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2.
Int J Mol Sci ; 22(17)2021 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-34502279

RESUMO

The endoplasmic reticulum (ER) is an extensive network of intracellular membranes. Its major functions include proteosynthesis, protein folding, post-transcriptional modification and sorting of proteins within the cell, and lipid anabolism. Moreover, several studies have suggested that it may be involved in regulating intracellular auxin homeostasis in plants by modulating its metabolism. Therefore, to study auxin metabolome in the ER, it is necessary to obtain a highly enriched (ideally, pure) ER fraction. Isolation of the ER is challenging because its biochemical properties are very similar to those of other cellular endomembranes. Most published protocols for ER isolation use density gradient ultracentrifugation, despite its suboptimal resolving power. Here we present an optimised protocol for ER isolation from Arabidopsis thaliana seedlings for the subsequent mass spectrometric determination of ER-specific auxin metabolite profiles. Auxin metabolite analysis revealed highly elevated levels of active auxin form (IAA) within the ER compared to whole plants. Moreover, samples prepared using our optimised isolation ER protocol are amenable to analysis using various "omics" technologies including analyses of both macromolecular and low molecular weight compounds from the same sample.


Assuntos
Arabidopsis/citologia , Arabidopsis/metabolismo , Retículo Endoplasmático/metabolismo , Ácidos Indolacéticos/metabolismo , Metabolômica/métodos , Proteínas de Arabidopsis/análise , Proteínas de Arabidopsis/metabolismo , Metaboloma , Células Vegetais , Proteômica/métodos , Plântula/citologia , Plântula/metabolismo
3.
Cells ; 10(8)2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34440822

RESUMO

Plant mitochondrial transcription is initiated from multiple promoters without an apparent motif, which precludes their identification in other species based on sequence comparisons. Even though coding regions take up only a small fraction of plant mitochondrial genomes, deep RNAseq studies uncovered that these genomes are fully or nearly fully transcribed with significantly different RNA read depth across the genome. Transcriptomic analysis can be a powerful tool to understand the transcription process in diverse angiosperms, including the identification of potential promoters and co-transcribed genes or to study the efficiency of intron splicing. In this work, we analyzed the transcriptional landscape of the Arabidopsis mitochondrial genome (mtDNA) based on large-scale RNA sequencing data to evaluate the use of RNAseq to study those aspects of the transcription process. We found that about 98% of the Arabidopsis mtDNA is transcribed with highly different RNA read depth, which was elevated in known genes. The location of a sharp increase in RNA read depth upstream of genes matched the experimentally identified promoters. The continuously high RNA read depth across two adjacent genes agreed with the known co-transcribed units in Arabidopsis mitochondria. Most intron-containing genes showed a high splicing efficiency with no differences between cis and trans-spliced introns or between genes with distinct splicing mechanisms. Deep RNAseq analyses of diverse plant species will be valuable to recognize general and lineage-specific characteristics related to the mitochondrial transcription process.


Assuntos
Arabidopsis/genética , Mitocôndrias/genética , Splicing de RNA , Transcrição Genética , Arabidopsis/citologia , Proteínas de Arabidopsis/genética , DNA Mitocondrial/genética , Genes de Plantas/genética , Genoma Mitocondrial/genética , Íntrons , Regiões Promotoras Genéticas , RNA de Plantas/genética , Análise de Sequência de RNA
4.
Nat Commun ; 12(1): 4682, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34344886

RESUMO

A key impediment to studying water-related mechanisms in plants is the inability to non-invasively image water fluxes in cells at high temporal and spatial resolution. Here, we report that Raman microspectroscopy, complemented by hydrodynamic modelling, can achieve this goal - monitoring hydrodynamics within living root tissues at cell- and sub-second-scale resolutions. Raman imaging of water-transporting xylem vessels in Arabidopsis thaliana mutant roots reveals faster xylem water transport in endodermal diffusion barrier mutants. Furthermore, transverse line scans across the root suggest water transported via the root xylem does not re-enter outer root tissues nor the surrounding soil when en-route to shoot tissues if endodermal diffusion barriers are intact, thereby separating 'two water worlds'.


Assuntos
Raízes de Plantas/metabolismo , Água/metabolismo , Arabidopsis/anatomia & histologia , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/metabolismo , Transporte Biológico , Hidrodinâmica , Modelos Biológicos , Mutação , Raízes de Plantas/anatomia & histologia , Raízes de Plantas/citologia , Raízes de Plantas/genética , Brotos de Planta/metabolismo , Estômatos de Plantas/metabolismo , Análise Espectral Raman , Xilema/metabolismo
5.
Dokl Biochem Biophys ; 499(1): 233-237, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34426918

RESUMO

The effect of T-DNA insertion in the 3'-UTR region of Arabidopsis thaliana At3g58450 gene encoding the Germination-Related Universal Stress Protein (GRUSP) was studied. It was found that under a long-day condition this mutation delays transition to flowering of grusp-115 transgenic line that due to a reduced content of endogenous bioactive gibberellins GA1 and GA3 in comparison to the wild-type plants (Col-0). Exogenous GA accelerated flowering of both lines but did not change the time of difference in the onset of flowering between Col-0 and grusp-115. In addition to changes in GA metabolism, grusp-115 evidently has disturbances in realization of the signal that induces flowering. This is confirmed by the results of gene expression of the floral integrator FLOWERING LOCUS T (FT) and the floral repressor FLOWERING LOCUS C (FLC), which are key flowering regulators and acting opposite. We hypothesize that the formation of grusp-115 phenotype can also be affected by a low expression level of FT due to up-regulated FLC expression.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Flores/crescimento & desenvolvimento , Giberelinas/metabolismo , Arabidopsis/citologia , Fenótipo , Transdução de Sinais
6.
Science ; 373(6550)2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34210850

RESUMO

The plant male germline undergoes DNA methylation reprogramming, which methylates genes de novo and thereby alters gene expression and regulates meiosis. Here, we reveal the molecular mechanism underlying this reprogramming. We demonstrate that genic methylation in the male germline, from meiocytes to sperm, is established by 24-nucleotide small interfering RNAs (siRNAs) transcribed from transposons with imperfect sequence homology. These siRNAs are synthesized by meiocyte nurse cells (tapetum) through activity of CLSY3, a chromatin remodeler absent in other anther cells. Tapetal siRNAs govern germline methylation throughout the genome, including the inherited methylation patterns in sperm. Tapetum-derived siRNAs also silence germline transposons, safeguarding genome integrity. Our results reveal that tapetal siRNAs are sufficient to reconstitute germline methylation patterns and drive functional methylation reprogramming throughout the male germline.


Assuntos
Arabidopsis/citologia , Arabidopsis/genética , Epigênese Genética , Herança Paterna , Pólen/genética , RNA Interferente Pequeno/genética , Metilação de DNA , Meiose/genética , Mitose/genética
7.
Nat Commun ; 12(1): 4327, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34267202

RESUMO

Trivalent rare earth elements (REEs) are widely used in agriculture. Aerially applied REEs enter leaf epidermal cells by endocytosis and act systemically to improve the growth of the whole plant. The mechanistic basis of their systemic activity is unclear. Here, we show that treatment of Arabidopsis leaves with trivalent lanthanum [La(III)], a representative of REEs, triggers systemic endocytosis from leaves to roots. La(III)-induced systemic endocytosis requires AtrbohD-mediated reactive oxygen species production and jasmonic acid. Systemic endocytosis impacts the accumulation of mineral elements and the development of roots consistent with the growth promoting effects induced by aerially applied REEs. These findings provide insights into the mechanistic basis of REE activity in plants.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Lantânio/farmacologia , NADPH Oxidases/metabolismo , Arabidopsis/citologia , Proteínas de Arabidopsis/genética , Ciclopentanos/metabolismo , Endocitose/fisiologia , Regulação da Expressão Gênica de Plantas , Proteínas de Fluorescência Verde/genética , Minerais/metabolismo , NADPH Oxidases/genética , Oxilipinas/metabolismo , Células Vegetais/efeitos dos fármacos , Folhas de Planta/citologia , Folhas de Planta/efeitos dos fármacos , Raízes de Plantas/citologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Transdução de Sinais
8.
Nat Plants ; 7(6): 826-841, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34112988

RESUMO

The leaf epidermis is a dynamic biomechanical shell that integrates growth across spatial scales to influence organ morphology. Pavement cells, the fundamental unit of this tissue, morph irreversibly into highly lobed cells that drive planar leaf expansion. Here, we define how tissue-scale cell wall tensile forces and the microtubule-cellulose synthase systems dictate the patterns of interdigitated growth in real time. A morphologically potent subset of cortical microtubules span the periclinal and anticlinal cell faces to pattern cellulose fibres that generate a patch of anisotropic wall. The subsequent local polarized growth is mechanically coupled to the adjacent cell via a pectin-rich middle lamella, and this drives lobe formation. Finite element pavement cell models revealed cell wall tensile stress as an upstream patterning element that links cell- and tissue-scale biomechanical parameters to interdigitated growth. Cell lobing in leaves is evolutionarily conserved, occurs in multiple cell types and is associated with important agronomic traits. Our general mechanistic models of lobe formation provide a foundation to analyse the cellular basis of leaf morphology and function.


Assuntos
Arabidopsis/citologia , Células Vegetais , Folhas de Planta/citologia , Folhas de Planta/crescimento & desenvolvimento , Arabidopsis/crescimento & desenvolvimento , Fenômenos Biomecânicos , Forma Celular , Parede Celular/fisiologia , Celulose/metabolismo , Análise de Elementos Finitos , Microscopia Eletrônica de Transmissão , Microtúbulos/metabolismo , Modelos Biológicos , Mutação , Células Vegetais/metabolismo , Plantas Geneticamente Modificadas , Plasmodesmos
9.
EMBO J ; 40(15): e107455, 2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34152631

RESUMO

Plant microRNAs (miRNAs) guide cytosolic post-transcriptional gene silencing of sequence-complementary transcripts within the producing cells, as well as in distant cells and tissues. Here, we used an artificial miRNA-based system (amiRSUL) in Arabidopsis thaliana to explore the still elusive mechanisms of inter-cellular miRNA movement via forward genetics. This screen identified many mutant alleles of HASTY (HST), the ortholog of mammalian EXPORTIN5 (XPO5) with a recently reported role in miRNA biogenesis in Arabidopsis. In both epidermis-peeling and grafting assays, amiRSUL levels were reduced much more substantially in miRNA-recipient tissues than in silencing-emitting tissues. We ascribe this effect to HST controlling cell-to-cell and phloem-mediated movement of the processed amiRSUL, in addition to regulating its biogenesis. While HST is not required for the movement of free GFP or siRNAs, its cell-autonomous expression in amiRSUL-emitting tissues suffices to restore amiRSUL movement independently of its nucleo-cytosolic shuttling activity. By contrast, HST is dispensable for the movement and activity of amiRSUL within recipient tissues. Finally, HST enables movement of endogenous miRNAs that display mostly unaltered steady-state levels in hst mutant tissues. We discuss a role for HST as a hitherto unrecognized regulator of miRNA movement in relation to its recently assigned nuclear function at the nexus of MIRNA transcription and miRNA processing.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Carioferinas/metabolismo , MicroRNAs/metabolismo , Arabidopsis/citologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Transporte Biológico/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Carioferinas/genética , Mutação , Floema/citologia , Floema/genética , Células Vegetais , Raízes de Plantas/citologia , Raízes de Plantas/genética , Plantas Geneticamente Modificadas , Interferência de RNA , RNA de Plantas , Xilema/citologia , Xilema/genética
10.
Science ; 372(6547): 1176-1181, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-34112688

RESUMO

How eukaryotic cells assess and maintain sizes specific for their species and cell type remains unclear. We show that in the Arabidopsis shoot stem cell niche, cell size variability caused by asymmetric divisions is corrected by adjusting the growth period before DNA synthesis. KIP-related protein 4 (KRP4) inhibits progression to DNA synthesis and associates with mitotic chromosomes. The F BOX-LIKE 17 (FBL17) protein removes excess KRP4. Consequently, daughter cells are born with comparable amounts of KRP4. Inhibitor dilution models predicted that KRP4 inherited through chromatin would robustly regulate size, whereas inheritance of excess free KRP4 would disrupt size homeostasis, as confirmed by mutant analyses. We propose that a cell cycle regulator, stabilized by association with mitotic chromosomes, reads DNA content as a cell size-independent scale.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Proteínas Inibidoras de Quinase Dependente de Ciclina/metabolismo , DNA de Plantas/metabolismo , Meristema/citologia , Células Vegetais/fisiologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Divisão Celular Assimétrica , Ciclo Celular , Pontos de Checagem do Ciclo Celular , Divisão Celular , Tamanho Celular , Cromatina/metabolismo , Cromossomos de Plantas/metabolismo , Proteínas Inibidoras de Quinase Dependente de Ciclina/genética , Replicação do DNA , Proteínas F-Box/metabolismo , Fase G1 , Mitose , Modelos Biológicos , Mutação , Fase S
11.
Int J Mol Sci ; 22(9)2021 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-34064353

RESUMO

The lipid bilayer matrix of the thylakoid membrane of cyanobacteria and chloroplasts of plants and algae is mainly composed of uncharged galactolipids, but also contains anionic lipids sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG) as major constituents. The necessity of PG for photosynthesis is evident in all photosynthetic organisms examined to date, whereas the requirement of SQDG varies with species. In plants, although PG and SQDG are also found in non-photosynthetic plastids, their importance for the growth and functions of non-photosynthetic organs remains unclear. In addition, plants synthesize another anionic lipid glucuronosyldiacylglycerol (GlcADG) during phosphorus starvation, but its role in plant cells is not elucidated yet. To understand the functional relationships among PG, SQDG, and GlcADG, we characterized several Arabidopsis thaliana mutants defective in biosynthesis of these lipids. The mutants completely lacking both PG and SQDG biosynthesis in plastids showed developmental defects of roots, hypocotyls, and embryos in addition to leaves, which suggests that these lipids are pleiotropically required for the development of both photosynthetic and non-photosynthetic organs. Furthermore, our analysis revealed that SQDG, but not GlcADG, is essential for complementing the role of PG, particularly in photosynthesis under PG-deficient conditions such as phosphorus starvation.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/metabolismo , Cloroplastos/metabolismo , Diglicerídeos/metabolismo , Glicolipídeos/metabolismo , Fosfatidilgliceróis/metabolismo , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/metabolismo , Cloroplastos/genética , Cianobactérias/genética , Cianobactérias/metabolismo , Galactolipídeos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Hipocótilo/citologia , Hipocótilo/crescimento & desenvolvimento , Hipocótilo/metabolismo , Mutação , Células Vegetais/metabolismo , Folhas de Planta/citologia , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Sementes/citologia , Sementes/crescimento & desenvolvimento , Sementes/metabolismo
12.
Nat Plants ; 7(6): 730-738, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34059805

RESUMO

Seeds are a key life cycle stage for many plants. Seeds are also the basis of agriculture and the primary source of calories consumed by humans1. Here, we employ single-nucleus RNA-sequencing to generate a transcriptional atlas of developing Arabidopsis thaliana seeds, with a focus on endosperm. Endosperm, the primary site of gene imprinting in flowering plants, mediates the relationship between the maternal parent and the embryo2. We identify transcriptionally uncharacterized nuclei types in the chalazal endosperm, which interfaces with maternal tissue for nutrient unloading3,4. We demonstrate that the extent of parental bias of maternally expressed imprinted genes varies with cell-cycle phase, and that imprinting of paternally expressed imprinted genes is strongest in chalazal endosperm. Thus, imprinting is spatially and temporally heterogeneous. Increased paternal expression in the chalazal region suggests that parental conflict, which is proposed to drive imprinting evolution, is fiercest at the boundary between filial and maternal tissues.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Impressão Genômica , Sementes/genética , Arabidopsis/citologia , Arabidopsis/metabolismo , Ciclo Celular/genética , Núcleo Celular/genética , Endosperma/genética , Regulação da Expressão Gênica de Plantas , Sementes/citologia , Sementes/metabolismo , Análise de Sequência de RNA , Análise de Célula Única
13.
PLoS Genet ; 17(5): e1009561, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33999950

RESUMO

The DEFECTIVE EMBRYO AND MERISTEMS 1 (DEM1) gene encodes a protein of unknown biochemical function required for meristem formation and seedling development in tomato, but it was unclear whether DEM1's primary role was in cell division or alternatively, in defining the identity of meristematic cells. Genome sequence analysis indicates that flowering plants possess at least two DEM genes. Arabidopsis has two DEM genes, DEM1 and DEM2, which we show are expressed in developing embryos and meristems in a punctate pattern that is typical of genes involved in cell division. Homozygous dem1 dem2 double mutants were not recovered, and plants carrying a single functional DEM1 allele and no functional copies of DEM2, i.e. DEM1/dem1 dem2/dem2 plants, exhibit normal development through to the time of flowering but during male reproductive development, chromosomes fail to align on the metaphase plate at meiosis II and result in abnormal numbers of daughter cells following meiosis. Additionally, these plants show defects in both pollen and embryo sac development, and produce defective male and female gametes. In contrast, dem1/dem1 DEM2/dem2 plants showed normal levels of fertility, indicating that DEM2 plays a more important role than DEM1 in gamete viability. The increased importance of DEM2 in gamete viability correlated with higher mRNA levels of DEM2 compared to DEM1 in most tissues examined and particularly in the vegetative shoot apex, developing siliques, pollen and sperm. We also demonstrate that gamete viability depends not only on the number of functional DEM alleles inherited following meiosis, but also on the number of functional DEM alleles in the parent plant that undergoes meiosis. Furthermore, DEM1 interacts with RAS-RELATED NUCLEAR PROTEIN 1 (RAN1) in yeast two-hybrid and pull-down binding assays, and we show that fluorescent proteins fused to DEM1 and RAN1 co-localize transiently during male meiosis and pollen development. In eukaryotes, RAN is a highly conserved GTPase that plays key roles in cell cycle progression, spindle assembly during cell division, reformation of the nuclear envelope following cell division, and nucleocytoplasmic transport. Our results demonstrate that DEM proteins play an essential role in cell division in plants, most likely through an interaction with RAN1.


Assuntos
Arabidopsis/citologia , Arabidopsis/genética , Genes Essenciais , Genes de Plantas/genética , Células Germinativas/metabolismo , Alelos , Proteínas de Arabidopsis/metabolismo , Divisão Celular , Sobrevivência Celular/genética , Evolução Molecular , Dosagem de Genes , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Células Germinativas/citologia , Meiose , Família Multigênica , Especificidade de Órgãos , Pólen/crescimento & desenvolvimento , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo , Sementes , Transgenes , Proteína ran de Ligação ao GTP/metabolismo
14.
Plant Signal Behav ; 16(8): 1922796, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-33938395

RESUMO

In the physiological range, the phytohormone auxin inhibits the growth of underground tissues. In the roots of Arabidopsis thaliana, cell size inhibition has been shown to be accompanied by auxin-mediated reduction of vacuole size. A tonoplast-localized protein family (Networked 4) with actin-binding capacity was demonstrated to modulate the compactness of the vacuole. Overexpression of NET4A led to smaller, more spherical and compact vacuoles, which occupied less cellular space compared to wild type. This reduction of vacuolar occupancy is similar to the observed auxin-induced decrease in occupancy, albeit there are enormous morphological differences. Here, we show that a net4a net4b double mutant and a NET4A overexpressor line are still sensitive to auxin-induced vacuolar constrictions. However, the overexpressor showed a partial auxin resistance accompanied by more compact vacuoles, thereby indicating an additional regulatory mechanism. Furthermore, we show that other NET superfamily members do not compensate for the loss of NET4A and NET4B expression on the transcriptional level. This leads us to hypothesize that regulation of vacuole size is a general mechanism to regulate cell expansion and that other players besides NET4 must participate in regulating the vacuole-cytoskeleton interface.


Assuntos
Citoesqueleto de Actina , Actinas/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Processos de Crescimento Celular , Raízes de Plantas/citologia , Vacúolos , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Arabidopsis/ultraestrutura , Ácidos Indolacéticos/metabolismo , Microtúbulos , Células Vegetais , Desenvolvimento Vegetal , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Raízes de Plantas/ultraestrutura
15.
Nat Commun ; 12(1): 2650, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976192

RESUMO

Live cell imaging using fluorescent DNA markers are an indispensable molecular tool in various biological and biomedical fields. It is a challenge to develop DNA probes that avoid UV light photo-excitation, have high specificity for DNA, are cell-permeable and are compatible with cutting-edge imaging techniques such as super-resolution microscopy. Herein, we present N-aryl pyrido cyanine (N-aryl-PC) derivatives as a class of long absorption DNA markers with absorption in the wide range of visible light. The high DNA specificity and membrane permeability allow the staining of both organelle DNA as well as nuclear DNA, in various cell types, including plant tissues, without the need for washing post-staining. N-aryl-PC dyes are also highly compatible with a separation of photon by lifetime tuning method in stimulated emission depletion microscopy (SPLIT-STED) for super-resolution imaging as well as two-photon microscopy for deep tissue imaging, making it a powerful tool in the life sciences.


Assuntos
Núcleo Celular/química , DNA/química , Corantes Fluorescentes/química , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Organelas/química , Animais , Arabidopsis/citologia , Benzimidazóis/química , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Células Cultivadas , DNA/genética , DNA/metabolismo , Fluorescência , Células HeLa , Humanos , Camundongos , Microscopia Confocal/métodos , Estrutura Molecular , Células NIH 3T3 , Organelas/metabolismo , Coloração e Rotulagem/métodos , Imagem com Lapso de Tempo/métodos
16.
PLoS Genet ; 17(5): e1008919, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34003859

RESUMO

An essential component of the homologous recombination machinery in eukaryotes, the RAD54 protein is a member of the SWI2/SNF2 family of helicases with dsDNA-dependent ATPase, DNA translocase, DNA supercoiling and chromatin remodelling activities. It is a motor protein that translocates along dsDNA and performs multiple functions in homologous recombination. In particular, RAD54 is an essential cofactor for regulating RAD51 activity. It stabilizes the RAD51 nucleofilament, remodels nucleosomes, and stimulates the homology search and strand invasion activities of RAD51. Accordingly, deletion of RAD54 has dramatic consequences on DNA damage repair in mitotic cells. In contrast, its role in meiotic recombination is less clear. RAD54 is essential for meiotic recombination in Drosophila and C. elegans, but plays minor roles in yeast and mammals. We present here characterization of the roles of RAD54 in meiotic recombination in the model plant Arabidopsis thaliana. Absence of RAD54 has no detectable effect on meiotic recombination in otherwise wild-type plants but RAD54 becomes essential for meiotic DSB repair in absence of DMC1. In Arabidopsis, dmc1 mutants have an achiasmate meiosis, in which RAD51 repairs meiotic DSBs. Lack of RAD54 leads to meiotic chromosomal fragmentation in absence of DMC1. The action of RAD54 in meiotic RAD51 activity is thus mainly downstream of the role of RAD51 in supporting the activity of DMC1. Equivalent analyses show no effect on meiosis of combining dmc1 with the mutants of the RAD51-mediators RAD51B, RAD51D and XRCC2. RAD54 is thus required for repair of meiotic DSBs by RAD51 and the absence of meiotic phenotype in rad54 plants is a consequence of RAD51 playing a RAD54-independent supporting role to DMC1 in meiotic recombination.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Arabidopsis/metabolismo , Quebras de DNA de Cadeia Dupla , DNA Helicases/metabolismo , Meiose , Rad51 Recombinase/metabolismo , Reparo de DNA por Recombinação , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA Helicases/deficiência , DNA Helicases/genética , Proteínas de Ligação a DNA , Genes Essenciais , Meiose/genética , Mutação , Rad51 Recombinase/genética , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Proteínas Repressoras
17.
Nat Plants ; 7(6): 739-747, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34031540

RESUMO

Spatiotemporal control of cell division is essential for the growth and development of multicellular organisms. In plant cells, proper cell plate insertion during cytokinesis relies on the premitotic establishment of the division plane at the cell cortex. Two plant-specific cytoskeleton arrays, the preprophase band (PPB) and the phragmoplast, play important roles in division-plane orientation and cell plate formation, respectively1. Microtubule organization and dynamics and their communication with membranes at the cortex and cell plate are coordinated by multiple, mostly distinct microtubule-associated proteins2. How division-plane selection and establishment are linked, however, is still unknown. Here, we report members of the Arabidopsis IQ67 DOMAIN (IQD) family3 as microtubule-targeted proteins that localize to the PPB and phragmoplast and additionally reside at the cell plate and a polarized cortical region including the cortical division zone (CDZ). IQDs physically interact with PHRAGMOPLAST ORIENTING KINESIN (POK) proteins4,5 and PLECKSTRIN HOMOLOGY GTPase ACTIVATING (PHGAP) proteins6, which are core components of the CDZ1. The loss of IQD function impairs PPB formation and affects CDZ recruitment of POKs and PHGAPs, resulting in division-plane positioning defects. We propose that IQDs act as cellular scaffolds that facilitate PPB formation and CDZ set-up during symmetric cell division.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Dinitrobenzenos , Regulação da Expressão Gênica de Plantas , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Mutação , Filogenia , Células Vegetais/efeitos dos fármacos , Células Vegetais/metabolismo , Plantas Geneticamente Modificadas , Prófase , Domínios Proteicos , Sulfanilamidas , Tabaco/genética , Proteínas de Transporte Vesicular/metabolismo
18.
Nature ; 592(7854): 433-437, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33790463

RESUMO

Upon gamete fusion, animal egg cells secrete proteases from cortical granules to establish a fertilization envelope as a block to polyspermy1-4. Fertilization in flowering plants is more complex and involves the delivery of two non-motile sperm cells by pollen tubes5,6. Simultaneous penetration of ovules by multiple pollen tubes (polytubey) is usually avoided, thus indirectly preventing polyspermy7,8. How plant egg cells regulate the rejection of extra tubes after successful fertilization is not known. Here we report that the aspartic endopeptidases ECS1 and ECS2 are secreted to the extracellular space from a cortical network located at the apical domain of the Arabidopsis egg cell. This reaction is triggered only after successful fertilization. ECS1 and ECS2 are exclusively expressed in the egg cell and transcripts are degraded immediately after gamete fusion. ECS1 and ESC2 specifically cleave the pollen tube attractor LURE1. As a consequence, polytubey is frequent in ecs1 ecs2 double mutants. Ectopic secretion of these endopeptidases from synergid cells led to a decrease in the levels of LURE1 and reduced the rate of pollen tube attraction. Together, these findings demonstrate that plant egg cells sense successful fertilization and elucidate a mechanism as to how a relatively fast post-fertilization block to polytubey is established by fertilization-induced degradation of attraction factors.


Assuntos
Arabidopsis/metabolismo , Endopeptidases/metabolismo , Fertilização , Óvulo Vegetal/metabolismo , Tubo Polínico/metabolismo , Pólen/metabolismo , Arabidopsis/citologia , Arabidopsis/enzimologia , Proteínas de Arabidopsis/metabolismo , Fusão Celular , Óvulo Vegetal/enzimologia , Pólen/enzimologia
19.
Plant Cell ; 33(4): 1151-1160, 2021 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-33793916

RESUMO

The seeds of flowering plants contain three genetically distinct structures: the embryo, endosperm, and seed coat. The embryo and endosperm need to interact and exchange signals to ensure coordinated growth. Accumulating evidence has confirmed that embryo growth is supported by the nourishing endosperm and regulated by signals originating from the endosperm. Available data also support that endosperm development requires communication with the embryo. Here, using single-fertilization mutants, Arabidopsis thaliana dmp8 dmp9 and gex2, we demonstrate that in the absence of a zygote and embryo, endosperm initiation, syncytium formation, free nuclear cellularization, and endosperm degeneration occur as in the wild type in terms of the cytological process and time course. Although rapid embryo expansion accelerates endosperm breakdown, our findings strongly suggest that endosperm development is an autonomously organized process, independent of egg cell fertilization and embryo-endosperm communication. This work confirms both the altruistic and self-directed nature of the endosperm during coordinated embryo-endosperm development. Our findings provide insights into the intricate interaction between the two fertilization products and will help to distinguish the physiological roles of the signaling between endosperm and embryo. These findings also open new avenues in agro-biotechnology for crop improvement.


Assuntos
Arabidopsis/crescimento & desenvolvimento , Endosperma/crescimento & desenvolvimento , Sementes/citologia , Sementes/crescimento & desenvolvimento , Arabidopsis/citologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Endosperma/citologia , Endosperma/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Membrana/genética , Células Vegetais , Plantas Geneticamente Modificadas , Sementes/genética , Zigoto/crescimento & desenvolvimento
20.
Plant Cell ; 33(4): 1361-1380, 2021 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-33793856

RESUMO

Aluminum (Al) toxicity and inorganic phosphate (Pi) limitation are widespread chronic abiotic and mutually enhancing stresses that profoundly affect crop yield. Both stresses strongly inhibit root growth, resulting from a progressive exhaustion of the stem cell niche. Here, we report on a casein kinase 2 (CK2) inhibitor identified by its capability to maintain a functional root stem cell niche in Arabidopsis thaliana under Al toxic conditions. CK2 operates through phosphorylation of the cell cycle checkpoint activator SUPPRESSOR OF GAMMA RADIATION1 (SOG1), priming its activity under DNA-damaging conditions. In addition to yielding Al tolerance, CK2 and SOG1 inactivation prevents meristem exhaustion under Pi starvation, revealing the existence of a low Pi-induced cell cycle checkpoint that depends on the DNA damage activator ATAXIA-TELANGIECTASIA MUTATED (ATM). Overall, our data reveal an important physiological role for the plant DNA damage response pathway under agriculturally limiting growth conditions, opening new avenues to cope with Pi limitation.


Assuntos
Alumínio/toxicidade , Arabidopsis/citologia , Arabidopsis/efeitos dos fármacos , Caseína Quinase II/metabolismo , Fosfatos/metabolismo , Alumínio/farmacocinética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Caseína Quinase II/genética , Peptídeos e Proteínas de Sinalização Intercelular , Fosfatos/farmacologia , Fosforilação , Células Vegetais/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
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