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1.
Nucleic Acids Res ; 2024 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-39021337

RESUMO

Trichoderma reesei is an economically important enzyme producer with several unique meiotic features. spo11, the initiator of meiotic double-strand breaks (DSBs) in most sexual eukaryotes, is dispensable for T. reesei meiosis. T. reesei lacks the meiosis-specific recombinase Dmc1. Rad51 and Sae2, the activator of the Mre11 endonuclease complex, promote DSB repair and chromosome synapsis in wild-type and spo11Δ meiosis. DNA methyltransferases (DNMTs) perform multiple tasks in meiosis. Three DNMT genes (rid1, dim2 and dimX) differentially regulate genome-wide cytosine methylation and C:G-to-T:A hypermutations in different chromosomal regions. We have identified two types of DSBs: type I DSBs require spo11 or rid1 for initiation, whereas type II DSBs do not rely on spo11 and rid1 for initiation. rid1 (but not dim2) is essential for Rad51-mediated DSB repair and normal meiosis. rid1 and rad51 exhibit a locus heterogeneity (LH) relationship, in which LH-associated proteins often regulate interconnectivity in protein interaction networks. This LH relationship can be suppressed by deleting dim2 in a haploid rid1Δ (but not rad51Δ) parental strain, indicating that dim2 and rid1 share a redundant function that acts earlier than rad51 during early meiosis. In conclusion, our studies provide the first evidence of the involvement of DNMTs during meiotic initiation and recombination.

3.
Int J Mol Sci ; 22(2)2021 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-33430325

RESUMO

Heat stress (HS) is a major abiotic stress that negatively impacts crop yields across the globe. Plants respond to elevated temperatures by changing gene expression, mediated by transcription factors (TFs) functioning to enhance HS tolerance. The involvement of Group I bZIP TFs in the heat stress response (HSR) is not known. In this study, bZIP18 and bZIP52 were investigated for their possible role in the HSR. Localization experiments revealed their nuclear accumulation following heat stress, which was found to be triggered by dephosphorylation. Both TFs were found to possess two motifs containing serine residues that are candidates for phosphorylation. These motifs are recognized by 14-3-3 proteins, and bZIP18 and bZIP52 were found to bind 14-3-3 ε, the interaction of which sequesters them to the cytoplasm. Mutation of both residues abolished 14-3-3 ε interaction and led to a strict nuclear localization for both TFs. RNA-seq analysis revealed coordinated downregulation of several metabolic pathways including energy metabolism and translation, and upregulation of numerous lncRNAs in particular. These results support the idea that bZIP18 and bZIP52 are sequestered to the cytoplasm under control conditions, and that heat stress leads to their re-localization to nuclei, where they jointly regulate gene expression.


Assuntos
Proteínas 14-3-3/genética , Arabidopsis/genética , Resposta ao Choque Térmico/genética , RNA Longo não Codificante/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Núcleo Celular/genética , Regulação da Expressão Gênica de Plantas/genética , Fatores de Transcrição/genética
4.
PLoS Genet ; 16(4): e1007881, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32310948

RESUMO

Meiotic double-strand breaks (DSBs) are generated by the evolutionarily conserved SPO11 complex in the context of chromatin loops that are organized along axial elements (AEs) of chromosomes. However, how DSBs are formed with respect to chromosome axes and the SPO11 complex remains unclear in plants. Here, we confirm that DSB and bivalent formation are defective in maize spo11-1 mutants. Super-resolution microscopy demonstrates dynamic localization of SPO11-1 during recombination initiation, with variable numbers of SPO11-1 foci being distributed in nuclei but similar numbers of SPO11-1 foci being found on AEs. Notably, cytological analysis of spo11-1 meiocytes revealed an aberrant AE structure. At leptotene, AEs of wild-type and spo11-1 meiocytes were similarly curly and discontinuous. However, during early zygotene, wild-type AEs become uniform and exhibit shortened axes, whereas the elongated and curly AEs persisted in spo11-1 mutants, suggesting that loss of SPO11-1 compromised AE structural maturation. Our results reveal an interesting relationship between SPO11-1 loading onto AEs and the conformational remodeling of AEs during recombination initiation.


Assuntos
Endodesoxirribonucleases/metabolismo , Recombinação Homóloga , Meiose , Zea mays/citologia , Zea mays/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Pareamento Cromossômico , Quebras de DNA de Cadeia Dupla , Endodesoxirribonucleases/genética , Genes de Plantas/genética , Meiose/genética , Mutação , Fenótipo , Zea mays/genética
5.
Development ; 147(11)2020 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-32345744

RESUMO

Precise guided pollen tube growth by the female gametophyte is a prerequisite for successful sexual reproduction in flowering plants. Cysteine-rich proteins (CRPs) secreted from the embryo sac are known pollen tube attractants perceived by pollen tube receptor-like kinases. How pre-mRNA splicing facilitates this cell-to-cell communication is not understood. Here, we report a novel function of Pre-mRNA PROCESSING factor 8 paralogs, PRP8A and PRP8B, as regulators of pollen tube attraction. Double mutant prp8a prp8b ovules cannot attract pollen tubes, and prp8a prp8b pollen tubes fail to sense the ovule's attraction signals. Only 3% of ovule-expressed genes were misregulated in prp8a prp8b Combination of RNA sequencing and the MYB98/LURE1.2-YFP reporter revealed that the expression of MYB98, LUREs and 49 other CRPs were downregulated, suggesting loss of synergid cell fate. Differential exon usage and intron retention analysis revealed autoregulation of PPR8A/PRP8B splicing. In vivo, PRP8A co-immunoprecipitates with splicing enhancer AtSF3A1, suggesting involvement of PRP8A in 3'-splice site selection. Our data hint that the PRP8A/PRP8B module exhibits spliceosome autoregulation to facilitate pollen tube attraction via transcriptional regulation of MYB98, CRPs and LURE pollen tube attractants.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Tubo Polínico/metabolismo , Proteínas de Ligação a RNA/metabolismo , Spliceossomos/metabolismo , Sequência de Aminoácidos , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Microscopia de Fluorescência , Mutagênese , Plantas Geneticamente Modificadas/metabolismo , Tubo Polínico/crescimento & desenvolvimento , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Sítios de Splice de RNA , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Alinhamento de Sequência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
6.
Plant Signal Behav ; 13(12): e1547575, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30444162

RESUMO

Maize, Zea mays, the second-most-widely-grown crop, yields 20 % of all consumed calories worldwide.1 Despite its agronomic importance, research progress is limited by costly transformation. We recently described the Trojan horse method as a useful tool to study maize proteins in situ that circumvents time- and space-consuming whole plant transformation. The Trojan horse approach uses the protein-folding and secretory properties of the corn smut fungus Ustilago maydis to secrete maize proteins from fungal cells into the maize apoplast. Here, we discuss the timing and location of U. maydis during infection and the protein secretion site in relation to anther anatomy. This spatiotemporal analysis enables the study of apoplastic anther proteins in various premeiotic anther developmental stages, and could be adapted for larger screens.

7.
Plant Cell ; 30(3): 528-542, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29449414

RESUMO

Small proteins are crucial signals during development, host defense, and physiology. The highly spatiotemporal restricted functions of signaling proteins remain challenging to study in planta. The several month span required to assess transgene expression, particularly in flowers, combined with the uncertainties from transgene position effects and ubiquitous or overexpression, makes monitoring of spatiotemporally restricted signaling proteins lengthy and difficult. This situation could be rectified with a transient assay in which protein deployment is tightly controlled spatially and temporally in planta to assess protein functions, timing, and cellular targets as well as to facilitate rapid mutagenesis to define functional protein domains. In maize (Zea mays), secreted ZmMAC1 (MULTIPLE ARCHESPORIAL CELLS1) was proposed to trigger somatic niche formation during anther development by participating in a ligand-receptor module. Inspired by Homer's Trojan horse myth, we engineered a protein delivery system that exploits the secretory capabilities of the maize smut fungus Ustilago maydis, to allow protein delivery to individual cells in certain cell layers at precise time points. Pathogen-supplied ZmMAC1 cell-autonomously corrected both somatic cell division and differentiation defects in mutant Zmmac1-1 anthers. These results suggest that exploiting host-pathogen interactions may become a generally useful method for targeting host proteins to cell and tissue types to clarify cellular autonomy and to analyze steps in cell responses.


Assuntos
Zea mays/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Interações Hospedeiro-Patógeno , Zea mays/genética , Zea mays/microbiologia
8.
Plant Biotechnol J ; 15(2): 249-256, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27510270

RESUMO

The capacity of the bread wheat (Triticum aestivum) genome to tolerate introgression from related genomes can be exploited for wheat improvement. A resistance to powdery mildew expressed by a derivative of the cross-bread wheat cv. Tähti × T. militinae (Tm) is known to be due to the incorporation of a Tm segment into the long arm of chromosome 4A. Here, a newly developed in silico method termed rearrangement identification and characterization (RICh) has been applied to characterize the introgression. A virtual gene order, assembled using the GenomeZipper approach, was obtained for the native copy of chromosome 4A; it incorporated 570 4A DArTseq markers to produce a zipper comprising 2132 loci. A comparison between the native and introgressed forms of the 4AL chromosome arm showed that the introgressed region is located at the distal part of the arm. The Tm segment, derived from chromosome 7G, harbours 131 homoeologs of the 357 genes present on the corresponding region of Chinese Spring 4AL. The estimated number of Tm genes transferred along with the disease resistance gene was 169. Characterizing the introgression's position, gene content and internal gene order should not only facilitate gene isolation, but may also be informative with respect to chromatin structure and behaviour studies.


Assuntos
Triticum/genética , Ascomicetos/patogenicidade , Sequência de Bases , Pão , Mapeamento Cromossômico , Cromossomos de Plantas/genética , Cromossomos de Plantas/metabolismo , Simulação por Computador , DNA de Plantas/genética , Resistência à Doença , Genes de Plantas , Marcadores Genéticos , Repetições de Microssatélites , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Translocação Genética , Triticum/microbiologia
9.
Methods Mol Biol ; 1110: 217-30, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24395259

RESUMO

Plants typically produce numerous flowers whose meiotic chromosomes are relatively easy to observe, making them excellent structures for studying the cellular processes underlying meiosis. In recent years, breakthroughs in light and electron microscopic technologies for small chromosomes, combined with molecular genetic methods, have resulted in major advances in the understanding of meiosis in the model plant Arabidopsis thaliana. In this chapter, we summarize protocols for basic cytology, fluorescence in situ hybridization, immunofluorescence, electron microscopy, and isolation of male meiocytes for the analysis of Arabidopsis meiosis.


Assuntos
Arabidopsis/citologia , Separação Celular/métodos , Cromossomos de Plantas/metabolismo , Meiose , Arabidopsis/ultraestrutura , Hibridização in Situ Fluorescente , Indóis/metabolismo , Microscopia Eletrônica de Transmissão , Microtecnologia , Complexo Sinaptonêmico/ultraestrutura
10.
Plant J ; 76(4): 592-602, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24033746

RESUMO

Male fertility in flowering plants relies on proper division and differentiation of cells in the anther, a process that gives rise to four somatic layers surrounding central germinal cells. The maize gene male sterility32 (ms32) encodes a basic helix-loop-helix (bHLH) transcription factor, which functions as an important regulator of both division and differentiation during anther development. After the four somatic cell layers are generated properly through successive periclinal divisions, in the ms32 mutant, tapetal precursor cells fail to differentiate, and, instead, undergo additional periclinal divisions to form extra layers of cells. These cells become vacuolated and expand, and lead to failure in pollen mother cell development. ms32 expression is specific to the pre-meiotic anthers and is distributed initially broadly in the four lobes, but as the anther develops, its expression becomes restricted to the innermost somatic layer, the tapetum. The ms32-ref mac1-1 double mutant is unable to form tapetal precursors and also exhibits excessive somatic proliferation leading to numerous, disorganized cell layers, suggesting a synergistic interaction between ms32 and mac1. Altogether, our results show that MS32 is a major regulator in maize anther development that promotes tapetum differentiation and inhibits periclinal division once a tapetal cell is specified.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Diferenciação Celular , Divisão Celular , Flores/crescimento & desenvolvimento , Zea mays/genética , Divisão Celular/fisiologia , Teste de Complementação Genética , Fenótipo , Proteínas de Plantas/fisiologia , Zea mays/citologia , Zea mays/crescimento & desenvolvimento
11.
Methods Mol Biol ; 990: 167-74, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23559213

RESUMO

Transmission electron microscopy (TEM) of thin sections of meiocytes embedded in plastic resin allows analysis of structures in meiotic nuclei, such as the synaptonemal complex, at very high resolution while preserving the spatial organization of chromosomes.


Assuntos
Núcleo Celular/ultraestrutura , Meiose/genética , Microscopia Eletrônica de Transmissão/métodos , Microtomia/métodos , Arabidopsis/citologia , Arabidopsis/genética , Cromossomos de Plantas , Células Germinativas Vegetais , Complexo Sinaptonêmico
12.
G3 (Bethesda) ; 3(2): 231-49, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23390600

RESUMO

Proper regulation of anther differentiation is crucial for producing functional pollen, and defects in or absence of any anther cell type result in male sterility. To deepen understanding of processes required to establish premeiotic cell fate and differentiation of somatic support cell layers a cytological screen of maize male-sterile mutants has been conducted which yielded 42 new mutants including 22 mutants with premeiotic cytological defects (increasing this class fivefold), 7 mutants with postmeiotic defects, and 13 mutants with irregular meiosis. Allelism tests with known and new mutants confirmed new alleles of four premeiotic developmental mutants, including two novel alleles of msca1 and single new alleles of ms32, ms8, and ocl4, and two alleles of the postmeiotic ms45. An allelic pair of newly described mutants was found. Premeiotic mutants are now classified into four categories: anther identity defects, abnormal anther structure, locular wall defects and premature degradation of cell layers, and/or microsporocyte collapse. The range of mutant phenotypic classes is discussed in comparison with developmental genetic investigation of anther development in rice and Arabidopsis to highlight similarities and differences between grasses and eudicots and within the grasses.


Assuntos
Alelos , Zea mays/genética , Proliferação de Células , Genes de Plantas , Meiose , Mutação , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , Pólen/genética , Pólen/crescimento & desenvolvimento , Zea mays/crescimento & desenvolvimento
13.
Development ; 139(14): 2594-603, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22696296

RESUMO

To ensure fertility, complex somatic and germinal cell proliferation and differentiation programs must be executed in flowers. Loss-of-function of the maize multiple archesporial cells 1 (mac1) gene increases the meiotically competent population and ablates specification of somatic wall layers in anthers. We report the cloning of mac1, which is the ortholog of rice TDL1A. Contrary to prior studies in rice and Arabidopsis in which mac1-like genes were inferred to act late to suppress trans-differentiation of somatic tapetal cells into meiocytes, we find that mac1 anthers contain excess archesporial (AR) cells that proliferate at least twofold more rapidly than normal prior to tapetal specification, suggesting that MAC1 regulates cell proliferation. mac1 transcript is abundant in immature anthers and roots. By immunolocalization, MAC1 protein accumulates preferentially in AR cells with a declining radial gradient that could result from diffusion. By transient expression in onion epidermis, we demonstrate experimentally that MAC1 is secreted, confirming that the predicted signal peptide domain in MAC1 leads to secretion. Insights from cytology and double-mutant studies with ameiotic1 and absence of first division1 mutants confirm that MAC1 does not affect meiotic cell fate; it also operates independently of an epidermal, Ocl4-dependent pathway that regulates proliferation of subepidermal cells. MAC1 both suppresses excess AR proliferation and is responsible for triggering periclinal division of subepidermal cells. We discuss how MAC1 can coordinate the temporal and spatial pattern of cell proliferation in maize anthers.


Assuntos
Flores/crescimento & desenvolvimento , Flores/metabolismo , Oryza/metabolismo , Zea mays/crescimento & desenvolvimento , Zea mays/metabolismo , Proliferação de Células , Flores/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Reprodução/genética , Reprodução/fisiologia , Zea mays/genética
14.
Theor Appl Genet ; 125(3): 609-23, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22534789

RESUMO

Introgression of several genomic loci from tetraploid Triticum militinae into bread wheat cv. Tähti has increased resistance of introgression line 8.1 to powdery mildew in seedlings and adult plants. In our previous work, only a major quantitative trait locus (QTL) on chromosome 4AL of the line 8.1 contributed significantly to resistance, whereas QTL on chromosomes 1A, 1B, 2A, 5A and 5B were detected merely on a suggestive level. To verify and characterize all QTLs in the line 8.1, a mapping population of double haploid lines was established. Testing for seedling resistance to 16 different races/mixtures of Blumeria graminis f. sp. tritici revealed four highly significant non-race-specific resistance QTL including the main QTL on chromosome 4AL, and a race-specific QTL on chromosome 5B. The major QTL on chromosome 4AL (QPm.tut-4A) as well as QTL on chromosome 5AL and a newly detected QTL on 7AL were highly effective at the adult stage. The QPm.tut-4A QTL accounts on average for 33-49 % of the variation in resistance in the double haploid population. Interactions between the main QTL QPm.tut-4A and the minor QTL were evaluated and discussed. A population of 98 F(2) plants from a cross of susceptible cv. Chinese Spring and the line 8.1 was created that allowed mapping the QPm.tut-4A locus to the proximal 2.5-cM region of the introgressed segment on chromosome 4AL. The results obtained in this work make it feasible to use QPm.tut-4A in resistance breeding and provide a solid basis for positional cloning of the major QTL.


Assuntos
Ascomicetos/patogenicidade , Mapeamento Cromossômico/métodos , Genes de Plantas , Doenças das Plantas/microbiologia , Triticum/genética , Pão , Cruzamento , Cromossomos de Plantas/genética , Cromossomos de Plantas/metabolismo , Cruzamentos Genéticos , DNA de Plantas/genética , Resistência à Doença/imunologia , Marcadores Genéticos , Haploidia , Desequilíbrio de Ligação , Repetições de Microssatélites , Doenças das Plantas/imunologia , Locos de Características Quantitativas , Reprodutibilidade dos Testes , Plântula/microbiologia
15.
Proc Natl Acad Sci U S A ; 108(15): 6151-6, 2011 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-21444821

RESUMO

Giardia intestinalis, a human intestinal parasite and member of what is perhaps the earliest-diverging eukaryotic lineage, contains the most divergent eukaryotic actin identified to date and is the first eukaryote known to lack all canonical actin-binding proteins (ABPs). We sought to investigate the properties and functions of the actin cytoskeleton in Giardia to determine whether Giardia actin (giActin) has reduced or conserved roles in core cellular processes. In vitro polymerization of giActin produced filaments, indicating that this divergent actin is a true filament-forming actin. We generated an anti-giActin antibody to localize giActin throughout the cell cycle. GiActin localized to the cortex, nuclei, internal axonemes, and formed C-shaped filaments along the anterior of the cell and a flagella-bundling helix. These structures were regulated with the cell cycle and in encysting cells giActin was recruited to the Golgi-like cyst wall processing vesicles. Knockdown of giActin demonstrated that giActin functions in cell morphogenesis, membrane trafficking, and cytokinesis. Additionally, Giardia contains a single G protein, giRac, which affects the Giardia actin cytoskeleton independently of known target ABPs. These results imply that there exist ancestral and perhaps conserved roles for actin in core cellular processes that are independent of canonical ABPs. Of medical significance, the divergent giActin cytoskeleton is essential and commonly used actin-disrupting drugs do not depolymerize giActin structures. Therefore, the giActin cytoskeleton is a promising drug target for treating giardiasis, as we predict drugs that interfere with the Giardia actin cytoskeleton will not affect the mammalian host.


Assuntos
Actinas/metabolismo , Citoesqueleto/metabolismo , Evolução Molecular , Giardia lamblia/metabolismo , Giardíase/microbiologia , Proteínas dos Microfilamentos/metabolismo , Actinas/genética , Animais , Células COS , Chlorocebus aethiops , Cistos/microbiologia , Técnicas de Silenciamento de Genes , Giardia lamblia/patogenicidade , Humanos , Intestinos/microbiologia , Coelhos
16.
J Exp Bot ; 62(5): 1533-44, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20926553

RESUMO

During meiotic prophase homologous chromosomes find each other and pair. Then they synapse, as the linear protein core (axial element or lateral element) of each homologous chromosome is joined together by a transverse central element, forming the tripartite synaptonemal complex (SC). Ten uncloned Zea mays mutants in our collection were surveyed by transmission electron microscopy by making silver-stained spreads of SCs to identify mutants with non-homologous synapsis or improper synapsis. To analyse the mutants further, zyp1, the maize orthologue of the Arabidopsis central element component ZYP1 was cloned and an antibody was made against it. Using antibodies against ZYP1 and the lateral element components AFD1 and ASY1, it was found that most mutants form normal SCs but are defective in pairing. The large number of non-homologous synapsis mutants defective in pairing illustrates that synapsis and pairing can be uncoupled. Of the ten mutants studied, only dsy2 undergoes normal homologous chromosome recognition needed for homologous pairing. The dsy2 mutation fails to maintain the SC. ZYP1 elongation is blocked at zygotene, and only dots of ZYP1 are seen at prophase I. Another mutant, mei*N2415 showed incomplete but homologous synapsis and ASY1 and AFD1 have a normal distribution. Although installation of ZYP1 is initiated at zygotene, its progression is slowed down and not completed by pachytene in some cells and ZYP1 is not retained on pachytene chromosomes. The mutants described here are now available through the Maize Genetics Cooperation Stock Center (http://maizecoop.cropsci.uiuc.edu/).


Assuntos
Pareamento Cromossômico/fisiologia , Cromossomos de Plantas/metabolismo , Troca Genética/fisiologia , Proteínas de Plantas/metabolismo , Complexo Sinaptonêmico/metabolismo , Zea mays/fisiologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/imunologia , Proteínas de Ciclo Celular/genética , Pareamento Cromossômico/genética , Cromossomos de Plantas/ultraestrutura , Imuno-Histoquímica , Meiose/genética , Microscopia Eletrônica de Transmissão , Mutação , Fenótipo , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Coloração pela Prata , Complexo Sinaptonêmico/genética , Complexo Sinaptonêmico/ultraestrutura , Zea mays/citologia , Zea mays/genética
17.
Plant Mol Biol ; 62(1-2): 99-110, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16897472

RESUMO

Nuclear reorganization and juxtaposition of homologous chromosomes at late leptotene/early zygotene are essential steps before chromosome synapsis at pachytene. We report the results of detailed studies, which demonstrate that nuclear reorganization and homolog juxtapositioning processes are defective in a null mutant, ask1-1. Our results from 4, 6-diamino-2-phenylindole (DAPI)-stained spreads showed that the "synizetic knot", which is typically found in wild type (WT) meiosis during late leptotene and zygotene, was missing in the ask1-1 mutant. Furthermore, ask1-1 meiocytes exhibited only limited homolog juxtaposition at centromere regions at early zygotene. Immunodetection of the cohesin protein SYN1 identified ask1 defects in cohesin distribution from zygotene to anaphase I. Analysis of meiotic chromosomes in ask1-1 and syn1 single mutants, as well as an ask1-1 syn1 double mutant indicate that ASK1 is required for normal SYN1 distribution during meiotic prophase I and suggest that ask1 associated defects may be primarily related to SYN1 mislocalization.


Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Proteínas de Ciclo Celular/metabolismo , Arabidopsis/citologia , Arabidopsis/genética , Mapeamento Cromossômico , Cromossomos de Plantas , Primers do DNA , Hibridização in Situ Fluorescente , Meiose , Mutagênese
18.
J Cell Sci ; 119(Pt 18): 3754-63, 2006 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-16940350

RESUMO

During early stages of meiotic prophase I the nucleus undergoes considerable reorganization, including the clustering of telomeres, the release of contacts between chromosomes and the nuclear membrane, the reorganization of the nucleolus, and chromatin remodeling. Using a light squashing technique for the analysis of meiotic chromosomes along with fluorescent in situ hybridization, transmission electron microscopy and immunolocalization studies with antibodies to modified histones, we demonstrate that ASK1 is essential for early nuclear reorganization events. A relatively large number of meiotic alterations have been identified in ask1-1 plants. We show that many of these defects, including alterations in homolog pairing, nucleolus migration and the missegregation of chromosomes, may arise from alterations in chromatin structure and the inability of chromosomes to resolve and release properly from the nuclear membrane and nucleolus during leptotene. These results raise the interesting possibility that ASK1 controls chromatin structure by targeting of either an early regulator of meiotic progression or possibly matrix attachment proteins for destruction.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Nucléolo Celular/metabolismo , Cromatina/metabolismo , Cromossomos de Plantas/metabolismo , Prófase Meiótica I/fisiologia , Membrana Nuclear/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/ultraestrutura , Cromossomos de Plantas/ultraestrutura , DNA Ribossômico/metabolismo , Histonas/metabolismo , Hibridização in Situ Fluorescente , Mutação/genética , Região Organizadora do Nucléolo/metabolismo , Proteínas Quinases Associadas a Fase S/metabolismo , Homologia de Sequência , Telômero/metabolismo
19.
Development ; 133(16): 3085-95, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16831835

RESUMO

In flowering plants, male fertility depends on proper cell differentiation in the anther. However, relatively little is known about the genes that regulate anther cell differentiation and function. Here, we report the analysis of a new Arabidopsis male sterile mutant, dysfunctional tapetum1 (dyt1). The dyt1 mutant exhibits abnormal anther morphology beginning at anther stage 4, with tapetal cells that have excess and/or enlarged vacuoles and lack the densely stained cytoplasm typical of normal tapetal cells. The mutant meiocytes are able to complete meiosis I, but they do not have a thick callose wall; they often fail to complete meiotic cytokinesis and eventually collapse. DYT1 encodes a putative bHLH transcription factor and is strongly expressed in the tapetum from late anther stage 5 to early stage 6, and at a lower level in meiocytes. In addition, the level of DYT1 mRNA is reduced in the sporocyteless/nozzle (spl/nzz) and excess microsporocytes1/extra sporogenous cell (ems1/exs) mutants; together with the mutant phenotypes, this suggests that DYT1 acts downstream of SPL/NZZ and EMS1/EXS. RT-PCR results showed that the expression levels of many tapetum-preferential genes are reduced significantly in the dyt1 mutant, indicating that DYT1 is important for the expression of tapetum genes. Our results support the hypothesis that DYT1 is a crucial component of a genetic network that controls anther development and function.


Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/crescimento & desenvolvimento , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Flores/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Fatores de Transcrição/fisiologia , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Sequência Conservada , Fertilidade/genética , Flores/genética , Sequências Hélice-Alça-Hélice , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Fenótipo , Proteínas Quinases/metabolismo , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/química , Fatores de Transcrição/genética
20.
Mol Biol Cell ; 17(3): 1331-43, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16394097

RESUMO

Recent studies of meiotic recombination in the budding yeast and the model plant Arabidopsis thaliana indicate that meiotic crossovers (COs) occur through two genetic pathways: the interference-sensitive pathway and the interference-insensitive pathway. However, few genes have been identified in either pathway. Here, we describe the identification of the PARTING DANCERS (PTD) gene, as a gene with an elevated expression level in meiocytes. Analysis of two independently generated transferred DNA insertional lines in PTD showed that the mutants had reduced fertility. Further cytological analysis of male meiosis in the ptd mutants revealed defects in meiosis, including reduced formation of chiasmata, the cytological appearance of COs. The residual chiasmata in the mutants were distributed randomly, indicating that the ptd mutants are defective for CO formation in the interference-sensitive pathway. In addition, transmission electron microscopic analysis of the mutants detected no obvious abnormality of synaptonemal complexes and apparently normal late recombination nodules at the pachytene stage, suggesting that the mutant's defects in bivalent formation were postsynaptic. Comparison to other genes with limited sequence similarity raises the possibility that PTD may present a previously unknown function conserved in divergent eukaryotic organisms.


Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Meiose/genética , Recombinação Genética/genética , Sequência de Aminoácidos , Proteínas de Arabidopsis/química , Cromossomos de Plantas/ultraestrutura , DNA Bacteriano/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Éxons/genética , Flores/citologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Íntrons/genética , Dados de Sequência Molecular , Mutagênese Insercional , Mutação/genética , Fenótipo , Infertilidade das Plantas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Complexo Sinaptonêmico/ultraestrutura
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