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1.
Mol Plant ; 2021 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-34740849

RESUMO

The signaling pathway of the gaseous hormone ethylene is involved in plant reproduction, growth, development, and stress responses. During reproduction, the two synergid cells of the angiosperm female gametophyte both undergo programmed cell death (PCD)/degeneration but in a different manner: PCD/degeneration of one synergid facilitates pollen tube rupture and thereby the release of sperm cells, while PCD/degeneration of the other synergid blocks supernumerary pollen tubes. Ethylene signaling was postulated to participate in some of the synergid cell functions, such as pollen tube attraction and the induction of PCD/degeneration. However, ethylene-mediated induction of synergid PCD/degeneration and the role of ethylene itself have not been firmly established. Here, we employed the CRISPR/Cas9 technology to knock out the five ethylene-biosynthesis 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) genes and created Arabidopsis mutants free of ethylene production. The ethylene-free mutant plants showed normal triple responses when treated with ethylene rather than 1-aminocyclopropane-1-carboxylic acid, but had increased lateral root density and enlarged petal sizes, which are typical phenotypes of mutants defective in ethylene signaling. Using these ethylene-free plants, we further demonstrated that production of ethylene is not necessarily required to trigger PCD/degeneration of the two synergid cells, but certain components of ethylene signaling including transcription factors ETHYLENE-INSENSITIVE 3 (EIN3) and EIN3-LIKE 1 (EIL1) are necessary for the death of the persistent synergid cell.

2.
Trends Plant Sci ; 26(10): 993-995, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34246552

RESUMO

Accurate communication at the stigma surface is required to promote plants' own pollen and reject foreign pollen. Liu et al. have now discovered an autocrine signaling pathway at the surface of arabidopsis stigmatic papillae, accumulating ROS. Downregulation of ROS production via an antagonistic peptide from the pollen coat promotes pollen hydration and germination.


Assuntos
Arabidopsis , Tubo Polínico , Arabidopsis/genética , Percepção , Polinização , Espécies Reativas de Oxigênio
3.
Nat Plants ; 7(8): 1143-1159, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34253868

RESUMO

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


Assuntos
Embriófitas/crescimento & desenvolvimento , Embriófitas/genética , Perfilação da Expressão Gênica , Magnoliopsida/crescimento & desenvolvimento , Magnoliopsida/genética , Organogênese Vegetal/genética , Reprodução/genética , Regulação da Expressão Gênica de Plantas , Variação Genética , Genótipo , Organogênese Vegetal/fisiologia , Fenótipo , Proteínas de Plantas/metabolismo , Reprodução/fisiologia , Análise de Sequência de RNA , Fatores de Transcrição/metabolismo
4.
Plant Cell ; 33(9): 3042-3056, 2021 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-34125904

RESUMO

In eukaryotes, homotypic fusion and vacuolar protein sorting (HOPS) as well as class C core vacuole/endosome tethering (CORVET) are evolutionarily conserved membrane tethering complexes that play important roles in lysosomal/vacuolar trafficking. Whether HOPS and CORVET control endomembrane trafficking in pollen tubes, the fastest growing plant cells, remains largely elusive. In this study, we demonstrate that the four core components shared by the two complexes, Vacuole protein sorting 11 (VPS11), VPS16, VPS33, and VPS18, are all essential for pollen tube growth in Arabidopsis thaliana and thus for plant reproduction success. We used VPS18 as a representative core component of the complexes to show that the protein is localized to both multivesicular bodies (MVBs) and the tonoplast in a growing pollen tube. Mutant vps18 pollen tubes grew more slowly in vivo, resulting in a significant reduction in male transmission efficiency. Additional studies revealed that membrane fusion from MVBs to vacuoles is severely compromised in vps18 pollen tubes, corroborating the function of VPS18 in late endocytic trafficking. Furthermore, vps18 pollen tubes produce excessive exocytic vesicles at the apical zone and excessive amounts of pectin and pectin methylesterases in the cell wall. In conclusion, this study establishes an additional conserved role of HOPS/CORVET in homotypic membrane fusion during vacuole biogenesis in pollen tubes and reveals a feedback regulation of HOPS/CORVET in the secretion of cell wall modification enzymes of rapidly growing plant cells.

5.
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
6.
Plant Physiol ; 186(2): 865-873, 2021 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-33638984

RESUMO

Reproductive isolation is a prerequisite to form and maintain a new species. Multiple prezygotic and postzygotic reproductive isolation barriers have been reported in plants. In the model plant, Arabidopsis thaliana conspecific pollen tube precedence controlled by AtLURE1/PRK6-mediated signaling has been recently reported as a major prezygotic reproductive isolation barrier. By accelerating emergence of own pollen tubes from the transmitting tract, A. thaliana ovules promote self-fertilization and thus prevent fertilization by a different species. Taking advantage of a septuple atlure1null mutant, we now report on the role of AtLURE1/PRK6-mediated signaling for micropylar pollen tube guidance. Compared with wild-type (WT) ovules, atlure1null ovules displayed remarkably reduced micropylar pollen tube attraction efficiencies in modified semi-in vivo A. thaliana ovule targeting assays. However, when prk6 mutant pollen tubes were applied, atlure1null ovules showed micropylar attraction efficiencies comparable to that of WT ovules. These findings indicate that AtLURE1/PRK6-mediated signaling regulates micropylar pollen tube attraction in addition to promoting emergence of own pollen tubes from the transmitting tract. Moreover, semi-in vivo ovule targeting competition assays with the same amount of pollen grains from both A. thaliana and Arabidopsis lyrata showed that A. thaliana WT and xiuqiu mutant ovules are mainly targeted by own pollen tubes and that atlure1null mutant ovules are also entered to a large extent by A. lyrata pollen tubes. Taken together, we report that AtLURE1/PRK6-mediated signaling promotes conspecific micropylar pollen tube attraction representing an additional prezygotic isolation barrier.

7.
Annu Rev Plant Biol ; 72: 641-676, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-33606951

RESUMO

Following fertilization in flowering plants (angiosperms), egg and sperm cells unite to form the zygote, which generates an entire new organism through a process called embryogenesis. In this review, we provide a comparative perspective on early zygotic embryogenesis in flowering plants by using the Poaceae maize and rice as monocot grass and crop models as well as Arabidopsis as a eudicot model of the Brassicaceae family. Beginning with the activation of the egg cell, we summarize and discuss the process of maternal-to-zygotic transition in plants, also taking recent work on parthenogenesis and haploid induction into consideration. Aspects like imprinting, which is mainly associated with endosperm development and somatic embryogenesis, are not considered. Controversial findings about the timing of zygotic genome activation as well as maternal versus paternal contribution to zygote and early embryo development are highlighted. The establishment of zygotic polarity, asymmetric division, and apical and basal cell lineages represents another chapter in which we also examine and compare the role of major signaling pathways, cell fate genes, and hormones in early embryogenesis. Except for the model Arabidopsis, little is known about embryopatterning and the establishment of the basic body plan in angiosperms. Using available in situ hybridization, RNA-sequencing, and marker data, we try to compare how and when stem cell niches are established. Finally, evolutionary aspects of plant embryo development are discussed.


Assuntos
Magnoliopsida , Linhagem da Célula , Desenvolvimento Embrionário , Regulação da Expressão Gênica de Plantas , Sementes
9.
Plant Reprod ; 34(1): 47-60, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33258014

RESUMO

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


Assuntos
Magnoliopsida , Tubo Polínico , Óvulo Vegetal , Peptídeos , Tabaco , Zea mays
10.
Methods Mol Biol ; 2200: 371-390, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33175388

RESUMO

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


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Mutação/genética , Sementes/genética
11.
J Chem Phys ; 153(21): 214114, 2020 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-33291918

RESUMO

We derive an electron-vibration model Hamiltonian in a quantum chemical framework and explore the extent to which such a Hamiltonian can capture key effects of nonadiabatic dynamics. The model Hamiltonian is a simple two-body operator, and we make preliminary steps at applying standard quantum chemical methods to evaluate its properties, including mean-field theory, linear response, and a primitive correlated model. The Hamiltonian can be compared to standard vibronic Hamiltonians, but it is constructed without reference to potential energy surfaces through direct differentiation of the one- and two-electron integrals at a single reference geometry. The nature of the model Hamiltonian in the harmonic and linear-coupling regime is investigated for pyrazine, where a simple time-dependent calculation including electron-vibration correlation is demonstrated to exhibit the well-studied population transfer between the S2 and S1 excited states.

12.
Nat Plants ; 6(10): 1275-1288, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33020609

RESUMO

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


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas do Domínio Armadillo/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Transdução de Sinais , Proteínas rho de Ligação ao GTP/metabolismo , Arabidopsis/citologia , Arabidopsis/enzimologia , Arabidopsis/crescimento & desenvolvimento , Polaridade Celular , Proteínas de Ligação a DNA/metabolismo , Evolução Molecular , Raízes de Plantas/citologia , Raízes de Plantas/metabolismo , Tricomas/citologia , Tricomas/metabolismo
13.
Plant Physiol ; 184(4): 1640-1657, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32989009

RESUMO

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


Assuntos
Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Germinação/genética , Magnoliopsida/crescimento & desenvolvimento , Magnoliopsida/genética , Pólen/crescimento & desenvolvimento , Pólen/genética , Evolução Biológica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Germinação/fisiologia , Tubo Polínico/genética , Tubo Polínico/crescimento & desenvolvimento , Proteômica , Transcriptoma
14.
Methods Mol Biol ; 2166: 3-21, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32710400

RESUMO

To understand the development and differentiation processes within a tissue and a cell, analysis of the cell type-specific gene expression pattern as well as the subcellular localization of the produced RNAs is essential. The simplest and fastest method to visualize RNA molecules is in situ hybridization (ISH) on whole-tissue samples. Over the past 40 years, various labeling and visualization techniques have been established to analyze either the expression domain of genes in tissues (using the classical chromogenic detection system) or the specific subcellular localization of mRNAs (using fluorescently labeled probes). By using the Arabidopsis root tip as an example tissue, we describe and compare classic in situ hybridization techniques. The protocols described can be easily transferred to almost all other tissues or model organism with slight modifications.


Assuntos
Corantes Fluorescentes/química , Hibridização in Situ Fluorescente/métodos , Sondas RNA/metabolismo , RNA/genética , Coloração e Rotulagem/métodos , Fixação de Tecidos/métodos , Arabidopsis/metabolismo , Corantes Fluorescentes/metabolismo , Peroxidase do Rábano Silvestre/metabolismo , Raízes de Plantas/metabolismo
15.
Plant Cell ; 32(5): 1449-1463, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32152189

RESUMO

Transcript elongation factors associate with elongating RNA polymerase II (RNAPII) to control the efficiency of mRNA synthesis and consequently modulate plant growth and development. Encountering obstacles during transcription such as nucleosomes or particular DNA sequences may cause backtracking and transcriptional arrest of RNAPII. The elongation factor TFIIS stimulates the intrinsic transcript cleavage activity of the polymerase, which is required for efficient rescue of backtracked/arrested RNAPII. A TFIIS mutant variant (TFIISmut) lacks the stimulatory activity to promote RNA cleavage, but instead efficiently inhibits unstimulated transcript cleavage by RNAPII. We could not recover viable Arabidopsis (Arabidopsis thaliana) tfIIs plants constitutively expressing TFIISmut. Induced, transient expression of TFIISmut in tfIIs plants provoked severe growth defects, transcriptomic changes and massive, transcription-related redistribution of elongating RNAPII within transcribed regions toward the transcriptional start site. The predominant site of RNAPII accumulation overlapped with the +1 nucleosome, suggesting that upon inhibition of RNA cleavage activity, RNAPII arrest prevalently occurs at this position. In the presence of TFIISmut, the amount of RNAPII was reduced, which could be reverted by inhibiting the proteasome, indicating proteasomal degradation of arrested RNAPII. Our findings suggest that polymerase backtracking/arrest frequently occurs in plant cells, and RNAPII-reactivation is essential for correct transcriptional output and proper growth/development.


Assuntos
Arabidopsis/enzimologia , Arabidopsis/genética , RNA Polimerase II/metabolismo , Elongação da Transcrição Genética , Sequência de Aminoácidos , Arabidopsis/crescimento & desenvolvimento , Núcleo Celular/metabolismo , Proliferação de Células , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Genética , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/metabolismo , Transcriptoma/genética
16.
Methods Mol Biol ; 2122: 191-203, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31975304

RESUMO

Compared with small model plants like Arabidopsis containing ovules with few cell layers, embryo sac and embryo development of model crop plants such as maize and other grasses are difficult to image. Multiple layers of tissue usually surround the deeply embedded embryo sac and developing embryo. Moreover, reliable cell biological marker lines labeling, for example, nuclei, plasma membrane, cell walls, or cells of a specific identity are often not available. The introduction of markers to study mutants is difficult and time-consuming and may require several generations of backcrosses. In this chapter, we therefore present an easy protocol to image maize ovaries and developing embryo sacs before and after fertilization allowing also high-throughput mutant analysis. The laborious embedding of samples and preparation of thin sections are omitted in this fixing-Feulgen staining-clearing (FFC) method. Optical sectioning through multiple layers of tissue is possible allowing 3D reconstructions of the whole embryo sac if necessary. The advantage of staining cell nuclei using the FFC method described here compared, for example, with DAPI staining is a wide range of Schiff's type reagents available for the Feulgen reaction. Depending on the reagent of choice, various conditions such as different excitation/emission filters or even white light can be applied for imaging. Moreover, in order to better visualize cell division, nuclei polarity as well as cell extent and integrity, periodic acid staining (PAS) of cell walls can be combined with Feulgen staining.


Assuntos
Corantes de Rosanilina/análise , Sementes/embriologia , Coloração e Rotulagem/métodos , Zea mays/embriologia , Microscopia/métodos , Sementes/ultraestrutura , Fixação de Tecidos/métodos , Zea mays/ultraestrutura
17.
Methods Mol Biol ; 2072: 141-156, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31541444

RESUMO

The study of heritable genetic changes that do not implicate alterations in the DNA sequence-epigenetics-represents one of the most prolific and expanding fields in plant biology during the last two decades. With a focus on DNA methylation and histone modifications, recent advances also reported the identification of epigenetic regulatory mechanisms that control reproductive development in cereal crop plants. One of the most powerful methods to selectively study interactions between epigenetic factors or specific proteins bound to genomic DNA regions is called chromatin immunoprecipitation (ChIP). ChIP can be widely used to determine the presence of particular histones with posttranslational modifications at specific genomic regions or whether and where specific DNA-binding proteins including transcription factors interact with candidate target genes. ChIP is also an exciting tool to study and compare chromatin states under normal and stress conditions. Here, we present a detailed step-by-step ChIP assay to investigate epigenetic chromatin marks during vegetative and reproductive development in cereals. However, the method described here can be used for all plant tissues and plant species.


Assuntos
Imunoprecipitação da Cromatina , Grão Comestível/genética , Epigênese Genética , Epigenômica , Desenvolvimento Vegetal/genética , Reprodução/genética , Imunoprecipitação da Cromatina/métodos , Sequenciamento de Cromatina por Imunoprecipitação , Metilação de DNA , Grão Comestível/metabolismo , Epigenômica/métodos , Código das Histonas , Histonas/metabolismo , Reação em Cadeia da Polimerase , Processamento de Proteína Pós-Traducional
18.
Front Plant Sci ; 10: 1469, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31824527

RESUMO

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

19.
Curr Biol ; 29(19): 3256-3265.e5, 2019 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-31564495

RESUMO

In angiosperms, two sperm cells are transported and delivered by the pollen tube to the ovule to achieve double fertilization. Extensive communication takes place between the pollen tube and the female tissues until the sperm cell cargo is ultimately released. During this process, a pollen tube surface-located receptor complex composed of ANXUR1/2 (ANX1/2) and Buddha's Paper Seal 1/2 (BUPS1/2) was reported to control the maintenance of pollen tube integrity by perceiving the autocrine peptide ligands rapid alkalinization factor 4 and 19 (RALF4/19). It was further hypothesized that pollen-tube rupture to release sperm is caused by the paracrine RALF34 peptide from the ovule interfering with this signaling pathway. In this study, we identified two Arabidopsis pollen-tube-expressed glycosylphosphatidylinositol-anchored proteins (GPI-APs), LORELEI-like-GPI-anchored protein 2 (LLG2) and LLG3, as co-receptors in the BUPS-ANX receptor complex. llg2 llg3 double mutants exhibit severe fertility defects. Mutant pollen tubes rupture early during the pollination process. Furthermore, LLG2 and LLG3 interact with ectodomains of both BUPSs and ANXURs, and this interaction is remarkably enhanced by the presence of RALF4/19 peptides. We further demonstrate that the N terminus (including a YISY motif) of the RALF4 peptide ligand interacts strongly with BUPS-ANX receptors but weakly with LLGs and is essential for its biological function, and its C-terminal region is sufficient for LLG binding. In conclusion, we propose that LLG2/3 serve as co-receptors during BUPS/ANX-RALF signaling and thereby further establish the importance of GPI-APs as key regulators in plant reproduction processes.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas Ligadas por GPI/genética , Tubo Polínico/crescimento & desenvolvimento , Transdução de Sinais , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas Ligadas por GPI/metabolismo , Ligantes
20.
Trends Plant Sci ; 24(11): 978-981, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31607472

RESUMO

RALFs are secreted peptides that are perceived by various CrRLK1L-LRE/LLG receptor complexes. The mechanistic basis of this perception has now been elucidated showing that the co-receptor LLG binds RALF23 to nucleate a FER receptor complex. This interaction likely occurs in other tissues where RALFs meet CrRLK1L receptors and LRE/LLG co-receptors.


Assuntos
Proteínas de Arabidopsis , Proteínas de Transporte , Peptídeos
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