética , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Genes Homeobox , Dados de Sequência Molecular , Especificidade de Órgãos/genética , Óvulo Vegetal/ultraestrutura , Fenótipo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ligação Proteica , Alinhamento de Sequência , Thalictrum/ultraestrutura , Técnicas do Sistema de Duplo-Híbrido
13.
Planta ; 241(1): 209-27, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25262422

RESUMO

Calcium (Ca(2+)) plays essential roles in plant sexual reproduction, but the sites and the mechanism of Ca(2+) mobile storage during pollen-pistil interactions have not been fully defined. Because the Ca(2+)-buffering protein calreticulin (CRT) is able to bind and sequester Ca(2+), it can serve as a mobile intracellular store of easily releasable Ca(2+) and control its local concentration within the cytoplasm. Our previous studies showed an enhanced expression of Petunia hybrida CRT gene (PhCRT) during pistil transmitting tract maturation, pollen germination and tube outgrowth on the stigma, gamete fusion, and early embryogenesis. Here, we demonstrate that elevated expression of CRT results in the accumulation of this protein in response to anthesis, pollination, sperm cells deposition within the receptive synergid and fertilization, when the level of exchangeable Ca(2+) changes dynamically. CRT localizes mainly to the endoplasmic reticulum and Golgi compartments in the pistil transmitting tract cells, germinated pollen/tubes, and sporophytic/gametophytic cells of the ovule and corresponds with loosely bound Ca(2+). Additionally, the immunogold research shows, for the first time, highly selective CRT distribution in specific nuclear sub-domains. On the basis of our results, we discuss the possible functions of CRT with respect to the critical role of Ca(2+) homeostasis during key events of the multi-step process of generative reproduction in angiosperms.


Assuntos
Cálcio/metabolismo , Calreticulina/metabolismo , Flores/metabolismo , Petunia/metabolismo , Proteínas de Plantas/metabolismo , Western Blotting , Calreticulina/ultraestrutura , Fertilização , Flores/ultraestrutura , Cinética , Microscopia Eletrônica de Transmissão , Óvulo Vegetal/metabolismo , Óvulo Vegetal/ultraestrutura , Petunia/ultraestrutura , Polinização , Fatores de Tempo
14.
J Vis Exp ; (88): e51530, 2014 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-24998753

RESUMO

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues. Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.


Assuntos
Arabidopsis/química , Arabidopsis/ultraestrutura , Cromatina/química , Óvulo Vegetal/química , Óvulo Vegetal/ultraestrutura , Análise de Célula Única/métodos , Arabidopsis/metabolismo , Núcleo Celular/química , Núcleo Celular/ultraestrutura , Cromatina/metabolismo , DNA de Plantas/análise , Hibridização in Situ Fluorescente/métodos
15.
Plant J ; 80(1): 185-95, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25041411

RESUMO

The pollen tube is the most rapidly growing cell in the plant kingdom and has the function to deliver the sperm cells for fertilization. The growing tip region of the cell behaves in a chemotropic manner to respond to the guidance cues emitted by the pistil and the female gametophyte, but how it perceives and responds to these directional triggers is virtually unknown. Quantitative assessment of chemotropic behavior can greatly be enhanced by the administration of pharmacological or other biologically active agents at subcellular precision, which is a technical challenge when the target area moves as it grows. We developed a laminar flow based microfluidic device that allows for continuous administration of two different solutions with a movable interface that permits the dynamic targeting of the growing pollen tube apex over prolonged periods of time. Asymmetric administration of calcium revealed that rather than following the highest calcium concentration as would be expected with simple chemotropic behavior, the pollen tube of Camellia targets an optimal concentration suggesting the presence of two superimposed mechanisms. Subcellular application of pectin methyl esterase (PME), an enzyme that modifies the growth behavior by rigidifying the pollen tube cell wall, caused the tube to turn away from the agent - providing important evidence for a previously proposed conceptual model of the growth mechanism.


Assuntos
Cálcio/metabolismo , Camellia/crescimento & desenvolvimento , Tubo Polínico/crescimento & desenvolvimento , Camellia/ultraestrutura , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Parede Celular/metabolismo , Fertilização , Flores/crescimento & desenvolvimento , Flores/ultraestrutura , Modelos Biológicos , Óvulo Vegetal/crescimento & desenvolvimento , Óvulo Vegetal/ultraestrutura , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Tubo Polínico/ultraestrutura , Polinização
16.
Methods Mol Biol ; 1110: 253-61, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24395261

RESUMO

Ovules are the major female reproductive organs in higher plants. Furthermore, ovules of Arabidopsis thaliana are successfully used as model system to study plant organogenesis. Here we describe two microscopic techniques to analyze ovule development in Arabidopsis. Both methods involve fixed specimens and allow rapid, easy, and reproducible morphological comparisons between wild-type and mutant ovule development.


Assuntos
Arabidopsis/citologia , Microscopia Confocal/métodos , Microscopia Eletrônica de Varredura/métodos , Óvulo Vegetal/citologia , Arabidopsis/ultraestrutura , Óvulo Vegetal/ultraestrutura , Propídio/metabolismo , Coloração e Rotulagem , Fixação de Tecidos
17.
Acta Biol Hung ; 64(3): 319-27, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24013893

RESUMO

Embryo sac cells are highly differentiated in plants. The central cell is one of the most important cells of the embryo sac. It forms endosperm by fusion with a sperm cell. Ultrastructure of the central cell in the mature embryo sac of Castilleja wightii was investigated in this study. Nucleolus which had a lot of vacuole in a large secondary nucleus and numerous dictyosomes, vesicles, mitochondria, amyloplasts in cytoplasm were seen in this cell. Also free ribosomes in the form of polysomes and large lipid bodies were detected in the cytoplasm. Numerous vacuoles of different size were observed and some of them had autophagic function. Both smooth and rough endoplasmic reticulums were seen. Although invaginations were seen in the plasmalemma of the central cell to the inside of the embryo sac, a thick cuticular layer was observed outer side on the cell wall. The aim of this study was to contribute studies about the ultrastructure of embryo sacs.


Assuntos
Acanthaceae/ultraestrutura , Óvulo Vegetal/ultraestrutura , Scrophulariaceae/ultraestrutura , Especificidade da Espécie
18.
Plant Reprod ; 26(2): 125-37, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23539301

RESUMO

Expression datasets relating to the Arabidopsis female gametophyte have enabled the creation of a tool set which allows simultaneous visual tracking of each specific cell type (egg, synergids, central cell, and antipodals). This cell-specific, fluorescent labeling tool-set functions from gametophyte cellularization through fertilization and early embryo development. Using this system, cell fates were tracked within Arabidopsis ovules following molecular manipulations, such as the ablation of the egg and/or synergids. Upon egg cell ablation, it was observed that a synergid can switch its developmental fate to become egg/embryo-like upon loss of the native egg. Also, manipulated was the fate of the somatic ovular cells, which can become egg- and embryo-like, reminiscent of adventitious embryony. These advances represent initial steps toward engineering synthetic apomixis resulting in seed derived wholly from the maternal plant. The end goal of applied apomixis research, fixing important agronomic traits such as hybrid vigor, would be a key benefit to agricultural productivity.


Assuntos
Apomixia/genética , Arabidopsis/genética , Arabidopsis/ultraestrutura , Regulação da Expressão Gênica no Desenvolvimento , Sementes/genética , Sementes/ultraestrutura , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fertilização , Fluorescência , Regulação da Expressão Gênica de Plantas , Marcadores Genéticos , Mutação , Especificidade de Órgãos , Óvulo Vegetal/genética , Óvulo Vegetal/metabolismo , Óvulo Vegetal/ultraestrutura , Fenótipo , Sementes/metabolismo
19.
Plant Cell ; 25(3): 1093-107, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23482856

RESUMO

Protein S-acylation, commonly known as palmitoylation, is a reversible posttranslational modification that catalyzes the addition of a saturated lipid group, often palmitate, to the sulfhydryl group of a Cys. Palmitoylation regulates enzyme activity, protein stability, subcellular localization, and intracellular sorting. Many plant proteins are palmitoylated. However, little is known about protein S-acyl transferases (PATs), which catalyze palmitoylation. Here, we report that the tonoplast-localized PAT10 is critical for development and salt tolerance in Arabidopsis thaliana. PAT10 loss of function resulted in pleiotropic growth defects, including smaller leaves, dwarfism, and sterility. In addition, pat10 mutants are hypersensitive to salt stresses. We further show that PAT10 regulates the tonoplast localization of several calcineurin B-like proteins (CBLs), including CBL2, CBL3, and CBL6, whose membrane association also depends on palmitoylation. Introducing a C192S mutation within the highly conserved catalytic motif of PAT10 failed to complement pat10 mutants, indicating that PAT10 functions through protein palmitoylation. We propose that PAT10-mediated palmitoylation is critical for vacuolar function by regulating membrane association or the activities of tonoplast proteins.


Assuntos
Aciltransferases/metabolismo , Arabidopsis/enzimologia , Arabidopsis/crescimento & desenvolvimento , Plantas Tolerantes a Sal/enzimologia , Aciltransferases/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Brefeldina A/farmacologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Contagem de Células , Membrana Celular/metabolismo , Ativação Enzimática , Pleiotropia Genética , Microscopia Eletrônica de Varredura , Óvulo Vegetal/metabolismo , Óvulo Vegetal/ultraestrutura , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/fisiologia , Mutação Puntual , Pólen/metabolismo , Pólen/ultraestrutura , Ligação Proteica , Transporte Proteico , Plantas Tolerantes a Sal/genética , Plantas Tolerantes a Sal/fisiologia , Cloreto de Sódio/farmacologia , Estresse Fisiológico , Vacúolos/metabolismo
20.
Methods Mol Biol ; 959: 127-35, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23299671

RESUMO

Ovules are the major female reproductive organs in higher plants. In addition, ovules of Arabidopsis thaliana are successfully used as model system to study plant organogenesis. Here we describe two microscopic techniques to analyze Arabidopsis ovule development from the organ to the cellular level in a rapid and reproducible fashion. Both methods are of great value when comparing the morphology of wild-type and mutant ovule development.


Assuntos
Arabidopsis/crescimento & desenvolvimento , Óvulo Vegetal/crescimento & desenvolvimento , Arabidopsis/ultraestrutura , Regulação da Expressão Gênica de Plantas , Óvulo Vegetal/ultraestrutura
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